CN107648609A - Inhibitory action of the IL 35 to T effector cell autophagy in pyemia - Google Patents

Abstract

The invention discloses application of the inhibitor of IL 35 in the preparation for promoting T effector cell autophagy is prepared.In addition, the invention also discloses the method for promoting T effector cell autophagy.The innovative point of the present invention is to be found that IL 35 has correlation with T effector cell autophagy, can promote T effector cell autophagy by suppressing IL 35, strengthen cell immune response.The achievement in research of the present invention provides new method for internal, external depression effect T cell autophagy and new drug target is provided for clinical treatment diseases associated with inflammation, infectious diseases, tumour and autoimmune disease.

Description

Inhibitory effect of IL-35 on autophagy of effector T cells in sepsis

technical field

The invention belongs to the field of biomedicine, and relates to an inhibitor of autophagy of effector T cells and a regulating method, in particular to the application of an IL-35 inhibitor in the preparation of a preparation for promoting autophagy of effector T cells.

Background technique

T cells, also known as T lymphocytes, are derived from pluripotent stem cells in the bone marrow. During the embryonic and nascent stages of the human body, some pluripotent stem cells or precursor T cells in the bone marrow migrate to the thymus, differentiate and mature in the thymus, and become immune-active T cells.

Mature T cells are distributed through the bloodstream to settle in the thymus-dependent area of peripheral immune organs, and are recirculated through blood and lymph, playing an important regulatory role in the cellular immune response. The recycling of T cells is conducive to extensive exposure to antigenic substances entering the body, strengthening the immune response, and maintaining long-term immune memory. There are many different markers on the cell membrane of T cells, mainly surface antigens and receptors.

According to the stage of activation, T cells are divided into different subgroups: including naive T cells (naive T cells), effector T cells (effector T cells) and memory T cells (memory T cells). According to the surface antigen receptor (T cellantigen receptor, TCR) type, T cells are divided into TCRαβ ⁺ T cells and TCRγδ ⁺ T cells. According to whether CD4 or CD8 molecules are expressed, T cells can be divided into CD4 ⁺ T cells and CD8 ⁺ T cells. According to their different immune effects, T cells are divided into helper T cells (Th), cytotoxic T cells (cytotoxic T cells, Tc or CTL), and regulatory T cells (regulatory T cells, Treg).

The restriction and balance between effector T cells and regulatory T cells determine the state of T cell immune response, and even determine the overall trend of cellular immunity. Regulatory T cells exert an inhibitory effect through direct contact with effector T cells, and can also release inhibitory cytokines such as transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and interleukin-35 (IL-10). -35) to regulate the immune response.

IL-35 is a newly discovered inhibitory cytokine of the IL-12 family, which plays an important or even key role in the immune suppression of Treg. Under the action of exogenous IL-35, human and mouse naive T cells were transformed into CD4 ⁺ Foxp3 ^- regulatory T cells iTR35, which had strong immunosuppressive activity. Regulatory T cells need to fully exert their immunosuppressive effect under the action of IL-35. It has also been reported that IL-35 is also involved in the process of suppressing tumor antigen-specific effector T cells by CD8 ⁺ cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) ⁺ regulatory T cells.

The specific molecular mechanism of IL-35 immunosuppressive effect is still unclear. The exploration of this issue is the basis for further intervention and regulation of the immunosuppressive effect of IL-35. The applicant initially explored the possible mechanism of IL-35 inhibiting the proliferation and function of effector T cells from the perspective of autophagy.

Contents of the invention

In order to solve the problems in the prior art, the present invention provides a method for regulating autophagy of effector T cells. The present invention finds through experiments that activated effector T cells stimulated by IL-35 lead to inhibition of T cell autophagy, based on which it is deduced that the autophagy of effector T cells can be promoted by inhibiting the expression or activity of IL-35, thereby Improve IL-35-mediated immunosuppressive status.

Specifically, the present invention adopts the following technical solutions:

The invention provides an application of an IL-35 inhibitor in the preparation of a preparation for promoting autophagy of effector T cells.

According to one aspect of the present invention, the IL-35 inhibitor includes an inhibitor capable of inhibiting the expression of IL-35.

Further, the inhibitor capable of suppressing the expression of IL-35 includes an inhibitor capable of suppressing the expression level of IL-35 gene mRNA, or an inhibitor capable of suppressing the expression level of IL-35 protein.

According to another aspect of the present invention, the IL-35 inhibitor includes an inhibitor capable of inhibiting the expression of IL-35 upstream substances or downstream substances in the signaling pathway where IL-35 is located.

Further, the inhibitor capable of inhibiting the expression of IL-35 upstream substances or downstream substances in the signaling pathway where IL-35 is located includes the ability to inhibit the mRNA expression level of IL-35 upstream substances or downstream substances in the signaling pathway where IL-35 is located Inhibitors, or inhibitors capable of inhibiting the protein expression levels of IL-35 upstream substances or downstream substances in the signaling pathway where IL-35 is located.

According to yet another aspect of the present invention, the IL-35 inhibitor includes an inhibitor capable of inhibiting IL-35 activity.

Further, the inhibitors capable of inhibiting the activity of IL-35 include inhibitors that inhibit the activity of IL-35 itself, or inhibitors that inhibit the activity of substances upstream or downstream of IL-35 in the signaling pathway where IL-35 is located.

In conclusion, the type of IL-35 inhibitor of the present invention is not limited, as long as the inhibitor is effective in promoting autophagy of effector T cells. The types of the inhibitors in the present invention include, but are not limited to: interfering RNA, negative regulatory miRNA, negative regulatory transcription regulators, inhibitory targeting small molecule compounds, and protein-specific antibodies.

Further, the interfering RNA includes siRNA, shRNA and corresponding constructs.

Further, the protein-specific antibody includes monoclonal antibody or polyclonal antibody to IL-35 protein.

Antibodies of the invention include intact antibodies or fragments thereof. Antibodies or fragments thereof of any structure, size, immunoglobulin class, origin, etc. can be used as long as it binds the target protein. Antibodies or fragments thereof of the invention may be monoclonal or polyclonal. An antibody fragment refers to a part of an antibody (partial fragment) or a peptide containing a part of an antibody that retains the antigen-binding activity of the antibody. Antibody fragments may include F(ab') ₂ , Fab', Fab, single chain Fv (scFv), disulfide-bonded Fv (dsFv) or polymers thereof, dimerized V regions (diabodies), or containing CDR peptides.

Antibodies can be obtained by methods known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing animals with antigens, immune cells are obtained from the immunized animals and fused with myeloma cells to obtain hybridomas. Antibodies are then harvested from the hybridoma cultures. Finally, a monoclonal antibody against a protein can be obtained by subjecting the obtained antibody to antigen-specific purification using the protein or a part thereof used as the antigen. Polyclonal antibodies can be prepared by immunizing animals with the same antigens as above, collecting blood samples from the immunized animals, separating serum from the blood, and subjecting the serum to antigen-specific purification using the above antigens. Antibody fragments can be obtained by treating the obtained antibody with an enzyme or by using the sequence information of the obtained antibody.

The present invention also provides a method for promoting autophagy of effector T cells, the method comprising administering the aforementioned IL-35 inhibitor.

The present invention also provides a drug for promoting autophagy of effector T cells, the active ingredient of which is the IL-35 inhibitor described above in the present invention.

The inhibitor of the present invention can be formulated into a drug that promotes autophagy of effector T cells by any means known in the art. The inhibitor of the present invention can be formulated into the medicament of the present invention with one or more pharmaceutically acceptable carriers, diluents, fillers, binding agents and other excipients. These substances are added to aid in the stability of the formulation or to help increase the activity or its bioavailability.

The inhibitors of the present invention can be administered alone, or in various combinations, and in combination with other autophagy-promoting effector T cells.

The medicine of the present invention can be prepared into various dosage forms as required. Including but not limited to: tablet, solution, granule.

The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are all identical except for possible small amounts of spontaneous mutation. Thus, the modifier "monoclonal" refers to the nature of the antibody as not being a mixture of discrete antibodies. Preferably, the monoclonal antibodies include monovalent or single-chain antibodies, double-chain antibodies, chimeric antibodies, porcine antibodies and derivatives, functional equivalents and homologues of the above-mentioned antibodies, as well as antibody fragments and antibodies containing Any polypeptide of an antigen binding domain. An antibody is any specific binding factor encompassing a binding domain with the desired specificity, thus the term encompasses antibody fragments, derivatives, humanized antibodies and functional equivalents and homologues of antibodies homologous thereto , also includes any polypeptide, whether natural or synthetically produced, that contains an antigen-binding domain. Examples of antibodies are immunoglobulin subtypes (such as IgG, IgE, IgM, IgD and IgA) and their subtype subclasses; also fragments comprising an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and Diabodies. Chimeric molecules comprising an antigen binding domain fused to another polypeptide or equivalents are also included. Cloning and expression of chimeric antibodies are described in EP.A.0120694 and EP.A.0125023. Antibodies can be modified in many ways, and recombinant DNA techniques can be used to generate other antibodies or chimeric molecules that retain the specificity of the original antibody. This technique may involve introducing DNA encoding the variable or complementarity determining regions (CDRs) of an antibody immunoglobulin into the constant or constant plus framework regions of a different immunoglobulin, see EP.A.184187, GB2188638A or EP .A.239400. Genetic mutations or other alterations can also be made to hybridoma cells or other antibody-producing cells, which may or may not alter the binding specificity of the antibodies produced. The "monoclonal antibody" used in the present invention can also be produced by the hybridoma method, because the DNA sequence encoding the murineized antibody of the present invention can be conventionally known to those skilled in the art, such as artificially synthesizing according to the amino acid sequence of IL-35 or using It is amplified by PCR method, so recombinant DNA method can also be used, and various methods well known in the art can be used to link the sequence into a suitable expression vector. Finally, the transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention, and then the monoclonal antibody of the present invention is purified by those skilled in the art using well-known conventional separation and purification means. Antibodies comprise a geometry of polypeptide chains linked together by disulfide bridges, two polypeptide backbones called light chains and heavy chains that make up all of the major structural classes (isotypes) of antibodies. Both heavy and light chains can be further divided into subregions called variable and constant regions. The heavy chain includes a single variable region and three different constant regions, and the light chain includes a single variable region (different from that of the heavy chain) and a single constant region (different from that of the heavy chain). The variable regions of the heavy and light chains are responsible for the binding specificity of the antibody.

The term "polyclonal antibody" describes a composition of different (multiple) antibody molecules capable of binding to or reacting with several different specific antigenic determinants/epitopes on the same or different antigens, wherein the Each individual antibody is capable of reacting with a specific epitope. Usually, the variability of polyclonal antibodies is located in the so-called polyclonal antibody variable regions, especially the CDR1, CDR2 and CDR3 regions. In the present invention, polyclonal antibodies can be produced in one pot from polyclonal cell lines, or can be a mixture of different polyclonal antibodies. A mixture of monoclonal antibodies is not equally considered polyclonal as they are produced in separate batches and not necessarily from the same cell line, which can cause differences in, for example, post-translational modifications. However, if a monoclonal antibody mixture provides the same antigen/epitope coverage as a polyclonal antibody of the invention, it can be considered an equivalent of a polyclonal antibody. When it is stated that a member of a polyclonal antibody specifically binds to or has specific reactivity against an antigen/antigenic site/epitope, it is meant herein that the binding constant is lower than 100 nM, preferably lower than 10 nM, even more preferably lower than 1nM.

The term "interfering RNA" in this paper refers to a double-stranded RNA molecule that can degrade specific mRNAs by targeting mRNAs with homologous complementary sequences. This process is the RNA interference pathway (RNA interference pathway).

Description of drawings

Figure 1 shows the flow cytometry detection of LC3-II expression in activated CD4 ⁺ CD25 ^- T cells, where: A: cell scatter plot; B: negative control; C: no anti-CD3/CD28 Monoclonal antibody treatment; D: anti-CD3/CD28 monoclonal antibody treatment for 24 hours; E: anti-CD3/CD28 monoclonal antibody treatment for 48 hours; F: anti-CD3/CD28 monoclonal antibody treatment for 72 hours; G: anti-CD3/ CD28 monoclonal antibody treatment for 96h;

Figure 2 shows the statistical histogram of LC3-II expression in activated CD4 ⁺ CD25 ^- T cells detected by flow cytometry;

Figure 3 shows the flow cytometry of LC3-II expression in CD4 ⁺ CD25 ^- T cells detected by flow cytometry under the action of different doses of recombinant IL-35, wherein, A: cell scatter plot; B: negative control; C: No anti-CD3/CD28 monoclonal antibody; D: anti-CD3/CD28 monoclonal antibody treatment; E: anti-CD3/CD28 monoclonal antibody and 25ng/ml IL-35 treatment simultaneously; F: anti-CD3/CD28 Simultaneous treatment of monoclonal antibody and 50ng/ml IL-35; G: simultaneous treatment of anti-CD3/CD28 monoclonal antibody and 100ng/ml IL-35;

Figure 4 shows the statistical histogram of LC3-II expression in CD4 ⁺ CD25 ^- T cells detected by flow cytometry under the action of different doses of recombinant IL-35;

Figure 5 shows the flow cytometry of the formation of autophagic vesicles, autophagosomes and autolysosomes in CD4 ⁺ CD25 ^- T cells detected by flow cytometry combined with CytoID kit, where, A: cell scattergram; B : negative control; C: no anti-CD3/CD28 monoclonal antibody; D: anti-CD3/CD28 monoclonal antibody treatment; E: anti-CD3/CD28 monoclonal antibody and 50ng/ml IL-35 treatment simultaneously;

Figure 6 shows the statistical histogram of the formation of autophagic vesicles, autophagosomes and autolysosomes in CD4 ⁺ CD25 ^- T cells detected by flow cytometry combined with CytoID kit;

Figure 7 shows the western blot of the expression level of LC3-I/II in CD4 ⁺ CD25 ^- T cells detected by Western blot. The effect of -I/II expression level; B: The effect of bafilomycin on the expression level of LC3-I/II under the background of the joint action of anti-CD3/CD28 monoclonal antibody and IL-35;

Figure 8 shows the results of detecting the expression level of Beclin 1 in CD4 ⁺ CD25 ^- T cells by Western blot, where A: western blot; B: statistical histogram;

Fig. 9 shows the results of detecting the formation of autophagosomes in mouse spleen CD4 ⁺ CD25 ^- T cells by laser confocal microscopy, wherein, A: fluorescence image; B: statistical histogram.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the embodiment, usually according to conventional conditions, such as people such as Sambrook, molecular cloning: the condition described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the experimental reagent manufacturer suggested conditions.

Example 1 CD4 ⁺ CD25 ^- T cell culture

Procedure: C57BL/6 mice (6-8w, 18-20g) were sacrificed by neck dislocation, and the spleen was obtained. Separation of whole spleen cells, lymphocyte separation medium to separate mononuclear cells, CD4 ⁺ CD25 ⁺ Regulatory T Cell Isolation Kit (mouse, 130-091-041, Miltenyi Biotec Company) sorting to obtain CD4 ⁺ CD25 ^- T cells, flow cytometry Check the purity of T cells. Mouse CD4 ⁺ CD25 ^- T cells were placed in RPMI 1640 medium (containing penicillin 100 U/ml, streptomycin 100 μg/ml and L-glutamine) plus 10% fetal bovine serum (FBS, heat inactivated) at 37°C, 5% cultured in a CO ₂ incubator.

Example 2 Acquisition of recombinant IL-35

Mouse recombinant IL-35 [IL-35(mouse):Fc(human)(rec.), CHI-MF-11135-C025, Chimerigen Laboratories]. purchased from Jiangsu Futai Biotechnology Co., Ltd.

Example 3 Effect of recombinant IL-35 on autophagy of CD4 ⁺ CD25 ^- T cells

1. Screening for the optimal dosage of recombinant IL-35

Anti-CD3 monoclonal antibody (2 μg/ml) was pretreated and bound to the culture plate. Anti-CD28 monoclonal antibody (2 μg/ml) was added to RPMI 1640+10% FBS culture system, and the expression of LC3-II in CD4 ⁺ CD25 ^- T cells was detected by flow cytometry after 24, 48, 72 and 96 hours of culture. The results in Figure 1 and Figure 2 show that: compared with the control group (resting effector T cells), LC3-II in T cells increased after co-stimulation with anti-CD3/CD28 monoclonal antibodies, and reached the peak level after 72 hours (78.0033±3.74873% vs. 48h: 40.0933±2.25538%, P<0.01), and the expression level did not increase until 96h (78.0033±3.74873% vs. 96h: 73.0367±1.58279%, P=0.30), therefore, 72h is costimulation affecting the expression of LC3-II in cells most notably time. At this time point, the effect of different doses of recombinant IL-35 on the content of LC3-II in T cells was further observed. At the same time as anti-CD3/CD28 monoclonal antibody co-stimulation, 25, 50 and 100 ng/ml recombinant IL-35 was used to treat effector T cells for 72 hours. The results in Figure 3 and Figure 4 showed that: 50 ng/ml recombinant IL-35 had an effect on effector T cells. The inhibitory effect of LC3-II was the most obvious.

Flow cytometry detection of LC3-II steps: after anti-CD3/CD28 monoclonal antibody co-stimulation and recombinant IL-35 effect effector T cells, the cells were collected, resuspended in PBS, and 0.025% digitonin (ab141501, Abcam Company) was used to rupture the membrane in an ice bath 3 min, washed three times with PBS, fixed cells with 4% paraformaldehyde at room temperature for 30 min, washed twice with PBS, 1:1000 LC3 primary antibody (MAP1LC3A/LC3A Rabbit anti-Human Polyclonal (aa81-111) (FITC) Antibody, LS-C262516, (Lifespanbioscience Company) were incubated overnight at 4°C, washed twice with PBS, and detected by flow cytometry.

2. Detection of autophagy-related indicators of CD4 ⁺ CD25 ^- T cells after recombinant IL-35 treatment

(1) Detection of the formation of autophagic vesicles, autophagosomes and autolysosomes in CD4 ⁺ CD25 ^- T cells cultured in vitro

Cyto-ID kit detection (Cyto-ID ^TM Autophagy Detection Kit, ENZ-51031-K200, Enzo lifescience company)

Steps: pretreatment with anti-CD3 monoclonal antibody (2 μg/ml) and binding to the culture plate. Add anti-CD28 monoclonal antibody (2 μg/ml) to RPMI 1640+10% FBS culture system, and after 72 hours of culture, use flow cytometry combined with Cyto-ID kit to detect autophagic vesicles and autophagosomes in CD4 ⁺ CD25 ^- T cells and autophagy lysosomes, the specific operation steps of the kit are carried out according to the detection process provided by the manufacturer. The results in Figure 5 and Figure 6 show that, under the background of co-stimulation with anti-CD3/CD28 monoclonal antibody, after recombinant IL-35 treatment, compared with the untreated group, autophagic vesicles, autophagosomes and autophagosomes in CD4 ⁺ CD25 ^- T cells The number of phagolysosomes decreased, which indicated that IL-35 interfered with the formation of autophagic vesicles, autophagosomes and autolysosomes in CD4 ⁺ CD25 ^- T cells.

Detection using confocal laser microscopy

Procedure: Under the background of co-stimulation with anti-CD3/CD28 monoclonal antibody, CD4 ⁺ CD25 ^- T cells were treated with recombinant IL-35 for 72 hours, the cells were collected, and resuspended in PBS. Cells were fixed with 4% paraformaldehyde at room temperature for 30 min, washed twice with PBS, membrane permeated with 0.2% Triton-X100 at room temperature for 20 min, washed twice with PBS, blocked with 1% BSA for 30 min, and 1% BAS LC3 primary antibody was prepared at a ratio of 1:400 (LC3B, 2775s, Cell Signaling Technology Company) working solution, incubate overnight at 4°C, wash 3 times with PBS, incubate with fluorescent secondary antibody at room temperature for 1 hour, wash 3 times with PBS, absorb 1×10 ⁶ cells and drop them on the adherent glass slide, drop into 20 μl DAPI, the coverslips were stored in the dark for 6 hours, and then detected by laser confocal microscopy. The results in Figure 9 show that: under the background of co-stimulation with anti-CD3/CD28 monoclonal antibody, after treatment with recombinant IL-35 for 72 hours, compared with the untreated group, the number of autophagosomes in CD4 ⁺ CD25 ^- T cells decreased, indicating that IL-35 Interfering with the formation of autophagosomes (LC3puncta) in T cells (P=0.046vs.anti-CD3/CD28group).

(2) Detect the expression of autophagy-related proteins in CD4 ⁺ CD25 ^- T cells cultured in vitro

Western blot detection

Steps: BCA method was used to quantitatively analyze the total protein of mouse T cells. In polyacrylamide gel electrophoresis (SDS-PAGE), the loading amount of cell protein was 50 μg, and 30 μl of concentrate was taken to load, and electrophoresis was performed on 100 g/L polyacrylamide gel. The primary antibody was rabbit anti-mouse monoclonal antibody (1:2000), the second antibody was horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000), and then luminescence was developed and fixed. Optical density values were analyzed using Image J 1.46r software.

The results in Figure 7 show that after 72 hours of co-stimulation with anti-CD3/CD28 monoclonal antibodies, the expression of LC3-II in CD4 ⁺ CD25 ^- T cells was significantly up-regulated (P=0.003vs.control group, Figure 7A), accompanied by the up-regulation of LC3-I expression (P<0.05vs.control group, Figure 7A); Bafilomycin treatment on the basis of co-stimulation, the content of LC3-II in cells was further up-regulated (P<0.05vs.anti-CD3/CD28 group, Figure 7A), which suggested that co-stimulation caused The upregulation of intracellular LC3-II is not due to the blockage of autophagic flux, but to promote the process of autophagy. When treated with IL-35 in the background of costimulation, the intracellular LC3-II decreased (P<0.05vs.anti-CD3/CD28group, Figure 7A), which may be due to the obstruction of the autophagy process, or it may be due to the inhibition of autophagy Flow facilitation. On this basis, when Bafilomycin was administered to inhibit autophagic flow, no significant increase in LC3-II was observed (Fig. 7B), which suggested that the reduction of intracellular LC3-II caused by IL-35 was not due to the promotion of autophagic flow, but due to Inhibition of the autophagy process may also have a certain impact on the flow of autophagy.

The results in Figure 8 show that there is a certain expression of Beclin 1 in resting effector T cells, and after co-stimulation with anti-CD3/CD28, the content of Beclin 1 in the cells increases significantly (3.1955±0.19665vs.control group: 0.5298±0.01440, P<0.001) . In the background of anti-CD3/CD28 co-stimulation, IL-35 decreased the content of intracellular Beclin1 (0.6991±0.06103vs.anti-CD3/CD28group: 3.1955±0.19665, P=0.035).

The description of the above embodiments is only for understanding the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims (10)

1. Application of the 1.IL-35 inhibitor in the preparation for promoting T effector cell autophagy is prepared.
2. 2. application according to claim 1, it is characterised in that the IL-35 inhibitor includes that IL-35 expression can be suppressed Inhibitor.
3. 3. application according to claim 2, it is characterised in that the inhibitor that can suppress IL-35 expression includes energy Enough suppress the inhibitor of IL-35 mrna expressions, or the inhibitor of IL-35 protein expression levels can be suppressed.
4. 4. application according to claim 1, it is characterised in that the IL-35 inhibitor includes that IL-35 places can be suppressed Signal path in IL-35 upstream materials or downstream substrates expression inhibitor.
5. 5. application according to claim 4, it is characterised in that IL- in the signal path that can suppress where IL-35 The inhibitor of the expression of 35 upstream materials or downstream substrates includes IL-35 upstreams in the signal path where can suppressing IL-35 The inhibitor of the mRNA expressions of material or downstream substrates, or IL-35 upstreams in the signal path where IL-35 can be suppressed The inhibitor of the protein expression level of material or downstream substrates.
6. 6. application according to claim 1, it is characterised in that the IL-35 inhibitor includes that IL-35 activity can be suppressed Inhibitor.
7. 7. application according to claim 6, it is characterised in that the inhibitor that can suppress IL-35 activity includes suppression The activity of IL-35 upstream materials or downstream substrates in signal path where the activity of IL-35 processed in itself, or suppression IL-35 Inhibitor.
8. 8. according to the application any one of claim 1-7, it is characterised in that the type of the inhibitor includes interference RNA, negative regulation miRNA, the transcription regulatory factor of negative regulation type, suppressive targeting micromolecular compound, protein specific antibody.
9. 9. application according to claim 8, it is characterised in that the protein specific antibody includes the list of IL-35 albumen Clonal antibody or polyclonal antibody.
10. A kind of 10. method for promoting T effector cell autophagy, it is characterised in that methods described includes applying in claim 1-9 IL-35 inhibitor described in any one.