CX3CR1–CD8+ T cells are critical in antitumor efficacy but functionally suppressed in the tumor microenvironment
Takayoshi Yamauchi, Toshifumi Hoki, Takaaki Oba, Hidehito Saito, Kristopher Attwood, Michael S. Sabel, Alfred E. Chang, Kunle Odunsi, Fumito Ito
Takayoshi Yamauchi, Toshifumi Hoki, Takaaki Oba, Hidehito Saito, Kristopher Attwood, Michael S. Sabel, Alfred E. Chang, Kunle Odunsi, Fumito Ito
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Research Article Immunology

CX3CR1–CD8+ T cells are critical in antitumor efficacy but functionally suppressed in the tumor microenvironment

Abstract

Although blockade of the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) immune checkpoint has revolutionized cancer treatment, how it works on tumor-infiltrating CD8+ T cells recognizing the same antigen at various differentiation stages remains elusive. Here, we found that the chemokine receptor CX3CR1 identified 3 distinct differentiation states of intratumor CD8+ T cell subsets. Adoptively transferred antigen-specific CX3CR1–CD8+ T cells generated phenotypically and functionally distinct CX3CR1int and CX3CR1hi subsets in the periphery. Notably, expression of coinhibitory receptors and T cell factor 1 (Tcf1) inversely correlated with the degree of T cell differentiation defined by CX3CR1. Despite lower expression of coinhibitory receptors and potent cytolytic activity, in vivo depletion of the CX3CR1hi subset did not alter the antitumor efficacy of adoptively transferred CD8+ T cells. Furthermore, differentiated CX3CR1int and CX3CR1hi subsets were impaired in their ability to undergo proliferation upon restimulation and had no impact on established tumors upon second adoptive transfer compared with the CX3CR1– subset that remained effective. Accordingly, anti–PD-L1 therapy preferentially rescued proliferation and cytokine production of the CX3CR1– subset and enhanced antitumor efficacy of adoptively transferred CD8+ T cells. These findings provide a better understanding of the phenotypic and functional heterogeneity of tumor-infiltrating CD8+ T cells and can be exploited to develop more effective immunotherapy.

Authors

Takayoshi Yamauchi, Toshifumi Hoki, Takaaki Oba, Hidehito Saito, Kristopher Attwood, Michael S. Sabel, Alfred E. Chang, Kunle Odunsi, Fumito Ito

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Figure 6

In vitro proliferation and in vivo antitumor efficacy of antigen-specific CX3CR1+CD8+ T cells after ACT.

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In vitro proliferation and in vivo antitumor efficacy of antigen-specifi...
(A) Schematic illustration showing evaluation of in vitro proliferation of 3 subsets of Pmel-1 T cells determined by CX3CR1 expression. FACS-sorted cells were stimulated with hgp100 peptide with mitomycin-C–treated B6 splenocytes for 4 days. (B) Tcf7 mRNA expression in each subset quantitated by real-time RT-PCR. Gapdh was used as an internal control. Error bars represent the standard error of the value obtained in the experiments performed in triplicate. (C) Left shows representative microscopic pictures of each subset at day 4. Scale bar: 250 μm. Right shows total cell numbers after 4 days of stimulation of each subset. Error bars represent the SEM of the value obtained in the experiments performed in triplicate. (D) Schematic illustration showing evaluation of in vivo antitumor efficacy of CX3CR1– and CX3CR1+ subsets using adoptive transfer of each subset sorted from B16 tumor–bearing mice 7 days after adoptive transfer of Pmel-1 T cells. (E) Frequency of adoptively transferred Pmel-1 T cells in blood of B16 tumor–bearing mice infused with either CX3CR1– or CX3CR1+ as indicated. (F) Representative dot plot showing CX3CR1 expression of Pmel-1 T cells in blood of B16 tumor–bearing mice. Expression of CD27 and CX3CR1 on infused CX3CR1–CD8+ or CX3CR1+CD8+ T cells are shown. The right panel shows the percentage of CX3CR1 expression in Pmel-1 T cells in different treatments as indicated. (G) Tumor growth curves and survival curves in C57BL/6 mice bearing B16 melanomas established for 12 days in different treatment groups. (n ≥ 6 mice per group.) Mean (±SEM). **P < 0.01, and ***P < 0.005 by 1-way ANOVA with Tukey’s test (B and C), unpaired 2-tailed t test (D–F), or log-rank test (G).