Extended Data Fig. 6: Neuropeptide CGRP and ADM enhance Th1 differentiation through cAMP signalling.

(a-b) CGRP and ADM induce cAMP signalling in T cells. (a) Differentially expressed genes between CGRP and Vehicle group (after 4 h treatment) are presented in the heatmap. (b) Gene ontology enrichment analysis was performed with the upregulated genes in CGRP treatment group. (c) CGRP and ADM induce intracellular cAMP accumulation in CD4⁺ T cells. Naïve T cells were treated with CGRP or ADM for 30 min and intracellular cAMP was measured using ELISA (n = 3). (d-e) cAMP is involved in the CGRP-Ramp3 positive feedback loop. The expression of Ramp3 (d) and Calca (e) are significantly induced upon dbcAMP treatment for 4 h (n = 5). (f) Well differentiated T cells are less responsive to the CGRP / ADM treatment. Fold change of Calca, Ramp3 and Crem expression relative to control (n = 4). (g) dbcAMP treatment recapitulates CGRP and ADM effects in promoting IFNγ expression in CD8⁺ T cells. Cells were collected for intracellular cytokine staining after 3 days in vitro culture. Representative flow cytometry left panel, quantitation, right panel (n = 5). (h) Enhancement of Th1 differentiation by ADM is abrogated with CREB inhibitor 666-15 (50 μM). Cells were collected for intracellular cytokine staining after 3 days in vitro culture. Representative flow cytometry left panel, quantitation, right panel (n = 4-5). (i-j) Enhancement of Th1 differentiation by CGRP (i) and ADM (j) is blocked with adenylyl cyclase inhibitor SQ22536 (20 μM). Cells were collected for intracellular cytokine staining after 3 days in vitro culture (n = 7-8). Two-tailed Student’s t test. Data are presented as mean +/− SD (c-j). n = X samples pooled by ≥ 3 mice. Results shown are from one experiment, representative of at least three independent experiments.

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