The immune system plays a significant role in urothelial bladder cancer (UBC) progression, with CD8+ T cells being capable to directly kill tumor cells using perforin and granzymes. However, tumors avoid immune recognition by escape mechanisms. In this study, we aim to demonstrate tumor immune escape mechanisms that suppress CD8+ T cells cytotoxicity. 42 patients diagnosed with UBC were recruited. CD8+ T cells from peripheral blood (PB), sentinel nodes (SN), and tumor were analyzed in steady state and in vitro-stimulated conditions by flow cytometry, RT-qPCR, and ELISA. Mass spectrometry (MS) was used for identification of proteins from UBC cell line culture supernatants. Perforin was surprisingly found to be low in CD8+ T cells from SN, marked by 1.8-fold decrease of PRF1 expression, with maintained expression of granzyme B. The majority of perforin-deficient CD8+ T cells are effector memory T (TEM) cells with exhausted Tc2 cell phenotype, judged by the presence of PD-1 and GATA-3. Consequently, perforin-deficient CD8+ T cells from SN are low in T-bet expression. Supernatant from muscle invasive UBC induces perforin deficiency, a mechanism identified by MS where ICAM-1 and TGFß2 signaling were causatively validated to decrease perforin expression in vitro. Thus, we demonstrate a novel tumor escape suppressing perforin expression in CD8+ T cells mediated by ICAM-1 and TGFß2, which can be targeted in combination for cancer immunotherapy.
Mucosal associated invariant T cells (MAIT cells) are innate-like T cells (TC) which are known to be activated by several bacteria and viruses. However, activation of MAIT cells by moulds, such as the opportunistic human pathogen Aspergillus, is not well described. Stimulation of human PBMC with A. fumigatus, A. flavus, or A. terreus conidia revealed that in contrast to conventional CD4+ and CD8+ TC, MAIT cells responded already after 4 h of coincubation with upregulation of CD69. Furthermore, concurrent increase of CD107a expression and reduced intracellular expression of cytolytic proteins like perforin and granzyme indicated degranulation of intracellular vesicles. MAIT cell activation only occurred in the presence of APC and was dependent on cell-cell contact as separation of TC and APC abrogated MAIT cell activation. Furthermore, we observed that MAIT cell activation by moulds requires presentation of riboflavin metabolites and depends on TCR engagement as antibody blocking of MR1, the antigen presenting molecule for MAIT cells, prevented upregulation of CD69 and CD107a. In summary, we could demonstrate that MAIT cells are activated by Aspergillus conidia in a TCR-dependent manner by APC. These findings reveal MAIT cells as an interesting new target in antifungal defense.
