Soo-Jeet Teh

RasGRP1 transduces low-grade TCR signals which are critical for T cell development, homeostasis, and differentiation.

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.

TNF Receptor Type 2 (p75) Functions as a Costimulator for Antigen-Driven T Cell Responses In Vivo

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.

TNF Receptor 2-Deficient CD8 T Cells Are Resistant to Fas/Fas Ligand-Induced Cell Death

Apoptotic cell death plays a fundamental role in the maintenance of tissue homeostasis in complex biological systems. It is also a major mechanism for keeping immune reactions in check. Members of the TNF family of receptors and cytokines are implicated in the regulation of apoptotic signals that shape the immune system. In this study, we have examined the role of three members of the TNFR family, Fas (CD95), TNFR1 (p55), and TNFR2 (p75), in inducing cell death in Con A-activated CD4 and CD8 T cells. It was found that Con A-activated p55−/− CD4 or CD8 T cells were highly resistant to TNF-induced cell death. By contrast, although activated p75−/− CD4 or CD8 T cells were killed by TNF, they were more resistant to TNF-induced killing when compared with p75+/+ cells, particularly at higher concentrations of TNF. We also determined whether activated p55−/− and p75−/− T cells differ in their sensitivity to cell death induced by TCR cross-linking. We found that activated p55−/− CD4 or CD8 T cells were equally susceptible to TCR-induced cell death. More interestingly, the loss of the p75 receptor conferred resistance to TCR-induced death in activated CD8, but not CD4 T cells. This resistance to TCR-induced death in activated p75−/− CD8 T cells correlated with the resistance of these cells to Fas/Fas ligand-induced cell death.

A Population of In Vivo Anergized T Cells with a Lower Activation Threshold for the Induction of CD25 Exhibit Differential Requirements in Mobilization of Intracellular Calcium and Mitogen-Activated Protein Kinase Activation

Chronic exposure of mature T cells with specificity for self-Ags can lead to the induction of a nonfunctional state which is referred to as T cell anergy. It is unclear whether anergic T cells are destined for cell death and thereby harmless or whether they can contribute to the induction of autoimmunity and/or regulation of anti-self reactivity. We have begun to address this issue. In a recent study, we showed that a population of mature CD4−CD8− T cells that express a transgenic TCR specific for the Ld MHC class I molecule are rendered anergic in Ld-expressing mice. In this study, we show that this population of anergic T cells possess a lower activation threshold for the induction of CD25 and CD69 in response to stimulation by antigenic ligands. Furthermore, these anergic T cells undergo extensive proliferation when stimulated with a low-affinity ligand in the presence of an exogenous source of IL-2. Biochemical analysis of the early intracellular signaling events of these in vivo anergized T cells showed that they have a signaling defect at the level of ZAP-70 and linker for the activation of T cell (LAT) phosphorylation. They also exhibit a defect in mobilization of intracellular calcium in response to TCR signaling. However, these anergic T cells demonstrate no defect in SLP-76 phosphorylation and extracellular signal-regulated kinase 1/2 activation. These biochemical characteristics of the anergic T cells were associated with an elevated level of Fyn, but not Lck expression. The potential contributions of these anergic T cells in the induction and/or regulation of autoimmune responses are discussed.

TNFR2-Deficient Memory CD8 T cells Provide Superior Protection Against Tumor Cell Growth

TNF receptor-2 (TNFR2) plays a critical role in promoting the activation and survival of naive T cells during the primary response. Interestingly, anti-CD3 plus IL-2 activated TNFR2−/− CD8 T cells are highly resistant to activation-induced cell death (AICD), which correlates with high expression levels of prosurvival molecules such as Bcl-2, survivin, and CD127 (IL-7Rα). We determined whether the resistance of activated TNFR2−/− CD8 T cells to AICD contributes to more effective protection against tumor cell growth. We found that during a primary tumor challenge, despite initial inferiority in controlling tumor cell growth, TNFR2−/− mice were able to more effectively control tumor burden over time compared with wild-type (WT) mice. Furthermore, vaccination of TNFR2−/− mice with recombinant Listeria monocytogenes that express OVA confers better protection against the growth of OVA-expressing E.G7 tumor cells relative to similarly vaccinated WT mice. The enhanced protection against tumor cell growth was not due to more effective activation of OVA-specific memory CD8 T cells in vaccinated TNFR2−/− mice. In vitro studies indicate that optimally activated OVA-specific TNFR2−/− CD8 T cells proliferated to the same extent and possess similar cytotoxicity against E.G7 tumor cells as WT CD8 T cells. However, relative to WT cells, activated OVA-specific TNFR2−/− CD8 T cells were highly resistant to AICD. Thus, the enhanced protection against E.G7 in TNFR2−/− mice is likely due to the recruitment and activation of OVA-specific memory TNFR2−/− CD8 T cells and their prolonged survival at the tumor site.

p59fyn (Fyn) Promotes the Survival of Anergic CD4−CD8− αβ TCR+ Cells but Negatively Regulates Their Proliferative Response to Antigen Stimulation

T cell anergy is characterized by alterations in TCR signaling that may play a role in controlling the unresponsiveness of the anergic cell. We have addressed questions regarding the importance of the Src kinase p59fyn (Fyn) in this process by using Fyn null mice. We demonstrate that a mature population of CD4−CD8− αβ TCR+ anergic T cells lacking Fyn have a substantial recovery of their proliferation defect in response to Ag stimulation. This recovery cannot be explained by ameliorated production of IL-2, and the improved proliferation correlates with an enhanced ability of the Fyn−/− anergic T cells to up-regulate the high affinity IL-2 receptor. We also observe that anergic CD4−CD8− αβ TCR+ T cells have a heightened survival ability that is partially dependent on the elevated levels of Fyn and IL-2 receptor β-chain expressed by these cells. The enhanced survival correlates with an increased capacity of the anergic cells to respond to IL-15. We conclude that Fyn plays an important role in aspects of T cell anergy pertaining to TCR signaling and to cell survival.

Regulation of cytotoxic responses to alloantigens by Ia+ cells☆

Abstract Pretreatment of responder spleen cells with anti-Ia plus complement led to an enhancement of cytotoxic responses to alloantigens as well as to TNP-modified self antigens. This observation confirms previous reports that cytotoxic T lymphocytes (CTL) and their precursors (CLP) are Ia − . Furthermore, it suggests that the CTL responses to alloantigens or TNP-modified self-antigens are regulated by an Ia + suppressor cell. Absorption studies and studies with anti-Ia sera specific for either the entire I region or the I-E/C subregions suggest that the regulatory cell certainly expresses I-E/C-coded determinants although the possibility that it also expresses I-A/B/J-coded determinats cannot be ruled out. Cell-mixing studies suggest that the regulatory cell is Thy-1 − and requires cell division before it can suppress. A clonal assay for CLP was used to show that the enhancement of the CTL response to alloantigens cannot be accounted for on the basis of an increase in the number of CLP in the anti-Ia + C-treated group.