muris-only (clear) infected groups per 40 caecum crypts. Data display median ± SD of 5 animals per group. P values <0.05 were considered statistically significant. (ns = non significant). Co-infection increases CD4+ splenocyte frequencies and modifies the TH1/TH2 immune balance Flow cytometric

analysis Selumetinib demonstrated that co-infection according to either infection protocol (Figure 1A and B) did not impact lymphocyte composition in the spleen or MLN, since no significant differences between infection groups were observed for populations of CD3+ T cells or B220+ B cells (data not shown). However, analysis of ex-vivo lymphocyte subpopulations in BALB/c mice infected according to Figure 1A, revealed an increase in CD4+ T helper cell population in the spleens of mice co-infected according to the protocol in Figure 1A, when compared to BCG-only

infected mice (Figure 5A). Although no differences in the percentages of natural regulatory T cell (CD4+CD25+Foxp3+) populations were observed between infection groups in either the spleen or MLN (data not shown), co-infection significantly increased the percentage of IL-4-producing CD4+ and CD8+ splenocytes in comparison to M. bovis BCG-only infected controls (Figure 5B). IL-4-producing CD4+ and CD8+ MLN cells from co-infected mice were however significantly reduced in comparison to T. muris-only infected mice (Figure 5C). A marked decrease in CD8+IFNγ+ MLN cells was also observed in co-infected mice in comparison to mice infected only with T. muris, whereas frequencies of CD4+ IFNγ+ MLN cells were measured at similar levels between co-infected and T.muris-only infected

mice (Figure 5D). Figure 5 Co-infection affects the KPT-330 concentration frequency of CD4 + and Treg lymphocyte populations and alters ex vivo TH1/TH2 cell populations. (A) Percentages of CD4+ splenocytes in BCG-only (clear) and co-infected (black) BALB/c mice infected according to experimental design in Figure 1A. Data display median ± min-max, representing 2–3 individual experiments of 20 animals per group. (B) Percentages of IL-4 producing CD4+ and CD8+ splenocytes in BCG-only (clear) and co-infected (black) BALB/c mice infected according to the protocol in Figure 1B. Data display median ± min-max, representing 2–3 individual experiments of 8–10 animals per group. (C-D) Percentages of CD4+IL-4+, CD8+IL-4+ and DNA ligase CD4+IFN-γ+ MLN cell populations in T. muris-only (clear) and co-infected (black) BALB/c mice infected according to experimental design in Figure 1B. Data represents experiments with 8–10 animals per group. Percentages of (E) activated T cells (CD4+CD25+Foxp3-) and (F) inducible regulatory T cells (iTreg) (CD4+CD25-Foxp3+) in MLNs of T. muris-only (clear) and co-infected (black) BALB/c mice infected according to experimental design in Figure 1B. Data display median ± min-max, representing 2–3 individual experiments of 8–10 animals per group. P values <0.05 were considered statistically significant. (*p ≤ 0.05, **p ≤ 0.01, ns = non-significant).