To gain insight into how pathogens adapt to new hosts, Cryptococcus neoformans (H99W) was serially passaged in Galleria mellonella. The phenotypic characteristics of the passaged strain (P15) and H99W were evaluated. P15 grew faster in hemolymph than H99W, in vitro and in vivo, suggesting that adaptation had occurred. However, P15 was more susceptible to hydrogen peroxide in vitro, killed fewer mouse macrophages, and had less fungal burden in human ex vivo macrophages than H99W. Analysis of gene expression changes during Galleria infection showed only a few different genes involved in the reactive oxygen species response. As P15 sheds more GXM than H99W, P15 may have adapted by downregulating hemocyte hydrogen peroxide production, possibly through increased capsular glucuronoxylomannan (GXM) shedding. Hemocytes infected with P15 produced less hydrogen peroxide, and hydrogen peroxide production in response to GXM-shedding mutants was correlated with shed GXM. Histopathological examination of infected larvae showed increased numbers and sizes of immune nodules for P15 compared to H99W, suggesting an enhanced, but functionally defective, response to P15. These results could explain why this infection model does not always correlate with murine models. Overall, C. neoformans’ serial passage in G. mellonella resulted in a better understanding of how this yeast evolves under selection.

Innate CD8αα+ cells (iCD8α) are TCRneg intraepithelial lymphocytes (IEL) that possess immune functions such as phagocytosis, bacteria killing, and antigen processing and presentation. Recently our group and others reported that iCD8α cells promote IEL survival via osteopontin. Herein, we investigate whether iCD8α cells sustain intestinal homeostasis through maintaining IEL balance. We compared the number of IEL in the colon of WT and iCD8α-deficient mice (E8i−/−). E8i is an enhancer required for CD8α homodimer expression in IEL. Despite the dramatic reduction of CD8αα expression on TCRαβ+ and TCRgd+ IEL in E8i−/− mice, the total cell numbers were comparable to WT mice. E8i−/− mice had higher number of TCRβ+CD4+, and lower number of TCRβ+CD4+CD8α+ and TCRβ+CD8αα+ IEL. There were no differences in TCRβ+CD8αβ+ and TCRneg IEL. Then, we tested the role of iCD8α cells during intestinal inflammation in different colitis models. iCD8α-deficient mice had higher susceptibility to Citrobacter rodentium infection as indicated by increased weight loss, disease index, and bacterial load in the colon compared to WT mice. Similar results were obtained using DSS-induced colitis: E8i−/− mice presented increased shortening of colon and pathological scores. Finally, we performed adoptive transfer of naïve CD4 T cells into Rag-2−/− and E8i−/−Rag-2−/− mice. iCD8α-deficient mice rapidly developed chronic colitis, manifested by sever weight loss and bloody diarrhea. These mice also reconstituted with higher number of donor-derived CD4 T cells that mostly developed into pathogenic TCRβ+CD4+ IEL. Our results indicate that iCD8α cells have a critical role in the maintenance of IEL homeostasis and healthy intestines.