Extended Data Fig. 9: CGRP-RAMP3 axis enhances antigen-specific Th1 and Tc1 response during LCMV Armstrong infection.

(a) At steady state, there is no difference on Th1 or Tc1 differentiation between Ramp3^−/− and WT mice (WT: n = 5; Ramp3^−/−: n = 3). (b) At steady state, Ramp3^−/− mice have similar spleen size as WT mice (WT: n = 5; Ramp3^−/−: n = 3). (c) Th1 response was restrained in Ramp3^−/− mice upon LCMV Armstrong infection without affecting cell activation. At day 8 post Armstrong infection, splenocytes were restimulated with PMA / Ionomycin for intracellular cytokine expression analyses within CD4⁺ T cells. Representative flow cytometry, left, quantitation, right (WT: n = 3; Ramp3^−/−: n = 4). (d) Tc1 response was restrained in CD8⁺ T cells of Ramp3^−/− mice upon LCMV Armstrong infection without affecting cell activation. At Day8 post Armstrong infection, splenocytes were restimulated with PMA / Ionomycin for intracellular cytokine expression analyses within total CD44⁺CD8⁺ T cells. Representative flow cytometry, left, quantitation, right (WT: n = 3; Ramp3^−/−: n = 4). (e) Antigen-specific Th1 response was restrained in Ramp3^−/− mice upon LCMV Armstrong infection. At day 8 post Armstrong infection, splenocytes were restimulated with PMA / Ionomycin for intracellular cytokine expression analyses within antigen-specific CD4⁺ T cells (LCMV GP66-77 tetramer⁺). Representative flow cytometry, left panel, quantitation, right (WT: n = 3; Ramp3^−/−: n = 4). (f) Tc1 response was restrained in CD8⁺ T cells of Ramp3^−/− mice upon LCMV Armstrong infection. At Day8 post Armstrong infection, splenocytes were restimulated with PMA / Ionomycin for intracellular cytokine expression analyses within antigen-specific CD8⁺ T cells (LCMV GP_33-41 tetramer⁺). Representative flow cytometry, left, quantitation, right (WT: n = 3; Ramp3^−/−: n = 4). (g) Antigen-specific Th1 response was reduced in Ramp3^−/− mice during LCMV Armstrong infection. Splenocytes were collected from spleen at Day 8 post Armstrong infection, followed by restimulation with LCMV GP_61-80 for another 4 h for intracellular cytokine staining. Representative flow cytometry, left panel, quantitation, right (WT: n = 3; Ramp3^−/−: n = 4). (h) Ramp3^−/− mice show higher weight of spleen (WT: n = 4; Ramp3^−/−: n = 5). (i) RAMP1 did not show function in regulating T cell response during LCMV Armstrong infection. At Day8 post Armstrong infection, splenocytes were restimulated with PMA / Ionomycin (left) or LCMV GP_61-80 / LCMV GP_33-41 (right) for intracellular cytokine expression analyses (WT: n = 5; Ramp1^−/−: n = 4). Two-tailed Student’s t test. Data are presented as mean +/− SD. n = X mice per group. Results shown are from one experiment, representative of at least three independent experiments.

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