Our findings highlight the potential for MAIT cells to promote protective immunity in human influenza. = 16 patients; days 6C34) revealed significantly lower numbers of MAIT TP-472 cells in the fatal cases (= 0.014, nonparametric one-way ANOVA) (Fig. 56C88; median, 68.5), and age-matched controls (= 52C79; median, 72). (= 0.014; (and = 0.012, KruskalCWallis test. ( 0.05) (Fig. 1 and and Fig. S1) from PBMCs incubated with control (previously incubated in allantoic fluid) and H1N1/H3N2-infected A549 cells established that the IAV exposure induced IFN production in the MAIT cells (8.6 7.5%; = 22), at levels (Fig. 2 and = 22) and NK cells (20.8 13.5%; = 22). Indeed, significantly higher up-regulation of IFN in MAIT, NK, and T cells was found compared with classical MHC-restricted helper and killer T cells ( 0.001; Fig. 2= 22. *** 0.001, **** 0.0001, KruskalCWallis test. (and 0.001, Wilcoxon rank-sum test. Open in a separate window Fig. S1. The gating strategy for MAIT, NK, , CD4+, and CD8+ T cells using 12C14 parameter flow cytometery. Given the robust IFN production by MAIT, NK, and T cells, we next evaluated for any correlation between the high frequency of MAIT cells producing IFN during influenza infection and IFN production in NK or T cells within the same donor. Indeed, we observed strong correlations in IFN TP-472 production between MAIT/NK ( 0.01; = 0.569, Spearman rank correlation) and MAIT/ T cells ( 0.0001; = 0.888) (Fig. S2), suggesting that overall, these three subsets respond mutually during IAV infection. This does not imply that MAIT cells are dependent on NK or T cells to produce IFN, however. Open in a separate window Fig. S2. IFN production by MAIT cells is highly correlated with NK cell (= 22. Coculture of PBMCs with IAV-infected A549 cells did not result in significant expression of CD107a (minimally on NK cells), a known marker of degranulation for NK, MAIT, and T cells (Fig. 2 0.001; = 12). We further suggest that GzmB is an early marker of MAIT cell activation (Fig. 2= 8). MAIT Cell Activation Is Not Abrogated by MR1-Blocking Antibody. To understand MAIT cell activation during IAV infection, we first asked whether MAIT cell IFN production after exposure to IAV-infected epithelial cells is MR1-dependent. Several riboflavin derivatives from microbial species, including are presented by MR1 (5, 6, 17, 18); however, the addition of -MR1Cblocking monoclonal antibody (clone 26.5) to the IAV coculture system did not reduce the relative expression levels of IFN compared with coculture of PBMCs with 1% paraformaldehyde-fixed where -MR1 is known to inhibit cytokine production in MAIT cells (by approximately twofold) (Fig. 3(MOI 0.1) (10). ** 0.01, paired test. = 4. (and and = 3. In the absence of other PBMC subsets, FACS-purified CD161+V7.2+CD3+ MAIT cells cultured with IAV-infected A549 cells for TP-472 10 h in the presence of BFA did not make IFN (Fig. 3and and 0.05, Students test. = 5. (= 6. ( 0.05, one-way ANOVA. IL-18CDependent Activation of MAIT Cells During IAV Infection. Earlier studies (14, 19) have shown that MAIT cells respond robustly to cytokine-driven stimulation (IL-18, IL-12, and IL-7) and constitutively express high surface levels of the IL-18 and TP-472 IL-12 receptors (14). Given our findings indicating that MAIT cells make IFN and GzmB when stimulated in IAV A549-PBMC cocultures (Fig. 4 and = 4) by MAIT cells (Fig. 4 0.05), because robust IFN production was retained for cultures containing only the IL-12 p40/70 blocking antibody (Fig. 4 0.002). This suggests that monocytes are directly activated by exposure to IAV-infected epithelium, and in turn contribute to the induction of MAIT cells during influenza. Open in TP-472 a separate window Fig. 5. Monocytes are required for IFN up-regulation in MAIT cells after IAV infection. (= 0.0015, Students test. = 4. (= 0.007, Students test. = 5. (and 0.01), although IFN in the NK cells was reduced to background levels (Fig. 5 test, with a confidence level of 95%. 0.05 was considered significant. Note Added in Proof. An additional manuscript on the role of MAIT cells in viral infections has been published while our manuscript was in revision (36). Acknowledgments We thank Andrew Brooks for insightful discussions; Ted Hansen for the -MR1-26.5 blocking reagent; and Thakshila Amarasena, Sheilajen Alcantara, and Bernie McCudden for collecting blood. We thank all donors for donating blood for this study. This work was supported by National Health and Medical Research Council Program (NHMRC) Grant 1071916 (to P.C.D. and K.K.). L. Loh and S.J. were Rabbit polyclonal to ZNF317 supported by an NHMRC C. J. Martin fellowship. Z.W. was supported by an NHMRC AustraliaCChina.