HK1120548A - USE OF COMMON γ CHAIN CYTOKINES FOR THE VISUALIZATION, ISOLATION AND GENETIC MODIFICATION OF MEMORY T LYMPHOCYTES - Google Patents

Description

Application of common gamma chain cell factor in developing, separating and genetic modifying memory T lymphocyte

Brief introduction to the drawings

All components of antigen (Ag) -specific T-cells are tightly regulated by homeostatic mechanisms that ensure their persistence and functionality even in the absence of antigen. Upon encountering antigen, naive T cells undergo rapid clonal propagation to differentiate into effector T-cells (1, 2). The lifespan of effector T-cells is limited by cell death, which can occur upon encountering other antigens (activation-induced cell death) or due to the absence of survival factors. During the course of the immune response, memory T-cells are also generated. Memory T-cells can survive throughout life, providing long-lasting protection against recall pathogens (3). However, the frequency of antigen-specific memory T-cells in most biological samples is still below the detection limit for Ag/MHC (major histocompatibility complex) tetramer staining and functional assays (e.g., intracellular cytokine staining and ELISpot) (4, 5). In particular, Ag-specific CD4⁺T cells are mostly undetectable ex vivo and thus analyzed after multiple in vitro Ag-driven T cell propagation cycles. However, restimulation in vitro may favor terminal differentiation of cells, preventing their long-term survival. As a result, in vitro Ag re-stimulated T cells may also exhibit phenotypes that do not fully represent T cells found in vivo. For these reasons, there is a need for alternative strategies that improve the ex vivo detection of Ag-specific T cells, to better characterize ongoing immune responses, and to evaluate the immunocompetence of patients with immune-related disorders.

Some studies have demonstrated that the establishment and maintenance of T cell memory is controlled by cell-associated (Ag/MHC complexes) and soluble (cytokines) driven signals (3, 6, 7). The triggering of the TCR by both self and non-self Ag/MHC complexes regulates the conversion of naive cells to memory cells, the survival and proliferation of memory cells. MemoryThe lymphocyte population may be highly heterogeneous. Recently, class 2 memory T-cells have been identified: effector memory T-cells (CD45RA-CCR7-, CD62L-) and central memory T-cells, which are CD45 RA-negative cells characterized by the expression of CCR7 and CD62L (2 molecules required for the T-cell region leading to the second lymphoid organ). After antigenic stimulation, central memory T-cells produce low levels of effector cytokines such as IL-4 and IFN- γ, but high levels of IL-2, which maintain their rapid and consistent proliferation. Upon encountering antigen, central memory T-cells experience: 1) proliferation, leading to an auto-regenerative process aimed at increasing their population, and 2) differentiation, leading to the generation of effector memory T-cells, characterized by low proliferative potential, but capable of migrating to inflamed non-lymphoid tissues and mediating the effector phase of the immune response (8). Ag withdrawal is critical to avoid excessive TCR stimulation and activation-induced cell death and generation of central memory T cells. Cytokines that tightly regulate the survival, proliferation and apoptosis of human and murine T lymphocytes ensure proper T-cell homeostasis. Among the soluble factors, common gamma chain-binding cytokines (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) promote cell survival and homeostatic proliferation (2). More specifically, IL-2 maintains T-cell proliferation and apoptosis upon encountering antigen. TCR and IL-7-derived signals control proliferation and survival of naive and memory cells (7, 9-14). IL-7 can confer mature human naive and memory CD4 in the absence of TCR engagement⁺T cells are less susceptible to Fas-induced cell death (15). Furthermore, by inducing up-regulation of Bc1-2, it favors CD4 encountering Ag⁺T cell conversion to quiescent memory cells (9, 11). Finally, IL-15, in combination with anti-apoptotic activity, consistently acts to promote proliferation of naive and memory T cells (16). For these reasons, conventional gamma chain-binding cytokines have previously been used in combination with Ag-driven cell proliferation for the in vitro maintenance and propagation of Ag-specific T cell lines. In some cases, common gamma chain-binding cytokines are also used to improve the detection of Ag-specific T cells.

U.S. patent application US2005/0074822 relates to a method of detecting a population of antigen-specific T cells, wherein the cells are exposed to an antigen in the presence of a common gamma chain-binding cytokine. This approach does not allow for the propagation or enrichment of Ag-specific memory cells.

Disclosure of Invention

In contrast, in the present invention, the authors have investigated whether IL-7 and/or IL-15 would allow:

1) memory T cells accumulate in an antigen-free environment (and thus can be used in short term in vitro culture, without Ag-driven cell proliferation, to enrich a rare population of in vivo-pretreated antigen-specific T cells, each of which is specific for an antigen encountered in vivo), to identify pathogen/tumor/allergen/self-specific T cells. 2) Central memory cells multiply while maintaining their functional phenotype (and thus can be used to facilitate genetic modification of central memory lymphocytes by viral vectors).

To validate the invention, the authors used 3 unrelated preclinical animal models and validated on human samples. The first 2 models allowed counting Ag-specific T cells at the single cell level in the context of tumor disease (17) and dendritic cell-based vaccination (18). These models allow to validate the concept of T cell accumulation in vitro in antigen-free environment. The 3 rd model is based on the engraftment of human T cells in immunodeficient mice, allowing the assessment of the immunocompetence of genetically modified central memory T cells.

Model 1 to study tumor-specific T cell responses, the authors used a recently developed animal model (17). In this model, TS/A-LACK tumors (TS/A adenocarcinoma tumor cells expressing the Leishmania major-derived antigenic protein LACK) were grown in syngeneic BALB/c mice and LACK-specific T cells in peripheral lymphoid organs (lymph nodes, spleen or blood) were studied by flow cytometry using fluorescent LACK-peptide/MHC class II multimers. LACK-specific T cells can also be independently characterized by Ag-induced intracellular cytokine release in this model. TS/A-LACK-specific T cells can be followed in BALB/c mice and 16.2 β TCR transgenic mice, which express transgenic TCR β chains specific for LACK, making characterization of LACK-specific CD 4T-cell responses easier. Furthermore, TS/A cells naturally express the endogenous MuLV coat protein gp70, an immunodominant epitope of which has been described previously (AH-1, (19)). Thus, in addition to LACK-specific CD4T cell responses, AH-1-specific T cell responses could also be followed in TS/A-LACK tumor-bearing mice.

Model 2 to study vaccine-specific T cell responses, authors used IV pulsed bone marrow-derived Dendritic Cells (DCs) labeled with the virus SV 40-derived antigenic peptide, and vaccinated syngeneic C57BL/6 mice (18). In this model, marker IV-specific T cells were also characterized by antigen-specific cytokine secretion assays ex vivo to count marker IV-specific CD8T cell responses.

After the proposed short-term in vitro culture in the presence of IL-7 in the absence of Ag-driven cell stimulation, studies of Ag-specific CD4(LACK) and CD8(AH-1, marker IV) T cell responses were performed ex vivo. The optimal amount of IL-7 was used, with IL-2 or IL-15 as a control and IL-6, IL-10 and TNF- α as negative controls. Under all experimental conditions, the authors found that simple short-term culture of optimal amounts of IL-7 allowed the accumulation of in vivo pretreated Ag-specific T lymphocytes, without the need for Ag-driven cell proliferation and maintenance of the lymphocyte primitive phenotype. Most importantly, in some cases, short-term culture of IL-7 will reveal a rare population of Ag-specific T cells that cannot be detected by routine experimentation.

With respect to the results on validated human samples, T lymphocytes were derived from healthy donors and patients infected with mycobacterium tuberculosis, analyzed ex vivo after IL-7-driven short-term culture by antigen-specific cytokine release. In all cases, IL-7 favoured the accumulation of antigen-specific IL-2 and intermediate memory T cells producing IFN- γ by maintaining their proliferation and survival in vitro. IL-7 efficacy depends on antigen encounter in vivo, optimal cytokine levels and high cell density conditions, and can be prevented by anti-LFA-1 antibodies and cyclosporin A, IL-7 is significantly more effective on CD4 memory T cell proliferation than IL-2 and IL-15, whereas IL-15 and IL-2 favor CD8 memory T cell proliferation.

The results of this study showed that:

1) short term culture at high cell density and optimal IL-7 or IL-15 levels is suitable for maintaining and selectively propagating in vivo pre-conditioned memory CD4 and CD8T cell populations. These cells are best defined as secreting IL-2 and IFN-g and proliferating rapidly in response to IL-7(CD4) or IL-15(CD 8).

2) Short term culture in IL-7 (and to some extent IL-15 or IL-2) allows detection of rare Ag-specific CD4+ or CD8+ T cells that may not be detectable by conventional methods in vivo, without the need for Ag-driven propagation in vitro.

3) Short term culture in IL-7 (and to some extent IL-2 or IL-15) will increase the frequency and total number of Ag-specific T cells pretreated in vivo in an Ag-dependent manner.

4) Short term culture in IL-7 (and not IL-2) will maintain all lymphocyte subpopulations independent of their activation state, will not favor terminal differentiation of cells, and maintain the original phenotype of in vivo pretreated T cells.

5) Short-term culture in IL-7 in the absence of Ag allowed the accumulation of CD4 with Ag-specific response and long-term survival⁺Effector and central memory T lymphocytes.

6) IL-7/IL-15-proliferating cells have clinical relevance because they can delay tumor growth when metastasized into naive animals.

The proposed strategy has the advantages over the existing schemes:

A) the possibility of enriching a biological sample of antigen-specific memory T cells without TCR engagement (i.e. Ag-stimulation). Unlike existing protocols, which do not alter the surface and functional phenotype of cells, Ag-specific T cells can be counted in biological samples and their frequency in vivo assessed by combining the new protocol with surface unfolding of peptide/MHC I or MHC II multimer staining.

B) Revealing the possibility of rare antigen-specific T cells that would otherwise be undetectable ex vivo by routine counting. This is critical in all clinical conditions where the enumeration of those rare antigen-specific in vivo pretreated CD4 and CD8T cells has diagnostic and prognostic benefits, and currently relies on repeated, time-consuming in vitro Ag-driven cell proliferation.

C) Possibility of proliferating effector, central and intermediate memory T lymphocytes. There is currently no protocol available for maintaining central memory lymphocytes in vitro. The present invention has an impact on the strategy of adoptive immunotherapy. Indeed, although the available strategies require the transfer of large numbers of short-lived effector cells, comparable or even improved clinical outcomes may be achieved by transferring a limited number of recoverable, long-lived memory IL-7/IL-15-cultured cells.

In summary, the present invention has diagnostic and therapeutic relevance. On the one hand, it can help to identify rare populations of clinically relevant pathogen/tumor-specific T cells, and on the other hand, it can also improve existing adoptive immunotherapy strategies.

It is envisioned that the defined in vitro culture may be used to study several infectious and immune-mediated diseases, such as HIV, CMV, RSV, Flu, HBV, HPV, cancer, diabetes, rheumatoid arthritis, lyme arthritis, multiple sclerosis, celiac disease.

Model 3. another aspect of the invention relies on the concept that central memory cells can be propagated in vitro and can be genetically modified by viral vectors after TCR triggering in the presence of co-stimulation with gamma-cytokines and in culture, while maintaining their functional phenotype.

Cell therapy of T lymphocytes is thought to have great potential for curing cancer, infection, immunodeficiency and autoimmunity. In addition, it can be used to modulate immune responses that occur in the context of transplantation. The aim of the genetic modification is to broaden the treatment intervals of T lymphocytes by increasing their efficacy and/or limiting their toxicity. This can be achieved by transferring genes encoding novel receptors, biologically active products, resistance and control factors. It is expected that the control factors will provide selective in vivo elimination/inactivation of gene-modified cells if toxic/undesirable effects would occur. In the context of allogeneic hematopoietic cell transplantation (allogeneic-HCT), suicide gene therapy is the clearest example of how T-cells can be genetically modified using control factors to achieve therapeutic benefit. In allogeneic-HCT, immune recognition of host antigens by donor T-cells is a "sword-double" that results in specific beneficial effects: t cell 1) mediates direct antitumor effects (graft-versus-leukemia-GvL); 2) promoting the implantation of hematopoietic precursors; 3) the transplanted patient is provided with an intact immune system, thereby allowing for a reduction in the incidence and severity of post-transplant infection. Unfortunately, donor T-cells may also react with healthy host tissue, resulting in life-threatening graft versus host disease (GvHD) (20). Genetic modification of T-cells with retroviral vectors expressing the herpes simplex virus-Thymidine Kinase (TK) suicide gene confers selective sensitivity to the prodrug Ganciclovir (GCV). In patients, TK is infused⁺Lymphocytes and subsequent administration of GCV result in long-term regulation of anti-host reactivity for maintaining T-cell benefit and selective control of GvHD (21-23).

The success of T-cell therapy and T-cell gene therapy depends on the ability of T-cells to proliferate and survive for long periods in vivo. For this purpose, T-cells need to be properly directed to a second lymphoid organ where the antigen is properly predicted and the T-cells induced to acquire effector function. It is increasingly recognized that these attributes tend to segregate at an early stage of mature T-cell differentiation (more specifically, in the central memory compartment). Genetic modification of viral vectors may alter T-cell physiology. More specifically, genetic modification of Retroviral Vectors (RVs) requires cell proliferation. Currently, this is achieved by activation of polyclonal stimuli and culturing in the presence of high doses of recombinant human IL-2. The authors found that gene-modified human T lymphocytes generated with the current protocol (i.e. activated with soluble anti-CD 3 antibody and cultured in the presence of IL-2) were predominantly effector memory cells, which readily display effector function in vitro, but were difficult to engraft into a restricted immunodeficient host. Since the proliferation and persistence of human T cells is a key prerequisite for effective T-cell based gene therapy, the present invention provides methods for T cell culture and transduction that result in genetically modified central memory T cells. To this end, the authors combine:

-activating T cells with anti-CD 3 and anti-CD 28 antibody conjugated beads,

incubation with low doses of IL-7 and IL-15,

transduction with retroviral vectors.

The results indicate that it is feasible to produce gene-modified lymphocytes with beads in the presence of IL-7 and IL-15, these cells having a physiological CD4/CD8 ratio and a central memory function phenotype, as defined by the following conditions: i) lack of expression of CD45RA, presence of expression of CD62L, ii) co-expression of molecules CD27 and CD28, and iii) production of IL-2 in the absence of IFN- γ and/or IL-4.

Furthermore, the authors observed that infusion of genetically modified central memory T-cells in a restricted immunodeficient host i) would translocate and proliferate at significantly higher water levels than effector memory genetically modified T-cells, and ii) would be more effective than effector memory genetically modified lymphocytes in inducing an immune response to host antigens.

These results demonstrate that fully functional central memory recombinant lymphocytes can be obtained and used for the treatment of human diseases.

In the present invention, fully functional central memory recombinant lymphocytes refer to central memory T-cells with long-term survival potential that can be directed to peripheral lymphoid organs and differentiate into effector cells upon re-encountering antigen in vivo.

Thus, one subject of the present invention is an in vitro method of proliferating a rare population of antigen-specific memory T cells in a sample, comprising the step of exposing said sample to an effective amount of at least one cytokine receptor agonist capable of selectively proliferating said rare population of antigen-specific memory T cells. Preferably, the cytokine receptor agonist is a cytokine or a derivative thereof.

In a preferred embodiment, the at least one cytokine receptor agonist is an IL-7 receptor agonist or an IL-15 receptor agonist, preferably an IL-15 receptor agonist or an IL-7 receptor agonist, respectively, is also present.

In a preferred embodiment, the rare population of antigen-specific memory T cells comprises CD4⁺And/or CD8⁺And/or γ δ and/or NKT T cell populations.

In a preferred embodiment, the sample is a biological sample belonging to the group consisting of: blood and other liquid samples of biological origin, solid tissue samples, tissue cultures of cells derived from them and their progeny, cells isolated from biological samples i.e. PBMCs.

Another subject of the invention is an in vitro method for detecting rare populations of antigen-specific memory T cells in a sample, comprising the following steps:

a) exposing said sample to an effective amount of at least one cytokine receptor agonist capable of selectively proliferating rare antigen-specific memory T cell populations as described above;

b) incubating the sample with at least one ligand specific for one of the proliferating rare antigen-specific memory T cell populations;

c) detecting a proliferating rare population of antigen-specific memory T cells that bind to a specific ligand.

Preferably, the specific ligand is a specific antigen, or a derivative of one of the rare populations of antigen-specific memory T cells, more preferably, the specific antigen is associated with a microbial pathogen, including but not limited to mycobacteria, pneumocystis carinii, plasmodium falciparum, candida, toxoplasma, CMV, EBV, BPV, HCV, HBV, HIV. Alternatively, the antigen is a tumor-associated antigen. Alternatively, the antigen is an allergen. Or the antigen is a self-antigen.

In a preferred embodiment, the specific antigen is present as an antigen-MHC complex or a derivative thereof.

In a preferred embodiment, the detection of the expanded rare population of antigen-specific memory T cells is achieved by a binding assay. Alternatively, detection of the expanded rare population of antigen-specific memory T cells is achieved by a cytokine release assay. Alternatively, detection of the expanded rare population of antigen-specific memory T cells is achieved by a proliferation assay.

In a preferred embodiment, the cells are labeled with a fluorescent vital dye, the sample is then incubated with a specific ligand, and the detection step is performed by a dye dilution assay.

Another subject of the invention is a kit for carrying out the above-described method for detecting rare populations of antigen-specific memory T cells in a sample, comprising at least one cytokine receptor agonist; at least one ligand specific for a rare population of antigen-specific memory T cells; and (5) detecting the tool.

Another subject of the invention is an in vitro method for isolating a rare population of antigen-specific memory T cells in a sample, comprising the following steps:

a) exposing said sample to an effective amount of at least one cytokine receptor agonist as described above that selectively proliferates rare antigen-specific memory T cell populations;

b) incubating the sample with at least one ligand specific for one of the proliferating rare antigen-specific memory T cell populations;

c) isolating a proliferating rare population of antigen-specific memory T cells that bind to a specific ligand.

Preferably, the specific ligand is a specific antigen or a derivative of one of the rare antigen-specific memory T cell populations; more preferably, the specific antigen is associated with a microbial pathogen, including but not limited to mycobacteria, Pneumocystis carinii, Plasmodium falciparum, Candida, Toxoplasma, CMV, EBV, BPV, HCV, HBV, HIV. Or the antigen is a tumor-associated antigen. Or the antigen is an allergen. Or the antigen is a self-antigen.

In a preferred embodiment, the specific antigen is present as an antigen-MHC complex or a derivative thereof.

In a preferred embodiment, the isolation of the expanded rare population of antigen-specific memory T cells is achieved by a binding step. Or the expanded rare population of antigen-specific memory T cells by measuring cytokine and cytotoxic production, including but not limited to IL-2, IFN-g, IL-4, IL-5, IL-10, TNF-alpha, TGF-beta, granzyme ELISPOT assay, ELISA assay, flow cytometry cytokine detection assay.

Another subject of the present invention is the in vitro method for the diagnostic and/or prognostic clinical study of immune-, infectious-, cancer-, allergy-, autoimmune-related pathologies.

Another subject of the present invention is the use of the rare T cell population isolated according to the above method for the treatment and/or prevention of immune-, infectious-, cancer-, allergy-, autoimmune-related pathologies. In a specific embodiment, the rare population of T cells is genetically modified.

Another subject of the invention is an in vitro method for obtaining a population of genetically modified memory T cells, comprising the following steps:

a) activating lymphocytes with at least 2 specific activating receptor agonists capable of driving lymphocyte activation, including but not limited to agonist antibodies, recombinant ligands and derivatives thereof;

b) exposing activated lymphocytes to an effective amount of at least one cytokine receptor agonist capable of selectively proliferating a population of memory T cells;

c) inserting the foreign gene into the cell obtained in b) with the aid of an appropriate vector, and expressing.

Preferably, the population of memory T cells comprises CD4⁺And/or CD8⁺And/or γ δ and/or NKT T cell populations.

Preferably, the lymphocytes are derived from a biological sample belonging to: blood and other liquid samples of biological origin, solid tissue samples, tissue cultures of cells derived from them and their progeny, cells isolated from biological samples i.e. PBMCs.

Preferably, the specific lymphocyte activation receptor agonist is conjugated to a cell mimic support, more preferably the cell mimic support is a paramagnetic bead.

In a preferred embodiment, one of the lymphocyte activating receptor agonists is specific for the CD3 polypeptide, preferably the other lymphocyte activating receptor agonist is specific for the co-stimulatory receptor, CD 28.

In a preferred embodiment, the at least one cytokine receptor agonist is an IL-7 receptor agonist or an IL-15 receptor agonist, preferably an IL-15 receptor agonist or an IL-7 receptor agonist, respectively, is also present.

In a preferred embodiment, the vector is a viral vector.

In a preferred embodiment, the exogenous gene encodes a suicide gene, and/or a marker gene, and/or a biologically active molecule, and/or a receptor, and/or a soluble factor that resides within the cell or is released outside the cell, and/or a gene that confers resistance to a prodrug.

Another subject of the present invention is the use of a population of genetically modified memory T cells produced according to the above method for the treatment and/or prevention of cancer, infection, immunodeficiency or autoimmunity or transplantation of hematopoietic precursors or solid organs.

The invention will now be described, by way of non-limiting example, with reference to the following drawings:

FIG. 1 IL-7 favours tumor-specific memory CD4⁺T cells accumulate without the need for Ag-stimulation. By 3X 10⁵BALB/c mice (5 per group) were challenged with TS/A-LACK or TS/A tumor cells and sacrificed after 21 days. Cells from tumor-ducted LN (A-D) and ductless LN (E-H) populations were analyzed in vitro after 7 days of culture with IL-7 alone. A, C, E, G) staining the cells as described in materials and methods. For live CD4⁺，B220^-，CD8^-，CD11b^-，TOPRO-3^-After cell gating, a representative flow cytometry curve is shown. Indicates CD44^{Height of}I-A^d/LACK⁺ CD4⁺The frequency of the cells. B, D, F, H) lymphocytes were stimulated with LACK aAPC (see materials and methods), fixed, permeabilized, stained with anti-CD 4mAb, anti-IL-2, and anti-IFN- γ mAb, and analyzed by flow cytometry. Shows CD4⁺Representative dot plots of IL-2 and IFN- γ production. The frequency of cytokine-producing cells is reported in each quadrant. The experiment represents 6 independent assays. In some cases, LACK-specific IFN- γ release was detected in IL-7-treated LN cultures of TS/A tumor-bearing mice. Although the nature of these cells remains to be elucidated, these cells may be specific for the LACK homologue mammalian RACK (24).

FIG. 2 IL-7 and IL-2 cause tumor-specific CD4⁺Accumulation of T cells, but Ag, IL-15 and IL-6 do not. LN cell populations recovered from BALB/c mice bearing TS/a-LACK tumors were cultured in the Absence (APC) or presence of LACK peptide (Ag/APC) with irradiated splenocytes, or with IL-7, IL-2, IL-15 and IL-6 alone (n-5). After 7 days, cells were recovered and surface stained to determine the frequency of LACK-specific T cells undergoing Ag (a, C) or stimulated with LACK aAPC to assess LACK-specific intracellular cytokine release (B, D) as described in figure 1. A) For live CD4⁺，B220^-，CD8^-，CD11b^-，TO-PRO-3^-After cell gating, a representative flow cytometry curve is shown. Indicates CD44^{Height of}I-A^d/LACK⁺CD4⁺Frequency (A) and total number (C) of cells. B) The representative dot plot depicts CD4⁺IL-2 and IFN- γ production by T cells. CD4 is reported⁺Frequency (B) and total number (D) of LACK-specific cytokine-producing cells in T cells. The experiment represents 3-5 independent assays.

FIG. 3 IL-7 and IL-2 support in vivo pretreated tumor-specific CD4⁺Ag-independent proliferation of T cells. A) LN cell populations recovered from naive or TS/a-LACK tumor-bearing BALB/c mice (n ═ 5) were labeled with CFSE vital dye and cultured in usual medium for 1 week. Shows a live CD4⁺Representative dot plots of T cells, B-D) CFSE-labeled LN cells derived from TS/A-LACK-tumor-persuading LN were cultured for 7 days in the absence of (nil) or with IL-7, IL-2, IL-15, and IL-6. Cells were then stimulated with LACK aAPC (B, D) or control aAPC (c) and analyzed for intracellular cytokine release by flow cytometry as described in figure 1. In B and C live CD4 is shown⁺A representative dot plot of CFSE content and IL-2 or IFN- γ production of T cells. In D, CFSE for IL-2 and/or IFN-gamma production is described^dim CD4⁺Total number of T cells. The experiment represents 3 independent assays.

FIG. 4 IL-2 would mimic in the absence of AgIL-7 and enrichment of tumor-specific memory CD4 in cell culture⁺T cells, but IL-15 and IL-6, IL-10, TNF- α are not. TS/A-LACK and TS/A-tumor-leading LN1 weeks derived from 16.2 β transgenic mice were cultured without (-) or with the indicated recombinant cytokines alone and analyzed by flow cytometry as described in FIG. 1. A) For live CD4⁺，B220^-，CD8^-，CD11b^-，TOPRO-3^-After cell gating, representative dot plots are shown. Indicate I-A^d/LACK⁺CD4⁺The frequency of the cells. B-D) lymphocytes derived from TS/A-LACK- (B, C) and TS/A- (D) -tumor persuasion LN cultures were stimulated with LACK aAPC (B, D) or control aAPC (C) to detect intracellular IL-2, IFN- γ and IL-4. Shows CD4⁺Representative dot plots of IL-2 and IFN- γ production. In all experiments IL-4⁺Cells were within background levels. IL-2 is reported in each quadrant⁺、IFN-γ⁺Cytokine-frequency of producing cells. E) Tumor-ducted LN cells from 16.2 β mice bearing TS/a-LACK-tumors were labeled with CFSE and cultured for 1 week in the absence (-) or presence of indicated cytokines. Thereafter, cells were re-stimulated with LACK aAPC and analyzed by flow cytometry. Shows CD4⁺And a representative dot plot of the CFSE content and IL-2 and IFN-gamma production. The experiment represents 3 independent assays.

FIG. 5 IL-7 and IL-2 favour T cell survival and optimal expression of Bc 1-2. Cells from TS/A-LACK tumor ductal LN were labeled with CFSE vital dye and cultured in the absence (-) or presence of IL-7, IL-2, IL-15, and IL-6 alone. After 7 days, cells were recovered and stained with anti-CD 4mAb and TO-PRO-3(A) and anti-Bc 1-2mAb (B, C). A) Shows the total CD4⁺Representative dot plots of cells. Total CD4 is reported⁺ TOPRO-3⁺Frequency of (bracket) and CFSE dim, TO-PRO-3^-Frequency of cells (bold). B-C) are shown on live CD4⁺Post-physical gating events on T lymphocytes. C) Thin line: an isotype control; thin line shadow: anti-Bc 1-2Ab, cells cultured in medium; thick line: anti-Bc 1-2Ab, cytokine cultured cells. Fruit of Chinese wolfberryThe experiment represents 2 independent assays.

FIG. 6 is a graphical representation of the phenotype and subpopulations of lymphocytes maintained in IL-7 and IL-2. CFSE-labeled TS/A-LACK-tumor-Dredging LN cultures were maintained for 1 week in the presence of IL-7 or IL-2. Thereafter, cells were stained with anti-CD 4, CD44, CD25, CD127, and CD132 mabs, a) representative dot plots reported the expression levels of CD44, CD62L, CD25, CD127, and CD132 for live CD4 +. B) Shows CD4 derived from IL-7 (thin line) and IL-2 (thick line) cultures in the absence of Ag-stimulation⁺CFSE dim cells. C) Surface staining of CFSE-labeled cells was performed for surface levels of CD4 and CD62L and intracellular Bc 1-2. Shows a pair of CD4⁺Dot plots after CFSE dim cell gating. The experiment represents 3 independent assays.

FIG. 7 IL-7-cultured cells were comparable to those found at sacrifice. I-A as described by FIG. 1^dThe TS/A-LACK-tumor-leading LN derived from 16.2 β transgenic mice was analyzed ex vivo after short term culture in IL-7 or IL-2 in the absence of Ag-stimulation. A) Representative dot plots report live CD4⁺，B220^-，CD8^-，CD11b^-，TOPRO-3^-Expression levels of CD44, CD25, CD127, and CD132 by the cell. B) Shows a pair B220^-，CD8^-，CD11b^-，TOPRO-3^-After lymphocyte gating, CD4⁺、I-A^d/LACK⁺And CD4⁺、I-A^d/LACK^-Of the first and second image data. In vitro: a dashed line; thin line: IL-7; thick line: IL-2.

FIG. 8 is an in vivo-pretreated marker IV-specific memory CD8+ T cells enriched for IL-7-, IL-2-, or IL-15-driven cultures. Bone marrow-derived dendritic cells pulsed with labeled IV peptide, C57BL/6 mice were immunized. After 14 days, axillary, brachial and inguinal LN cells were recovered and analyzed ex vivo after 1 week of culture in the absence or presence of IL-7, IL-2, IL-15, IL-6. Reports of marker IV-specific cytokine-production CD45.1^- CD8⁺Total number of T cells. The experiment represents 2 independent assays.

FIG. 9 IL-7-, IL-2-, or IL-15-driven cultures rescue a significant number of CD8+ T cells. Bone marrow-derived dendritic cells pulsed with labeled IV peptide, C57BL/6 mice were immunized. After 14 days, axillary, brachial and inguinal LN cells were recovered and analyzed ex vivo after 1 week of culture in the absence or presence of IL-7, IL-2, IL-15, IL-6. CD45.1 is reported^- CD8⁺Total number of T cells. The experiment represents 2 independent assays.

FIG. 10 IL-7 favors tumor-specific memory CD8 that is otherwise undetectable ex vivo⁺Accumulation of T cells. A) By 3X 10⁵TS/A-LACK tumor cells challenged BALB/c mice (5 mice per group) and sacrificed after 21 days. Cells from tumor-derived LN populations were analyzed ex vivo (A) and after 7 days of culture in the absence or presence of IL-7, IL-2, IL-15, IL-6(B-C) (A-C). A) Representative dot plots depict KJ1.26^- CD8⁺IL-2 and IFN- γ production by T cells. KJ1.26 reported to produce AH-1-specific cytokines^- CD8⁺Frequency (A, B) and total number (C) of T cells.

FIG. 11 intermediate memory CD4 in the presence of IL-7⁺T cells accumulate in a cell-density dependent, CsA-sensitive manner. Cells derived from axillary, brachial and inguinal LNs of naive (A, B) and TS/A-LACK tumor-bearing (C-E) 16.2. beta. mice were labeled with CFSE and cultured in the presence of LACK peptide (Ag) (A, B) or IL-7(C-E), respectively, in the absence of (nil) or in the presence of the indicated inhibitors, in usual culture media for 7 days. At the end of the medium, cells were stimulated with L/28aAPC for 5h and intracellular cytokine release was measured. A-D) histogram shows an equivalent number of (7X 10)⁴)CD4⁺CFSE dilution curve of T cells. In A and C, the thin line reflects the CFSE curve for CD4+ T cells cultured in normal medium, while the thick line depicts the CFSE curve for CD4+ T cells cultured in Ag or IL-7, respectively. In B and D, the fine line: cells cultured without inhibitor, bold line: cells cultured in the presence of an inhibitor. E) Shown in live CD4⁺On T cellsGated dot plots. The percentage indicates the frequency of LACK-specific cytokine secreting cells.

FIG. 12 IL-7 supports a portion of human peripheral blood CD4⁺Rapid, Ag independent proliferation of T cells. Human PBMCs from healthy donors were labeled with CFSE vital stain and cultured for 7 days at the indicated cell density in the absence (nil) or presence of recombinant human IL-7(100 ng/ml). Depicting live CD4⁺Representative dot plots of T cells. Indicates CFSE^dim CD4⁺Frequency of T cells. B) CD4 cultured at a specified cell density⁺Histograms of T cells overlap (N: non-proliferating cells, S and F: slowly and rapidly proliferating cells).

IL-7 supports the accumulation of rapidly dividing cells in autologous serum. Human PBMCs from healthy donors were labeled with CFSE vital stain and cultured at the indicated cell density for 7 days in medium supplemented with 10% autologous serum in the absence (nil) or presence of recombinant human IL-7(100 ng/ml). Depicting live CD4⁺Representative dot plots of T cells. CFSE indicative of non-proliferation (N), and Slow- (S) and fast- (F) proliferation^dim CD4⁺Frequency of T cells.

FIG. 14 IL-7-driven accumulation of rapidly dividing CD4T cells is dose dependent. Human PBMCs from healthy donors were labeled with CFSE vital stain and cultured for 7 days in the presence of the indicated amount of recombinant IL-7. Depicting live CD4⁺Representative dot plots of T cells. CFSE indicative of non-proliferation (N), and Slow- (S) and fast- (F) proliferation^dim CD4⁺Frequency of T cells.

FIG. 15 IL-7 supports rapidly dividing IFN-. gamma. -producing memory CD4⁺Accumulation of T cells. Human PBMCs from healthy donors were labeled with CFSE vital stain and cultured for 7 days in the presence of the indicated amount of recombinant human IL-7. After 7 days, cells were harvested and restimulated with PMA and ionomycin for 6 hours. Cells were then surface stained, fixed, and stained with anti-IFN- γ mAb. Depicting live CD4⁺Representative dot plots of T cells.

FIG. 16. IL-2/IFN-. gamma.⁺ CD4⁺IL-7-driven accumulation of T cells is dose-dependent. Human PBMC from healthy donors were cultured for 7 days in the presence of the indicated amounts of recombinant human IL-7. After 7 days, cells were harvested and restimulated with PMA and ionomycin for 6 hours. Cells were then surface stained, fixed, and stained with anti-IFN- γ mAb. Depicting live CD4⁺Representative dot plots of T cells.

FIG. 17 IL-7 best propagated rapidly dividing memory CD4⁺T lymphocytes, while IL-15 drives rapidly dividing memory CD8⁺Accumulation of T lymphocytes. CFSE-labeled human PBMC were cultured in the presence of human recombinant IL-7, IL-2, IL-15, and IL-6 in usual medium (nil) for 7 days, then stained with anti-CD 4 and anti-CD 8mAb and analyzed by flow cytometry. Histogram overlay shows CD4 at the same number⁺(A) And CD8⁺(B) CFSE content in lymphocytes. Fast- (F), slow- (S), and non- (N) proliferating cells are indicated.

FIG. 18 optimal Bc1-2 expression on cultured human cells driven by IL-7, IL-2 and IL-15. Human PBMCs from healthy donors were labeled with CFSE vital stain and cultured for 7 days in the absence (nil) or presence of human recombinant IL-6, IL-7, IL-2 and IL-15. The cells were then stained with anti-CD 4mAb, fixed, and intracellular levels of Bc1-2 were determined by intracellular staining. Depicting a gated CD4⁺post-T cell events.

FIG. 19 IL-7-driven CD4T cell proliferation of human peripheral blood memory CD4T cells dependent on CsA and LFA-1/ICAM-dependent signaling. Using CFSE⁺Vital stain labeling of human PBMC from healthy donors, cultured in the absence (nil) or presence of the indicated inhibitor in the presence of human recombinant IL-7 for 7 days. Cells were then stained with anti-CD 4, anti-CD 45RA, and anti-CD 62L mabs and analyzed by flow cytometry. A) Histogram depicts live CD4⁺Overlap of CFSE curves for T cells. B) Dot plots depict live CD4T cells. Rapidly (F), slowly (S) and non (N) proliferating cells were determined electronically and shown in C. Indicating in the figuresA graph comparing naive and memory T cells is shown. Results represent 2 independent assays.

FIG. 20 enrichment of Mycobacterium tuberculosis-specific CD4 by IL-7-driven cultures⁺T cells. PBMCs from 3 TB patients (Pt. #1, FIG. 20A; Pt #2, FIG. 20B; and Pt #3, FIG. 20C) were analyzed for MTP-specific IFN- γ release by the ELISPOT assay at thaw (cryopreservation) and after 7 days of culture in the absence (nil) or in the presence of human recombinant IL-7 (culture). In control wells that were not pulsed (control), background IFN- γ release was measured. B) The total number of MTP-specific IFN-. gamma.producing cells is indicated.

FIG. 21 IL-7-driven culture promotes Mycobacterium tuberculosis-specific CD4⁺And (4) identifying T cells. Cryopreserved and IL-7 cultured PBMCs derived from 8 healthy donors and 5 TB-patients were analyzed for MTP-specific IFN- γ production by ELISPOT. Statistical significance was assessed by paired two-tailed T-test.

FIG. 22. Mycobacterium tuberculosis specific IL-2/IFN-gamma⁺ CD4⁺T cells accumulate in IL-7 driven cultures. Pt #1 cells were cultured in the absence (nil) or presence of human recombinant IL-7 (culture). A) MTP-specific IFN- γ release was detected by ELISPOT (also depicted in fig. 20A). B) Parallel group cultured cells were also restimulated with MTP-pulsed autologous irradiated PBMC for 6 hours, surface stained with anti-CD 4mAb, fixed, and further stained with anti-IFN- γ mAb. Depicting a gated CD4⁺post-T cell events.

FIG. 23 Mycobacterium tuberculosis specific CD4⁺T cells will proliferate in IL-7-driven cultures. A) Pt #1 cells were cultured in the absence (nil) or presence of human recombinant IL-7 (culture). MTP-specific IFN- γ release was detected by ELISPOT (also depicted in fig. 20A). B) Parallel cultures were also established with CFSE-labeled PBMC. After 7 days, cells were stimulated with MTP-pulsed autologous irradiated PBMCs and intracellular IFN- γ release was determined by flow cytometry. Showing the gated CD4⁺post-T cell events.

FIG. 24. Candida albicans specific IFN-. gamma.⁺Memory T cells accumulate in IL-7-driven cultures. A) Describes the Candida albicans-specific IFN- γ release, ELISPOT assay, of Pt #1 cells cryopreserved or cultured for 7 days in the absence (nil) or in the presence of human recombinant IL-7. Background IFN- γ release was measured in control wells (control) that were not pulsed. B) The total number of IFN-. gamma.producing cells is described.

FIG. 25 IL-7/IL-15 cultured memory cells delayed tumor growth after adoptive cell in vivo transfer. In the presence of IL-7 and IL-15 (both at 50ng/ml), at high cell densities (5X 10)⁶Cells/ml) lymph node cells derived from control (naive) and TS/a-LACK tumor-bearing mice were cultured for 7 days. Thereafter, 10 is⁷Cultured cells were transferred into naive BALB/c mice adoptively. After 48 hours, mice were stimulated with 300.000TS/A-LACK cells and tumor growth was monitored over time. Statistical significance was assessed by paired two-tailed T-test.

Figure 26 schematic of the proposed strategy and its diagnostic and therapeutic uses.

FIG. 27 activation of beads conjugated with anti-CD 3 and anti-CD 28 antibodies (ba CD3/CD28), and culture with IL-7 and IL-15, promoted T cell proliferation and efficiently produced genetically modified human lymphocytes with a fixed CD4/CD8 ratio. Stimulation of 5X 10 with aCD3⁶PBMC cultured with IL-2 or bagD 3/CD28 and with IL-7 and IL-15. On day 14, cells were counted by trypan blue exclusion. (A) The average value of T cell proliferation under 2 stimulation and culture conditions (n-4 donors) was reported. Cells were transduced with SFCMM3 retroviral vectors 48 and 72h after initial stimulation. On day 6, genetically modified cells were quantified by flow cytometry after staining with anti-LNGFR antibody. (B) The average value of transduction efficiency (in%) under 2 stimulation and culture conditions was reported (n-4 donors). On day 14, cells were analyzed by flow cytometry for Δ LNGFR expression and CD4 and CD8 expression. (C) The mean of the CD4/CD8 ratio in genetically modified cells generated with both protocols was reported (n-4 donors).

FIG. 28 activation with bagD 3/CD28 and incubation with IL-7 and IL-15 resulted in TK with a central memory phenotype⁺Human lymphocytes. On day 14, TKs produced with aCD3 and cultured with IL-2, or with bagD 3/CD28 and cultured with IL-7 and IL-15 were analyzed⁺Memory phenotype of the cell. After gated Δ LNGFR expression, cells were analyzed by flow cytometry for co-expression of CD45RA and CD 62L. (A) CD4 obtained from n-4 donors is reported⁺And (B) CD8⁺CD45RA of cells⁺CD62L⁺(black bar), CD45RA^-CD62L⁺(dark gray strip), CD45RA^-CD62L- (light gray bar) or CD45RA⁺CD62L^-Relative distribution average (y-axis,%) (white bar). Cells were also analyzed by flow cytometry for co-expression of CD27 and CD 28. (C) CD4 obtained from n-4 donors is reported⁺And (D) CD8⁺CD28 of cells⁺CD27⁺(black bar), CD28^-CD27⁺(dark gray strip), CD28^-CD27^-(light gray stripes) or CD28⁺CD27^-Relative distribution average (y-axis,%) (white bar).

FIG. 29. Central memory TK⁺Human lymphocytes are unpolarized cells. On day 14, TKs produced with aCD3 and cultured with IL-2, or with bagD 3/CD28 and cultured with IL-7 and IL-15 were analyzed⁺Cytokine production by cells. After gating for Δ LNGFR expression, cells were analyzed by flow cytometry for IFN- γ and IL-4 production. (A) CD4 is reported⁺And (B) CD8⁺IL-4 of cells⁺IFN-γ^-(white bar), IL-4⁺IFN-γ⁺(Grey stick) or IL-4^-IFN-γ⁺Relative distribution average (y-axis,%) (black bars).

FIG. 30 activation of beads conjugated with anti-CD 3 and anti-CD 28 antibodies, and culture with IL-7+/-IL-15 or IL-2, promoted T cell proliferation and efficiently produced genetically modified human lymphocytes with a fixed CD4/CD8 ratio. PBMCs were stimulated with anti-CD 3 and anti-CD 28 (designated "B") antibody conjugated beads and cultured in the absence of cytokines, IL-7+ IL-15, IL-2, or IL-7, or with soluble anti-CD 3 and IL-2. Cells were transduced with SFCMM3 retroviral vectors 48 and 72h after the initial stimulation. A) On day 6, genetically modified cells were quantified by flow cytometry after staining with anti-LNGFR antibody. The average values of transduction efficiency (in%) under different stimulation and culture conditions are reported. B) On day 10, cells were analyzed by flow cytometry for Δ LNGFR expression and expression of CD4 and CD 8. The mean value of the CD4/CD8 ratio of genetically modified cells generated with different protocols is reported. C) Cells were counted by trypan blue exclusion on days 6 and 9. The average value of T cell proliferation under different stimulation and culture conditions is reported (n-5 donors). P < 0.05 p < 0.01.

FIG. 31 activation of beads conjugated with anti-CD 3 and anti-CD 28 antibodies, and culture with IL-7+/-IL-15 or IL-2, resulted in transduced human lymphocytes with a central memory phenotype. On day 10, the memory phenotype of transduced lymphocytes produced with anti-CD 3 and anti-CD 28 antibody-conjugated beads and cultured in the absence of cytokines, IL-7+ IL-15, IL-2, or IL-7, or stimulated with soluble anti-CD 3 and cultured with IL-2, was analyzed. Cells were analyzed by flow cytometry for co-expression of CD45RA and CD 62L. CD45RA of CD3+ Δ LNGFR + cells was reported⁺CD62L⁺(naive cells), CD45RA^-CD62L⁺(Central memory cells), CD45RA^-CD62L^-(Effector memory cells) or CD45RA⁺CD62L^-Relative distribution average (y-axis,%) (n ═ 6 donors) of (final differentiation effectors). P < 0.05 p < 0.01.

FIG. 32 the kinetics of IL-2/15 receptor beta (CD122) expression was independent of activation, culture and transduction procedures. On days 0,2, 4, 9 and 13, after primary stimulation, the transduced lymphocytes produced with anti-CD 3 and anti-CD 28 antibody-conjugated beads and cultured in the absence of cytokines, IL-7+ IL-15, IL-2, or IL-7, or stimulated with soluble anti-CD 3 and cultured with IL-2 were analyzed for CD122 expression. On a) CD8+ and B) CD4+ cells, the% (n ═ 4 donors) of CD3+ cells (at days 0,2 and 4) and transduced cells (at days 9 and 13) expressing CD122 at different time points were reported.

FIG. 33 activation of beads conjugated with anti-CD 3 and anti-CD 28 antibodies and incubation with IL-7+/-IL-15 or IL-2 promotes strong and prolonged expression of IL-2 receptor alpha (CD25) on transduced lymphocytes. On days 0,2, 4, 9 and 13, after primary stimulation, transduced lymphocytes produced with anti-CD 3 and anti-CD 28 antibody-conjugated beads and cultured in the absence of cytokines, IL-7+ IL-15, IL-2, or IL-7, or stimulated with soluble anti-CD 3 and cultured with IL-2 were analyzed for CD25 expression. On a) CD8+ and B) CD4+ cells, the% (n ═ 4 donors) of CD3+ cells (at days 0,2 and 4) and transduced cells (at days 9 and 13) expressing CD25 at different time points were reported. P < 0.05 p < 0.01.

FIG. 34 activation of beads conjugated with anti-CD 3 and anti-CD 28 antibodies and incubation with IL-7 promoted strong and prolonged expression of IL-7 receptor alpha (CD127) on transduced lymphocytes. On days 0,2, 4, 9 and 13, after primary stimulation, the transduced lymphocytes produced with anti-CD 3 and anti-CD 28 antibody conjugated beads and cultured in the absence of cytokines, IL-7+ IL-15, IL-2, or IL-7, or stimulated with soluble anti-CD 3 and cultured with IL-2 were analyzed for CD127 expression. On a) CD8+ and B) CD4+ cells, the% (n ═ 4 donors) of CD3+ cells (at days 0,2 and 4) and transduced cells (at days 9 and 13) expressing CD127 at different time points were reported. P < 0.05 p < 0.01.

FIG. 35 activation of beads conjugated with anti-CD 3 and anti-CD 28 antibodies and culture with IL-7 maintained high allogenic proliferation potential and low sensitivity to apoptotic signals in transduced cells.

At day 9 post primary stimulation, transduced T cells, produced using CFSE, conjugated with anti-CD 3 and anti-CD 28 antibodies, and cultured with IL-7 or IL-2, or activated with soluble anti-CD 3 and cultured with IL-2, were co-cultured with irradiated allogeneic PBMC. Non-manipulated Peripheral Blood Lymphocytes (PBLs) from the same individual were stained with CFSE, co-cultured with the same irradiated allogeneic PBMC, and used as a control. After 7 days, cells were counted, stained with To-pro3, and the percentage of dividing and/or dying cells was evaluated by FACS analysis. A) Percentage of dividing cells. B) Total number of dead cells. Activation-induced cell death was calculated on dividing cells (white, lower part of the histogram) and negligible death was calculated on non-dividing cells (black, upper part of the histogram). P < 0.05 p < 0.01.

FIG. 36 self-recovery ability of central memory genetically modified cells generated with anti-CD 3 and anti-CD 28 antibody conjugated beads and cultured with IL-7 following allogeneic stimulation.

A) 7 days after the start of MLR, cells treated as described in FIG. 35 were analyzed for CCR7 and IL-7Ra expression. In the upper left quadrant, the percentage of dividing cells of CCR7+ (upper panel) and IL-7Ra + (lower panel) is shown. B) The cells were then challenged again in vitro with the same allogeneic stimulus under the same culture conditions as the first stimulus. The percentage of dividing cells after the second stimulation is shown.

FIG. 37 transduced lymphocytes generated with the bead CD3/CD28 antibody rapidly engraft into NOD/scid mice

Intravenous infusion of 20X 10 to restricted and human skin transplanted NOD/scid mice⁶Transduced lymphocytes produced with beads CD3/CD28 and cultured with IL7 or IL2, or produced with OKT3 and cultured with IL-2. Human chimerism was assessed weekly by flow cytometry after staining for human CD3 and mouse CD45. Quadrants and percentages were set according to isotype control staining. The kinetics of human chimerism (n-4 donor) is shown.

FIG. 38 transduced lymphocytes produced with the beads CD3/CD28 and cultured with I L-7 induced severe allogeneic GvHD in NOD/scid mice

Intravenous infusion of 20X 10 to restricted and human skin transplanted NOD/scid mice⁶Transduced lymphocytes produced with beads CD3/CD28 and cultured with IL7 or IL2, or produced with OKT3 and cultured with IL-2. Mice were monitored for allogeneic GvHD based on 1) weight loss, 2) GvHD clinical score (see materials and methods). To pairAccording to the following steps: animals that did not receive an infusion of lymphocytes.

FIG. 39 transduced lymphocytes produced with beads CD3/CD28 and cultured with IL-7 induced severe allogeneic GvHD in NOD/scid mice

3 weeks after human T-cell infusion, NOD/Scid chimeric mice were sacrificed and human skin was removed from both sides. All biopsies were subsequently analyzed by a blinded pathologist by hematoxylin-eosin (EE) and anti-human CD3 staining (α hCD 3). Representative fractions are reported.

Materials and methods

The protocol was approved by the ethical committee of San Raffaele Scientific Institute and was operated according to its guidelines.

Mouse and tumor cells

7-8-week old BALB/c and CD45.2⁺C57BL/6 mice were purchased from Charles River (Charles River Italiaa, Milano, Italy). Feeding CD45.1 in a specific pathogen-free device at the study site⁺C57BL/6, DO11.10 and 16.2. beta. transgenic (Tg) (BALB/C background) (25) mice. TS/A and TS/A-LACK mouse breast cancer has been previously described (17, 19, 26). On the right side of syngeneic mice (BALB/c), 4X 10, exponentially growing in 100. mu.l PBS, were injected subcutaneously⁵A tumor cell. Typically, 5 mice/group were used in each experiment.

Primary culture of T cells

20 days after tumor cell injection, mice were sacrificed and axillary, brachial and inguinal peripheral Lymph Node (LN) canalization and distal to the tumor growth site (no canalization LN) were removed. In experiments with Dendritic Cell (DC) vaccination, mice were sacrificed 14 days after DC administration and axillary, brachial and inguinal LNs were surgically excised. 5X 10 in the absence or presence of recombinant murine IL-7(200ng/ml), IL-2(20ng/ml), IL-6(45ng/ml), or IL-15(100ng/ml) (all from Peprotech)⁶Density of in 24 poresLN cells were cultured in complete medium in the plate. In parallel cultures, LACK-derived MHC II-restricted peptides (5. mu.M, (25)) and 5X 10 were used⁶The irradiated syngeneic splenocytes stimulate the cells in vitro. As a control, similar cultures were established from syngeneic naive mice. In some experiments, LN cells were labeled with the fluorescent dye CFSE (5- (and-6) -carboxyfluorescein diacetate, succinimidyl ester) at a final concentration of 1. mu.M according to the manufacturer's instructions.

Dendritic Cell (DC) preparation

Bone marrow-derived DCs were obtained as described previously (18). Briefly, CD45.2 was propagated in complete Iscove's medium containing 25ng/ml recombinant murine GM-CSF and 5ng/ml recombinant murine IL-4(Pharmingen, San Diego, Calif.)⁺C57BL/6 bone marrow precursor for 7 days. BMDCs were then matured in the presence of LPS (1. mu.g/ml, Sigma, Milan, Italy) at 37 ℃ for 8 hours and pulsed with 10. mu.g/ml of large T Ag-derived tagged IV peptide for 1 hour (18). DC maturation and purity were routinely assessed by flow cytometry after staining with mabs (all from Pharmingen) that recognize CD11c, MHC class II, B7.1, B7.2, and CD40 molecules. On the right side of the syngeneic C57BL/6 mice, 2X 10 in 200. mu.l PBS was injected subcutaneously⁵Pulsed mature DCs.

Flow cytometry analysis

I-A is described previously^dStaining of LACK multimers. Briefly, I-A was prepared by adding Alexa 488-conjugated protein A (Molecular Probes Inc., Eugene, 0.3. mu.g/sample) in PBS for 30 minutes at room temperature^dLACK dimer (MHC I-peptide complex, 3. mu.g/sample) multimerization. Free protein A binding sites were saturated by the addition of total IgG (1. mu.g/sample). First, 6X 10 incubations were performed with blocking buffer (5% rat serum + 95% 2.4G2 anti-FcR mAb-producing hybridoma cell culture supernatant, 20 min)⁵Cells were then stained with multimers (1h at 4 ℃ in PBS supplemented with 0.5% BSA). Then with anti-CD 4, anti-CD 44, anti-CD 11B, anti-B220, anti-CD 8a mAb (PharMingen, san Diego, Calif., USA) and TO-PRO-3(1nM, molecular)r Probes) stained the cells. By excluding all anti-CD 11b⁺anti-B220⁺anti-CD 8a⁺And TO-PRO-3⁺Event, Collection 3 × 10⁵ CD4⁺Or 10³ CD4⁺ I-A^d/LACK⁺An event. When indicated, cells were surface stained with anti-CD 4 or anti-CD 8mAb, and anti-CD 44, anti-CD 127, anti-CD 25, anti-CD 132 and anti-CD 62LmAb (all from PharMingen except the anti-CD 127Ab, A7R 34 clone from Bioscience), fixed, permeabilized, and further stained with anti-Bc 1-2mAb according to the manufacturer's instructions.

LACK-specific artificial antigen presenting cells (aAPC) and cytokine secretion assay

5 μm polystyrene sulfate rubber beads (Interfacial Dynamics) were coated with I-A^dLACK dimer (20. mu.g/ml) and anti-CD 28mAb (37.51; 2. mu.g/ml) (LACKaAPC) or anti-CD 28mAb alone (control aAPC). The coating of the protein was monitored by flow cytometry analysis. In general, 5X 10 at 37 ℃ is used⁶aAPC culture 5X 10⁵LN cells for 5 hours. Brefeldin a (5 μ g/ml, Sigma) was added to the culture for the last 2 hours. LACK aAPC induced cytokine release was comparable to LACK-pulsed syngeneic splenocytes (not shown). Splenocytes were derived from DO11.10 and CD45.1 in the presence of AH-1 and marker IV-induced cytokine production⁺C57BL/6 mice, and used as antigen presenting cells. Splenocytes (3X 10) were pulsed with 1. mu.M AH-1(19) and 10. mu.g/ml labeled IV (18) peptide at 37 deg.C⁷Cells/ml) for 1 hour, and then used to stimulate LN-derived syngeneic LN cells from tumor-bearing and DC-immunized mice, respectively. Thereafter, the cells were stained with anti-CD 8, and IL-2 and IFN-. gamma.as described above, and anti-clonotypes KJ1.26 mAb and anti-CD 45.1⁺The labeling stains the cells to exclude T cells of APC origin. CD4 was then collected on a FACS Calibur⁺，KJ 1.26^-Or CD8⁺CD45.1^-An event. Determination of Ag-specific IL-2 by multiplying the percentage by the total number of Trypan blue-negative LN cells⁺/IFN-γ⁺T cellsFor an overview of (1).

Human T cell culture and ELISPOT assay

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from tuberculosis patients (TB) and healthy donors by centrifugation of the blood through an Fycoll-Hypaque density gradient and either analyzed immediately or frozen for future analysis. When indicated, cells were stained with CFSE (1. mu.M). Cells were cultured for 7 days in the absence or presence of human IL-7(200ng/ml), IL-2(20ng/ml, IL-15(100ng/ml) or IL-6(45 ng/ml). Thieladin A (CsA) (0.5. mu.g/ml) or anti-LFA-1 (5. mu.g/ml) blocking antibody was added when indicated, then the cells were harvested, stained with CD4, CD8, CD3, CD56, CD45RA, CD62L (all from PharMingen), and analyzed by flow cytometry.

ELISPOT assays for IFN-. gamma.secretion were performed as previously reported (28). Briefly, in the presence of autologous irradiated PBMC (5X 10)⁴Cells/well) at 5 × 10⁴Cells/well, cells were seeded in duplicate into 96-well plates (MAIPS 4510; Millipore, Bedford, Mass.) precoated with anti-IFN- γ capture mAb (B-B1; Diaclone, Besancon, France) at 37 ℃ in air + 5% CO₂In (1), the MTP peptide population was incubated for 18 h. Biotinylated anti-IFN-. gamma.detection mAb (B-G1; Diaclone) was added for 4h, followed by streptavidin-alkaline phosphatase conjugate (Amersham Pharmacia Biotech eye GmbH, Freiburg, Germany) for 1 h. After the washing step, nitro blue tetrazolium-BCIP (5-bromo-4-chloro-3-indolyl phosphate; Sigma, St. Louis, Mo.) chromogenic substrate was added. Individual blot cells (SFC) were counted using an automated image analysis system ELISPOT reader (AID-GmbH, Strassberg, Germany). Specific reactions were tested using 6 synthetic Mycobacterium tuberculosis peptide populations (MTP; Primm srl, Milano, Italy) at a final concentration of 2. mu.g/ml for each peptide, 20 amino acids in length, > 70% purity, ESAT-6 and CFP-10 secreted protein sequences from Mycobacterium tuberculosis (28). PBMC stimulated in medium alone or with 5. mu.g/ml phytohemagglutinin (PHA-P; Sigma) were used as negative and positive controls, respectively.

In at leastIn some cases, MTP-specific IFN- γ release was analyzed at the single cell level by intracellular cytokine secretion assays. Briefly, 0.6X 10 restimulation was performed in the presence of human anti-CD 28 stimulating mAb (2. mu.g/ml)⁶CFSE-labeled cells were pulsed for 6 hours with HLA-DR-restricted MTP (4. mu.g/ml) at 3X 10⁶Autologous irradiated (5000rad) PBMCs, or no pulse treatment, served as negative controls. In the last 5 hours, brefeldin A (10. mu.g/ml) was added to the cells. Thereafter, cells were fixed, permeabilized, and stained with anti-CD 4, anti-IL-2, and anti-IFN-. gamma.mAbs for analysis on a FACS Calibur.

Classification of TB patients

5 HIV-seronegative patients with active TB (confirmed clinically and by culture) were collected at the infectious disease clinic of the s. They underwent the Tuberculin Skin Test (TST) and were administered 0.1ml (5 tuberculin units) of Biocinetest-PPD tuberculin (Chiron Italia srl, Milano, Italy) by the Mantoux method. After 48-72 hours, the size of the induration was evaluated (induration ≥ 10mm was positive). Peripheral blood was drawn and then any therapy was initiated with written consent. Healthy controls (n-8) were selected among HIV-seronegative individuals without a history of TB exposure, no infection and no negative TST response.

Activation, culture and retroviral transduction of human T-cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coats (Fresenius, Oslo, Norway) of healthy donors obtained after informed consent by Lymphoprep gradient separation. PBMC were cultured in RPMI1640 medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with antibiotics, glutamine, and 10% heat-inactivated FBS (BioWhittaker-Italia, Milano, Italy). PBMC were seeded into 6-well plates (1X 10)⁶Ml), with anti-aCD 3(OKT 330 ng/ml, OrthoBiotech, Raritan, NJ) or paramagnetic BACD3/CD28 (3: 1 beads/T-cells) (Dynabeads, Dynal Biotech, or Xcell therapeutics Inc., Seattle WA, USA, Invitrogen). Prior to culture, T-cells were enriched with bagD 3/CD 28. Recombinant IL-2(Chiron, Emeryv) was prepared with 600I U/ml humanille, CA), cultured cells activated with aCD 3.

Culturing cells activated with bagd 3/CD 28: 1. in the absence of cytokines; 2, cultured with human recombinant IL-7 at a minimum concentration of 5ng/ml (Peprotech, London, UK); 3. human recombinant IL-7 and IL-15 were used in culture, both at a minimum concentration of 5ng/ml (Peprotech, London, UK). On days 2 and 3, cells were transduced with SFCMM3 retroviral supernatant by centrifugation with 8. mu.g/ml polybrene (Sigma, St Louis, Mo) at 37 ℃ for 2h at 2400rpm (39-40). The SFCMM3 retroviral vector encodes the TK suicide gene under the LTR promoter and encodes a truncated form of the nerve growth factor (Δ LNGFR) under the SV40 promoter (27). Retroviral supernatants were supplied by Molmed s.p.a. On day 6 after T-cell activation, cell-sized beads for stimulation were removed from the T-cells according to the manufacturer's instructions. In some experiments, transduced lymphocytes were positively selected 7 days after T-cell activation according to the following method (the method is titled: positive immunoselection of transduced lymphocytes).

The cells were cultured for up to 14 days. On day 14, LNGFR determined by multiplying percent by trypan blue count flow cytometry⁺Cells, calculate multiplication. According to the original method, medium and cytokines were replaced every 3-4 days. At selected times,. DELTA.LNGFR determined by multiplying the percentage by Trypan blue count flow cytometry⁺Cells, calculate multiplication.

Positive immunoselection of transduced lymphocytes

At day 7 after T-cell activation, transduced lymphocytes were positively selected because transduced cells expressed Δ LNGFr on their surface, and transduced lymphocytes were separated from non-transduced lymphocytes by administering anti-LNGFr antibodies coated with magnetic beads.

Cells were collected from the plates, washed (1500rpm, 10min. at RT) at 5X 10⁶The final concentration of/ml was resuspended in WB. The anti-LNGFr antibody 20.1 was then added to the cell suspension (1ug/20 prepared)10⁶) Cells were spun at 10rpm for 30min at room temperature. The T-cells were then washed 1 time at 25X 10⁶The suspension was resuspended in WB at a concentration of/ml. Dynabead M-450 sheep anti-mouse IgG (5X 10) was then added⁶Bead/10⁶Positive cells) were spun at 10rpm for 30min at room temperature. The transduced cells were then magnetically selected. For this, the tube was placed near the magnet for 3min and the negative fraction was discarded. This operation was repeated 3 times in total. Finally, the cell fraction bound to the guide beads was removed from the magnet, washed, and washed at 1X 10⁶The cells, at a concentration of cells/ml, are resuspended in fresh medium containing a mixture of appropriate cytokines.

Flow cytometry of transduced T-cells

Flow cytometry was used to analyze surface phenotype, transduction efficiency, cell cycle, and cytokine production. The following antibodies (Pharmingen) were used: FITC-conjugated mabs against human CD4, CD8, CD45RA, CD27 and IFN- γ, (PE) -conjugated mabs against human CD4, CD8, LNGFR, CD62L, CD28 and I L-4, polymethine flavin chlorophyll-a protein (PerCP) -conjugated mAb (ly5.1) against mouse CD45, and Allophycocyanin (APC) -conjugated mabs against human CD 3. Following the isotype-matched fluorochrome-conjugated irrelevant mAb-stained controls, the samples were analyzed for data on a Facscalibur flow cytometer (Becton Dickinson, Mountain View, CA) using CellQuest software (Becton Dickinson).

Fluorochrome-conjugated antibodies against CD127, CD122, CCR7 and against mouse CD45 (Pharmingen, San Diego, CA, USA) were also used to stain lymphocytes.

Cytokine production

To determine cytokine production, cells were seeded into 24-well plates (1X 10)⁶Ml) and stimulated with 50ng/ml PMA (Sigma) and 1. mu.g/ml ionomycin (Sigma). After 4h, brefeldin A (Sigma) was added for an additional 2h (10. mu.g/ml). Then, the cells were stained with an appropriate fluorochrome-conjugated anti-surface labeled antibody and fixed with 1% paraformaldehyde at 4 ℃ for 10min. PBS 2% FBS containing 0.05% saponin (Sigma) at room temperatureAfter 20min incubation, intracellular staining was performed with appropriate fluorochrome-conjugated anti-cytokine antibodies.

CFSE dilution assay and Mixed Lymphocyte Reaction (MLR)

T-cell alloreactivity was analyzed 10 days after primary lymphocyte culture. On day 10, transduced T-cells were stained with CFSE and then cultured with irradiated allogeneic PBMC. CFSE consists of a fluorescein molecule containing a succinimide ester functional group and 2 acetate moieties, which can diffuse freely into cells, and intracellular esterases cleave the acetate group, converting it into a fluorescent, membrane-permeable dye. The dye is not transferred to adjacent cells. CFSE is confined by the cell in the cytoplasm and does not adversely affect cell function. The relative intensity of the dye decreases by-half in each cell division cycle.

CFSE staining procedure:

cells were washed 2 times in PBS (in the absence of serum) and adjusted to 2X 10⁷And/ml. CFSE was diluted to 1. mu.M in PBS and mixed with the cell suspension in a 1: 1 ratio. Vortex cells rapidly and mix for 8 min. At room temperature, FBS was then added in a 1: 1 ratio, and after 1 minute, the cells were centrifuged at 2000rpm for 2 min. The supernatant was then discarded and the cells were washed 2 times with a solution containing 10% FBS (PBS or culture medium).

MLR

At the end of the procedure, CFSE-stained transduced T-cells (responder cells) were cultured in 24-well plates containing 2000cGy irradiated allogeneic PBMC (stimulators) at a 1: 1 ratio. No cytokines were added to the cell culture. CFSE-stained transduced T-cells cultured in the absence of stimulus were used as negative controls. Cells cultured with soluble anti-CD 3 antibody were used as positive controls.

Reading number

At selected times after stimulation, cell samples were collected and stained with fluorochrome-conjugated anti-surface labeled monoclonal antibodies. Just prior To FACS collection, 1. mu.l of the To-Pro-3 solution was added To each FACS sample. To-Pro-3 is a deep red intercalating DNA dye, detectable in fluorescence 4, which provides the possibility of co-staining cells with FITC, PE and APC conjugated antibodies. Its function is to reveal the fraction of dead cells.

In vivo analysis

In the appropriate (scid, recombination activating gene)^-/-) And in the innate compartment (NOD, common gamma chain)^-/-) Are commonly used to study human lymphocyte biology in vivo. We used NOD/scid mice to test the activity of central memory genetically modified lymphocytes in vivo. 6-8 week old female NOD/scid mice were obtained from Charles-River Italiaa (Calco, Italy). The experimental protocol was obtained by the Institutional Animal research Committee (IACUC)]) Approval of (1). Mice were treated according to the following protocol:

allograft-host disease model

6-8 week old female NOD/scid mice were obtained from Charles-River Italiaa (Calco, Italy). 1 week prior to infusion, mice were transferred from laminar flow isolators to general cages, kept free of specific pathogens, and given free access to sterile water and irradiated feed. On the day before the experiment, mice were administered intraperitoneally with 1mg of blocking anti-mouse IL-2R β monoclonal antibody to neutralize residual NK activity. anti-IL 2R β antibody was produced as described from TM β -1 hybridoma kindly supplied by professor Tanaka (Osaka University, Japan). On day 0, mice received a single dose of 350cGy (gamma irradiation from linear accelerator) systemic irradiation and were immediately infused with unmodified PBL or human lymphocytes transduced with SFCMM3 retroviral vector (28). Unmodified PBLs were obtained from PBMC and contaminating monocytes, B-and NK-cells were removed using the Pan T-cell isolation kit (Miltenyi, Bergisch Gladbach, Germany). Cells were resuspended in 500. mu. l X-VIV015 medium and then the intraperitoneal infused mice were monitored for GvHD by calculating weight loss. For ethical reasons, Mor ibund mice were sacrificed. Each timeHuman chimerism was determined by flow cytometry after weekly blood draws from the tail vein. Human chimerism was calculated as follows: human chimerism (%) ═ huCD3⁺/(huCD3⁺+mCD45⁺)]×100。

Analysis of allogeneic GvHD

At 1, 2 and 3 weeks post T-cell infusion, mice were weighed and the allogeneic GvHD was evaluated according to the following score: weight loss (0 for weight loss < 10%, 1 for weight loss 10% -25%, 2 for weight loss > 25%), humpback (0-2), activity (0-2), fur texture (0-2), and skin integrity (0-2), max index 10. Weight loss was also estimated as an independent variable, as it was considered the most objective criterion (table 1).

Table 1: evaluation of clinical variant-GvHD in human T-cell infused mice

Standard of merit

Level 0

Level 1

Level 2

Weight lossLight and lightweight	＜10％	10-25％	＞25％
				Posture of a person	Is normal	Seeing hunchback only at rest	Serious humpback injury exercise
Degree of motion	Is normal	Mild to moderate remission	Being quiescent prior to stimulation
				Fur texture	Is normal	Mild to moderate creping	Severe creping/differential hair behavior
Skin integrity	Is normal	Peeling of paw/tail	Areas of apparent bare skin

Allogeneic GvHD model

In the allogeneic GvHD model, mice were transplanted with human skin and infused with allogeneic genetically modified lymphocytes to evaluate their ability to home to human skin and mediate allogeneic GvHD responses. 1 week prior to transplantation, mice were transferred from laminar flow isolators to general cages, kept free of specific pathogens, and given free access to sterile water and irradiated feed. Approximately 3 weeks prior to human T-cell infusion, mice were anesthetized intraperitoneally with 12-18mg tribromoethanol/mouse. They were then dehaired on the back and horizontally incised on the skin on both sides of the animal's back. The subcutaneous pocket is then opened and a small piece of human abdominal epidermis (derived from dermal fat and connective tissue) is introduced. At the end of the procedure, the wound is sutured. Since the mice gradually decreased in temperature during the procedure, the animals were placed in a heating box for about 30min and finally transferred to their cages.

Human T-cell infusion

To facilitate engraftment of human lymphocytes in NOD/scid mice, we functionally inactivated NK cells with an anti-mouse IL-2 receptor beta (TM β -1) antibody prior to lymphocyte transfer. Antibodies were produced from TM β -1 hybridomas kindly supplied by professor Tanaka (Osaka University, Japan). On day 0, mice received a single dose of 350cGy (gamma irradiation from a linear accelerator) of total body irradiation. The animals were then weighed and infused immediately with transduced human lymphocytes that had been collected on day 9 after the initial stimulation. Cells were infused intravenously in 250 μ L saline solution.

Analysis of T cell engraftment

At weeks 1, 2 and 3 after T-cell infusion, approximately 300 μ l of blood/mouse was harvested from a mini-incision in the tail vein and collected in tubes containing heparin. Red blood cells were lysed by exposure to ACK for 3min, and then stained for cytofluorometric analysis as described in the paragraph entitled "surface marker staining and cytofluorometric analysis".

Analysis of allogeneic GvHD

Mice were sacrificed 3 weeks after T-cell infusion, or earlier in the case of severe GvHD, and 2 human skins were removed from both sides. Formalin-fixed paraffin-embedded skin was cut into 4- μm thick sections, stained with hematoxylin and eosin, and subjected to morphological evaluation. Immunohistochemical assessment of the presence of human T lymphocytes was performed using an automated Dako immunostaining machine by the avidin/biotin peroxidase complex method with a monoclonal anti-human CD3 antibody (Dako, Glostrup, Denmark) diluted 1: 100. The staining reaction was revealed by the tetrahydrochloride chromogen method, and the sections were counterstained with hematoxylin. Photographs were taken with a Zeiss Axiocam HRC.

Statistical analysis

For each variable considered in this study, the mean, median and standard deviation were calculated. All statistical analyses were performed using Microsoft Excel 2003(Microsoft, Redmond, WA) and its insert form Statcel2(OMS publish, Saitama, Japan). Parameter data were subjected to Scheffe's F test (ANOVA) after analysis of variables, and non-parameter data were subjected to Mann-Whitney's U test after Kruskal-Wallis test.

Results and discussion

IL-7 favors rare tumor-specific CD4⁺Detection of T cells without Ag-driven cell proliferation

The counting of Ag-specific T cells may be critical for several clinical conditions where the presence of Ag-specific T cells has diagnostic and prognostic benefits. The authors have recently developed a preclinical mouse model of tumor-disease, expressing Leishmania major-derived model Ag LACK (TS/A-LACK) with TS/A tumor cells. Although LACK-specific naive CD4 could not be identified in non-manipulated BALB/c mice⁺T cells (29), the authors recently identified LACK-specific Ag-experienced CD44 in TS/A-LACK tumor-bearing mice by fluorescent MHC class II/Ag multimer staining and LACK-specific IL-2 and IFN- γ intracellular release^{Height of}CD4⁺T lymphocytes (20).

In an effort to improve the detection and cloning of Ag-specific T cells, the authors investigated CD4, a known aid to memory⁺Whether survival of T cells (7, 9-14) by IL-7 would increase tumor-specific CD4⁺Frequency of T cells. BALB/c mice were injected subcutaneously with control TS/A and TS/A-LACK tumor cells. Swelling in the middleAll mice had developed measurable tumors 20 days after tumor cell injection. Mice were sacrificed and tumor canals and no canalization LN were surgically excised. Although the former contained LACK-specific Ag-experienced CD44 capable of producing IL-2 and IFN-gamma^{Height of}CD4⁺T lymphocyte population, the latter still not being aware of tumors and the current LACK-specific naive CD4⁺T lymphocytes (31). LACK-specific CD4 was analyzed ex vivo after 7 days of culture in the presence of recombinant IL-7⁺Frequency of T cells, no further Ag stimulation was required (fig. 1). Ex vivo, leading LNs from TS/A-LACK-tumor bearing animals showed lower but significant CD4⁺I-A^d/LACK⁺Frequency of cells expressing high levels of CD44 (FIG. 1A) and capable of producing IL-2 and/or IFN- γ following LACK stimulation (FIG. 1B). CD4 after 7 days of incubation with IL-7 alone⁺I-A^d/LACK⁺ CD44^{Height of}And the frequency of LACK-specific cytokine-producing cells (fig. 1A, B), as well as their total number (not shown, and fig. 2, 4) increased significantly. Of the cytokine-secreting cells, IL-2 and IFN- γ -producing cells (which may represent intermediate memory T lymphocytes) (30-33), previously described as multifunctional, are the most abundant. The frequency of LACK-specific T cells in tumor-thinner LN neutralization TS/a of mice carrying control TS/a-tumors and in non-thinner LN of TS/a-LACK mice was comparable to that found in naive BALB/C mice ex vivo (fig. 1C, D and not shown), and within background measurements after culture in IL-7 (fig. 1C, D). Thus, IL-7 will enrich for in vivo-pretreated tumor-specific Ag-experienced CD4⁺LN cultures of T cells, thereby facilitating their enumeration, without the need for Ag-driven cell proliferation in vitro.

IL-7 and IL-2 contribute to tumor-specific CD4⁺Accumulation of T cells, but antigen stimulation does not

Restimulation of Ag is most commonly used for proliferation, and in some cases, for identifying Ag-specific CD4⁺T cells (34). In addition, IL-2 and IL-15, in addition to IL-7, also control memory T cell proliferation (13, 35-37). Thus, in the presence of unpulsed (APC) or with LACK-derived peptidesLN cells from TS/A-LACK tumor-bearing mice were cultured in the presence of shock-treated (Ag/APC) irradiated singenic APCs, or in the presence of optimal amounts of IL-7, IL-2, IL-15 and IL-6 as controls, and analyzed by flow cytometry. CD4 in cultured cells⁺ I-A^d/LACK⁺ CD44^{Height of}The frequency of T cells was slightly higher than that found ex vivo (fig. 1A), but no difference was detected in the control (APC) and Ag-stimulated culture (Ag/APC) (fig. 2A). Conversely, culturing cells in IL-7 resulted in LACK-specific CD4⁺The frequency (FIG. 2A, B) as well as the total number (FIG. 2C, D) of T cells increased beyond that found in control (not shown) and IL-6-driven cultures. Similar to IL-7, IL-2 is also enriched in CD4⁺ I-A^d/LACK⁺CD44^{Height of}LN cultures of T cells (FIG. 2A, C) and CD4 secreting IL-2 and IFN- γ following Ag-specific stimulation⁺LN cultures of cells (fig. 2B, D). In IL-15-driven cultures, CD4⁺ I-A^d/LACK⁺ CD44^{Height of}The frequency of T cells was still comparable to that of control cultures, but LACK-specific CD4 secreting cytokines⁺T cells were enriched in several independent experiments. In addition, IL-2/IFN-. gamma.secreting CD4 in LACK-specific cytokine-producing cells⁺T cells were most enriched and functioned better in IL-7-driven cultures (FIG. 2C, D).

IL-7 and IL-2 support in-pretreated tumor-specific CD4⁺Ag independent spontaneous proliferation and survival of T cells

The authors next investigated the LACK-specific CD4 that IL-7 and IL-2 contribute to in vivo pretreatment⁺The mechanism of accumulation of T cells. First, the authors analyzed the ability of these cytokines to support the in vitro propagation of these cells. LN cells were labeled with CFSE vital stain and cultured for 1 week in the absence or presence of recombinant cytokine. LN cells derived from naive mice were unable to proliferate, maintaining their original CFSE content, in the absence of exogenous cytokines (fig. 3A, left panel). In contrast, from TS/A-LACK-bearing tumorsMouse CD4⁺A detectable population of LN cells will proliferate and its CFSE content diluted in vitro in the absence of additional stimulation (fig. 3A, right panel). LACK-specific memory T cells identified by their ability to secrete IL-2 and IFN-gamma after in vitro LACK restimulation, are mainly comprised of CFSE that proliferates rapidly^dimIn cells (FIG. 3B, nil, MFI: 66), indicating that they are able to proliferate after the most recent tumor-Ag encounter in vivo. Higher frequencies of cells derived from tumor-ducted LN completed 1-3 cell division and LACK-specific T cells identified by their ability to secrete IL-2 and IFN-g in the presence of IL-7 after LACK-specific restimulation were significantly enriched and had lower CFSE content (MFI: 44) (FIGS. 3B and C). Indeed, LACK-specific CD4⁺T cells have undergone more than 3-4 cell cycles, which distinguishes them from cells that undergo slow homeostasis-like cell division (less than 4 cell cycles). In LN of naive control mice, IL-7-driven homeostatic cell division was also found (FIG. 5C). However, LACK-specific CD4⁺T cells were not enriched in these cultures (not shown). LACK-specific T cells found in tumor-leading LN and accumulated in response to IL-7 in vitro, probably represent recently pretreated unpolarized intermediate memory T cells, which were also found in chronically infected patients because of the ability to produce IL-2 and IFN-g following spontaneous in vitro proliferation and restimulation (32).

In addition to IL-7, IL-2 also supports a portion of CD4⁺T cells proliferate in vitro and increase the number of LACK-specific memory lymphocytes. In contrast, IL-15 and IL-6 were unable to support cell proliferation, nor did they enable LACK-specific CD4⁺T cells accumulated to a level exceeding that found in control (nil) cultures (fig. 3B, and D). Thus, IL-7 and IL-2 support in vitro proliferation of memory T cells undergoing Ag.

In parallel experiments, the authors confirmed similar results by analysis of LN cultures derived from 16.2 β transgenic mice harboring TS/A-and TS/A-LACK-tumors, containing a rather high frequency of I-A^d/LACK⁺ CD4⁺Naive T cells (25). Indeed, as in the case of cultures derived from tumor-bearing BALB/C mice, cultures derived from 16.2 β mice contained LACK-specific CD4 expressing high levels of CD44 (FIG. 4A) and capable of achieving IL-2 and IFN- γ LACK-specific release (FIGS. 4B, 4C)⁺. IL-7, IL-2 will enrich the culture of these cells, with lesser degrees of IL-15, but IL-6, TNF-. alpha.and IL-10 will not. LACK-specific CD4 capable of achieving IL-2 and IFN-gamma LACK-specific release⁺T cells were also contained within CFSE dim cells (fig. 4E). Even in this unrelated model, IL-7 and IL-2 will enrich for Ag-experienced CD4 by supporting their proliferation in vitro⁺LN cultures of T cells, without Ag-stimulation.

To further characterize the Ag-experienced CD4 promoting IL-7 and IL-2⁺Relative efficacy of accumulation of T cells, staining CFSE-labeled cells with the fluorescent dye TO-PRO-3 (which distinguishes between live and dead cells within proliferating cells), IL-7 preferably maintained only 15% TO-PRO-3⁺The culture survival rate of (1), cell death after week. In contrast, up TO 47% of the cells were maintained in IL-2, 60%, 57% and 73% of the cells cultured in the absence of exogenous cytokines or in the presence of IL-15 and IL-6 were TO-PRO-3⁺(FIG. 5A). Furthermore, although only 40% of CFSE dim cells repel dye in the presence of IL-2, up TO 72% of these cells are still TO-PRO-3 in the presence of IL-7^-. This indicates that cells actively proliferating in the presence of IL-7 alone are more viable and may be less sensitive to cell death than cells cultured in IL-2 alone.

The ability of IL-7 and IL-2 to favor T cell survival correlates with their ability to modulate the expression of the anti-apoptotic factor Bc1-2 (10, 38). Thus, the authors analyzed the level of Bc1-2 in CFSE-labeled LN cultures maintained in the absence and presence of recombinant cytokines. Under each culture condition, CFSE dim cells expressed optimal levels of Bc1-2 (FIG. 5B) in addition to IL-15, indicating that cells that had been pretreated in vivo had received a pro-survival signal. But instead of the other end of the tubeIL-7 uniquely favours the survival of CFSE dim and CFSE bright cells. In fact, up to 82.5% of CD4⁺T cells expressed optimal Bc1-2 levels in the presence of IL-7, while only 41.3%, 16.7%, 38%, and 10.5% of the cells cultured in control medium, IL-2, IL-15, and IL-6, respectively, maintained high Bc1-2 expression (FIG. 5B, C). Bc1-2 expression was even reduced in most cells in the presence of IL-2 and IL-15 (FIG. 5C). These findings together support the CD 4-favouring of IL-7 over IL-2 or IL-15⁺Superior ability of T lymphocytes to survive and suggest that IL-7 may uniquely retain all CD4⁺T lymphocyte subpopulations, regardless of their activation state.

The authors further analyzed the surface phenotype in tumor-persuasive LN-derived cultures in order to assess whether IL-7 would retain all subpopulations of tumor-specific CD4T + cells produced in vivo more strongly than IL-2, thereby assessing whether IL-7 is a major interesting tool for tracking and detecting rare in vivo Ag-experienced CD4+ T cells. TS/A-LACK tumor-Dredging LN cells were labeled with CFSE vital dye and cultured for 1 week in the presence of IL-7 or IL-2 alone. In CFSE dim cells cultured in IL-7 only, a portion of CD4 was compared to ex vivo cultured cells (FIG. 6A)⁺T cells maintained high or low expression of CD44 and CD62L (fig. 6A, B). These cells may represent naive, effector and central memory lymphocytes (8, 39-41). IL-7-treated CFSE dim cells express intermediate levels of CD25 and CD132 and down-regulate the surface expression of CD127 as previously reported (42). Differently, most CFSE dim cells cultured in IL-2 only express a fully activated surface phenotype (CD 44)^{Height of}、CD25^{Height of}、CD62L^{Is low in}，Bc1-2^{Is low in}FIG. 6A), and the culture is enriched with CD44^{Height of}T cells (fig. 6B and 7B). In cultures derived from 16.2 β transgenic mice, IL-7 and IL-2 were enriched for Ag-experienced CD4 with surface phenotype, although compared to that found ex vivo⁺T cells (FIG. 7A), IL-7 alone will be in I-A^d/LACK⁺And I-A^d/LACK^- CD4⁺CD44 in T cells maintained the original ratio before high and low cells (fig. 7B). When trying to tryThe ability to retain relative lymphocyte representation may be relevant when using short-term culture to determine the frequency of Ag-specific T cells in a biological sample. Furthermore, although in IL-2-cultured cells, most of the Bc1-2⁺The cells expressed low levels of the LN homing molecule CD62L, up to 53% of CFSEdim CD4 in the presence of IL-7⁺T cells maintained optimal expression of Bc1-2 and CD62L (FIG. 6C).

IL-7, IL-2 and IL-15 will proliferate Ag-specific memory CD8 in an Ag-independent manner⁺T cells

To further investigate the general usefulness of short-term Ag-independent culture in IL-7 for revealing Ag-specific T cells produced in vivo, the authors investigated whether Ag-specific CD8 could be followed in different tumor disease contexts⁺T lymphocytes. The main purpose is: 1) evaluation of CD8 used in the present invention to track Ag-experience in vivo⁺T cells (not just Ag-experienced CD4 in vivo)⁺T cells), 2) to evaluate the applicability of the invention for clinical situations (effective vaccination) different from the diagnosis of anti-tumor immune responses.

In an attempt to investigate whether IL-7 could be used to reveal Ag-specific T cells in clinical settings different from tumor disease, we analyzed Dendritic Cell (DC) -based vaccine-induced peptide-specific CD8⁺T cells. C57BL/6 mice were inoculated with bone marrow-derived DCs pulsed with MHC class I restricted labeled IV peptide (DC-Tag) derived from the SV40 large T antigen (18). After 14 days, LN cells were analyzed ex vivo and after cytokine-driven culture by Ag-specific intracellular cytokine release. As a control, LN cells were also derived from naive C57BL/6 mice. Label IV specific CD8 producing IFN-gamma or IL-2 and IFN-gamma only after re-stimulation with Ag⁺T cells, undetectable in naive mice, and detectable at low frequency (0% and 0.37%, respectively) in DC-vaccinated mice. Labeling IV-Teter in cultures derived from DC-vaccinated mice after 7 days of culture with IL-7, IL-2 and IL-15 without Ag restimulationHeterosexual CD8⁺The frequency (3.94%, 1.83% and 1.95% respectively) and total number of T cells (fig. 8) increased significantly, whereas cultures derived from naive mice did not (not shown).

In the same culture, the authors analyzed total CD8⁺T cell versus marker IV-specific CD8⁺Relative enrichment of T cells (fig. 9). Under all different conditions, culturing in IL-7, IL-2 and IL-15 alone (but IL-6 did not) or in general medium increased CD8 relative to ex vivo analysis by doubling their absolute numbers⁺Total number of T cells (fig. 9). In contrast, although the overall CD8⁺The T cells increased 2-fold and the marker IV specific CD8+ T cells had undergone a 5-7 fold increase (FIG. 8), which means that the in vivo marker IV-vaccine experienced CD8T cells with superior potency to the other CD8⁺The great advantage of T cell populations and are selectively enriched in culture in the presence of pro-survival cytokines (e.g., IL-7, IL-2 or IL-15). Thus, IL-7 can be used to unmask tumor-and vaccine-induced CD8⁺T cell responses even without Ag restimulation.

IL-7 revealed an otherwise undetectable antigen-specific CD8 ex vivo⁺T cells

TS/A cells naturally express the endogenous MuLV coat protein gp70, an immunodominant epitope of which was previously described (AH-1, (19)). Whether culture in IL-7 might also aid in the identification of rare Ag-specific CD8 in their study⁺In further attempts at T cells, the authors compared AH-1-specific CD8⁺T cell responses, ex vivo and in the absence of Ag restimulation, after 1 week of culture in the absence or presence of IL-7, IL-2, IL-15 and IL-6, respectively. Lymphocytes were analyzed by intracellular cytokine release following stimulation of both unpulsed and AH-1-pulsed syngeneic splenocytes (FIG. 10). AH-1-specific CD8 in tumor-bearing mice⁺T cells were undetectable ex vivo, as the frequency of cytokine-producing cells remained within background levels (fig. 10A), and correlated with that found in naive miceComparative (not shown). After 7 days, LN cultures derived from LN bearing TS/A-LACK tumors and maintained with or without recombinant cytokines contained variable frequency of IFN- γ producing cells that were restimulated independently of AH-1 (FIG. 10B). In the presence of IL-7, the cultures were enriched with AH-1-specific CD8 that produce IFN- γ alone or IL-2 and IFN- γ after Ag restimulation⁺T cells (fig. 10B). Although IL-2 also increases AH-1-specific CD8⁺Frequency and total number of T cells (fig. 10B and C), which could not be increased by IL-15-and IL-6, when the frequency remained comparable to that found in cultures derived from naive mice, and within background levels (data not shown). Thus, IL-7 and IL-2 could reveal tumor-specific CD8 that was otherwise undetectable ex vivo⁺T cells, do not require Ag-driven cell proliferation in vitro.

Interleukin-7 can cooperate with cyclosporin A-sensitive signal to selectively reproduce memory CD4⁺T cells.

Memory CD4⁺The in vivo proliferation and survival of T cells is dependent on IL-7 as well as TCR-driven events (9). In vitro, TCR-driven human memory T cell proliferation requires intact ERK activity, whereas cytokine-driven homeostatic cell division is dependent on p38 and is insensitive to cyclosporine a (csa) (13). Thus, the authors analyzed the requirement for IL-7-driven intermediate memory T cell accumulation by culturing lymphocytes in the presence of blocking antibodies or inhibitors of selected signaling pathways. The cells are derived from LNs of naive or TS/A-LACK-tumor-bearing 16.2 β mice, which are naive CD4 with considerable frequency of LACK-specificity⁺T cell transgenic mice (25), labeled with CFSE, were cultured in the presence of optimal amounts of IL-7 and the indicated inhibitors. As a control, Ag-driven T cell proliferation was significantly inhibited by anti-MHC class II mAb and CsA and partially inhibited by anti-ICAM-1, anti-LFA-1 mAb (fig. 11A, B) with Antigen Presenting Cells (APCs) treated with LACK-pulses in the absence or presence of selected inhibitors (fig. 11A, B). As expected, rapamycin (mTOR inhibitor) only delayed Ag-floodingMotile T cell division (fig. 11A, B), whereas SB202190 (a p38 inhibitor) did not inhibit Ag-stimulated T cell division as previously reported (13). A fraction of CD4 in cultures derived from control naive mice in the presence of IL-7⁺T cells underwent 1-2 cycles of slow homeostatic cell division (FIG. 11C, nil, thin line), while rapidly proliferating CD4⁺ CFSE^dimCell populations accumulated in tumor-derived dredging LN cultures (fig. 11C, nil, bold line). In addition, LACK-specific T cells that release IFN-. gamma. (FIG. 11E) and IL-2 (not shown) were found to be predominantly present in rapidly proliferating CD4T cells. Addition of anti-MHC class II mAbs to IL-7-driven tumor-Dredging LN cultures against rapidly proliferating CD4⁺ CFSE^dimThe accumulation of cells had a slight effect (FIG. 11D, bold line), reducing the frequency of LACK-specific IFN-. gamma.producing cells by 50% (FIG. 11E). anti-ICAM-1 or anti-LFA-1 mAbs and CsA inhibit IL-7-driven fast-dividing CFSE^dimCD4⁺T cell accumulation and LACK-specific IFN-. gamma.producing cells accumulation did not alter IL-7-driven homeostatic cell division (FIG. 11D, bold line and FIG. 11E). LACK-specific CD4 despite SB202190 or PP2 only partially inhibiting IL-7-driven rapid proliferation⁺T accumulation (FIG. 11D, E), rapamycin completely abolished IL-7-driven slow and fast cell division (FIG. 11D, bold line) and IL-7-driven LACK-specific T cell accumulation (FIG. 11E).

Together, these findings indicate that IL-7 can drive rapidly proliferating IL-2/IFN- γ through a synergistic effect with cell-derived CsA sensitive signaling⁺Intermediate memory CD4⁺Accumulation of T cells, possibly mediated by adhesion molecules and/or self-peptide/MHC interactions.

IL-7 supports the selective accumulation of rapidly dividing peripheral blood human CD4 memory T lymphocytes

IL-7 and IL-15 have previously been reported to support slow homeostatic-like cell division of central memory and effector human memory T cells (13). The authors investigated whether high density culture conditions and optimal IL-7 levels would also reveal peripheral blood-derived T-lymphocytesRapidly dividing intermediate memory T cell populations in the blast cells. For this purpose, PBMCs were derived from healthy donors, labeled with CFSE, and cultured at different cell densities for 7 days in the absence or presence of optimal amounts of IL-7 (fig. 12). At low cell density (10)⁶Cells/24 wells/ml), and in the absence of IL-7, all cells did not proliferate, maintaining the original CFSE content. At high cell density (5X 10)⁶Cells/24 wells/ml), a trace, but measurable, rapidly proliferating CFSE was present in control cultures maintained in fetal bovine serum (fig. 12, nil, top panel) or autologous serum (fig. 13)^dimCD4⁺A population of T cells capable of undergoing more than 4 cell divisions in culture for 7 days. In the presence of IL-7, a higher proportion of CD4T cells proliferated and completed 1-6 cell divisions (FIG. 12, IL-7, lower panel). In high cell density cultures (FIG. 12B) and optimal IL-7 levels (FIG. 14), cells capable of rapid proliferation over 5 cell cycles were best revealed. These rapidly dividing cells contain predominantly intermediate memory cells, since most of them produce IFN-. gamma. (FIG. 15) and IL-2 (not shown) following stimulation with PMA and ionomycin. IL-2/IFN-gamma at concentrations greater than 50ng/ml⁺T cells best accumulated frequency (fig. 16) and total number (not shown).

In addition to IL-7, IL-2 and IL-15 also support human CD4 as reported elsewhere (13)⁺In vitro proliferation of T cells (fig. 17A) and up-regulation of Bc1-2 expression (fig. 18). However, most cells that proliferate in response to IL-2 and IL-15 complete 1-4 cycles of slow homeostatic-like cell division, and rapidly proliferating memory T cells do not accumulate. When CD8 was assayed in the same culture⁺In T cells, IL-7 primarily supports a portion of CD8⁺Slow steady state division of T lymphocytes, while IL-2 and IL-15 allow accumulation of rapidly dividing CD8⁺T lymphocyte populations (fig. 17B), demonstrating similar effects of these cytokines on 2 different T cell subsets.

IL-7-driven T cell proliferation of peripheral blood human T lymphocytes sensitive to cyclosporin A

It was reported that IL-7 and IL-15-driven slow homeostatic-like cell division of human memory T cells is not CsA sensitive, but rather dependent on p 38-dependent signaling (13).

The authors next investigated the signaling required for IL-7-driven intermediate memory fast proliferating human T cell proliferation. IL-7-driven rapid proliferation of CD4, as in the case of mouse cells⁺T cell accumulation was sensitive to CsA and RAPA, and less sensitive to SB (fig. 19A) and PP2 (not shown). CsA failed to prevent cytokine-driven slow proliferation, consistent with previous reports (13). Proliferation of rapidly proliferating memory cells was also dependent on LFA-1-dependent interactions, as addition of anti-LFA-1 mAb inhibited their accumulation (fig. 19B). Rapidly proliferating CD4T cells propagated under high cell density conditions and in IL-7-driven cultures from CD45RA^-、CD62L^-T lymphocytes (Effective memory) and CD45RA^-、CD62L⁺(central memory) CD4T lymphocytes. Note that it appears to be the largest fraction of spontaneously proliferating CD4T cells, CD45RA^-、CD62L⁺Memory T cells, enriched primarily by ex vivo propagation methods, and most sensitive to CsA inhibition (fig. 19B and C).

Thus, the authors identified CD45RA capable of spontaneous in vitro rapid proliferation in peripheral blood of healthy donors^-、CD62L⁺Memory CD4⁺A population of T cells. These memory CDs 4⁺Accumulation of T cells benefits from IL-7, is cell density-dependent, and CsA-sensitive.

IL-7-driven short-term cultures will aid Mycobacterium tuberculosis-specific CD4 in human subjects⁺Enumeration of T cells

It has been established that IL-7 supports the in vitro propagation of rapidly proliferating memory T cells which may be programmed in vivo, and the authors investigated whether this could be used in clinical studies of immune-related pathologies. For this purpose, cryopreserved PBMC samples from mycobacterium tuberculosis-infected (TB) patients were analyzed by MTP-specific ELISPOT assay at thawing or after 1 week of culture with optimal IL-7 amounts (43). Based on their clinical history, signs of acute TBPatients were selected (confirmed clinically and in culture), their positive response to TST, and their ability to respond to MTP in the ELISPOT-IFN- γ assay. Cryopreserved PBMCs from uninfected healthy donors were also analyzed as controls. Upon thawing, Pt #1 showed a considerable amount of IFN-. gamma.after MTP-specific restimulation⁺Blot (FIG. 20A) (cryopreservation: 950 SFC. times.10)⁶PBMC). IFN-. gamma.after culturing the cells in control Medium (Nil)⁺Multiplication of the number of MTP-specific cells (1890 SFC × 10)⁶PBMC) reflecting the high frequency of CD4 in cultured cells⁺T cells (numbers in parentheses in fig. 20A). IFN-gamma in the presence of IL-7⁺Number of MTP-specific cells (3930 SFC. times.10)⁶PBMC) was 4 times that of the cryopreserved/thawed samples, 2 times that of the control cultures. Pt #2 and Pt #3 had detectable MTP-specific T cells at the time of sample collection (not shown), but not in the cryopreserved/thawed samples (fig. 20B, 20C). After IL-7-driven culture, IFN-. gamma.was revealed⁺MTP-specific blotting. IL-7 resulted in an increase in absolute numbers of MTP-specific T cells in all TB-patients analyzed, and as a result, the difference in the number of MTP-specific IFN- γ producing cells was significant in healthy donors and TB patients (fig. 21). MTP-specific T cells in response to IL-2 accumulation are predominantly composed of IL-2/IFN- γ⁺Memory CD4T cells were representative, as determined by MTP-induced intracellular cytokine secretion (fig. 22). Furthermore, MTP-specific IL-2/IFN-. gamma.was prevented in the presence of CsA⁺Proliferation of memory CD4T cells (fig. 23).

IL-7-driven short-term cultures will aid in the enumeration of Candida antigen-specific human T lymphocytes

In addition to MTP-specific T cells, the authors also investigated whether IL-7 would enhance the identification of T lymphocytes specific for Candida albicans-derived antigens. For this purpose, PBMCs from Pt #1 were analyzed by ELISPOT assays with irradiated autologous PBMCs that were not pulsed or were candida albicans-derived Ag-pulsed, either at thawing or after 7 days of culture in normal media or in the presence of IL-7. In the case of Mycobacterium tuberculosis-specific T cell responses, Candida albicans-specific T cells capable of releasing IFN- γ were also enriched in IL-7 short-term cultures (FIG. 24A, B).

Adoptive cell therapy of IL-7/IL-15 cultured memory T cells will delay tumor growth in vivo

Having determined that IL-7 determines the accumulation of memory CD4T cells that are pretreated in vivo, and that IL-15 best drives the proliferation of CD8 memory T cells, the authors evaluated whether the proliferating population is clinically relevant. For this purpose, lymph nodes were derived from TS/A-LACK tumor-bearing mice and at optimal cell density (5X 10)⁶Cells/ml) and optimal cytokine amounts (50ng/ml) for 7 days. As a control, naive T cells derived from control mice were also cultured under the same conditions. Thereafter, 10 carrying a comparable frequency of CD4 and CD8T cells will be⁷Cultured cells were transferred into naive BALB/c mice adoptively. After 48 hours, mice were challenged with 300.000TS/A-LACK cells and tumor growth was monitored over time. As shown in FIG. 25, TS/A-LACK tumors formed rapidly in control mice and in mice with cytokine-treated naive T cell adoptive transfer. In contrast, tumor growth was significantly delayed in mice that were adoptively metastatic with cytokine-treated tumor-bearing mouse-derived T cells (fig. 25). This suggests that IL-7/IL-15-driven cultures determine the propagation of clinically relevant memory T cell populations.

Generation of Gene-modified Central memory human T-cells

Retroviral transduction of T lymphocytes requires cell proliferation. The authors activated PBMC with aCD3 or bacD3/CD 28. Cells were activated with bagd 3/CD28 and cultured with IL-7 and IL-15 or with aCD3, and with IL-2 (fig. 27A). On day 2, cells were transduced with SFCMM3 retroviral vector by spin seeding. Transduction was performed simultaneously in the same manner. Activation with baCD3/CD28 in the presence of IL-7 and IL-15 promoted higher T cell proliferation and resulted in higher transduction efficiency (fig. 27A) than activation with aCD3 in the presence of IL-2 (fig. 27B). In addition, at the end of the culture period, the physiological CD4/CD8 ratio was analyzed and found to be better maintained in transduced cells activated with baCD3/CD28 and cultured with IL-7 and IL-15 than in transduced cells activated with aCD3 and cultured with IL-2 (fig. 27C).

Polyclonal activation required for retroviral transduction of T lymphocytes enriches memory cells

To determine the relative distribution of memory subsets in human T-cells transduced with retroviral vectors, the authors analyzed CD45RA/CD62L co-expression. On day 14, transduced T cells activated with aCD3 and cultured with IL-2 were predominantly CD45RA^-CD62L^-I.e., effector memory cells. In contrast, transduced CD4 activated with bagD 3/CD28 and cultured with IL-7 and IL-15⁺Highly enriched T-cells CD45RA^-CD62L⁺I.e., central memory cells (fig. 28A). Transduced CD8 was also observed in the case of cells activated with bagD 3/CD28 and cultured with IL-7 and IL-15⁺CD45RA of cells^-CD62L⁺Enrichment of central memory cells (fig. 28B). To better define the memory phenotype, we also analyzed CD28/CD27 co-expression. Although for transduced CD4⁺Cell, the relative distribution of the subpopulations under 2 conditions did not differ (fig. 28C), for transduced CD8⁺Cells activated with bagd 3/CD28 and cultured with IL-7 and IL-15 favoured CD28⁺CD27⁺T-cells (FIG. 28D).

Functional association of gene-modified central memory human T-cells

There is a difference in the ability of central and effector memory human T lymphocytes to produce effector cytokines identified by surface phenotype. On day 14, the authors restimulated 2 gene-modified T-cell populations and analyzed their cytokine production. CD4 stimulated with aCD3 and cultured with IL-2⁺T-cells will efficiently produce the prototype effector cytokine IFN- γ. In clear contrast, most of CD4 stimulated with bagD 3/CD28, IL-7 and IL-15⁺T-cells were unpolarized cells and did not produce IFN-. gamma.or IL-4 (FIG. 29A). Using CD8⁺Similar results were obtained with T-cells (FIG. 29B).

GvHD potential of gene-modified central memory human T-cells

Different xenograft models have been proposed to study human lymphocyte-induced GvHD. To evaluate the relative anti-host reactivity of two suicide gene-modified central and effector memory human T lymphocytes in vivo, these populations were infused into NOD/scid mice restricted with non-lethal irradiation and anti-NK antibodies. Control mice were infused with human purified syngeneic PBL. The authors observed that central memory gene-modified lymphocytes engrafted more efficiently than their effector memory counterparts (human chimerism at week 1 for effector memory genetically modified cells: 0, 45% range 0,2-1,1 on average; 4, 5% range 4,1-5,2 on average for central memory genetically modified cells). Table 2.5 of 6 mice infused with effector memory gene-modified T-cells showed reduced human chimerism after 1 week and showed no GvHD. On the other hand, in most mice infused with central memory suicide gene-modified T-cells, persistent human chimerism was observed, with 4 out of 6 mice developing severe GvHD.

TABLE 2 Implantation and graft versus host disease

	PBL	Effective memory TK+Cells	Central memory TK+Cells
				Human chimerism in% (range)Enclose)a	3.6(2.5-5)	0.45(0.2-1.1)	4.5(4.1-5.2)
Incidence of GvHDb	6/6	1/6	4/6

^aMean (range) 1 week after infusion

^bDefined as > 10% weight loss from the original body weight

Establishment of Gene-modified Central memory human T cells

To determine the minimum requirements needed to obtain many gene-modified central memory human T-cells suitable for clinical applications, we compared the following 5T-cell transduction conditions:

1. soluble anti-CD 3 antibody (OKT-3) + high dose interleukin 2(600 UI/ml);

2. anti-CD 3/CD28 cell size beads without any cytokines;

3. anti-CD 3/CD28 cell size beads + low dose IL-2(200 IU/ml);

4. anti-CD 3/CD28 cell size beads + low dose IL-7(5 ng/ml);

5. anti-CD 3/CD28 cell size beads + low dose IL-7(5ng/ml) + low dose IL-15(5 ng/ml).

Cells were transduced and cultured according to the methods described in the materials and methods section. These experiments yielded the following results:

1. activation with anti-CD 3/CD28 beads allowed higher transduction efficiency than activation with soluble anti-CD 3 antibody

As shown in fig. 30A, the retroviral transduction efficiency was significantly higher after stimulation with cell-size magnetic beads than after stimulation with soluble anti-CD 3 antibody. This result is independent of the use of cytokines in the culture mix.

2. Activation with CD3/CD28 beads followed by culture in the presence of cytokines (IL2.IL7+ IL15 or IL7) maintained the physiological CD4/CD8 ratio in transduced T lymphocytes

To evaluate our transduction method's ability to maintain physiological CD4/CD8 ratio, we analyzed the CD4/CD8 ratio of transduced cells produced by different methods 10 days after primary stimulation. As shown in figure 30B, stimulation with magnetic beads alone, followed by culture in cytokines (IL2, IL7 or IL7+ IL15) maintained a physiological CD4/CD8 ratio in transduced T-cells, whereas the average CD4/CD8 ratio observed with other culture conditions did not exceed 1/1.

3. Activation with CD3/CD28 beads, followed by culture in the presence of cytokines (IL2, IL7+ IL15 or IL7) induced a significantly higher proliferation rate of transduced cells than other culture conditions.

Ex vivo gene transfer methods designed for clinical applications must meet relevant criteria relating to feasibility: in clinical applications of gene therapy protocols, one major potential issue relates to cell number and cell proliferation in vitro, as shown in FIG. 30C, with statistically significant differences in the number of cells obtained from genetically modified cells stimulated with beads and cultured with cytokines compared to other conditions (bead alone and OKT3+ IL-2). These results indicate that transduced lymphocytes obtained with anti-CD 3/CD 28-conjugated beads and cultured with IL-7, IL-7+ IL-15 or IL-2, will rapidly multiply in vitro to a number suitable for clinical use.

4. Activation with anti-CD 3/CD28 beads predominantly results in central memory CD8⁺And CD4⁺Transduced lymphocytes

We investigated the immunophenotype of transduced cells obtained by different culture conditions by FACS staining of CD3, CD4, CD8, CD45RA and CD62L 10 days after primary stimulation. We found that a very high proportion (about 80%) of CD8 stimulated with anti-CD 3/CD28 beads⁺And CD4⁺The T-cell is CD45RA^-CD62L⁺: the map corresponds to central memory T-lymphocytes. In contrast, OKT 3-stimulated T-cells exhibited 60% CD8⁺And 45% CD4⁺Effector memory T-cells (CD45 RA)^-CD62L^-) And 30% CD8⁺And 50% CD4⁺Central memory TK⁺Lymphocytes (fig. 31).

Gamma-chain receptor expression during culture of transduced lymphocytes

During T-cell stimulation, cytokine receptor expression is tightly regulated. We analyzed the kinetics of expression of gamma-chain cytokine receptors during different T cell culture and transduction methods as a measure of T cell function and potential. To this end, we performed cytofluorimetric analysis after staining the transduced cells with fluorochrome-labeled antibodies against CD122(IL-2/15 receptor normal beta chain), CD25(IL-2 receptor alpha chain) and CD127(IL-7 receptor alpha chain) at different time points after the first stimulation.

1. IL-2/15 receptor beta chain (CD122) expression was not altered in different transduction methods

During the course of the immune response, IL-2/IL-15 receptor beta expression increases following T cell activation, and then decreases to intermediate expression levels, which remain throughout the memory-cell phase (13).

FIG. 32 shows the kinetics of CD122 expression in transduced lymphocytes during 13 days of culture. With respect to CD122 expression on T cells cultured in 5 methods, we did not observe any differences. In all cases, T-cells, in particular CD4⁺Cells, up-regulating CD122 after activation, peak at about day 4 when nearly 100% of the genetically modified cells express the molecule. The cells then slowly down-regulate the CD122 phenotypeTo the same level of expression as was observed 13 days after the start of the T-cell culture before stimulation, at which time the cells had reached a quiescent state.

2. Stimulation with the bead CD3/CD28 promotes strong and prolonged expression of IL-2 receptor alpha in transduced T-cells

IL-2 receptor alpha (CD25) is a relevant activation marker for T-lymphocytes. Under physiological conditions naive T cells do not express CD 25; however, its expression is rapidly up-regulated by T-cell activation and usually decreases before the proliferation peak of the response.

In bead-activated transduced cells, flow cytometry analysis showed that most T-cells experienced a significant increase in IL-2 receptor alpha expression at day 2 post-stimulation, independent of the subsequent culture conditions. In contrast, only 40% of transduced cells activated with soluble OKT3 up-regulated the receptor (fig. 33), indicating that most T cells were not properly activated by this stimulation protocol. As long as CD25 is expressed, T cells can proliferate IL-2, thereby actively participating in the immune response. In transduced cells produced after bead activation, CD25 expression remained high in most cells up to day 13. In contrast, cells that up-regulated CD25 after stimulation with soluble anti-CD 3 antibody reached a peak of expression 2 days later than cells activated with beads, and then rapidly down-regulated CD25 molecules.

3. Transduced cells activated with anti-CD 3/CD28 beads + IL-7 showed maximal expression of IL-7 receptor alpha, a marker for long-term survival of memory T-cells

Under physiological conditions, IL-7 receptor alpha (CD127) is constitutively expressed by naive T cells. Its expression is down-regulated by T-cell activation (equivalent to CD25, in a mirror image), and such down-regulation may promote cell death. In contrast, CD127 expression increases as the immune response progresses, reaching high levels in memory T cells. IL-7 is a potent survival factor for memory T-lymphocytes: triggering of the receptor by IL7 promotes T cell survival and proliferation and protects cells from apoptosis through different intracellular signaling pathways. We analyzed ourKinetics of CD127 expression in transduced cells. After stimulation (about day 1 to day 6), IL-7 receptor alpha undergoes deeper down-regulation. From day 7, its expression gradually increased, and interestingly, we observed a significant difference in the proportion of CD127 + transduced cells obtained with beads CD3/CD28 and IL-7 relative to all other conditions (fig. 34). From day 9, more than 80% of CD8 that had been stimulated with cell-size beads and IL-7⁺And CD4⁺ TK⁺The cells are IL-7 receptor alpha positive. This suggests that IL-7 is responsible for maintaining high levels of CD127 expression in most T cells after activation, providing a particularly long-term survival advantage for transduced cells.

Transduced lymphocytes generated with beads + IL-7 have the highest alloreactivity potential

From the results shown above, activation with anti-CD 3/CD28 magnetic beads and the helper effect of cytokines (especially IL-7) are important factors in generating central memory-transduced T-lymphocytes with high survival potential.

Our next goal was to investigate whether these CM transduced T-lymphocytes could indeed elicit a potent and effective immune response. We solved this problem in vitro by stimulating transduced cells with alloantigens, and we obtained the following results:

1. transduced lymphocytes generated with beads + IL-7 have the highest proliferative potential when stimulated with alloantigens

Transduced T cells generated under each of the 5 conditions were stained with CFSE and co-cultured with irradiated allogeneic PBMC. After 1 week, we counted the number of cells and analyzed CFSE dilution by FACS to evaluate the percentage of dividing cells. As shown in FIG. 35A, we found that it is in CD3⁺And CD8⁺Statistically significant differences between conditions containing IL-7 and other methods in T-cells. Indeed, a high percentage (40%) of transduced cells generated with beads CD3/CD28+ IL-7 had divided within 1 week after allogeneic stimulation. In contrast, only 20% of the drugs are usedTransduced cells produced by OKT3 divide under the same culture conditions.

2. Transduced lymphocytes generated with beads + IL-7 have the lowest sensitivity to death

To maintain T-lymphocyte homeostasis, an allogeneic challenge results in massive T-cell activation, often followed by a broad apoptotic program, and IL-2 is a major role in so-called "activation-induced cell death" (AICD). Effective and durable immunological memory is developed after the primary immune response, and a mechanism against AICD is required. To investigate the sensitivity of transduced cells To AICD, we stained allo-stimulated CFSE with To-Pro3⁺Lymphocytes, the To-Pro3 is a fluorescent dye that selectively binds To dead cells. We counted the number of dead cells in the dividing and non-dividing transduced cell populations in order to evaluate activation-induced cell death and neglect death, respectively (fig. 35B). Transduced cells cultured in IL2 (independent of activation signal) demonstrated high sensitivity to AICD and negligible death. In contrast, transduced cells produced with beads CD3/CD28+ IL-7 showed the lowest mortality rate, comparable to unmodified lymphocytes. This finding can be in high proportion to CD127 (33% CD8)⁺And 52% of CD4⁺) Is associated with persistent expression on genetically modified lymphocytes generated with the beads CD3/CD28 and IL-7.

Based on this finding, transduced lymphocytes generated with the beads CD3/CD28+ IL-2, which demonstrated high sensitivity to cell death, showed the lowest proportion of expressed CD127⁺Cells of the cell (30%). The FACS plots in figure 36A show a representative example of CD127 detection in CFSE-stained transduced T-lymphocytes 10 days after allogeneic stimulation.

3. Transduced lymphocytes generated with beads + IL-7 retain a central memory phenotype following allogeneic stimulation

Immunological memory is ensured by the self-recovering capacity of memory cells, which divide after encountering antigen again, producing effector cells (capable of eliminating pathogens directly) and memory cells (capable of growing)To protect the host). To confirm whether central memory genetically modified lymphocytes have this self-recovery capacity, we analyzed the expression of CCR7 (a marker for central memory cells) on CFSE-stained cells on day 10 post-allogeneic stimulation. As shown in FIG. 36A, our data indicate 59% of the CD3 produced with beads CD3/CD28 and IL-7⁺Genetically modified lymphocytes, which have undergone at least one division, are positive for CCR7 expression. In contrast, only 17% of CD3 modified with OKT3⁺Genetically modified lymphocytes express CCR 7. Culture in the presence of IL-7 is necessary to maintain the self-recovery capacity of central memory lymphocytes, since only 36% of the CD3 produced with the beads CD3/CD28 and IL-2⁺Genetically modified lymphocytes maintain expression of CCR7 following allogeneic stimulation. According to this result, when the cells were challenged again in vitro with the same allogeneic stimuli, more than 70% of the transduced cells produced with CD3/CD28 beads and IL-7 and only 50% of the transduced cells produced with OKT3 proliferated the next week, following the same culture conditions as the first stimuli (fig. 36B).

Transduced lymphocytes generated with beads + IL-7 have the highest potential for in vivo alloreactivity

To evaluate the in vivo efficacy of central memory transduced T-cells, we created a new chimeric model based on NOD/Scid mice transplanted with human skin. Based on the results obtained in vitro, we decided to study the efficacy of genetically modified central memory lymphocytes produced with the beads CD3/CD28+ IL-7, CD3/CD28+ IL-2 and to compare them with the functional activity of genetically modified effector memory lymphocytes produced with OKT-3 and IL-2 currently used in clinical trials. After infusion of transduced cells, allogeneic T-cell reactivity was determined by clinical observation, while allogeneic GvHD was evaluated histologically and correlated with an analysis of the infiltration of transduced cells into human skin. The results of these studies are summarized below:

genetically modified cells generated with CD3/CD28 and IL-7 will rapidly engraft into skin-transplanted NOD/Scid mice

The number of human T-lymphocytes in peripheral blood of mice increased from week 1 to week 2 after infusion. Transduced cells produced with beads engrafted to a greater extent than transduced cells produced with OKT-3. The difference was more evident at 2 weeks post T cell infusion (figure 37).

Bead CD3/CD28 activation and IL-7 stimulation confer the highest anti-alloantigen reactivity on transduced cells

Allogenic GvHD was monitored by measuring weight loss according to the clinical scores described in materials and methods. Infused NOD/Scid mice gradually lost their body weight, some of them eventually died of allogeneic GvHD, or were sacrificed for ethical reasons. Most allo-reactive T-cells were those stimulated with anti-CD 3/CD28 beads and cultured with IL-7, followed by transduced cells generated with CD3/CD28 beads and IL-2. Stimulation with soluble anti-CD 3 antibody did not produce strong allo-reactive T-cells, as mice infused with these lymphocytes did not show significant weight loss, nor did they show other clinical allo-GvHD signs (fig. 38).

Bead CD3/CD28 activation and IL-7 stimulation confer the highest anti-alloantigen reactivity on transduced cells

Our NOD/Scid human skin chimeric mouse model consists of NOD/Scid mice that have undergone a skin graft, where 2 pieces of human abdominal epidermis are inserted into 2 subcutaneous pockets on the back of the mice, and the genetically modified cells are subsequently infused intravenously. Human skin transplantation allows the study of T-cell reactivity against alloantigens by histological studies. In fact, transplanted human skin contains not only epidermal and mesenchymal cells, but also some antigen presenting cells, which attract circulating lymphocytes and possibly promote their activation. 3 weeks after human T-cell infusion, we sacrificed NOD/Scid chimeric mice and bilaterally removed human skin. All biopsies were subsequently analyzed by a blinded pathologist by hematoxylin-eosin (EE) and anti-human CD3 staining. Only in mice that have been infused with "bead + IL-7" stimulated T-lymphocytes, we observed massive human T-cell infiltration in the human skin background. Under "OKT-3" conditions, the level of T-cell tissue infiltration (if present) is clearly absent. Fig. 39 shows a representative example of these results.

Conclusion

Although it is generally accepted that T cells play a central role in the generation of immunity against pathogens, tumors, and immunodeficiencies, as well as in autoimmune disorders, it is difficult to use them as diagnostic and prognostic markers for human immune activity. In addition, there is currently a lack of technology suitable for propagating unpolarized multifunctional intermediates and central memory lymphocytes.

The results described in the present invention will identify new culture conditions that:

A) allowing accumulation of memory T cells pretreated in vivo independent of Ag;

B) leading the propagation of central memory lymphocytes, regardless of lymphocyte polyclonal stimulation and genetic manipulation.

The results indicate that IL-7, and IL-15 can be used to enrich biological samples, such as peripheral LN, blood and tumors, Ag (tumor/pathogen/allergen/autoantigen) -specific CD4 for in vivo pretreatment⁺Or CD8⁺T cells. IL-7 is better able to maintain primitive lymphocyte phenotype and characterization and is better able to favor survival of all T lymphocyte subpopulations than IL-2, allowing detection and propagation of rare CD4⁺T memory lymphocytes.

Enumeration of Ag-specific CD4 in the context of chronic viral infection, autoimmune disease, vaccination or immunotherapy⁺Or CD8⁺The capacity of T cells will provide a direct measure of the patient's immune activity or disease and assist the clinician in selecting the most appropriate therapy. Furthermore, the possibility of enriching in vivo-pretreated memory T cells without altering their phenotype may improve their characterization, as well as their utilization of immune responses in adoptive immunotherapy strategies. Finally, genetically modified central memory T-cells can be activated and homeostatic at CD3/CD28And culturing the cell factor to obtain the cell factor. When infused into a restricted immunodeficient host, genetically modified central memory T-cells i) will migrate into and multiply with significantly higher water levels than effector memory genetically modified T-cells, and ii) will induce immune responses against host and alloantigens more efficiently than effector memory genetically modified lymphocytes.

These results demonstrate that fully functional central memory gene-modified lymphocytes are available and useful for curing human diseases.

Reference to the literature

1.Jenkins，M.K.，A.Khoruts，E.Ingulli，D.L.Mueller，S.J.McSorley，R.L.Reinhardt，A.Itano，and K.A.Pape.2001.In vivo activation of antigen-specific CD4 T cells.Annu RevImmunol 19：23-45.

2.Sprent，J.，and C.D.Surh.2002.T cell memory.Annu Rev Immunol 20：551-579.

3.Marrack，P.，J.Bender，D.Hildeman，M.Jordan，T.Mitchell，M.Murakami，A.Sakamo to，B.C.Schaefer，B.Swanson，and J.Kappler.2000.Homeostasis of alpha betaTCR+T cells.Nat Immunol 1：107-111.

4.Novak，E.J.，A.W.Liu，G.T.Nepom，and W.W.Kwok.1999.MHC class II tetramersidentify peptide-specific human CD4(+)T cells proliferating in response to influenza Aantigen. J Clin Invest 104：R63-67.

5.Mallone，R.，and G.T.Nepom.2004.MHC Class II tetramers and the pursuit of antigen-specific T cells：define，deviate，delete.Clin Immunol 110：232-242.

6.Boursalian，T.E.，and K.Bottomly.1999.Survival of naive CD4 T cells：roles ofrestricting versus selecting MHC class II and cytokine milieu.J Immunol 162：3795-3801.

7.Seddon，B.，and R.Zamoyska.2002.TCR and IL-7 receptor signals can operateindependently or synergize to promote lymphopenia-induced expansion of naive T cells.J Immunol 169：3752-3759.

8.Sallusto，F.，D.Lenig，R.Forster，M.Lipp，and A.Lanzavecchia.1999.Two subsets ofmemory T lymphocyteswith distinct homing potentials and effector functions.Nature401：708-712.

9.Seddon，B.，P.Tomlinson，and R.Zamoyska.2003.Interleukin 7 and T cell receptorsignals regulate homeostasis of CD4 memory cells.Nat Immunol 4：680-686.

10.Kondrack，R.M.，J.Harbertson，J.T.Tan，M.E.McBreen，C.D.Surh，and L.M.Bradley.2003.Interleukin 7 regulates the survival and generation of memory CD4 cells.J ExpMed 198：1797-1806.

11.Li，J.，G.Huston，and S.L.Swain.2003.IL-7promotes the transition of CD4 effectors topersistent memory cells.J Exp Med 198：1807-1815.

12.Lenz，D.C.，S.K.Kurz，E.Lemmens，S.P.Schoenberger，J.Sprent，M.B.Oldstone，and D.Homann.2004.IL-7 regulates basal homeostatic proliferation of antiviral CD4+T cellmemory.Proc Natl Acad Sci U S A 101：9357-9362.

13.Geginat，J.，F.Sallusto，and A.Lanzavecchia.2001.Cytokine-driven proliferation anddifferentiation of human naive，central memory，and effector memory CD4(+)T cells.JExpMed194：1711-1719.

14.Rivino，L.，M.Messi，D.Jarrossay，A.Lanzavecchia，F.Sallusto，and J.Geginat.2004.Chemokine receptor expression identifies Pre-T helper(Th)1，Pre-Th2，and nonpolarizedcells among human CD4+ central memory T cells.J Exp Med 200：725-735.

15.Jaleco，S.，L.Swainson，V.Dardalhon，M.Burjanadze，S.Kinet，and N.Taylor.2003.Homeostasis of naive and memory CD4+T cells：IL-2and IL-7 differentially regulate thebalance between proliferation and Fas-mediated apoptosis.J Immunol 171：61-68.

16.Jameson，S.C.2002.Maintaining the normi T-cell homeostasis.Nat Rev Immunol 2：547-556.

17.Benigni，F.，V.S.Zimmermann，S.Hugues，S.Caserta，V.Basso，L.Rivino，E.Ingulli，L.Malherbe，N.Glaichenhaus，and A.Mondino.2005.Phenotype and homing of CD4tumor-specific T cells is modulated by tumor bulk.J Immunol 175：739-748.

18.Degl′Innocenti，E.，M.Grioni，A.Boni，A.Camporeale，M.T.Bertilaccio，M.Freschi，A.Monno，C.Arcelloni，N.M.Greenberg，and M.Bellone.2005.Peripheral T cell toleranceoccurs early during spontaneous prostate cancer development and can be rescued bydendritic cell immunization.Eur J Immunol 35：66-75.

19.Rosato，A.，S.D.Santa，A.Zoso，S.Giacomelli，G.Milan，B.Macino，V.Tosello，P.Dellabona，P.L.Lollini，C.De Giovanni，and P.Zanovello.2003.The cytotoxic T-lymphocyte response against a poorly immunogenic mammary adenocarcinoma isfocused on a single immunodominant class I epitope derived from the gp70 Env productof an endogenous retrovirus.Cancer Res 63：2158-2163.

20.Appelbaum，F.R.2001.Haematopoietic cell transplantation as immunotherapy.Nature411：385-389.

21.Bonini，C.，G.Ferrari，S.Verzeletti，P.Servida，E.Zappone，L.Ruggieri，M.Ponzoni，S.Rossini，F.Mavilio，C.Traversari，and C.Bordignon.1997.HSV-TK gene transfer intodonor lymphocytes for control of allogeneic graft-versus-1eukemia Science 276：1719-1724.

22.Fehse，B.，F.A.Ayuk，N.Kroger，L.Fang，K.Kuhlcke，M.Heinzelmann，T.Zabelina，A.A.Fauser，and A.R.Zander.2004.Evidence for increased risk of secondary graftfailure after in vivo depletion of suicide gene-modified T lymphocytes transplanted inconjunction with CD34+-enriched blood stem cells.Blood 104：3408-3409.

23.Tiberghien，P.，C.Ferrand，B.Lioure，N.Milpied，R.Angonin，E.Deconinck，J.M.Certoux，E.Robinet，P.Saas，B.Petracca，C.Juttner，C.W.Reynolds，D.L.Longo，P.Herve，and J.Y.Cahn.2001.Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft.Blood 97：63-72.

24.Mougneau，E.，F.Altare，A.E.Wakil，S.Zheng，T.Coppola，Z.E. Wang，R.Waldmann，R.M.Locksley，and N.Glaichenhaus.1995.Expression cloning of a protectiveLeishmania antigen.Science 268：563-566.

25. Malherbe，L.，C.Filippi，V.Julia，G.Foucras，M.Moro，H.Appel，K.Wucherpfennig，J.C.Guery，and N.Glaichenhaus.2000.Selective activation and expansion of high-affinity CD4+T cells in resistant mice upon infection with Leishmania major.Immunity13：771-782.

26.Lollini，P.L.，C.de Giovanni，V.Eusebi，G.Nicoletti，G.Prodi，and P. Nanni.1984.High-metastatic clones selected in vitro from a recent spontaneous BALB/c mammaryadenocarcinoma cell line.Clin Exp Metastasis 2：251-259.

27.Verzeletti，S.，C.Bonini，S.Marktel，N.Nobili，F.Ciceri，C.Traversari，and C.Bordignon.1998.Herpes simplex virus thymidine kinase gene transfer for controlledgraft-versus-host disease and graft-versus-leukemia：clinical follow-up and improved newvectors.Hum Gene Ther 9：2243-2251.

28.Bondanza，A.，V.Valtolina，Z.Magnani，M.Ponzoni，K.Fleischhauer，M.Bonyhadi，C.Traversari，F.Sanyito，S.Toma，M.Radrizzani，S.La Seta-Catamancio，F.Ciceri，C.Bordignon，and C.Bonini.2005.Suicide gene therapy of graft-versus-host diseaseinduced by central memory human T lymphocytes.Blood

29.Stetson，D.B.，M.Mohrs，V.Mallet-Designe，L.Teyton，and R.M.Locksley.2002.Rapidexpansion and IL-4 expression by Leishmania-specific naive helper T cells in vivo.Immunity 17：191-200.

30.Iezzi，G.，K.Karjalainen，and A.Lanzavecchia.1998.The duration of antigenicstimulation determines the fate of naive and effector T cells.Immunity8：89-95.

31.Gett，A.V.，F.Sallusto，A.Lanzavecchia，and J.Geginat.2003.T cell fitness determinedby signal strength.Nat Immunol 4：355-360.

32.Harari，A.，S.Petitpierre，F.Vallelian，and G.Pantaleo.2004.Skewed representation offunctionally distinct populations of virus-specific CD4 T cells in HIV-l-infected subjectswith progressive disease：changes after antiretroviral therapy.Blood 103：966-972.

33.Harari，A.，F.Vallelian，P.R.Meylan，and G.Pantaleo.2005.Functional heterogeneity ofmemory CD4 T cell responses in different conditions of antigen exposure andpersistence.J Immunol 174：1037-1045.

34.Jackson，H.M.，N.Dimopoulos，Q.Chen，T.Luke，T.Yee Tai，E.Maraskovsky，L.J.Old，I.D.Davis，J.Cebon，and W.Chen.2004.A robust human T-cell culture method suitablefor monitoring CD8+and CD4+T-cell responses from cancer clinical trial samples.JImmunol Methods 291：51-62.

35.Unutmaz，D.，F.Baldoni，and S.Abrignani.1995.Human naive T cells activated bycytokines differentiate into a split phenotype with functional features intermediatebetween naive and memoryT cells.Int Immunol7：1417-1424.

36.Unutmaz，D.，P.Pileri，and S.Abrignani.1994.Antigen-independent activation of naiveand memory resting T cells by a cytokine combination.J Exp Med 180：1159-1164.

37.Dudley，M.E.，J.R.Wunderlich，T.E.Shelton，J.Even，and S.A.Rosenberg.2003.Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapyfor melanoma patients.J Immunother 26：332-342.

38.Hassan，J.，and D.J.Reen.1998.IL-7 promotes the survival and maturation but notdifferentiation of human post-thymic CD4+ T cells.Eur J Immunol 28：3057-3065.

39.Reinhardt，R.L.，A.Khoruts，R.Merica，T.Zell，and M.K.Jenkins.2001.Visualizing thegeneration of memory CD4 T cells in the whole body.Nature 410：101-105.

40.Roman，E.，E.Miller，A.Harmsen，J.Wiley，U.H.Von Andrian，G.Huston，and S.L.Swain.2002.CD4 effector T cell subsets in the response to influenza：heterogeneity，migration，and function.J Exp Med 196：957-968.

41.Sallusto，F.，J.Geginat，and A.Lanzavecchia.2004.Central memory and effectormemory T cell subsets：function，generation，and maintenance.Annu Rev Immunol22：745-763.

42.Park，J.H.，Q.Yu，B.Erman，J.S.Appelbaum，D.Montoya-Durango，H.L.Grimes，and A.Singer.2004.Suppression of IL7Ralpha transcription by IL-7 and other prosurvivalcytokines：a novel mechanism for maximizing IL-7-dependent T cell survival.Immunity21：289-302.

43.Scarpellini，P.，S.Tasca，L.Galli，A.Beretta，A.Lazzarin，and C.Fortis.2004.Selectedpool of peptides from ESAT-6 and CFP-10 proteins for detection of Mycobacteriumtuberculosis infection.J Clin Microbiol2：3469-3474

Claims (42)

1. An in vitro method of proliferating a rare population of antigen-specific memory T cells in a sample, comprising the step of exposing the sample to an effective amount of at least one cytokine receptor agonist capable of selectively proliferating the rare population of antigen-specific memory T cells.

2. The in vitro method of claim 1, wherein said cytokine receptor agonist is a cytokine or a derivative thereof.

3. The in vitro method according to claim 1 or 2, wherein said at least one cytokine receptor agonist is an IL-7 receptor agonist or an IL-15 receptor agonist.

4. The in vitro method according to claim 3, wherein said IL-15 receptor agonist or IL-7 receptor agonist, respectively, is also present.

5. The in vitro method of claims 1-4, wherein said rare population of antigen-specific memory T cells comprises CD4⁺And/or CD8⁺And/or γ δ and/or NKT T cell populations.

6. The in vitro method according to claims 1 to 5, wherein said sample is a biological sample belonging to the group consisting of: blood and other liquid samples of biological origin, solid tissue samples, tissue cultures of cells derived therefrom and their progeny, cells isolated from biological samples.

7. An in vitro method for detecting a rare population of antigen-specific memory T cells in a sample, comprising the steps of:

a) exposing the sample to an effective amount of at least one cytokine receptor agonist of any one of claims 1-6 that is capable of selectively proliferating rare antigen-specific memory T cell populations;

b) incubating the sample with at least one ligand specific for one of the proliferating rare antigen-specific memory T cell populations;

c) detecting a proliferating rare population of antigen-specific memory T cells that bind to a specific ligand.

8. The in vitro method according to claim 7, wherein said specific ligand is a specific antigen, or a derivative of one of said rare antigen-specific memory T cell populations.

9. The in vitro method according to claim 8, wherein said specific antigen is associated with a microbial pathogen including but not limited to mycobacteria, Pneumocystis carinii, Plasmodium falciparum, Candida, Toxoplasma, CMV, EBV, BPV, HCV, HBV, HIV.

10. The in vitro method according to claim 8, wherein said antigen is a tumor-associated antigen.

11. The in vitro method according to claim 8, wherein said antigen is an allergen.

12. The in vitro method according to claim 8, wherein said antigen is a self-antigen.

13. The in vitro method according to claims 8 to 12, wherein said specific antigen is present as an antigen-MHC complex or a derivative thereof.

14. The in vitro method according to claims 7 to 13, wherein the detection of said expanded rare population of antigen specific memory T cells is achieved by a binding assay.

15. The in vitro method according to claims 7-13, wherein the detection of said expanded rare population of antigen-specific memory T cells is achieved by a cytokine release assay.

16. The in vitro method according to claims 7-13, wherein said detection of said expanded rare population of antigen-specific memory T cells is effected by means of a proliferation assay.

17. The in vitro method according to claims 7 to 13, wherein the cells are labeled with a fluorescent vital dye, the sample is then incubated with a specific ligand and the detection step is carried out by a dye dilution assay.

18. A kit for carrying out the method for detecting rare populations of antigen-specific memory T cells in a sample according to claims 7-17, comprising at least one cytokine receptor agonist; at least one ligand specific for a rare population of antigen-specific memory T cells; and (5) detecting the tool.

19. An in vitro method of isolating rare populations of antigen-specific memory T cells in a sample, comprising the steps of:

a) exposing the sample to an effective amount of at least one cytokine receptor agonist of any one of claims 1-6 that is capable of selectively proliferating rare antigen-specific memory T cell populations;

b) incubating the sample with at least one ligand specific for one of the proliferating rare antigen-specific memory T cell populations;

c) isolating a proliferating rare population of antigen-specific memory T cells that bind to a specific ligand.

20. The in vitro method according to claim 19, wherein said specific ligand is a specific antigen or a derivative of one of said rare antigen-specific memory T cell populations.

21. The in vitro method according to claim 19, wherein said specific antigen is associated with a microbial pathogen including but not limited to mycobacteria, pneumocystis carinii, plasmodium falciparum, candida, toxoplasma, CMV, EBV, BPV, HCV, HBV, HIV.

22. The in vitro method according to claim 19 or 20, wherein said antigen is a tumor-associated antigen.

23. The in vitro method according to claim 19 or 20, wherein said antigen is an allergen.

24. The in vitro method according to claim 19 or 20, wherein said antigen is a self-antigen.

25. The in vitro method according to claims 19 to 24, wherein said specific antigen is present as an antigen-MHC complex or a derivative thereof.

26. The in vitro method according to claims 19-25, wherein the isolation of said expanded rare population of antigen-specific memory T cells is achieved by a binding step.

27. The in vitro method according to claims 19-25, wherein the isolation of said proliferated rare population of antigen-specific memory T cells is achieved by measuring cytokine and cytotoxic production, including but not limited to the ELISPOT assay of IL-2, IFN-g, IL-4, IL-5, IL-10, TNF-a, TGF- β, granzyme, ELISA assay, flow cytometry cytokine detection assay.

28. The in vitro method according to any of the preceding claims for the diagnostic and/or prognostic clinical study of immune-, infectious-, cancer-, allergy-, autoimmune-related pathologies.

29. Use of a rare T cell population isolated according to the method of claims 18-26 for the treatment and/or prevention of immune-, infectious-, cancer-, allergy-, autoimmune-related pathologies.

30. Use of a rare population of T cells according to claim 29, which is genetically modified.

31. An in vitro method for obtaining a population of genetically modified memory T cells comprising the steps of:

a) activating lymphocytes with at least 2 specific activating receptor agonists capable of driving lymphocyte activation, including but not limited to agonist antibodies, recombinant ligands and derivatives thereof;

b) exposing activated lymphocytes to an effective amount of at least one cytokine receptor agonist capable of selectively proliferating a population of memory T cells;

c) inserting the foreign gene into the cell obtained in b) with the aid of an appropriate vector, and expressing.

32. The in vitro method of claim 31, wherein the memory T cell population comprises CD4⁺And/or CD8⁺And/or γ δ and/or NKT T cell populations.

33. The in vitro method of claim 31 or 32, wherein said lymphocytes are derived from a biological sample belonging to: blood and other liquid samples of biological origin, solid tissue samples, tissue cultures of cells derived therefrom and their progeny, cells isolated from biological samples.

34. The in vitro method according to claims 31 to 33, wherein said specific lymphocyte activation receptor agonist is conjugated to a cell mimic support.

35. The in vitro method of claims 31-34, wherein said cell mimicking support is a paramagnetic bead.

36. The in vitro method of claims 31-35, wherein one of the lymphocyte activating receptor agonists is specific for a CD3 polypeptide.

37. The in vitro method according to claims 31 to 36, wherein one of the lymphocyte activating receptor agonists is specific for the co-stimulatory receptor, CD 28.

38. The in vitro method according to claims 31 to 37, wherein at least one cytokine receptor agonist is an IL-7 receptor agonist or an IL-15 receptor agonist.

39. The in vitro method according to claims 31 to 38, wherein an IL-15 receptor agonist or an IL-7 receptor agonist, respectively, is also present.

40. The in vitro method of claims 31 to 39, wherein said vector is a viral vector.

41. The in vitro method according to claims 31 to 40, wherein said exogenous gene encodes a suicide gene, and/or a marker gene, and/or a biologically active molecule, and/or a receptor, and/or a soluble factor that resides inside the cell or is released outside the cell, and/or a gene conferring resistance to a prodrug.

42. Use of a population of genetically modified memory T cells produced according to the method of claims 31-41 for the treatment and/or prevention of cancer, infection, immunodeficiency or autoimmunity or transplantation of hematopoietic precursors or solid organs.