T cell receptor sequencing of activated CD8 T cells in the blood identifies tumor-infiltrating clones that expand after PD-1 therapy and radiation in a melanoma patient

Abstract

PD-1-targeted therapy has dramatically changed advanced cancer treatment. However, many questions remain, including specificity of T cells activated by PD-1 therapy and how peripheral blood analysis correlates to effects at tumor sites. In this study, we utilized TCR sequencing to dissect the composition of peripheral blood CD8 T cells activated upon therapy, comparing it with tumor-infiltrating lymphocytes. We report on a nonagenarian melanoma patient who showed a prominent increase in peripheral blood Ki-67 + CD8 T cells following brain stereotactic radiation and anti-PD-1 immunotherapy. Proliferating CD8 T cells exhibited an effector-like phenotype with expression of CD38, HLA-DR and Granzyme B, as well as expression of the positive costimulatory molecules CD28 and CD27. TCR sequencing of peripheral blood CD8 T cells revealed a highly oligoclonal repertoire at baseline with one clonotype accounting for 30%. However, the majority of dominant clones-including a previously identified cytomegalovirus-reactive clone-did not expand following treatment. In contrast, expanding clones were present at low frequencies in the peripheral blood but were enriched in a previously resected liver metastasis. The patient has so far remained recurrence-free for 36 months, and several CD8 T cell clones that expanded after treatment were maintained at elevated levels for at least 8 months. Our data show that even in a nonagenarian individual with oligoclonal expansion of CD8 T cells, we can identify activation of tumor-infiltrating CD8 T cell clones in peripheral blood following anti-PD-1-based immunotherapies.

Keywords: CD8 T cells; Immunotherapy; Melanoma; PD-1; T cell repertoire.

Fig. 1

CD8 T cell activation in peripheral blood. a Scheme shows treatment and blood sampling schedule. b Proliferation of CD4 and CD8 T cells. c Ki-67 and Bcl-2 expression on CD8 T cells prior to treatment and post cycle 1. Numbers represent frequency among total CD8 T cells. d Phenotypic analysis of proliferating CD8 T cells on day 21 post treatment initiation. Numbers represent frequency among Ki-67 + CD8 T cells. Contour plots show total CD8 T cells (gray contour plot) and Ki-67 + CD8 T cells (red dots). C: cycle, Pembro: Pembrolizumab, d: days

Fig. 2

Analysis of tumor-infiltrating T cells in liver metastasis. a Experimental scheme. b Immunohistochemical (IHC) analysis of tumor-infiltrating CD4 and CD8 T cells in a highly infiltrated tumor site (hotspot). c Frequency of unique T cell clones. Dotted line indicates cut-off of 5 TCRβ copies

Fig. 3

TCR repertoire analysis in peripheral blood CD8 T cells. a Scheme shows treatment and sampling schedule. b Clonal composition and clonality score of peripheral blood CD8 T cells at the indicated time points. c Frequency of the ten most prevalent CD8 T cell clones at baseline. Asterisk indicates TCRβ previously shown to recognize the CMV-derived epitope pp65_265−275. C: cycle, Pembro: Pembrolizumab, d: days

Fig. 4

Expanding tumor-infiltrating clones are enriched in tumor and among activated peripheral blood CD8 T cells. a Frequency of tumor-infiltrating CD8 T cell clones in peripheral blood. Expanding clones (red) increased in frequency of at least 1.5-fold upon treatment initiation, whereas non-expanding clones (blue) showed less than 1.5-fold increase in frequency. Dotted line indicates limit of detection. b Ratio of expanding and non-expanding CD8 T cell clones in blood to tumor. p value was calculated using Mann–Whitney test. c Relationship of the ratio of clonal frequency in blood to tumor prior to treatment initiation and peripheral blood frequency of 131 tumor-infiltrating CD8 T cell clones. Expanding T cell clones are shown as red dots, non-expanding clones as blue dots, and previously identified CMV-reactive clones are depicted as orange open circles. Dotted line indicates a suggested blood/tumor ratio cut-off of 3 that would separate mainly non-expanding clones enriched in the peripheral blood. d Gates used for sorting of activated (HLA-DR/CD38) + and non-activated (HLA-DR/CD38)- CD8 T cells and subsequent separation based on PD-1 expression on day 21 post treatment initiation (post cycle 1). e Cumulative frequency of expanding tumor-infiltrating clones among the indicated CD8 T cell populations in the peripheral blood on day 21 post treatment initiation. f Frequency of expanding tumor-infiltrating clones in PD-1^hi and PD-1^lo-activated CD8 T cell subsets