CD4⁺ malignant T cells contain the leukemic stem cell population in murine ITK-SYK⁺ PTCLs and drive an inflammatory phenotype including granulocyte expansion. (A) Schematic representation of ITK-SYK. blue, kinase domain. (B) Representative images of CD3-stained spleens of GFP control vs ITK-SYK⁺ mice. (C) Percentage of CD11b⁺Ly6G⁺ granulocytes within GFP⁺ or GFP⁻ PB cells, the spleen, or BM cells of control BALB/c mice that received a transplantation with BM transduced with empty pMIG-vector (GFP mice; left) or with pMIG/ITK-SYK-vector (ITK-SYK mice; right) (n = 5 per group). Blood samples were taken on day 43 after transplantation and analyzed via flow cytometry (fluorescence-activated cell sorting [FACS]). (D) Representative images for CD11b staining showing granulocytes/macrophages within the BM of control mice and ITK-SYK⁺ mice. (E) Representative gating strategy for ITK-SYK⁺ malignant T-cell subtypes before sorting and secondary transplantation (day 43 after transplantation): GFP⁺ cells represent ITK-SYK–expressing cells, followed by CD3⁺ gating, and then distribution into CD4⁺, CD8⁺, CD4⁺CD8⁺, and CD4⁻CD8⁻ cells. (F,G) Overall survival (OS) of lethally irradiated BALB/c mice that received transplantation with thymic or splenic ITK-SYK/GFP⁺ T-cell subsets (CD4⁺, CD8⁺, CD4⁺CD8⁺, or CD4⁻CD8⁻ T cells) mixed with 1 × 10⁶ untreated BALB/c BM support cells. Thymic T cells transplanted were between 4000 and 15 000 per mouse (CD4⁺, n = 9; CD8⁺, n = 7; CD4⁺CD8⁺, n = 2; CD4⁻CD8⁻, n = 2) and spleen T cells were between 20 000 and 50 000 per mouse (CD4⁺, n = 4; CD8⁺, n = 9; CD4⁺CD8⁺, n = 2; CD4⁻CD8⁻, n = 5). Engraftment of ITK-SYK–expressing CD4⁺ and CD8⁺ T cells was independent of the number of transplanted cells, and the OS was monitored for 12 months. Lines representing thymic CD8⁺ and CD4⁺CD8⁺ T cells are hidden behind the line representing thymic CD4⁻CD8⁻ cells. (H) Representative images of tails and ears of mice that received retransplantation with ITK-SYK⁺ CD4⁺ cells from the thymus or spleen. (I-L) Comparative analysis of mice that either received transplantation with control BM only (gray bar, n = 5) or combined with sorted ITK-SYK⁺ CD4⁺ T cells from the thymus (n = 5) or spleen (n = 4) of ITK-SYK⁺ mice (red bars). (I) Comparison of phenotypic score (supplemental Table 1) using weight loss, and tail, ear, and skin infiltration as indicators for TCL manifestation and inflammation. Comparison of (J) spleen weight, (K) percentage of lymphocytes in the PB, and (L) percentage of granulocytes in the PB at the time of death or after 12 months at termination of the experiment. n.d, not detected; PH, pleckstrin-homology domain; ReTx, retransplantation; TH, Tec-homology domain.