Summary of the publications
2001-2004
Naoki Kobayashi, Hiroshi Takata, Shumpei Yokota, and Masafumi Takiguchi, Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation. Eur. J. Immunol., 34(12): 3370-3378, 2004.
Multi-color flow cytometric analysis on human CD8+ T cell subsets revealed that CXCR4 is predominantly expressed on CD8+ T cells with the naive CD27+CD28+CD45RA+ phenotype, and is down-regulated during differentiation into those with an effector phenotype. The downregulation of CXCR4 expression during peripheral differentiation was supported by the fact that the expression of CXCR4 on CD8+ T cells was negatively correlated with that of perforin. The analysis of CCR5, CCR7, and CXCR4 co-expression further showed that CD8+ T cells expressing a high level of CXCR4 are CCR7+CCR5? naive or central memory subsets, and those expressing a low level of CXCR4 were included in the CCR7?CCR5+/? memory/effector and effector subsets. Epstein Barr virus-specific CD8+ T cells, which mostly express the memory phenotype, expressed CXCR4, while human cytomegalovirus-specific CD8+ T cells, which mostly express the effector phenotype, partially expressed this receptor, showing that the expression of CXCR4 is also down-regulated during differentiation of viral antigenspecific CD8+ T cells. The classification of human CD8+ T cells based on the expression of these chemokine receptors should prove useful for studies that clarify the differentiation of human CD8+ T cells.
Takamasa Ueno, Hiroko Tomiyama, Mamoru Fujiwara, Shinichi Oka, and Masafumi Takiguchi, Functionally impaired HIV-specific CD8 T cells show high-affinity T cell receptor-ligand interactions. J. Immunol. 173: 5451-5457, 2004.
We eventually isolated two different clonotypic CD8 T cell subsets recognizing an HIV Pol-derived epitope peptide (IPLTEEAEL) in association with HLA-B35 from a chronic HIV-infected patient. By kinetic analysis experiments, the subsets showed a >3-fold difference in half-lives for the HLA tetramer in complex with the Pol peptide. In functional assays in vitro and ex vivo, both subsets showed substantial functional avidity toward peptide-loaded cells. However, the high affinity subset did not show cytolytic activity, cytokine production, or proliferation activity toward HIV-infected cells, whereas the moderate affinity one showed potent activities. Furthermore, using ectopic expression of each of the TCR genes into primary human CD8 T cells, the CD8 T cells transduced with the high affinity TCR showed greater binding activity toward the tetramer and impaired cytotoxic activity toward HIVinfected cells, corroborating the results obtained with parental CD8 T cells. Taken together, these data indicate that impaired responsiveness of T cells toward HIV-infected cells can occur at the level of TCR-ligand interactions, providing us further insight into the immune evasion mechanisms by HIV.
Center for AIDS Research Best Paper Award 2004
Hiroshi Takata, Hiroko Tomiyama, Mamoru Fujiwara, Naoki Kobayashi, and Masafumi Takiguchi, Cutting edge: Expression of Chemokine Receptor CXCR1 on Human Effector CD8+ T Cells. J. Immunol. 173: 2231-2235, 2004.
IL-8 is a potent inflammatory cytokine that induces chemotaxis of neutrophils expressing CXCR1 and CXCR2,
thus indicating its involvement in the migration of these cells to inflammatory sites where bacteria proliferate.
Presently, we showed that CXCR1+ cells were predominantly found among CD8+ T cells having effector phenotype, and that the expression of CXCR1 was positively correlated with that of perforin, suggesting that CXCR1 is expressed on effector CD8+ T cells. Indeed, human CMVspecific CD8+ T cells from healthy individuals, which mostly express the effector phenotype and have cytolytic function, expressed CXCR1, whereas EBV-specific CD8+ T cells, which mostly express the memory phenotype and have no cytolytic function, did not express this receptor. The results of a chemotaxis assay showed that the migration of CXCR1+CD8+ T cells was induced by IL-8. These results suggest that the IL-8-CXCR1 pathway plays an important role in the homing of effector CD8+ T cells. The Journal of Immunology, 2004.
Hiroko Tomiyama, Hiroshi Takata, Tomoko Matsuda, and Masafumi Takiguchi, Phenotypic classification of human CD8+ T cells reflecting their function: An inverse correlation between quantitative expression of CD27 and cytotoxic effector function. Eur. J. Immunol. 34: 999-1010, 2004.
Phenotypic classification of human CD8+ T cells using three cell surface markers, CD27, CD28 and CD45RA, was recently suggested to be useful for identification of naive, memory and effector CD8+ T cells. However, it still remains unclear whether such classification precisely reflects functional classification of CD8+ T cells. To clarify this, we characterized each CD27CD28CD45RA subset of total and human cytomegalovirus (HCMV)-specific CD8+ T cells by analyzing the expression of perforin and two chemokine receptors, CCR5 and CCR7, as well as their function. An inverse correlation between perforin and CD27 expression was found in all four CD28CD45RA subsets. Therefore, to achieve a phenotypic classification of CD8+ T cells that moreprecisely reflects their function, the CD27+ subset was divided into CD27low and CD27high subsets based on the expression level of CD27. Functional and flow cytometric analyses of CD27CD28CD45RA subsets showed that this phenotypic classification reflects functional classification of CD8+ T cells. HCMV-specific CD8+ T cells from healthy HCMV-seropositive individuals were predominantly found in effector and memory/effector subsets, indicating that HCMV-specific effector CD8+ T cells are actively induced by HCMV replication in healthy HCMV carriers. Phenotypic analyses of CD8+ T cells using this classification will enable the characterization of antigen-specific CD8+ T cells.
Center for AIDS Research Best Paper Award 2002
Ueno Takamasa, Hiroko Tomiyama, and Masafumi Takiguchi, Single T cell receptor-mediated recognition of an identical HIV-derived peptide presented by multiple HLA class I molecules. J. Immunol. 169:4961-4969, 2002.
A dual specific human CTL clone harboring one and two inframe transcripts of TCR was previously reported to recognize an HIV Pol-derived nonapeptide (IPLTEEAEL) endogenously presented by both syngeneic HLA-B*3501 and HLA-B*5101. In the current study, a retrovirus-mediated TCR transfer of individual - and -chains to TCR-negative hybridoma showed that V12.1 TCR in complex with V5.6 were responsible for the peptide-specific response in the context of both HLA-B*3501 and HLA-B*5101, confirming single TCR-mediated dual specificity. The second TCR- chain was not somehow expressed on the cell surface. Remarkably, the V12.1/V5.6 TCR also recognized the same peptide presented by allogeneic HLA class I molecules that share the similar peptide-binding motifs, such as HLA-B*5301 and HLA-B*0702. The sensitivity of peptide recognition by the V12/V5.6 TCR appeared to be comparable when the peptide was presented by syngeneic and allogeneic HLA class I molecules, with changes in T cell responsiveness caused largely by peptide-binding capacity. Moreover, the CTL clone bearing V12.1/V5.6 TCR showed substantial cytolytic activity against the peptide-loaded cells expressing HLA-B*3501, HLA-B*5101, HLA-B*5301, or HLA-B*0702, providing further evidence that a single TCR complex can recognize the same peptide presented by a broad range of HLA class I molecules. A TCR with fine specificity for an HIV Ag but broad specificity to multiple HLA molecules may provide an advantage to the generation of allorestricted, peptide-specific T cells, and thus could be a potent candidate for immunotherapy against HIV infection.
Hiroko Tomiyama, Hirofumi Akari, Akio Adachi and Masafumi Takiguchi, Different effects of Nef-mediated HLA class I down-regulation on HIV-1-specific CD8+ T cell cytolytic activity and cytokine production. J. Virol. 76:7535-7543, 2002.
A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4+ T cells. We confirmed this effect by using a nef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4+ T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4+ T cells infected with NL-M20A. Interestingly, CTL clones stimulated with NL-432-infected CD4+ T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4+ T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4+ T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8+ T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8+ T cells are able to partially but not completely control HIV-1 replication in vivo.
Hiroko Tomiyama, Tomoko Matsuda, and Masafumi Takiguchi, Differentiation of Human CD8+ T Cells from a Memory to Memory/Effector Phenotype. J. Immunol. 168: 5538-5550, 2002.
Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA-CD8+ T cells with different CD28/CD27 phenotypes showed that CD28+CD45RA-CD8+ and CD27+CD45RA-CD8+ T cells have characteristics of memory T cells, whereas CD28-CD45RA-CD8+ and CD27-CD45RA-CD8+ T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8+ T cells into memory/effector CD8+ T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8+ T cells. Three subsets of CD45RA-CD8+ T cells were observed in both total CD8+ T cells and EBV- or HCMV-specific CD8+ T cells: CD27+CD28+, CD27+CD28-, and CD27-CD28-. A significant number of the CD27-CD28+ subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8+ T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27+CD28+CD45RA-CD27+CD28-CD45RA-CD27-CD28-CD45RA-. This was supported by the observation that the frequency of CCR5+ cells and CCR7+ cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27+CD28+ memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5-CCR7+, CCR5+CCR7+, and CCR5+CCR7-. These findings suggested the following differentiation pathway: CD27+CD28+CD45RA- (CCR5-CCR7+CCR5+CCR7+CCR5+CCR7-)CD27+CD28-CD45RA-CD27-CD28-CD45RA-. The presence of a CD27-CD28+ subset with a CCR5+CCR7- phenotype implies a specialized role for this subset in the differentiation of CD8+ T cells.
Katsuhiko Fukada, Yuji Sobao, Hiroko Tomiyama, Shinichi Oka and Masafumi Takiguchi, Functional Expression of the Chemokine Receptor CCR5 on Virus Epitope-Specific Memory and Effector CD8+ T Cells. J.　Immunol. 168:　2225-2232, 2002.
Because the chemokine receptor CCR5 is expressed on Th1 CD4+ cells, it is important to investigate the expression and function of this receptor on other T cells involved in Th1 immune responses, such as Ag-specific CD8+ T cells, which to date have been only partially characterized. Therefore, we analyzed the expression and function of CCR5 on virus-specific CD8+ T cells identified by HLA class I tetramers. Multicolor flow cytometry analysis demonstrated that CCR5 is expressed on memory (CD28+CD45RA-) and effector (CD28-CD45RA- and CD28-CD45RA+) CD8+ T cells but not on naive (CD28+CD45RA+) CD8+ T cells. CCR5 expression was much lower on two effector CD8+ T cells than on memory CD8+ T cells. Analysis of CCR7 and CCR5 expression on the different types of CD8+ T cells showed that memory CD8+ T cells have three phenotypic subsets, CCR5+CCR7-, CCR5+CCR7+, and CCR5-CCR7+, while naive and effector CD8+ T cells have CCR5-CCR7+ and CCR5+CCR7- phenotypes, respectively. These results suggest the following sequence for differentiation of memory CD8+ T cells: CCR5-CCR7+CCR5+CCR7+CCR5+CCR7-. CCR5+CD8+ T cells effectively migrated in response to RANTES, suggesting that CCR5 plays a critical role in the migration of Ag-specific effector and differentiated memory CD8+ T cells to inflammatory tissues and secondary lymphoid tissues. This is in contrast to CCR7, which functions as a homing receptor in migration of naive and memory CD8+ T cells to secondary lymphoid tissues.
Katsuhiko Fukada, Hiroko Tomiyama, Chantapong Wasi, Tomoko Matsuda, Shigeru Kusagawa, Hironori Sato, Shinichi Oka, Yutaka Takebe, and Masafumi Takiguchi, Cytotoxic T Cell Recognition of HIV-1 Cross-Clade and Clade-Specific Epitopes in HIV-1-infected Thais and Japanese. AIDS. 16:701-711, 2002.
OBJECTIVE: To identify and characterize cytotoxic T-cell (CTL) epitopes for HIV-1 clade E using eight known HLA-A*1101-restricted HIV-1 clade B epitopes.
METHODS: Induction of clade E-specific CTL was examined by stimulating peripheral blood mononuclear cells (PBMC) from clade E-infected Thai individuals with the clade E-specific peptide corresponding to the clade B epitopes. Cross-clade and clade-specific CTL recognition for these epitopes was analysed using CTL clones and bulk CTL specific for these epitopes. To clarify the presentation of these epitopes in HIV-1-infected T cells, CTL recognition for the clade E-specific and cross-clade epitopes was investigated using CD4CXCR4 cells infected with an HIV-1 clade E clone.
RESULTS: Three epitopes, which are identical among clades A-E, were recognized as cross-clade CTL epitopes in both individuals. Clade B and E sequences corresponding to three epitopes were recognized as clade-specific epitopes in clade B-infected and clade E-infected individuals, respectively. In contrast, clade E-specific peptides corresponding to two other clade B epitopes failed to elicit clade E-specific CTL. CTL specific for the three cross-clade and three clade E-specific epitopes effectively lysed target cells infected with HIV-1 clade E virus.
CONCLUSIONS: These six epitopes are found to be processed naturally in HIV-1 clade E-infected cells. We show here that a strategy utilizing HIV-1 clade B epitopes is very useful for identifying clade E CTL epitopes.
Yuji Sobao, Hiroko Tomiyama, Saburo Nakamura, Hisahiko Sekihara, Katsuaki Tanaka, and Masafumi Takiguchi, Visual demonstration of HCV-specific memory CD8+ T cell expansion in patients with acute hepatitis C. Hepatology. 33:287-294, 2001.

Hepatitis C virus (HCV)-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from patients infected with HCV were quantitatively analyzed by flow cytometry using an HLA-B*3501-HCV epitope tetrameric complex. In chronic hepatitis C, tetramer+CD8+ T cells were detected at frequencies ranging from 0.05% to 0.12% of total CD8+ T cells. The number of tetramer+CD8+ T cells in acute phase PBMCs from patients with acute hepatitis C was about 3 to 5 times higher than in recovery phase PBMCs from the same patients and in PBMCs from patients with chronic hepatitis C. Expanding tetramer+CD8+ T cells in PBMCs from patients with acute hepatitis C express a CD28+CD45RA- memory T-cell phenotype. In contrast, tetramer+CD8+ T cells in PBMCs from patients with chronic hepatitis C did not predominantly express this phenotype. These tetramer+CD8+ T cells did not have perforin in their cytoplasma. The present study visually showed that a high number of circulating HCV-specific CD8+ T cells in acute phase PBMCs from patients with acute hepatitis C are mostly memory T cells. (H 2001;33:287-294.)

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