ITKi alter functional polarization of PTCL cells. (A) Flow cytometry dot plots showing PD-1 and CXCR5 expression of unstimulated cells and cells following 4 days’ stimulation with anti-CD3/anti-CD28/IL-12 in the absence (DMSO) or presence of ITKi (ibrutinib or ONO7790500) for patients 1 and 2. Numbers indicate the percentage of CD4⁺ T cells within the gate. (B) Column plots for patients 1 and 2 showing Tfh-like (CD4⁺CXCR5^hiPD-1^hi) cells as a percentage of total CD4⁺ cells relative to the percentage obtained with anti-CD3/anti-CD28 and IL-12 in the absence of drug (DMSO) (red columns). Blue columns represent drug treatment with ibrutinib or ONO7790500 as indicated. Drug treatment caused significant reduction in percentage of CD4⁺CXCR5^hiPD-1^hi cells. Ibrutinib (paired Student t test, patient 1 P = .017; patient 2, P = .016) and ONO7790500 (patient 1, P = .010; patient 2, P = .027). (C) Flow cytometry dot plots showing CD4 and IL-17A expression of unstimulated cells and cells following 2 days’ stimulation under Th17 polarizing conditions in the absence (DMSO) or presence of ITKi (ibrutinib or ONO7790500) for patients 1 and 2. Numbers indicate the percentage of CD4⁺ T cells within the gate. (D) Column plots for patients 1 and 2 showing CD4⁺IL-17A⁺ cells as a percentage of total CD4⁺ cells. Blue columns represent drug treatment with ibrutinib or ONO7790500 as indicated. CD4⁺IL-17A⁺ cells as a percentage of total CD4⁺ cells, without stimulation (Unstim), is represented by the pink column. Drug treatment caused significant reduction in CD4⁺IL-17A⁺ cells. Ibrutinib (paired Student t test, patient 1, P = .017; patient 2, P = .016) and ONO7790500 (patient 1, P = .013; patient 2, P = .013). (E) Flow cytometry dot plots showing FoxP3 and CD25 expression of unstimulated cells and cells following 2 days’ stimulation under Treg polarizing culture conditions in the absence (DMSO) or presence of ITKi (ibrutinib or ONO7790500) for patients 1 and 2. Numbers indicate the percentage of CD4⁺ T cells within the gate. (F) Column plots for patients 1 and 2 showing CD4⁺CD25⁺FoxP3⁺ Treg-like cells as a percentage of total CD4⁺ cells. Blue columns represent drug treatment with ibrutinib or ONO7790500 as indicated. Unstimulated cells are represented by the pink column. Drug treatment caused a significant increase in percentage of Treg-like cells. Ibrutinib (paired Student t test, patient 1, P = .045; patient 2, P = .043) and ONO7790500 (patient 1, P = .021; patient 2, P = .034). *P < .05.