The TCR competent T-PLL cell shows aberrant effector responses. (A) Patterns of Ca²⁺ efflux upon CD3/28 crosslinking in 12 primary T-PLL; 4 representative examples (and number/cohort): TP017 (strong response, CD28-enhanced), TP093 (strong response, CD28-inhibited), TP018 (weak response, CD28-enhanced), TP046 (weak response, CD28-inhibited). (B) ITK inhibition blocks the stimulation-induced (CD3/CD28 crosslinking, phorbolmyristylacetate/ionomycin) increase in cell viability (CellTiter-Glo) in T-PLL cells (unpaired Student t test, SEM). Inhibitors: PRN-694 (covalent binding; relevant IC50s: ITK 0.3nM; RLK 1.4nM; JAK3 30nM) and BMS-509744 (reversible binding; IC50: ITK 15nM).⁵²  C) Cytokine secretion of anti-CD3/CD28-stimulated healthy-donor derived CD4⁺ naïve and CD4⁺ memory T cells (each n = 5) vs T-PLL cells (n = 10) in relation to their unstimulated controls (11-analyte human cytokine array). Overall, secretory responses of healthy memory T cells and T-PLL cells were more similar and higher as compared with healthy-donor CD4⁺ naïve T cells. Particularly, releases of IL-2, TNFα/β, GM-CSF, IL-10, IFNγ, and IL-1β were strongly increased (unpaired Student t test, SEM). (D) Upon TCR crosslinking T-PLL cells (5 cases) enter the cell cycle (propidium iodide (PI) staining and flow cytometry) more readily than pan–T cells (n = 4) or CD4⁺ memory T cells (n = 5) from healthy donors, which was more pronounced in conditions of combined CD3/CD28 costimulation (unpaired Student t test, SEM). (E) Enhanced pERK1/2 response to TCR crosslinking in leukemic Lck^pr-hTCL1A^tg mice upon enrichment of TCL1A-transgene expressing memory T cells (see Figure 1F) as compared with preleukemic mice (and each to age-matched C57BL/6J wild-type controls); pooled splenocyte lysates of 3 mice; for sample purities see supplemental Figure 6D.