Fig. 5. CGRP attenuates the anti-tumour immunity of RAMP1⁺ CD8⁺ T cells.

a–c, Splenocyte CD8⁺ T cells were FACS purified from Ramp1^WT (CD45.1⁺) or Ramp1^−/− (CD45.2⁺) mice, expanded and stimulated (CD3 and CD28 + IL-2) in vitro. Eight-week-old female Rag1^−/− mice were transplanted (i.v., 2.5 × 10⁶ cells) with activated Ramp1^−/− or Ramp1^WT CD8⁺ T cells or a 1:1 mix of Ramp1^−/− and Ramp1^WT CD8⁺ T cells. One week after transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5 × 10⁵ cells, i.d.). Ten days after B16F10 inoculation, we observed greater tumour growth (a) in Ramp1^WT transplanted mice. Intratumoral Ramp1^−/− (CD45.2⁺) and Ramp1^WT (CD45.1⁺) CD8⁺ T cells were FACS purified, immunophenotyped (b) and RNA sequenced (c). Ramp1^−/− CD8⁺ T cells showed a lower proportion of PD-1⁺LAG3⁺TIM3⁺ CD8⁺ T cells (b) as well as reduced transcript expression of exhaustion markers (c). d, In silico analysis of The Cancer Genome Atlas (TCGA) data⁴⁰ was used to correlate the survival rate of 459 patients with melanoma with the relative RAMP1 expression (primary biopsy bulk RNA sequencing). In comparison to patients with low RAMP1 expression, higher RAMP1 levels correlate with decreased patient survival. e, In silico analysis of single-cell RNA sequencing of human melanoma⁴¹ reveals that intratumoral RAMP1-expressing CD8⁺ T cells strongly overexpress several immune checkpoint receptors (PD-1 (also known as PDCD1) TIM3, LAG3, CTLA4) in comparison to Ramp1-negative CD8⁺ T cells. Data are shown as mean ± s.e.m. (a), slopegraph (b), as a heat map showing normalized gene expression (log₁₀(10³ × TPM) (c), as a Mantel–Cox regression (d) or as a violin plot (e). n as follows: a–c: n = 5 per group; d: high (n = 45), low (n = 68); e: RAMP1⁻ CD8 (n = 1,732), RAMP1⁺ CD8 (n = 25). Experiments were independently repeated two (a,b) times with similar results. The sequencing experiment was not repeated (c). P values were determined by two-way ANOVA with post-hoc Bonferroni (a), two-sided unpaired Student’s t-test (b) or Mantel–Cox regression (d).

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