Enhanced proliferation and cytokine production in DGKζ and Cbl-b deficient mice. Enhanced proliferation and cytokine production in DGKζ and Cbl-b deficient mice. (A) A total of 1 × 105 MACS-purified T cells were CFSE-labeled and incubated with varying concentrations of plate-bound anti-CD3 and 5 μg/ml anti-CD28 Abs at 37°C for 72 h. Cells were surface-stained for viability, CD8, and CD4 and evaluated for proliferation as assessed by dilution of CFSE within CD8+-gated cells. (B) Using ELISA, IFN-γ was measured from culture supernatants of (A) after 24 or 72 h of stimulation. (C) Intracellular granzyme B was measured from purified T cells gated from CD8+ populations after 24 and 72 h of stimulation with 0.3 μg/ml anti-CD3 stimulation. Representative of three iterations. For (D)–(F), 5 × 104 Stemcell Technologies–purified naive CD8+ T cells were incubated with 0.3 μg/ml anti-CD3 Ab at 37°C for 72 h. Cells were surface-stained for viability and evaluated for proliferation as assessed by dilution of CFSE within CD8+-gated cells (D). (E and F) Using ELISA, IL-2 and IFN-γ production were measured from culture supernatants of (D) after 72 h of stimulation. Representative of two iterations. Erin M. Wesley et al. ImmunoHorizons 2018;2:107-118 Copyright © 2018 The Authors