ITKi perturb in vitro functional polarization of tonsil CD4⁺T cells. (A) Flow cytometry dot plot showing PD-1 and CXCR5 expression of untreated tonsillar CD4⁺ T cells. (B) Flow cytometry dot plots showing PD-1 and CXCR5 expression of tonsillar CD4⁺ T cells treated with anti-CD3/anti-CD28 and IL-12. There was either no inhibitor included in the culture (dimethyl sulfoxide [DMSO]) or ibrutinib or ONO7790500. The numbers indicate the percentage of CD4⁺ T cells within the gate. (C) Percentage of CD4⁺CXCR5^hiPD-1^hi cells relative to cells stimulated with anti-CD3/anti-CD28 and IL-12 (red column). The percentage of cells without stimulation (Unstim; pink) or in stimulated cells treated with ITKi (shades of blue as shown in the legend) is indicated. Mean ± standard error of the mean (SEM). ITKi repress the fraction of CXCR5^hiPD-1^hi cells: ibrutinib (paired Student t test, P = .0048), PF-6465469 (P = .0068), BMS509744 (P = .026), and ONO7790500 (P = .037). (D) Flow cytometry dot plots showing IL-17A and FoxP3 expression following polarizing culture in the absence (DMSO) or presence of either ibrutinib or ONO7790500. The numbers indicate the percentage of CD4⁺ T cells within the quadrant. (E) Column charts show the percentage of CD4⁺FoxP3⁺ or CD4⁺IL-17A⁺ (mean ± SEM). No stimulation (pink), stimulation (red), and stimulation with either ibrutinib (light blue) or ONO7790500 (dark blue) are indicated. Th17-like cells were significantly reduced by ITKi (paired Student t test, ibrutinib, P = .02; ONO7790500, P = .03), whereas Treg-like cells were increased (ibrutinib, P = .04; ONO7790500, P = .03). (F) Flow cytometry dot plots showing IL-4 expression following polarizing culture in the absence (DMSO) or presence of either ibrutinib or ONO7790500. The numbers indicate the percentage of total CD4⁺ T cells within the quadrant. (G) Column chart shows the percentage of CD4⁺IL-4⁺ (mean ± SEM). No stimulation (pink), stimulation (red), and stimulation with either ibrutinib (light blue) or ONO7790500 (dark blue) are indicated. CD4⁺IL-4⁺ cells were significantly reduced by ITKi (ibrutinib, P = .01; ONO7790500, P = .01). For the flow cytometry experiments (A-G), the results shown are representative of 3 separate experiments. (H-I) Western blots showing total ITK and phosphorylated ITK in cells stimulated by anti-CD3/anti-CD28 in the absence or presence of 4 ITKi (PF-6465469, ibrutinib, ONO7790500, BMS509744) as indicated. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, is a loading control. Patient 1 (H) and patient 2 (I). (J-K) Immunofluorescence microscopy of lymph node sections taken from patients 1 and 2. Magnification is indicated at the top right. Images at ×80 are taken from the area and are indicated by the white rectangle in the ×20 image. (J) Sections were stained with anti-CD4 and anti-BCL6. (K) Sections were stained with anti-CD4, anti-FoxP3, and anti-RORγt. *P < .05, **P < .01.