Transcriptional and functional analyses of neoantigen-specific CD4 T cells during a profound response to anti-PD-L1 in metastatic Merkel cell carcinoma

Abstract

BackgroundMerkel cell carcinoma (MCC) often responds to PD-1 pathway blockade, regardless of tumor-viral status (~80% of cases driven by the Merkel cell polyomavirus (MCPyV)). Prior studies have characterized tumor-specific T cell responses to MCPyV, which have typically been CD8, but little is known about the T cell response to UV-induced neoantigens.MethodsA patient in her mid-50s with virus-negative (VN) MCC developed large liver metastases after a brief initial response to chemotherapy. She received anti-PD-L1 (avelumab) and had a partial response within 4 weeks. Whole exome sequencing (WES) was performed to determine potential neoantigen peptides. Characterization of peripheral blood neoantigen T cell responses was evaluated via interferon-gamma (IFNγ) ELISpot, flow cytometry and single-cell RNA sequencing. Tumor-resident T cells were characterized by multiplexed immunohistochemistry.ResultsWES identified 1027 tumor-specific somatic mutations, similar to the published average of 1121 for VN-MCCs. Peptide prediction with a binding cut-off of ≤100 nM resulted in 77 peptides that were synthesized for T cell assays. Although peptides were predicted based on class I HLAs, we identified circulating CD4 T cells targeting 5 of 77 neoantigens. In contrast, no neoantigen-specific CD8 T cell responses were detected. Neoantigen-specific CD4 T cells were undetectable in blood before anti-PD-L1 therapy but became readily detectible shortly after starting therapy. T cells produced robust IFNγ when stimulated by neoantigen (mutant) peptides but not by the normal (wild-type) peptides. Single cell RNAseq showed neoantigen-reactive T cells expressed the Th1-associated transcription factor (T-bet) and associated cytokines. These CD4 T cells did not significantly exhibit cytotoxicity or non-Th1 markers. Within the pretreatment tumor, resident CD4 T cells were also Th1-skewed and expressed T-bet.ConclusionsWe identified and characterized tumor-specific Th1-skewed CD4 T cells targeting multiple neoantigens in a patient who experienced a profound and durable partial response to anti-PD-L1 therapy. To our knowledge, this is the first report of neoantigen-specific T cell responses in MCC. Although CD4 and CD8 T cells recognizing viral tumor antigens are often detectible in virus-positive MCC, only CD4 T cells recognizing neoantigens were detected in this patient. These findings suggest that CD4 T cells can play an important role in the response to anti-PD-(L)1 therapy.

LAMP1 targeting of the large T antigen of Merkel cell polyomavirus results in potent CD4 T cell responses and tumor inhibition

“…However, significant numbers of antigen-specific CD8 T cells were not observed after vaccination with ITI-3000 ( Figure 2D ). This agrees with previous studies using human PBMC and MCC tumor-infiltrating cells that demonstrate strong CD4 T cell-mediated immune responses to the LT antigen ( 16 , 24 , 42 , 43 ).…”

Circulating cancer-specific CD8 T cell frequency is associated with response to PD-1 blockade in Merkel cell carcinoma

“…Multiplex immunohistochemistry was performed with a panel of antibodies including CD8 (clone 144B/Dako/fluor 520 opal/concentration 0.2 μg/mL), CD3 (Sp7/Thermo/fluor opal 650), HLA-DR (EP96/BSB/0.125 μg/mL/fluor opal 690), CD56 (123.C3/BSB 1 μg/mL/fluor 540), and PD-L1 (E1L3N 0.5 μg/mL) using a modified Akoya Opal Multiplex IHC assay. 71 We also attempted PD-1 staining with antibody clone D4W2J. However, non-specific staining was observed, and PD-1 was thus excluded from analysis.…”

Precision nanoscale patterning of TLR ligands for improved cancer immunotherapy

“… 4 , 5 For cancer vaccines, a Th1-skewed response generating neoantigen-specific CD8 cytotoxic T cells is considered desirable. 6 …”

“… 1 Most approved adjuvants, used in pathogen vaccines, induce a Th2 response deemed ill-suited for cancer vaccines. 4 , 6 Ligands for pattern-recognition receptors such as Toll-like receptors (TLRs) on APCs have been shown to induce Th1-polarized immune responses, though this appears to additionally depend on their spatial presentation context. 2 , 7 For example, studies on poly(lactic-co-glycolic acid) nanoparticles showed that a higher density of CpGs, a ligand for TLR9, on the nanoparticle induced Th1-polarization, while a lower density favored Th2.…”