p.m. versus 3000 c.p.m.; P < 0·03). From these data, along with

those shown in Figs 2 and 3, we speculate that eosinophils not only present antigens to CD4+ T cells in an MHC class II pathway, but also present antigens to CD8+ T cells by using their MHC class I molecules. To test this hypothesis, experiments were performed to determine whether the induction of C. neoformans-primed T-cell proliferation was caused by the presentation of find more antigens by eosinophils in conjunction with MHC class I and MHC class II molecules. C. neoformans-pulsed eosinophils were treated with anti-MHC class I or anti-MHC class II mAbs before incubation with C. neoformans-primed CD4+ and CD8+ T cells. The blocking of MHC molecules on the eosinophil surface was found to suppress the ability of C. neoformans-pulsed eosinophils to stimulate C. neoformans-primed T-cell proliferation (Fig. 6d). Moreover, the suppression seen BMS-354825 purchase in the lymphocyte proliferation was more pronounced with anti-MHC class II, which coincided

with the higher proliferation of CD4+ T cells shown in Fig. 6c. In conclusion, C. neoformans-pulsed eosinophils stimulated C. neoformans-primed MSCs and T cells (CD4+ as well as CD8+) in an MHC class II- or class I-dependent manner. This stimulation of proliferation, however, was not observed for naive T cells or when C. neoformans-pulsed Mφ were used as APCs. To characterize and differentiate the T-cell profile seen after co-culture with C. neoformans-pulsed eosinophils, C. neoformans-primed purified T cells (CD4+ and CD8+) were analyzed Etofibrate by flow cytometry to determine the intracellular expression levels of IFN-γ and IL-4 after 4 days of culture with C. neoformans-pulsed eosinophils or medium alone. Figure 7 shows a significant increase in the percentage of IFN-γ-producing cells when T cells were incubated with C. neoformans-pulsed eosinophils compared with T cells cultured in medium alone (6·56% versus 1·61%; P < 0·02). With regard to the IL-4-producing T-cell population, the percentage

with C. neoformans-pulsed eosinophils (2·42%) was similar to that for medium alone (2·35%). These results allowed us to conclude that C. neoformans-pulsed eosinophils were able to induce the expansion of IFN-γ-producing Th1 cells, but not of IL-4-producing Th2 cells. To analyze the production of cytokines by CD4+ and CD8+ T cells in supernatants, the concentrations of IFN-γ, TNF-α, IL-4, IL-10 and IL-13 were measured after 4 days of culture. The results presented in Fig. 8(a,b) show that there was a significant increase in the production of IFN-γ and TNF-α generated by C. neoformans-primed T cells cultured with C. neoformans-pulsed eosinophils compared to the cytokine production by T cells cultured in medium alone, with fixed yeasts of C. neoformans or with unpulsed eosinophils. In contrast, no differences in the levels of IL-4, IL-13 or IL-10 were detected in supernatants of C. neoformans-primed T cells cultured with C.