MECHANISTIC STUDIES ON DC-INDUCED T CELL HYPORESPONSIVENESS USING A TCR TRANGENIC MODEL

Abstract

P1007 Aims: Two populations of tolerogenic dendritic cells (DC) have been identified in mice; immature myeloid DC express low MHC and costimulatory molecules, while liver-derived B220+ lymphoid DC are phenotypically mature. To better understand the underlying mechanisms, it was attempted to characterize the antigen-specific T cell responses, using TCR transgenic DO11.10 (H2d) mice with CD4+ T cells expressing transgenic TCR specific for chicken oval albumin (OVA) which can be identified by specific mAb KJ1.26. Methods and Results: Immature myeloid and B220+ lymphoid DC were propagated from BALB/c (H-2d) mouse liver in IL-3 plus CD40L, and GM-CSF, respectively (bone marrow-derived mature myeloid DC were used as controls). Cell divisions were analyzed by CFSE dilution. T cells isolated from DO11.10 mouse lymph nodes (LN) were labeled with CFSE and cultured with irradiated DC (T/DC ratio of 10/1) in the present of 0.3 mM OVA. Responses of OVA specific T cells were analyzed in KJ1.26+ cell population. KJ1.26+ cell divisions were mild following stimulation with immature myeloid DC, reaching ∼16% KJ1.26+ cells at the end of culture, suggesting relatively low T cell proliferation. KJ1.26+ T cell division in B220+ DC group was very active, similar to that cultured with mature myeloid DC, and generated final ∼57% and ∼34% KJ1.26+ cells, respectively, indicating distinct responses to immature myeloid and B220+ DC. Combined observations of CFSE dilution and apoptotic death by anti-annexin V revealed that >90% of death occurred in divided KJ1.26+ cells in all groups, suggesting an activation induced cell death (AICD) nature. Annexin V+ cells in divided KJ1.26+ cells was 90% in liver B220+ DC, 66% in mature myeloid DC and 35% in immature myeloid DC group, suggesting the profound capability of B220+ DC to promote AICD in OVA-specific CD4+ T cells. To examine this in vivo, 2.5 x 105 DC pulsed with OVA were injected into footpad of BALB/c mice that had been adoptive transferred with 2.5 x 106 CFSE labeled DO11.10 T cells 24 h before. T cells isolated from the popliteal LN on d3 were analyzed in KJ1.26+ population. Few divided cells were identified in immature myeloid DC group, and only 0.6% cell were KJ1.26+. In contrast, active cell divisions were observed in injection with B220+ DC mice, and KJ1.26+ cells achieved 1.9% associated with elevated apoptosis activity, compared with 1.6% in mature DC injection group with less apoptotic activity. Conclusion: These data suggest that, distinct from immature myeloid DC, B220+ DC induce hyporesponsiveness by promotion of AICD in antigen-specific T cells.