Frequency, transcriptomic phenotype, and clinical relevance of CX3CR1+ CD8+ T cells in human melanomas. A, Representative histogram showing CX3CR1 expression of CD8+ T cells in human melanomas (blue). Isotype-matched controls are shown in red. Gating strategy is shown in Supplementary Fig. S2. The right shows frequency of CX3CR1+ subset in CD8+ T cells (n = 7). B–F, Single-cell profiling of melanoma-infiltrating T and NK cells from patients treated with ICI therapy in the Sade-Feldman et al. data. Final cell-type annotations of total cells and subsequent filtering of T and NK- cells are shown in Supplementary Fig. S4A and S4B. B, UMAP plots of melanoma-infiltrating T and NK cells from responders and nonresponders before and after ICI therapy. C, Expression patterns of indicated genes in T- and NK-cell clusters in melanoma. Expression levels are color-coded: gray, not expressed; purple, expressed. D, Heatmap displaying the top 10 significantly enriched genes found in each cluster. E, Violin plots show expression of PDCD1, TIGIT, and HAVCR2 in CX3CR1+ and CX3CR1− populations. Differential expression is determined by Wilcoxon rank sum test. F, Proportions of cell clusters observed in individual melanomas from responders (R) and nonresponders (NR) at pretreatment (Pre) and post-treatment (Post).
ARTICLE ABSTRACT
Recent progress in single-cell profiling technologies has revealed significant phenotypic and transcriptional heterogeneity in tumor-infiltrating CD8+ T cells. However, the transition between the different states of intratumoral antigen-specific CD8+ T cells remains elusive. Here, we sought to examine the generation, transcriptomic states, and the clinical relevance of melanoma-infiltrating CD8+ T cells expressing a chemokine receptor and T-cell differentiation marker, CX3C chemokine receptor 1 (CX3CR1). Analysis of single-cell datasets revealed distinct human melanoma-infiltrating CD8+ T-cell clusters expressing genes associated with effector T-cell function but with distinguishing expression of CX3CR1 or PDCD1. No obvious impact of CX3CR1 expression in melanoma on the response to immune checkpoint inhibitor therapy was observed while increased pretreatment and on-treatment frequency of a CD8+ T-cell cluster expressing high levels of exhaustion markers was associated with poor response to the treatment. Adoptively transferred antigen-specific CX3CR1− CD8+ T cells differentiated into the CX3CR1+ subset in mice treated with FTY720, which inhibits lymphocyte egress from secondary lymphoid tissues, suggesting the intratumoral generation of CX3CR1+ CD8+ T cells rather than their trafficking from secondary lymphoid organs. Furthermore, analysis of adoptively transferred antigen-specific CD8+ T cells, in which the Cx3cr1 gene was replaced with a marker gene confirmed that CX3CR1+ CD8+ T cells could directly differentiate from the intratumoral CX3CR1− subset. These findings highlight that tumor antigen–specific CX3CR1− CD8+ T cells can fully differentiate outside the secondary lymphoid organs and generate CX3CR1+ CD8+ T cells in the tumor microenvironment, which are distinct from CD8+ T cells that express markers of exhaustion.
Intratumoral T cells are composed of heterogeneous subpopulations with various phenotypic and transcriptional states. This study illustrates the intratumoral generation of antigen-specific CX3CR1+ CD8+ T cells that exhibit distinct transcriptomic signatures and clinical relevance from CD8+ T cells expressing markers of exhaustion.
