A novel assay to assess antigen-specific cytokine release from stimulated CD8(+) T cells derived ... more A novel assay to assess antigen-specific cytokine release from stimulated CD8(+) T cells derived from the mucosal and peripheral blood compartments has been developed and standardized using the influenza A virus matrix protein (MP) peptide, GILGFVFTL. This technology is based on the capacity for the human leucocyte antigen (HLA)-A2:Ig dimeric protein to stimulate CD8(+) T cells in a major histocompatibility complex (MHC) class I-restricted fashion without the necessity for antigen presenting cells (APC). This assay has been optimized utilizing a 9-amino acid residue (9mer) peptide, the optimal peptide length for presenting an epitope to CD8(+) T cells. Compared to existing assays, this more sensitive and specific methodology requires fewer cells, enabling easier and more accurate monitoring of the CD8(+) T-cell response in biological compartments, such as the mucosa during the course of viral infection and may be utilized to assess epitope-specific CD8(+) T-cell responses in vaccine trials.
Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for... more Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to en...
Cerebral malaria is an important cause of morbidity and mortality in many parts of the world. It ... more Cerebral malaria is an important cause of morbidity and mortality in many parts of the world. It has been suggested that cerebral malaria is associated with reduced perfusion due to the blockage of blood vessels by parasitized erythrocytes; although, no quantitative validation of this has been done. We infected C57BL/6 mice with the ANKA strain of Plasmodium berghei and on day 6 of infection we investigated alterations in brain function using arterial spin labeling MRI and proton MRS. MR images did not demonstrate signs of damage. However, there was a significant reduction in cerebral blood flow (P<0.012) and the ratio of N-acetyl-aspartate (NAA) to creatine (Cr) (P<0.01) relative to non-infected mice. The NAA/Cr ratios were significantly correlated with cerebral perfusion (r=0.87) suggesting a relationship between impaired oxygen delivery and neuronal dysfunction. Pathological examination revealed accumulations of damaged axons providing a correlate for the decreased NAA/Cr ratio in infected mice. This murine model will permit non-invasive studies of neurologic function during malarial infection.
Subcutaneous administration in mice of recombinant Sindbis viruses expressing a class I major his... more Subcutaneous administration in mice of recombinant Sindbis viruses expressing a class I major histocompatibility complex-restricted 9-mer epitope of the Plasmodium yoelii circumsporozoite protein or the nucleoprotein of influenza virus induces a large epitope-specific CD8(+) T-cell response. This immunization also elicits a high degree of protection against infection with malaria or influenza A virus.
Although the roles of CD8+ T cells and a major preerythrocytic antigen, the circumsporozoite (CS)... more Although the roles of CD8+ T cells and a major preerythrocytic antigen, the circumsporozoite (CS) protein, in contributing protective antimalaria immunity induced by radiation-attenuated sporozoites, have been shown by a number of studies, the extent to which these players contribute to antimalaria immunity is still unknown. To address this question, we have generated C57BL/6 (B6) transgenic (Tg) mice, expressing K(d) molecules under the MHC-I promoter, called MHC-I-K(d)-Tg mice. In this study, we first determined that a single immunizing dose of IrPySpz induced a significant level of antimalaria protective immunity in MHC-I-K(d)-Tg mice but not in B6 mice. Then, by depleting various T-cell subsets in vivo, we determined that CD8+ T cells are the main mediator of the protective immunity induced by IrPySpz. Furthermore, when we immunized (MHC-I-K(d)-Tg × CS-Tg) F1 mice with IrPySpz after crossing MHC-I-K(d)-Tg mice with PyCS-transgenic mice (CS-Tg), which are unable to mount PyCS-specific immunity, we found that IrPySpz immunization failed to induce protective antimalaria immunity in (MHC-I-K(d)-Tg × CS-Tg) F1 mice, thus indicating the absence of PyCS antigen-dependent immunity in these mice. These results indicate that protective antimalaria immunity induced by IrPySpz in MHC-I-K(d)-Tg mice is mediated by CS protein-specific, K(d)-restricted CD8+ T cells.
Since the discovery of circumsporozoite protein (CSP), a major sporozoite surface antigen, by Rut... more Since the discovery of circumsporozoite protein (CSP), a major sporozoite surface antigen, by Ruth Nussenzweig and Victor Nussenzweig in the early 1980s, the role of CSP in protection against malaria has been extensively investigated. Several monoclonal antibodies against CSP have been generated to date, with some of them mediating antimalarial protection upon passive transfer into animals. Genetically engineered transgenic mosquitoes producing the anti-CSP antibody have recently been generated to reduce malarial transmission. A monoclonal anti-CSP antibody was produced in mice by adeno-associated virus vector, which protected them from malaria. Phase III trials with RTS,S vaccine that targets CSP of Plasmodium falciparum have shown modest efficacy. Polyclonal anti-CSP antibodies derived from children who received the RTS,S vaccine failed to block malarial transmission through mosquitoes, but passive transfer of monoclonal antibodies raised from RTS,S-vaccinated recipient conferred protection against malaria in mice. Taken together, these findings may imply CSP as an antimalarial target.
It has been shown that human immunodeficiency virus (HIV)-1 infection induces the production of e... more It has been shown that human immunodeficiency virus (HIV)-1 infection induces the production of endogenous lipids required for effective viral production, and the cluster of differentiation (CD)1 molecule CD1d is downregulated by HIV-1 infection. However, the role of endogenous lipid presentation and the implications of CD1 downregulation by HIV-1 infection have not yet been characterized. In this study, we observed downregulation of both CD1c and CD1d expression through a Vpu-dependent and Nef-independent mechanism, and the concomitant HIV-1-induced production of host cholesterol decreased the extent of CD1c and CD1d modulation. While the modest downregulation of CD1c by HIV-1 infection decreased the ability of CD1c-restricted T cells to respond and secrete interferon-γ, the cholesterol upregulation in the same cells by HIV-1 infection appears to limit the downregulation of CD1c. The two conflicting HIV-1-mediated changes in CD1c expression appear to minimize the modulation of CD1c expression, thus leading the host to maintain a CD1c-restricted T-cell response against HIV-1.
We have raised a number of monoclonal antibodies (mAb) against idiotopes (Id) on monoclonal anti-... more We have raised a number of monoclonal antibodies (mAb) against idiotopes (Id) on monoclonal anti-murine class II (anti-Ak or Ek) antibodies. Two anti-Id mAb among 31 were found to cross-react with some T cells but not with macrophages and B cells of H-2k animals. They were able to block syngeneic mixed lymphocyte reaction (SMLR) and antigen-induced major histocompatibility complex (MHC)-restricted T cell proliferation of H-2k but not of other haplotypes. These results indicated that antibodies were recognizing Id associated with the MHC restriction site of T cells. The injection of these anti-Id mAb into H-2k mice resulted in the stimulation of self-class II-reactive T cell clones as determined by SMLR. They were also able to stimulate in non-H-2k strains to prime H-2k alloreactive T cell clones. Some animals developed anti-class II (anti-Iak) antibodies by the injection of anti-Id. These results indicated that certain anti-Id (anti-anti-class II) antibodies cross-reacted with T cell receptors which carried class II restriction specificity and were involved in allorecognition. These antibodies were found to have an ability to alter the T cell repertoire in vivo by stimulating such MHC-restricted clones.
A novel assay to assess antigen-specific cytokine release from stimulated CD8(+) T cells derived ... more A novel assay to assess antigen-specific cytokine release from stimulated CD8(+) T cells derived from the mucosal and peripheral blood compartments has been developed and standardized using the influenza A virus matrix protein (MP) peptide, GILGFVFTL. This technology is based on the capacity for the human leucocyte antigen (HLA)-A2:Ig dimeric protein to stimulate CD8(+) T cells in a major histocompatibility complex (MHC) class I-restricted fashion without the necessity for antigen presenting cells (APC). This assay has been optimized utilizing a 9-amino acid residue (9mer) peptide, the optimal peptide length for presenting an epitope to CD8(+) T cells. Compared to existing assays, this more sensitive and specific methodology requires fewer cells, enabling easier and more accurate monitoring of the CD8(+) T-cell response in biological compartments, such as the mucosa during the course of viral infection and may be utilized to assess epitope-specific CD8(+) T-cell responses in vaccine trials.
Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for... more Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to en...
Cerebral malaria is an important cause of morbidity and mortality in many parts of the world. It ... more Cerebral malaria is an important cause of morbidity and mortality in many parts of the world. It has been suggested that cerebral malaria is associated with reduced perfusion due to the blockage of blood vessels by parasitized erythrocytes; although, no quantitative validation of this has been done. We infected C57BL/6 mice with the ANKA strain of Plasmodium berghei and on day 6 of infection we investigated alterations in brain function using arterial spin labeling MRI and proton MRS. MR images did not demonstrate signs of damage. However, there was a significant reduction in cerebral blood flow (P<0.012) and the ratio of N-acetyl-aspartate (NAA) to creatine (Cr) (P<0.01) relative to non-infected mice. The NAA/Cr ratios were significantly correlated with cerebral perfusion (r=0.87) suggesting a relationship between impaired oxygen delivery and neuronal dysfunction. Pathological examination revealed accumulations of damaged axons providing a correlate for the decreased NAA/Cr ratio in infected mice. This murine model will permit non-invasive studies of neurologic function during malarial infection.
Subcutaneous administration in mice of recombinant Sindbis viruses expressing a class I major his... more Subcutaneous administration in mice of recombinant Sindbis viruses expressing a class I major histocompatibility complex-restricted 9-mer epitope of the Plasmodium yoelii circumsporozoite protein or the nucleoprotein of influenza virus induces a large epitope-specific CD8(+) T-cell response. This immunization also elicits a high degree of protection against infection with malaria or influenza A virus.
Although the roles of CD8+ T cells and a major preerythrocytic antigen, the circumsporozoite (CS)... more Although the roles of CD8+ T cells and a major preerythrocytic antigen, the circumsporozoite (CS) protein, in contributing protective antimalaria immunity induced by radiation-attenuated sporozoites, have been shown by a number of studies, the extent to which these players contribute to antimalaria immunity is still unknown. To address this question, we have generated C57BL/6 (B6) transgenic (Tg) mice, expressing K(d) molecules under the MHC-I promoter, called MHC-I-K(d)-Tg mice. In this study, we first determined that a single immunizing dose of IrPySpz induced a significant level of antimalaria protective immunity in MHC-I-K(d)-Tg mice but not in B6 mice. Then, by depleting various T-cell subsets in vivo, we determined that CD8+ T cells are the main mediator of the protective immunity induced by IrPySpz. Furthermore, when we immunized (MHC-I-K(d)-Tg × CS-Tg) F1 mice with IrPySpz after crossing MHC-I-K(d)-Tg mice with PyCS-transgenic mice (CS-Tg), which are unable to mount PyCS-specific immunity, we found that IrPySpz immunization failed to induce protective antimalaria immunity in (MHC-I-K(d)-Tg × CS-Tg) F1 mice, thus indicating the absence of PyCS antigen-dependent immunity in these mice. These results indicate that protective antimalaria immunity induced by IrPySpz in MHC-I-K(d)-Tg mice is mediated by CS protein-specific, K(d)-restricted CD8+ T cells.
Since the discovery of circumsporozoite protein (CSP), a major sporozoite surface antigen, by Rut... more Since the discovery of circumsporozoite protein (CSP), a major sporozoite surface antigen, by Ruth Nussenzweig and Victor Nussenzweig in the early 1980s, the role of CSP in protection against malaria has been extensively investigated. Several monoclonal antibodies against CSP have been generated to date, with some of them mediating antimalarial protection upon passive transfer into animals. Genetically engineered transgenic mosquitoes producing the anti-CSP antibody have recently been generated to reduce malarial transmission. A monoclonal anti-CSP antibody was produced in mice by adeno-associated virus vector, which protected them from malaria. Phase III trials with RTS,S vaccine that targets CSP of Plasmodium falciparum have shown modest efficacy. Polyclonal anti-CSP antibodies derived from children who received the RTS,S vaccine failed to block malarial transmission through mosquitoes, but passive transfer of monoclonal antibodies raised from RTS,S-vaccinated recipient conferred protection against malaria in mice. Taken together, these findings may imply CSP as an antimalarial target.
It has been shown that human immunodeficiency virus (HIV)-1 infection induces the production of e... more It has been shown that human immunodeficiency virus (HIV)-1 infection induces the production of endogenous lipids required for effective viral production, and the cluster of differentiation (CD)1 molecule CD1d is downregulated by HIV-1 infection. However, the role of endogenous lipid presentation and the implications of CD1 downregulation by HIV-1 infection have not yet been characterized. In this study, we observed downregulation of both CD1c and CD1d expression through a Vpu-dependent and Nef-independent mechanism, and the concomitant HIV-1-induced production of host cholesterol decreased the extent of CD1c and CD1d modulation. While the modest downregulation of CD1c by HIV-1 infection decreased the ability of CD1c-restricted T cells to respond and secrete interferon-γ, the cholesterol upregulation in the same cells by HIV-1 infection appears to limit the downregulation of CD1c. The two conflicting HIV-1-mediated changes in CD1c expression appear to minimize the modulation of CD1c expression, thus leading the host to maintain a CD1c-restricted T-cell response against HIV-1.
We have raised a number of monoclonal antibodies (mAb) against idiotopes (Id) on monoclonal anti-... more We have raised a number of monoclonal antibodies (mAb) against idiotopes (Id) on monoclonal anti-murine class II (anti-Ak or Ek) antibodies. Two anti-Id mAb among 31 were found to cross-react with some T cells but not with macrophages and B cells of H-2k animals. They were able to block syngeneic mixed lymphocyte reaction (SMLR) and antigen-induced major histocompatibility complex (MHC)-restricted T cell proliferation of H-2k but not of other haplotypes. These results indicated that antibodies were recognizing Id associated with the MHC restriction site of T cells. The injection of these anti-Id mAb into H-2k mice resulted in the stimulation of self-class II-reactive T cell clones as determined by SMLR. They were also able to stimulate in non-H-2k strains to prime H-2k alloreactive T cell clones. Some animals developed anti-class II (anti-Iak) antibodies by the injection of anti-Id. These results indicated that certain anti-Id (anti-anti-class II) antibodies cross-reacted with T cell receptors which carried class II restriction specificity and were involved in allorecognition. These antibodies were found to have an ability to alter the T cell repertoire in vivo by stimulating such MHC-restricted clones.
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Papers by Moriya Tsuji