Extended Data Fig. 5: Neuropeptide CGRP and ADM modulate T cell differentiation through Ramp3.

(a, b) CGRP and ADM promote Th1 / CD8⁺ T cell differentiation but antagonize Th2 differentiation. (a) Naïve CD8⁺ T cells were in vitro differentiated under Tc0 condition (anti-CD3 and anti-CD28) with / without CGRP Representative flow cytometry left panel, quantitation, right panel (n = 4). (b) ADM treatment show similar effects as CGRP in both CD4⁺ and CD8⁺ T cells (n = 4-5). (c) CGRP and ADM also inhibit IL-5 expression under Th2 condition after 4 days in vitro culture. Representative flow cytometry, left, quantitation, right (n = 8). (d) Naïve CD4⁺ T cells were cultured with Th1 / Th2 dichotomous condition for 3 days before analysing cytokine expression by flow cytometry, (representative flow cytometry, left panel, quantitation, right panel) (n = 5). (e) Cytokine secretion was analysed with LegendPlex after 3 days Th1 / Th2 dichotomous culture (n = 5). (f) CGRP induces the expression of Calca and Ramp family in CD4⁺ T cells. Isolated naïve CD4⁺ T cells were treated with the neuropeptide for 4 h and collected for qPCR analysis (n = 6). (g) Isoform specific expression analyses of Calca detected with qPCR in T cells (n = 4). (h) Calcitonin is detected in T cell lysate upon CGRP treatment for 72 h (n = 4). (i) The promotion of Th1 differentiation by CGRP is abrogated by BIBN4096BS (n = 8). (j). The promotion of Th1 differentiation by CGRP is abrogated in Calcrl knockout T cells (n = 5). (k) Gene expressions of Calcrl were detected by qPCR (n = 3). Two-tailed Student’s t test. Data are presented as mean +/− SD. n = X mice (h) / samples pooled by ≥ 3 mice (other panels) per group. Results shown are from one experiment, representative of at least three independent experiments.

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