These results suggest that CD4+ T cells are unique among T-lineage cells in that they are independent of γc signals in their differentiation high throughput screening assay and homeostasis — if prosurvival signals are provided. Collectively, these results unveil novel requirements for γc signaling in T-lineage cell specification

and differentiation that are distinct from its prosurvival effects. Thymocytes and resting T cells do not express detectable levels of Pim1 unless signaled by TCR or cytokines [16, 19]. However, Eμ enhancer driven Pim1Tg mice express Pim1 in all lymphocytes and independently of signaling [18, 19, 21, 26] (Supporting Information Fig. 1A and B). In such Pim1Tg mice, we found that ectopic Pim1 expression did not affect

thymocyte differentiation (Fig. 1A), but that it significantly increased overall thymocyte numbers (Fig. 1B). Increased cell numbers were not associated with aberrant differentiation of immature CD4, CD8 double negative (DN) thymocytes as we did not find significant differences in DN1-DN4 stage differentiation (Fig. 1C and Supporting Information Fig. 1C). Also, Pim1Tg positive selection was comparable with that of WT mice (Fig. 1D). Thus, transgenic Pim1 improved total thymocyte numbers without affecting thymocyte differentiation or selection. To assess whether Pim1 also improved peripheral T-cell numbers, next we analyzed LN cells in WT and Pim1Tg mice. Pim1 significantly increased both CD4+ and CD8+ LNT numbers (Fig. 1E and F). Importantly, T-cell numbers increased in the absence of T-cell activation, LY294002 datasheet as Pim1Tg T cells did not upregulate CD69 (Supporting

Information Fig. 1D) and freshly isolated Pim1Tg CD4+ T cells did not express proinflammatory cytokines (Fig. 1G and Supporting Information Fig. 1E). Such effects were intrinsic to Pim1Tg T cells, as adoptively transferred WT T cells did not show increased proliferation in Pim1Tg hosts compared with control WT host mice (Fig. 1H). Thus, Pim1 expands the size of the peripheral T-cell pool, and it likely does it so by providing survival through inactivation of proapoptotic Bad [19], but without direct upregulation of antiapoptotic molecule HSP90 mRNA expression (Supporting Information Fig. 1F). Collectively, Pim1 is a potent prosurvival factor that promotes thymopoiesis and peripheral T-cell homeostasis. To assess the extent to which Pim1 overexpression can replace γc signaling, we generated Pim1TgγcKO mice. γcKO mice do not generate meaningful number of thymocytes [4, 5]. Pim1TgγcKO mice, however, had significantly increased thymocyte numbers compared with those in γcKO mice (Fig. 2A). Transgenic Bcl-2 also improved thymocyte numbers in γcKO mice, but its effect was much weaker than Pim1 (Fig. 2A).