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We examined the role of the antiapoptotic molecule Bcl-2 in combating the proapoptotic molecule Bim in control of naive and memory T cell homeostasis using Bcl-2(-/-) mice that were additionally deficient in one or both alleles of Bim. Naive T cells were significantly decreased in Bim(+/-)Bcl-2(-/-) mice, but were largely restored in Bim(-/-)Bcl-2(-/-) mice. Similarly, a synthetic Bcl-2 inhibitor killed wild-type, but not Bim(-/-), T cells. Further, T cells from Bim(+/-)Bcl-2(-/-) mice died rapidly ex vivo and were refractory to cytokine-driven survival in vitro. In vivo, naive CD8(+) T cells required Bcl-2 to combat Bim to maintain peripheral survival, whereas naive CD4(+) T cells did not. In contrast, Bim(+/-)Bcl-2(-/-) mice generated relatively normal numbers of memory T cells after lymphocytic choriomeningitis virus infection. Accumulation of memory T cells in Bim(+/-)Bcl-2(-/-) mice was likely caused by their increased proliferative renewal because of the lymphopenic environment of the mice. Collectively, these data demonstrate a critical role for a balance between Bim and Bcl-2 in controlling homeostasis of naive and memory T cells.

CD28 provides important signals that lower the threshold of T cell activation, augment the production of IL-2, and promote T cell survival. The recent identification of a second family of costimulatory molecules within the TNFR family has reshaped the "two-signal" model of T cell activation. In this study the role of p75 as a T cell costimulatory molecule in controlling cell fate during TCR/CD28-mediated stimulation was examined. We found that p75-deficient T cells possess a profound defect in IL-2 production in response to TCR/CD28-mediated stimulation. Examination of key signaling intermediates revealed that TCR proximal events such as global tyrosine phosphorylation and ZAP70 phosphorylation, as well as downstream MAPK cascades are unperturbed in p75-deficient T cells. In contrast, p75 is nonredundantly coupled to sustained AKT activity and NF-kappaB activation in response to TCR/CD28-mediated stimulation. Moreover, p75-deficient T cells possess a defect in survival during the early phase of T cell activation that is correlated with a striking defect in Bcl-x(L) expression. These data indicate discrete effects of p75 on the intracellular signaling milieu during T cell activation, and reveal the synergistic requirement of TCR, CD28, and p75 toward optimal IL-2 induction and T cell survival. We propose that p75 acts as one of the earliest of the identified costimulatory members of the TNFR family, and is functionally linked to CD28 for initiating and determining T cell fate during activation.

Thymic selection adjusts the reactivity of the peripheral T cell repertoire to maximize recognition of pathogens and minimize stimulation by innocuous substances and self-antigen. The study of molecules implicated in T cell activation often involves the generation of knockout (-/-) mice. In many instances, knockout animals display revealing phenotypes. But should a lack of phenotype be interpreted as a lack of function? Bcl-xgamma was shown previously to affect T cell activation in vitro, and here we note that overexpression of this molecule increases cell cycling after T cell receptor ligation by antibody. It was therefore surprising that Bcl-xgamma(-/-), Bcl-xgamma transgenic, and WT T cells displayed similar levels of sensitivity to antigen according to ex vivo stimulation. Bcl-xgamma could be demonstrated to influence competitiveness and selection of thymocytes in a manner that counteracted the effects of Bcl-xgamma mutation on T cell activation. These findings suggest that thymic selection can overcome genetic defects in T cell activation to generate a T cell repertoire of normal reactivity.

Allogeneic hematopoietic stem cell transplantation (AHSCT) leads to a prolonged state of immunodeficiency characterized by low peripheral naive T-cell counts. To identify the mechanisms leading to this defect we quantitatively and qualitatively analyzed thymic function through quantification of T-cell receptor excision circle (TREC) frequencies (both the signal-joint TREC [sjTREC] and 6 different DbetaJbeta TRECs, by-products of T-cell receptor [TCR] alpha and beta gene rearrangement, respectively), in conjunction with immunophenotype and spectratype analyses in a cohort of patients sampled from 1 to 10 years following AHSCT. In this cohort, reduced thymic function was associated only with ongoing clinical chronic graft-versus-host disease (cGVHD). Nonetheless, the diversity of thymic production remained unchanged irrespective of the patient's cGVHD status. Interestingly, increased homeostatic proliferation was found in the naive T-cell compartment of cGVHD- patients who underwent transplantation. However, reduced expression of both the interleukin-7 receptor alpha (IL-7Ralpha) (CD127) chain and the antiapoptotic protein Bcl-2 was observed. Taken together, these data indicate that the inability to reconstitute the naive T-cell compartment for several years after AHSCT, in the absence of cGVHD, is a consequence of impaired naive T-cell survival rather than thymic dysfunction.

The molecular basis of CD28-dependent costimulation of T cells is poorly understood. Bcl-xgamma is a member of the Bcl-x family whose expression is restricted to activated T cells and requires CD28-dependent ligation for full expression. We report that Bcl-xgamma-deficient (Bcl-xgamma-/-) T cells display defective proliferative and cytokine responses to CD28-dependent costimulatory signals, impaired memory responses to proteolipid protein peptide (PLP), and do not develop PLP-induced experimental autoimmune encephalomyelitis (EAE). In contrast, enforced expression of Bcl-xgamma largely replaces the requirement for B7-dependent ligation of CD28. These findings identify the Bcl-xgamma cytosolic protein as an essential downstream link in the CD28-dependent signaling pathway that underlies T cell costimulation.

The molecular events triggered by MHC recognition and how they lead to the emergence of mature CD4 and CD8 lineage thymocytes are not yet understood. To address these questions, we have examined what signals are necessary to drive the development of CD8 lineage thymocytes in TCRalpha(-) mice in which TCR/MHC engagement cannot occur. We find that the combination of constitutive Notch activity and constitutive Bcl-2 expression are necessary and sufficient to allow the appearance of mature CD8 lineage thymocytes in TCRalpha(-) mice. In addition, Notch activity alone in TCRalpha(-) mice can induce the up-regulation of HES1, suggesting that thymocytes are competent to respond to Notch signaling in the absence of MHC recognition. These data indicate that survival and lineage commitment represent distinct, parallel pathways that occur as a consequence of MHC recognition, both of which are necessary for the development of mature CD8 lineage T cells.