Merkel polyomavirus-specific T cells fluctuate with merkel cell carcinoma burden and express therapeutically targetable PD-1 and Tim-3 exhaustion markers

Abstract

Purpose: The persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins in Merkel cell carcinoma (MCC) provides a unique opportunity to characterize immune evasion mechanisms in human cancer. We isolated MCPyV-specific T cells and determined their frequency and functional status.

Experimental design: Multiparameter flow cytometry panels and HLA/peptide tetramers were used to identify and characterize T cells from tumors (n = 7) and blood (n = 18) of patients with MCC and control subjects (n = 10). PD-1 ligand (PD-L1) and CD8 expression within tumors were determined using mRNA profiling (n = 35) and immunohistochemistry (n = 13).

Results: MCPyV-specific CD8 T cells were detected directly ex vivo from the blood samples of 7 out of 11 (64%) patients with MCPyV-positive tumors. In contrast, 0 of 10 control subjects had detectable levels of these cells in their blood (P < 0.01). MCPyV-specific T cells in serial blood specimens increased with MCC disease progression and decreased with effective therapy. MCPyV-specific CD8 T cells and MCC-infiltrating lymphocytes expressed higher levels of therapeutically targetable PD-1 and Tim-3 inhibitory receptors compared with T cells specific to other human viruses (P < 0.01). PD-L1 was present in 9 of 13 (69%) MCCs and its expression was correlated with CD8-lymphocyte infiltration.

Conclusions: MCC-targeting T cells expand with tumor burden and express high levels of immune checkpoint receptors PD-1 and Tim-3. Reversal of these inhibitory pathways is therefore a promising therapeutic approach for this virus-driven cancer.

Figure 1. CD8 T cells specific for MCPyV were detected in the majority of MCC patients and tracked with tumor burden and with anti-T-Ag antibodies

A. MCPyV-specific T cell frequencies among patients or control subjects. Dashed line indicates the threshold of tetramer detection. Solid line indicates the median among patients with detectable MCPyV-specific T cells. All analyses were from the first available blood draw of subjects who were HLA-A*24 or HLA-A*23 positive. Representative flow cytometry plots are shown cases that were tetramer-positive or tetramer-negative. **p<0.01, Fisher’s exact test. B–F. MCPyV-specific T cells (gray bars, percent of CD3⁺CD8⁺ cells) and anti-T-Ag antibody titers (black line) measured in serial blood draws from four MCC patients at indicated times in their disease course. Days since diagnosis of primary tumor are indicated. Clinical extent of disease at time of blood draw is as indicated: (−) = none through (+++) = heavy burden. G. CMV-specific (black dashed line) or EBV-specific (gray dashed line) T cells were measured in serial blood draws from MCC patients (circle, w678; X, w334; triangle, w672; square, w131) at the indicated times.

Figure 2. MCPyV-specific T cells and MCC TIL express multiple inhibitory receptors and activation markers

A. Evaluation of three inhibitory receptors: PD-1, Tim-3, CTLA-4 and three activation markers: CD28, CD69, CD137 as assessed by flow cytometry. Representative histograms are gated on CD3+CD8+ TIL or CD3+CD8+Tetramer+ PBMC from blood. B. Summary data for all MCC patients’ samples analyzed: tumor infiltrating lymphocytes (TIL) from MCC tumors (n=7); tetramer-positive PBMC specific for MCPyV (n=5), CMV (n=7), EBV (n=5). The horizontal line indicates the mean. Statistical comparisons were made between MCC TIL and MCPyV-specific PBMC and between all virus-specific T cells in the blood. *p<0.05, **p<0.01, Wilcoxon rank sum test.

Figure 3. Co-expression of PD-1 and Tim-3 inhibitory receptors is elevated among MCPyV-specific T cells and MCC-infiltrating lymphocytes

A. Co-expression of inhibitory receptors from four representative samples analyzed with SPICE software (35). Pie chart indicates number of co-expressed markers. Outer arcs correspond to the extent of indicated surface marker expression on CD3⁺CD8⁺ TIL or CD3⁺CD8⁺Tetramer⁺ PBMC as assessed by flow cytometry. B. Comparison of the fraction of cells that co-express PD-1, Tim-3 and CTLA-4 in MCC CD8+ TIL (n=7), PBMC specific for MCPyV (n=5), CMV (n=7), EBV (n=5) and all CD3⁺CD8⁺ T cells (n=11). The mean and SEM are shown. **p<0.01, Wilcoxon’s rank sum test. C. CD3⁺CD8⁺ TIL(n=7) assessed for PD-1 and Tim-3 expression by flow cytometry. Appropriate isotype antibody controls are included in the rightmost panel. Three distinct populations of PD-1 expression are often detected. Relative expression is indicated on the first plot as (−) = negative, (+) = positive, (++) = high-positive.

Figure 4. PD-1 is highly expressed on MCPyV-specific T cells and its expression is maintained throughout the MCC disease course

A. Median fluorescence intensity (MFI) of CD3⁺CD8⁺PD-1⁺ T cells specific for MCPyV (n=5), CMV (n=7) and EBV (n=5) measured in the first available blood draw from 12 MCC patients. Most MCC patients only had detectable tetramer-positive T cells for one of these viruses. Line indicates median. Tet⁺ = tetramer-positive. *p<0.05, **p<0.01, Wilcoxon’s rank sum test. B. Percent PD-1 expression among CD3⁺CD8⁺ T cells specific for MCPyV (solid lines, left panel), CMV (dashed lines, right panel) or EBV (dotted lines, right panel) measured in serial blood draws from MCC patients (diamond, w447; circle, w678; X, w334; triangle, w672; square, w131) at indicated times following diagnosis. Sizes of the black diamonds or circles on the solid black line represent relative disease burden among MCC patients with MCPyV-specific T cells (smallest data points represent no detectable disease burden). Not all patients had T cells that were reactive to each tetramer, but all tetramer-positive T cells results are shown.

Figure 5. Culture with inhibitory receptor blocking agents augments MCPyV-specific T cell function

A. PBMC from case w678 were analyzed directly ex vivo for ELISPOT-based IFN-γ cytokine production by CD8 T cells exposed to EBV or MCPyV peptide in the presence of indicated blockers or IgG isotype control antibody. A representative experiment is shown and similar findings were obtained in a separate experiment. Net SFU (spot forming units)is the average SFU of indicated duplicate wells minus the average SFU in the negative control (media only) wells. Error bars represent mean +/− SEM. B. PBMC from case w678 were cultured for 7 days as described in Methods and were assessed for IFN-γ production in an ELISPOT assay. A representative experiment is shown and analogous findings were obtained in a separate experiment. Error bars represent mean +/− SEM. *p<0.01, Student’s t-test.

Figure 6. PD-L1 expression in MCC tumors correlates with CD8 lymphocyte infiltration

A. Among 35 MCC tumors, CD8α and PD-L1 mRNA expression were closely correlated. B. Correlation in an independent set of MCC tumors (compared with Figure 6A) between protein expression of PD-L1 and intratumoral CD8 infiltration in 13 MCC tumors. *p<0.05, Wilcoxon’s rank sum test. C. Immunohistochemical analysis of CD8 infiltration (left) and PD-L1 expression (right) in two representative MCC tumors as assessed on serial sections of the same tumors. Scale bar: 100μm.