Annual Report 2004

Department of Microbiology and Immunology
Division of Immunology

Self-defense against invaded pathogenic microorganisms and foreign antigenic molecules is strictly controlled by the immune system and inflammation. Our major research interests are to elucidate cells and effector molecules in innate and acquired immunity and inflammation. In particular, we are focused on cellular and molecular mechanisms of development and activation of B cells and IgH class switch recombination under the influence of T cells, cytokines and adaptor proteins. We are also interested in elucidating cellular mechanisms of preferential induction of Th1 cells upon immunization with Mycobacterium-derived, Peptide-25 and its derivatives.
In 2003, Professor Dr. Fritz Melchers, Basel University, who were invited by the University of Tokyo as the Eminent Scientist of JSPS, has been joining us for our research projects. Dr. Melchers always encouraged us and suggested important issues for each project. We would like to appreciate his enormous contributions.


Mechanisms of preferential induction of Th1 response upon immunization with Mycobacteria peptide

The Ag85B of Mycobacterium (M.) tuberculosis and M. Bovis BCG is immunogenic in C57BL/6 mice with Ag85B to expand TCRVイ11+ CD4+ Th1 cells in conjunction with APCs in an I-Ab-restricted manner. We identified the major antigenic epitope (Peptide-25) for Ag85B-specific Vイ11+ T cells as the 15-mer peptide, covering amino acid residues 240-254 of Ag85B.

a. Role of MHC/peptide-TCR interaction in the Peptide-25-dependent Th1 development

Haruyuki Ariga, Makiyo Nakada, Takeshi Tokunaga, Yoko Shimohakamada, Ai Kariyone, Toshiki Tamura and Kiyoshi Takatsu

Activated CD4+ Th cells can be classified into two subsets, Th1 and Th2, on the basis of cytokine production profiles. Th1 cells play a critical role in the induction of the cell-mediated immune responses that are important for the eradication of intracellular pathogens and the development of organ-specific autoimmune diseases. In addition to the T cell antigen receptor (TCR) activation signals, other factors such as the cytokine environment, type of APC, genetic background and co-stimulatory molecules expressed by activated APC may also be involved in the determination of the differentiation of naive CD4+ T cells into Th1 cells. However, it is unclear whether the TCR signaling events exert a direct influence on Th1 differentiation. To elucidate cellular and molecular mechanisms of the induction of Th1 cells by Peptide-25, transgenic mice that express the TCR-Vア5-Vイ11 for recognition of Peptide-25, in conjunction with I-Ab molecules, were generated (P25 TCR-Tg), and the differential activities of naive CD4+ T cells from P25 TCR-Tg were examined.
Naive CD4+ T cells from P25 TCR-Tg preferentially differentiated into Th1 cells upon Peptide-25 stimulation in the presence of T and NK cells depleted I-Ab splenic APC under neutral condition. In contrast, a mutant of Peptide-25 could induce solely Th2 differentiation. Peptiode-25-induced Th1 differentiation was observed even in the presence of neutralizing monoclonal antibodies to IFN-ウ and IL-12. Furthermore, naive CD4+ T cells from STAT1 deficient P25 TCR-Tg also differentiated into Th1 cells upon Peptide-25 stimulation. Moreover, Peptide-25-loaded I-Ab-transfected Chinese hamster ovary cells (Peptide-25-I-Ab-CHO) that do not express co-stimulatory molecules, such as CD40/80/86 and ICAM-1 on their surface, and not produce any IFN-ウ and IL-12 effectively induced Th1 differentiation of naive CD4+ T cells from P25 TCR-Tg. Within 3 hr after the TCR stimulation with Peptide-25-I-Ab-CHO, IFN-ウ and IL-12 independent transient T-bet up-regulation and suppression of GATA-3 expression were observed. These results imply that direct interaction between Peptide-25/I-Ab and TCR primarily influences determination of the fate of naive CD4+ T cells in differentiation toward the Th1 subsets.

b. Adjuvant activity of Peptide-25 for enhancing anti-tumor immune response
Takeshi Kikuchi, Ai kariyone, Wen Xu, Toshiki Tamura and Kiyoshi Takatsu
CD8+ cytotoxic T cells (CTL) play an important role in the protection against tumor growth. Tumor cells are thought to express an array of antigens recognizable by CTLs that principally contribute to tumor rejection. It still remains unclear, however, whether CD4+ helper T cells together with CTLs mediate efficient immune responses leading to tumor rejection.
As the immunization of C57BL/6 mice with Peptide-25 emulsified in incomplete Freund adjuvant (IFA) induces Th1 response to Peptide-25, we examined adjuvant activity of Peptide-25 for CTL generation to ovalbumin (OVA) as a model tumor antigen. Results revealed that co-immunization of C57BL/6 mice with OVA and Peptide-25 could induce higher OVA specific-IgG2a and IFN-ウ production than OVA alone immunization. Intriguingly, the OVA-specific CTL generation was also enhanced when mice were co-immunized with OVA plus Peptide-25. The adjuvant effect of Peptide-25 was not observed in CD4 deficient or IFN-ウ deficient mice. Co-immunization of OVA and Peptide-25 could prevent in vivo growth of E.G7-OVA cell (EL4 thymoma transfected with cDNA encoding chicken OVA) leading to prolonged survival. Furthermore, the enhancement of CTL generation by Peptide-25 was also observed when class I-binding OVA peptide (SIINFEKL) was used in place of intact OVA. Moreover, CTL generation specific for class I-binding B16 melanoma peptide (SVDFFVWL) was enhanced by co-immunization with Peptide-25. To elucidate the mechanisms of this adjuvant activity of Peptide-25, we examined the dendritic cells (DC) activation by Peptide-25. Results revealed that Peptide-25 stimulation alone did not enhance the expression of surface molecules. When we co-cultured DC with CD4+ T cells from P25 TCR-Tg mice together with Peptide-25, expressions of MHC class I and ICAM-1 were enhanced and led to induce IL-12 p40 production. Such DC showed more effective OVA presentation to OVA specific CD8+ T cells and enhanced proliferation of the cells.
These results indicate that Peptide-25 exerts potent adjuvant activity and provides efficient help for CTL induction against neo-tumor antigen when concomitantly immunized.