Two important Ras-guanyl nucleotide exchange factors, Sos and RasGRP1, control Ras activation in thymocytes. However, the relative contribution of these two exchange factors to Ras/ERK activation and their resulting impact on positive and negative selection is unclear. We have produced two lines of RasGRP1(-/-) TCR transgenic mice to determine the effect of RasGRP1 in T cell development under conditions of defined TCR signaling. Our results demonstrate that RasGRP1 is crucial for thymocytes expressing weakly selecting TCRs whereas those that express stronger selecting TCRs are more effective at utilizing RasGRP1-independent mechanisms for ERK activation and positive selection. Analysis of RasGRP1(-/-) peripheral T cells also revealed hitherto unidentified functions of RasGRP1 in regulating T cell homeostasis and sustaining antigen-induced developmental programming.
Naive T cells require costimulation for robust Ag-driven differentiation and survival. Members of the TNFR family have been shown to provide costimulatory signals conferring survival at distinct phases of the T cell response. In this study, we show that CD4 and CD8 T cells depend on TNFR type 2 (p75) for survival during clonal expansion, allowing larger accumulation of effector cells and conferring protection from apoptosis for a robust memory pool in vivo. We demonstrate using the MHC class I-restricted 2C TCR and MHC class II-restricted AND TCR transgenic systems that TNFR2 regulates the threshold for clonal expansion of CD4 and CD8 T cell subsets in response to cognate Ag. Using a novel recombinant Listeria monocytogenes (rLM) expressing a secreted form of the 2C agonist peptide (SIY) to investigate the role of TNFR2 for T cell immunity in vivo, we found that TNFR2 controls the survival and accumulation of effector cells during the primary response. TNFR2−/− CD8 T cells exhibit loss of protection from apoptosis that is correlated with diminished survivin and Bcl-2 expression. Null mutant mice were more susceptible to rLM-SIY challenge at high doses of primary infection, correlating with impaired LM-specific T cell response in the absence of TNFR2-mediated costimulation. Moreover, the resulting memory pools specific for SIY and listeriolysin O epitopes derived from rLM-SIY were diminished in TNFR2−/− mice. Thus, examination of Ag-driven T cell responses revealed a hitherto unknown costimulatory function for TNFR2 in regulating T cell survival during the differentiation program elicited by intracellular pathogen in vivo.
The transport of calcium ions (Ca(2+)) to the cytosol is essential for immunoreceptor signaling, regulating lymphocyte differentiation, activation, and effector function. Increases in cytosolic-free Ca(2+) concentrations are thought to be mediated through two interconnected and complementary mechanisms: the release of endoplasmic reticulum Ca(2+) "stores" and "store-operated" Ca(2+) entry via plasma membrane channels. However, the identity of molecular components conducting Ca(2+) currents within developing and mature T cells is unclear. Here, we have demonstrated that the L-type "voltage-dependent" Ca(2+) channel Ca(V)1.4 plays a cell-intrinsic role in the function, development, and survival of naive T cells. Plasma membrane Ca(V)1.4 was found to be essential for modulation of intracellular Ca(2+) stores and T cell receptor (TCR)-induced rises in cytosolic-free Ca(2+), impacting activation of Ras-extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) pathways. Collectively, these studies revealed that Ca(V)1.4 functions in controlling naive T cell homeostasis and antigen-driven T cell immune responses.
Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Rα, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Rα Y449XXM motif in mice by knock-in mutagenesis (IL-7Rα449F). Thymic precursors were reduced in number in IL-7Rα449F mice, but in marked contrast to IL-7Rα−/− knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Rα signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Rα449F mice. Furthermore, we show that Bcl-2 is IL-7Rα Y449 independent and insufficient for IL-7–mediated maintenance of CD8 memory.
The vast majority of mature T cells are either of the CD4+ 8-or the CD4-8+ phenotype. The CD4 cell surface glycoprotein is expressed primarily on the surface of helper T cells, whose TCR recognize antigen on APCs in the context of class II MHC molecules. The CD8 molecule is primarily expressed on T cells, in particular CTL, whose TCR recognize antigen in the context of class I MHC molecules.The TCR on helper T cells and CTL has been identified as a disulfide-linked ado heterodimer with a molecular weight of 80,000-90,000 (reviewed in reference 1). The binding of CD4 and CD8 molecules to nonpolymorphic regions of class II and class I MHC molecules, respectively, enhances the binding of the TCR to its ligand and may also contribute to signals leading to T cell activation (2, 3).We have constructed TCR transgenic mice with the aim to study the selection ofthe T cell repertoire. To this end, u and a transgenes obtained from an HYspecific, H-21)6-restricted cytolytic CD4-8 + T cell clone were injected into fertilized eggs . We have reported that in female ca/(3 transgenic mice a large proportion of T cells is male specific and expresses both ce and (3 transgenes . In male mice we found that cells with an abnormal CD4/CD8 phenotype accumulated in the periphery, with the vast majority of a//3 T cells being CD4 -8 -and a minority being CD4-8+ . The latter expressed, however, low levels of CD8 accessory molecules. Due to the lack of an appropriate antibody we could not directly address the question as to whether or not these cells expressed the transgenic receptor. Here we describe the use ofmAbs that detect cells expressing both a and a transgenes . Using these antibodies we find that in male H-2D b mice the vast majority of peripheral T cells express both a and a transgenes . Our studies indicate that despite the fact that precursor T cells, which express a low density of the transgenic TCR, are largely deleted in the thymus of male transgenic mice (4), the majority of T cells spared by the deletion express a high density of the transgenic TCR but a low density of CD8 molecules.
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