Extended Data Fig. 7: CGRP-Ramp3-cAMP loop enhances Th1 differentiation through epigenomic remodelling and activating STAT1.

(a) Th1 pioneer program was induced by CGRP and ADM. Isolated naïve CD4⁺ T cells were treated with CGRP or ADM for 4 h and collected for qPCR analysis (n = 2-6). (b) Th1 pioneer program was induced by cAMP in a dose dependent manner. Isolated naïve CD4⁺ T cells were treated with dbcAMP for 4 h and collected for qPCR analysis (n = 4-5). (c) Categories of all cis-element open chromatin regions detected in ATAC-seq data of Th1 and Th2 cells. (d) Shared and unique Th1/Th2 differentially accessible chromatin regions were identified in the CGRP treated and control group. (e) Categories of differential cis-element open chromatin regions between Th1 and Th2 cells with or without CGRP treatment. (f) Overlapping of the genes proximal to differentially accessible regions and genes with differential expression between Th1 and Th2 cells. (g) CREB binding signals from CUT&Tag assay on in vitro differentiated Th1 cells. (h) Immunostaining of pSTAT1 in differentiated Th1 cells upon CGRP treatment for 8 h. Scale bar: 10 μm. Signal intensity of pSTAT1 is quantified (n = 36-159 sections from 2-3 mice). (i) The enhancement of Th1 differentiation by CGRP/ADMs is abrogated in Atf3 knockout T cells (n = 3). (j) Luciferase activity in 293 T cells transfected with luciferase reporters for the indicated cis-regulatory elements of Stat1 and plasmids encoding ATF3 (n = 3). Two-tailed Student’s t test. Data are presented as mean +/− SD (a, b and h-j). n = X mice (i) / samples (a, b and j) pooled by ≥ 3 mice per group. (k) Model graph of CGRP-RAMP3 axis in modulating T cell differentiation. Two-tailed Student’s t test. Data are presented as mean +/− SD. n = X samples pooled by ≥ 3 mice. Results shown are from one experiment, representative of at least three independent experiments.

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