Mucosal-Associated Invariant T (MAIT) Cell Dysfunction and PD-1 Expression in Prostate Cancer: Implications for Immunotherapy

Abstract

Prostate cancer is the second most common cancer in men worldwide. Despite an abundance of prostate-specific antigens, immunotherapies have yet to become a standard of care, potentially limited by T-cell dysfunction. Up to 10% of human circulating T-cells, and a significant fraction in the urogenital tract, are mucosal-associated invariant T (MAIT) cells. MAIT cells express stereotyped T-cell receptors that recognize riboflavin metabolites derived from microbes presented by MR-1. We evaluated the number, phenotype and function of circulating MAIT cells, alongside two other innate-like T (ILT) -cell subsets, in men with prostate cancer and age- and sex-matched controls. MAIT cells in men with prostate cancer circulated at similar frequencies to controls, but their cytokine production and proliferation was impaired. In contrast, the function of two other ILT-cell populations (natural killer T-cells and Vγ9Vδ2 T-cells) was not impaired. In both patients and controls, MAIT cells expressed high levels of the immune checkpoint molecule PD-1 at rest, while upregulation of PD-1 in response to the MR-1 ligand 5-amino-6D-ribitylaminouracil (5-A-RU) was greater in patients. 5-A-RU also induced upregulation of PD-L1 and -L2 RNA in primary mononuclear cells. We confirmed that circulating MAIT cell number and function were preserved before and during anti-PD1 therapy with pembrolizumab in a cohort of patients with melanoma. In vitro, 5-A-RU enhanced mononuclear cell cytotoxicity against the PD-L1 positive prostate cancer cell line PC3 in an MR-1-dependent manner. Addition of pembrolizumab enhanced this cytotoxicity, and was associated with increased MAIT cell expression of CD107a and IFN-γ. We conclude that prostate cancer is associated with MAIT-cell dysfunction, and that this might be overcome through the application of potent MR-1 ligands with PD-1 blockade. These findings may have implications for the development of cancer immunotherapies that exploit MAIT cells.

Keywords: MR1 protein; PD-1 protein; gamma delta T cells; human; mucosal-associated invariant T cells; natural killer T-cells; pembrolizumab; prostate cancer.

Figure 1

Circulating ILT-cell frequencies are preserved in men with prostate cancer. Flow cytometric analysis was used to determine the proportion of MAIT (A), NKT (B), Vγ9Vδ2T (C), CD4⁺ (D) and CD8⁺ (E) T-cells between men with prostate cancer and healthy male controls. Mann-Whitney test. N = 20 healthy controls, 36 men with prostate cancer. ns, no significance.

Figure 2

5-A-RU/MG stimulated MAIT-cell proliferation, but not IFN-γ SFU, is impaired in men with prostate cancer. The proliferation of MAIT (A), NKT (B) and Vγ9Vδ2T (C) cells was measured at the end of 7 days in culture with stimuli as indicated and expressed as a fold change to media control. IFN-γ spot forming units (SFUs) determined by ELISpot assay are demonstrated for PBMCs cultured with 5-A-RU/MG (D), α-GalCer (E) and pamidronate (F). **P < 0.01, Mann-Whitney test. N = 20 healthy controls, 36 men with prostate cancer. ns, no significance.

Figure 3

MAIT-cell agonist induced cytokines are impaired in men with prostate cancer. Culture supernatant was aspirated after 72 hours from the above proliferation assay and the Log₂ fold change in cytokines above media control is displayed (A). Multiple t-test. Individual plots quantifying IFN- (B), MIP-1α (C) and MCP-1 (D) in 5-A-RU/MG stimulated and media control samples are also displayed. ***P < 0.001. N = 18 healthy controls (HC), 36 men with prostate cancer (PC).

Figure 4

A MAIT-cell specific agonist upregulates PD-1 ligands, circulating MAIT-cells express high levels of PD-1 and MAIT-cells are over-represented among PD-1+ T-cells. Immune checkpoint genes significantly upregulated by PBMCs cultured with 5-A-RU/MG are shown in (A). N = 6 healthy donors. Flow cytometric analysis was used to determine the surface expression of PD-1 on MAIT and Vα7.2TCR^-CD161⁺/Vα7.2TCR⁺CD161^- T-cells in unstimulated PBMCs. Representative histograms (B) and surface PD-1 median fluorescence intensity (MFI) (C) are shown. The proportion of MAIT-cells within the PD-1⁺ and PD-1^- fractions of PBMCs from healthy controls and men with prostate cancer is displayed in (D). N = 20 healthy controls (HC), 36 men with prostate cancer (PC). ns, no significance. **P < 0.01. ***P < 0.001. ND, non detectable. ns, no significance.

Figure 5

PD-1 upregulation is associated with impaired MAIT-cell proliferation in men with prostate cancer. At the end of 7-day culture in the above proliferation assay, the surface expression of PD-1 on stimulated and unstimulated MAIT-cells was compared (A) and the relative fluorescence intensity calculated (B) for healthy controls and men with prostate cancer. Participants were stratified into high and low responders according to the median MAIT-cell fold change for their respective groups and the PD-1 RFI compared (C). Mann-Whitney test. N = 20 healthy controls, 36 men with prostate cancer. ns, no significance. *P < 0.05. ***P < 0.001.

Figure 6

Pembrolizumab does not deplete MAIT-cells in vivo nor does it enhance MAIT-cell function ex vivo. The circulating frequency of MAIT-cells (A) and their surface expression of CD137 (B) and PD-1 (C) in unstimulated PBMCs was compared for patients with melanoma before and after their first dose of pembrolizumab. PBMCs were then cultured with 5-A-RU/MG for 7 days and the proliferation of MAIT-cells (D) and the upregulation of CD137 (E) and PD-1 (F) at the end of culture calculated. Mann-Whitney test. ns, no significance. *P < 0.05. **P < 0.01. No significant differences were observed for cytokines quantified in 72 hour culture supernatant for pre and post pembrolizumab samples (G). N, 10.

Figure 7

Anti-PD-1 enhances healthy donor MAIT-cell suppression of prostate tumour growth in vitro. PC3 cells were grown in a real-time impendence based cell killing assay for 12 hours in media control or 5-A-RU/MG prior to the addition of healthy donor PBMCs. The effects of anti-MR1 (A, B) and anti-PD-1 (C, D) on tumor cell growth were calculated as the area under the curve. PC3 cells were primed with 5-A-RU/MG or media control and co-cultured with healthy donor PBMCs overnight and the CD107a degranulation (E) and IFN-γ expression (F) were calculated by flow cytometry. Friedman test with Dunn’s multiple comparison test. ns = no significance. *P < 0.05. **P < 0.01. N = 6 healthy donors.