STAT6 inhibition impairs cytokine production by CD3⁺ MF and SS lymphocytes. SS peripheral blood CD4⁺ T cells were activated in vitro with CD3/CD28, treated with/without STAT6 inhibitor (AS1517499) for 5 days, and restimulated with PMA and ionomycin. The proportion of IL-4⁺, IL-5⁺, and IL-13⁺ cells was determined by intracellular cytokine staining. (A-B) Representative examples (A) and cytokine frequency (B) shown from multiple SS patients (n = 5, P = .002: Student t test). Mean percentage of positive cells ± standard deviation is shown. (C) Cellular lysates were obtained at day +5 from blood SS CD4⁺ T cells treated with/without AS1517499 (n = 5) or transfected with STAT6 or target control (crtl) siRNAs (n = 3). Immunoblots were performed with STAT6, phospho (p)STAT6, and GATA-3 antibodies. Actin was used as loading ctrl. (D-E) A representative example is shown. ScRNAseq from fresh MF skin tumors (n = 3; supplemental Figure 4): dot-plots showing the proportion of cells and the scaled average GATA-3 gene expression between MF and NS samples (D)  and by different cell types (E). (F) Violin plots show expression of GATA-3 by TOX⁺ and TOX⁻ lymphocytes from MF tumors. Ex vivo skin explants from the excised MF skin tumors and NS were cultured for 5 days with/without STAT6 inhibition. (G) Hematoxylin and eosin staining, (×200) from representative examples are shown. (H-I) Double-color immunofluorescence staining for Th2 cytokines and CD3 or Th2 cytokines (H) and TOX (I), as indicated (×1000). 4′,6-diamidino-2-phenylindole stains nuclei. Representative NS and MF skin explants out of 5 tumors and 5 controls tested are shown.