Figure 2 from Generation, Transcriptomic States, and Clinical Relevance of CX3CR1⁺ CD8 T Cells in Melanoma

The differentiation of antigen-specific CX3CR1⁻ CD8⁺ T cells into CX3CR1⁺ CD8⁺ T cells in vivo. A, Phenotypic analysis of Pmel-1 CD8⁺ T cells after ex vivo-activation with hgp100 peptide, IL-7, and IL-15. B, Experimental protocol. XRT, external beam radiation therapy. C, Representative contour plots of Pmel-1 CD8⁺ T cell population in the spleen with or without vaccination. The right panels show frequency of the CX3CR1⁺ subset in CD90.1⁺CD8⁺ T cells (n = 5). (D) Representative contour plots of Pmel-1 CD8⁺ T cell population in the spleen and tumors 10 days after vaccination. FMT, fluorescence minus two (CD27 and CX3CR1). E, Representative histogram showing PD-1 and TIGIT expressions in intratumoral CX3CR1⁻and CX3CR1⁺ CD8⁺ T cells 19 days after vaccination. Gating strategy is shown in Supplementary Fig. S3. F, Representative histogram showing granzyme A (GZMA) and granzyme B (GZMB) expressions in CX3CR1⁻and CX3CR1⁺ CD8⁺ T cells in the spleen and tumors 25 days after immune vaccination. G, Representative histogram showing TNFα or IFNγ expression in intratumoral CX3CR1⁻and CX3CR1⁺ CD8⁺ T cells 7 days after vaccination. E–G, The right panels show frequency of each T-cell subset based on CX3CR1 expression (n = 4–5). H, Representative histogram showing CXCR3 expression in intratumoral CX3CR1⁻and CX3CR1⁺ CD8⁺ T cells 10 days after vaccination. The right panels show kinetics and frequency of the CXCR3⁺subset based on CX3CR1 expression. Mean (±SEM) (n = 4). A and E–H, Isotype-matched controls are shown in gray. C and E–H, *, P < 0.05; **, P < 0.01; ***, P < 0.001, and ****, P < 0.0001 by two-tailed unpaired (C) or paired (E–H) t-test. N.S., not significant.