PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors
Alena Gros, Paul F. Robbins, Xin Yao, Yong F. Li, Simon Turcotte, Eric Tran, John R. Wunderlich, Arnold Mixon, Shawn Farid, Mark E. Dudley, Ken-ichi Hanada, Jorge R. Almeida, Sam Darko, Daniel C. Douek, James C. Yang, Steven A. Rosenberg
Alena Gros, Paul F. Robbins, Xin Yao, Yong F. Li, Simon Turcotte, Eric Tran, John R. Wunderlich, Arnold Mixon, Shawn Farid, Mark E. Dudley, Ken-ichi Hanada, Jorge R. Almeida, Sam Darko, Daniel C. Douek, James C. Yang, Steven A. Rosenberg
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Research Article Immunology

PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors

Abstract

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8+ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8+ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8+ lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8+ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8+ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8+PD-1+ compared with CD8+PD-1– TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8+ and the CD8+PD-1+ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8+ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.

Authors

Alena Gros, Paul F. Robbins, Xin Yao, Yong F. Li, Simon Turcotte, Eric Tran, John R. Wunderlich, Arnold Mixon, Shawn Farid, Mark E. Dudley, Ken-ichi Hanada, Jorge R. Almeida, Sam Darko, Daniel C. Douek, James C. Yang, Steven A. Rosenberg

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Figure 5

Expression of PD-1, rather than 4-1BB, more comprehensively identifies the repertoire of tumor-reactive cells in human tumors.

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Expression of PD-1, rather than 4-1BB, more comprehensively identifies t...
(A) Representative dot plots showing PD-1 by 4-1BB expression of CD8+ TILs infiltrating FrTu1913 and FrTu3713. (B) Expression of PD-1 by 4-1BB on CD8+ TILs infiltrating melanoma tumors. The frequency of each combination of markers is shown as mean ± SEM. ***P < 0.001, Wilcoxon signed-rank test (n = 24). (C) Response of FrTu1913-derived clones to TC1913. CD8+ TILs were sorted from FrTu1913 into PD-1–/4-1BB–, PD-1+/4-1BB–, and PD-1+/4-1BB+, and clones were established. Clones derived from PD-1–/4-1BB– (n = 96), PD-1+/4-1BB– (n = 57), and PD-1+/4-1BB+ (n = 65) were cocultured with TC1913; 4-1BB upregulation upon coculture is plotted for each clone. The line represents the median. *P ≤ 0.05, ****P ≤ 0.0001, Dunn test for multiple comparisons. Pie charts depict the percentage of tumor-reactive and non–tumor-reactive clones in each population. Greater than 1% CD8+4-1BB+ and greater than twice the background percentage 4-1BB compared with no stimulation control was considered positive. (D) Response of bulk-expanded FrTu3713-derived TILs to TC3713. CD8+PD-1–/4-1BB–, PD-1+/4-1BB–, and PD-1+/4-1BB+ TILs were sorted from FrTu3713 and expanded. Cells were either left unstimulated, cocultured with TC3713, or stimulated with plate-bound anti-CD3. 4-1BB upregulation was measured to assess tumor recognition. Representative dot plots of CD3+CD8+-gated cells are shown. (E) Lysis of TC3713 by TILs derived from FrTu3713.