Surprisingly however, the CD4+ Fer-1 in vitro T cells from lck-DPP kd mice secreted virtually no IL-2 (Fig. 4A). As expected, the activation of isolated CD8+ T cells resulted in accumulation of only small amounts IL-2, and no difference between mutant and WT cells was observed (Fig. 4A). IFN-γ is secreted mainly by activated CD8+ cells and differentiated Th1 cells. DPP2 kd CD8+ T cells produced slightly more IFN-γ than controls at 24 h of stimulation, but no significant difference was observed at the other time points tested (Fig. 4B). Of special significance is the observation that

IL-17 production, a cytokine secreted exclusively by differentiated Th17 cells, was upregulated in unseparated lymphocytes, as well as in isolated CD4+ and CD8+ T cells from lck-DPP kd mice, most notably at 72 h (Fig. 4C). Intracellular staining of the cells at 72 h after stimulation revealed that the majority of CD4+ T cells from lck-DPP2 kd mice produce IL-17A compared with littermate controls (Fig. 4D), which supports the ELISA data. IL-4, the signature Th2 cytokine, was produced at extremely low levels by both DPP2 kd and control cells, and no difference was discernable (data not shown). To determine whether CD4+ T cells Idasanutlin mw from lck-DPP2 kd mice indeed produced less IL-2, as opposed to increased usage of this cytokine by the highly proliferating

DPP2 kd T cells, il-2 transcripts were quantified by qRT-PCR. As shown in Fig. 5A left panel, il-2 steady-state mRNA levels were significantly decreased in activated CD4+ T cells from lck-DPP kd versus control mice, suggesting that DPP2 kd CD4+ T cells indeed have a defect in IL-2 production. In parallel, ifn-γ mRNA levels were measured by qRT-PCR in activated CD8+ T cells and were found to be significantly lower in the lck-DPP2 kd versus control cells (Fig. 5A, right panel). On the other STK38 hand, il-17 transcript levels were significantly upregulated in both CD4+ and CD8+ T cells from lck-DPP2 kd compared with control

mice (Fig. 5B). RORγt is a transcription factor required for Th17-cell differentiation. Stimulated T cells from lck-DPP kd mice were analyzed for RORγt transcript levels by qRT-PCR, they were upregulated in CD4+ (Fig. 5C), but not CD8+ (data not shown). Mice were immunized with OVA in CFA s.c. and boosted with OVA in incomplete Freund’s adjurant (IFA) s.c. two weeks later. Ten days after boosting, the draining lymph nodes were harvested, restimulated in vitro with OVA and pulsed for 8 h with [3H]-thymidine at various time points (Fig. 6A). Consistent with the anti-CD3 plus anti-CD28 stimulation results obtained with naïve T cells, OVA-immune DPP2 kd T cells were hyper-proliferative and responded to lower doses of OVA compared to those from littermate controls. These data demonstrate that immune T cells from lck-DPP2 kd mice have a lower threshold of activation, when restimulated in vitro with specific antigen.