Extended Data Fig. 6: The role of CD74 on antigen-presenting machinery of mast cells.

(a, b) Negative (a) and positive controls (b) of the CD74 flow cytometry staining. (c) Flow cytometric gating scheme showing CD74^hi, CD74^int, and CD74^neg mast cells. (d) Quantification of surface CD74 expression on CD74^hi, CD74^int, and CD74^neg mast cells. One representative quantification of mean fluorescence intensity (MFI) from six independent experiments was shown. (e) Composite data of activation marker CD69 expression by OT-II CD4⁺ T cells primed by CD74^hi, CD74^int, and CD74^neg mast cells pulsed with OVA peptide or vehicle (n = 6). (f) Activation marker CD69 expression by OT-I CD8⁺ T cells primed by CD74^hi, CD74^int, and CD74^neg mast cells pulsed with OVA protein or vehicle (n = 6). (g–i) OT-II CD4⁺ (g, n = 6), OT-I CD8⁺ T cell activation (h, n = 6), and IL-2 production measured in the culture supernatant of T cells (i, n = 6) after stimulation with CD74⁺ or CD74⁻ mast cells transferred with Cd74-siRNA (Si-Cd74) or negative-control siRNA (Si-NC) pulsed with relative OVA. (j) Growth curves and tumor weights at endpoint in orthotopic AT3 tumors treated as indicated (mice receiving the same treatment in siRNA replicates were merged, n = 6). Si-NC apMCs were transfected with negative-control siRNA. Si-Cd74 apMCs were transfected with Cd74-siRNA, and two Cd74-siRNAs were used. (k) Quantification of CD4⁺ and CD8⁺ T cell densities by IHC staining from FFPE tumor sections in (j, n = 6). (l) Representative images of CD4 and CD8 staining in tumors from different treatment groups in (k). (m) Flow cytometry of infiltration and functional states of CD8⁺ T cells in tumors from each treatment group in (j, n = 6). (n) Representative flow cytometry plots of CD8⁺ T cell cytotoxic molecules in tumors from different treatment groups in (m). Data are mean ± s.e.m. (e, f, g–m). Two-sided unpaired Student’s t-test (e–i, j for tumor weight, k, m), two-way ANOVA (j for tumor volume).

Source data