Interleukin-2–inducible T-cell kinase inhibitors modify functional polarization of human peripheral T-cell lymphoma cells by Sami Mamand, Matthew Carr, Rebecca L. Allchin, Matthew J. Ahearne, and Simon D. Wagner BloodAdv Volume 3(5):705-710 March 12, 2019 © 2019 by The American Society of Hematology

Sami Mamand et al. Blood Adv 2019;3:705-710 © 2019 by The American Society of Hematology

ITKi perturb in vitro functional polarization of tonsil CD4+ T cells. 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+CXCR5hiPD-1hi 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 CXCR5hiPD-1hi 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. Sami Mamand et al. Blood Adv 2019;3:705-710 © 2019 by The American Society of Hematology

ITKi alter functional polarization of PTCL cells. 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+CXCR5hiPD-1hi) 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+CXCR5hiPD-1hi 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. Sami Mamand et al. Blood Adv 2019;3:705-710 © 2019 by The American Society of Hematology