Protein Kinase B Regulates T Lymphocyte Survival, Nuclear Factor κb Activation, and Bcl-XL Levels in Vivo

The serine/threonine kinase protein kinase B (PKB)/Akt mediates cell survival in a variety of systems. We have generated transgenic mice expressing a constitutively active form of PKB (gag-PKB) to examine the effects of PKB activity on T lymphocyte survival. Thymocytes and mature T cells overexpressing gag-PKB displayed increased active PKB, enhanced viability in culture, and resistance to a variety of apoptotic stimuli. PKB activity prolonged the survival of CD4+CD8+ double positive (DP) thymocytes in fetal thymic organ culture, but was unable to prevent antigen-induced clonal deletion of thymocytes expressing the major histocompatibility complex class I–restricted P14 T cell receptor (TCR). In mature T lymphocytes, PKB can be activated in response to TCR stimulation, and peptide-antigen–specific proliferation is enhanced in T cells expressing the gag-PKB transgene. Both thymocytes and T cells overexpressing gag-PKB displayed elevated levels of the antiapoptotic molecule Bcl-XL. In addition, the activation of peripheral T cells led to enhanced nuclear factor (NF)-κB activation via accelerated degradation of the NF-κB inhibitory protein IκBα. Our data highlight a physiological role for PKB in promoting survival of DP thymocytes and mature T cells, and provide evidence for the direct association of three major survival molecules (PKB, Bcl-XL, and NF-κB) in vivo in T lymphocytes

Cellular FLICE-inhibitory protein is required for T cell survival and cycling

Fas-associated death domain (FADD) and caspase-8 are key signal transducers for death receptor–induced apoptosis, whereas cellular FLICE-inhibitory protein (cFLIP) antagonizes this process. Interestingly, FADD and caspase-8 also play a role in T cell development and T cell receptor (TCR)–mediated proliferative responses. To investigate the underlying mechanism, we generated cFLIP-deficient T cells by reconstituting Rag−/− blastocysts with cFLIP-deficient embryonic stem cells. These Rag chimeric mutant mice (rcFLIP−/−) had severely reduced numbers of T cells in the thymus, lymph nodes, and spleen, although mature T lymphocytes did develop. Similar to FADD- or caspase-8–deficient cells, rcFLIP−/− T cells were impaired in proliferation in response to TCR stimulation. Further investigation revealed that cFLIP is required for T cell survival, as well as T cell cycling in response to TCR stimulation. Interestingly, some signaling pathways from the TCR complex appeared competent, as CD3 plus CD28 cross-linking was capable of activating the ERK pathway in rcFLIP−/− T cells. We demonstrate an essential role for cFLIP in T cell function

Cellular FLICE-inhibitory protein is required for T cell survival and cycling

Fas-associated death domain (FADD) and caspase-8 are key signal transducers for death receptor–induced apoptosis, whereas cellular FLICE-inhibitory protein (cFLIP) antagonizes this process. Interestingly, FADD and caspase-8 also play a role in T cell development and T cell receptor (TCR)–mediated proliferative responses. To investigate the underlying mechanism, we generated cFLIP-deficient T cells by reconstituting Rag−/− blastocysts with cFLIP-deficient embryonic stem cells. These Rag chimeric mutant mice (rcFLIP−/−) had severely reduced numbers of T cells in the thymus, lymph nodes, and spleen, although mature T lymphocytes did develop. Similar to FADD- or caspase-8–deficient cells, rcFLIP−/− T cells were impaired in proliferation in response to TCR stimulation. Further investigation revealed that cFLIP is required for T cell survival, as well as T cell cycling in response to TCR stimulation. Interestingly, some signaling pathways from the TCR complex appeared competent, as CD3 plus CD28 cross-linking was capable of activating the ERK pathway in rcFLIP−/− T cells. We demonstrate an essential role for cFLIP in T cell function

Novel roles for CD4 in T cell activation

grantor: University of TorontoAccessory activation molecules influence the outcome of antigen recognition by the TCR/CD3 complex. CD4 is expressed on MHC class II restricted T cells, and plays critical roles in enhancing TCR/CD3-mediated activation. This positive function has been attributed to its adhesive properties to MHC class II and its role in localization and activation of associated Lck. Results from this thesis demonstrate that the role of CD4 extends beyond these contributions. The study presented in chapter 2 involves the characterization of an interaction between CD4 and the PTP, CD45. The object of this study was to determine how CD45 is directed to substrates at the antigen receptor complex upon stimulation of resting T cells. Results from chapter 2 demonstrate that CD45 is physically associated with CD4 in resting, primary lymph node T cells, and that CD4-CD45 complexes are disrupted as a consequence of antigen-mediated activation, suggesting a role for these complexes in the activation process. Moreover, CD45 precipitates with CD4 molecules which are associated with Lck, as well as with those which are not. Further, effects on CD4-associated membrane Lck are demonstrable. Since antigen presentation by MHC class II results in coaggregation of CD4 and TCR/CD3, the association described in this study provides a physical basis through which CD45 could be recruited to the antigen receptor complex upon T cell stimulation. While the characterization of CD4 function to date has focused predominantly on its role in the initiation of T cell activation, the study presented in chapter 3 demonstrates that ligation of CD4 inhibits ongoing responses of pre-activated T cells. Specifically, delayed addition of CD4 specific mAb is shown to inhibit antigen, or mAb-induced, responses of both primary T cells and T cell clonal variants. The antigen responses of the latter are independent of the adhesion provided by CD4, thus the observed inhibition is not due to blocking CD4-MHC interactions. Further, analysis of the clonal variants demonstrates that CD4 associated Lck is not essential for the inhibition observed. It is demonstrated that delayed addition of anti-CD4 results in a rapid reduction in steady state levels of IL-2 mRNA in both primary T cells and clonal variants.Ph.D

A novel role for CD4 in antigen-mediated T-cell activation /

A number of T-cell membrane molecules influence the outcome of antigen recognition by TCR/CD3 complex. CD4, by virtue of its non-covalent association with the protein tyrosine kinase lck has the capacity to enhance TCR$alpha beta$ signalling. The extracellular domain of CD4 interacts with monomorphic determinants of Major Histocompatibility complex class-II molecules such that antigen presented in association with MHC class-II to CD4$sp+$ T-cells results in the coaggregation of CD4 and TCR/CD3, thus juxtaposing lck and the antigen-receptor complex. Anti-CD4 antibodies abrogate both antigen and anti-TCR-induced T-cell activation. Studies using antigen specific T-cell clones that express either no CD4, wild type CD4 or mutated CD4 that cannot associate with lck (Db CYS) indicate that CD4 sequesters the majority of cellular lck and when not coaggregated with TCR/CD3, prevents the generation of prerequisite signals.Results presented in this thesis indicate that while CD4-associated lck is providing prerequisite signals for TCR/CD3 signalling, the contribution of CD4 must be more than simply providing a shuttle for lck. Specifically, anti-CD4 inhibits the antigen response of Db CYS CD4-expressing clones. This result cannot be accounted for either by CD4 sequestration of lck, or reduction of avidity of the interaction between the T-cell and the antigen presenting cell, since CD4$sp-$ variants exhibit an antigen response comparable to that of CD4$sp+$ variants. Rather, they suggest a novel role for the ectodomain of CD4 in antigen-induced T-cell activation

[Correspondència entre Madeleine Bonnard i Francesc Pujol]

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