IntroductionProtection against recurrent infections resulting from the same pathogen is a hallmark of adaptive immunity. After acute infection by an intracellular pathogen, naive CD8 ϩ T cells expressing epitope-specific T-cell receptors (TCRs) are activated. The effector phase of the response is short, with a rapid expansion of antigenspecific T cells and pathogen clearance. The expanded effector cells undergo a contraction phase, while approximately 5% to 10% of antigen-specific cells are maintained to establish a memory pool and provide long-term protection from reinfection by the same pathogen. [1][2][3] In the early stages of the effector response to acute lymphocytic choriomeningitis virus (LCMV) infection, activated CD8 ϩ cells differentiate into 2 subsets with distinct fates. These populations can be phenotypically identified by cell-surface expression of killer cell lectin-like receptor G1 (KLRG-1) and the receptor for interleukin-7 (IL-7R). 4 Short-lived effector cells (SLECs) express high levels of KLRG-1 and the transcription factors Blimp-1 and T-bet, and decreased levels of IL-7R. [5][6][7][8] SLECs are dependent on signals from the environment, including TCR signals, inflammatory cytokines such as IL-12 and IFN␥, and common ␥ chain cytokine signaling from IL-2 and IL-15. 4,9 Conversely, memory precursor (MP) cells express low levels of KLRG-1 and higher levels of IL-7R␣, CXCR3, and CD27. 4,10,11 While these cells possess effector function, they also have the potential to further differentiate into long-lived memory T cells after the resolution of infection. The molecular nature of the proximal signals involved in the SLEC/MP cell-fate decision and those required for normal homeostasis of these populations have not been extensively studied.Previous studies have shown that IL-15-and IL-7-generated signals are required for memory T-cell homeostasis. [12][13][14][15] Depending on the experimental system and the characteristics used to define the memory population, TCR signals have been shown to be required or dispensable. For example, H-2D b -restricted, malespecific (H-Y TCR-transgenic) memory CD8 ϩ T cells require expression of either H-2D b or H-2D d for survival. 16 The absence of all major histocompatibility complex (MHC) class I expression leads to the disappearance of the cells, suggesting that a tonic MHC-TCR signal is required. In addition, CD8 ϩ CD44 hi cells do not persist in mice after gene deletion of the TCR␣ chain. 17 In contrast, polyclonal CD8 ϩ T-cell populations containing memory CD8 ϩ T cells generated by viral infection persist indefinitely when transferred into MHC class I-deficient mice. 18 CD44 hi cells and TCR-transgenic memory T cells persist long term, even when the expression of the src family tyrosine kinase Lck or of TCR itself is substantially decreased by a bitransgenic tetracycline regulatory system. [19][20][21] An obstacle to characterizing the requirements for memory population generation and persistence is the heterogeneity of definitions for memory CD8 ϩ T cells o...

SUMMARY The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of wild-type SLP-76 protein in thymocytes. Following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T cell mediated autoimmune disease experimental autoimmune encephalomyelitis. Our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.

The intracellular signaling mechanisms regulating the generation and long-term persistence of memory T cells in vivo remain unclear. In this study, we used mouse models with conditional deletion of the key T cell receptor (TCR)-coupled adaptor molecule SH2-domain-containing phosphoprotein of 76 kDa , to analyze signaling mechanisms for memory CD4 T cell generation, maintenance, and homeostasis. We found that ablation of SLP-76 expression after T cell priming did not inhibit generation of phenotypic effector or memory CD4 T cells; however, the resultant SLP-76-deficient memory CD4 T cells could not produce recall cytokines in response to TCR-mediated stimulation and showed decreased persistence in vivo. In addition, SLP-76-deficient memory CD4 T cells exhibited reduced steady-state homeostasis and were impaired in their ability to homeostatically expand in vivo in response to the γ c cytokine IL-7, despite intact proximal signaling through the IL-7R-coupled JAK3/STAT5 pathway. Direct in vivo deletion of SLP-76 in polyclonal memory CD4 T cells likewise led to impaired steady-state homeostasis as well as impaired homeostatic responses to IL-7. Our findings demonstrate a dominant role for SLP-76-dependent TCR signals in regulating turnover and perpetuation of memory CD4 T cells and their responses to homeostatic cytokines, with implications for the selective survival of memory CD4 T cells following pathogen exposure, vaccination, and aging.immune memory | signal transduction | linker-adapter T he enhanced functional and survival properties of memory T cells enable them to provide long-lasting secondary responses to recall antigens. Memory T cells are generated following antigen activation of naive T cells (1) and differ from naive T cells in their rapid production of effector cytokines following antigenic stimulation through the T cell receptor (TCR). The TCR-coupled signaling pathways for naive T cell activation have been well defined (2) and include an initial phosphorylation of TCR/CD3 components, leading to activation of the ZAP-70 proximal kinase, and the coupling of proximal phosphorylation events to distal signaling through linker-adapter molecules such as the SH2-containing phosphoprotein of 76 kDa (SLP-76) and linker for activated T cells (LAT) (3). The specific TCR-coupled signaling events important for promoting memory T cell development and persistence remain undefined.Once generated, memory T cells have variable requirements for TCR engagement for their maintenance. Whereas memory CD8 T cells can persist and maintain recall function in the absence of MHC class I expression (4), memory CD4 T cells exhibit impaired functional responses when maintained in the absence of MHC class II expression (5, 6), although they can survive in MHC class II-deficient hosts (5,7,8). In addition, the presence of MHC class II or TCR signaling is associated with improved memory CD4 T cell survival and homeostasis (9-11), suggesting that memory CD4 T cells may depend on TCR signals during their long-term maintenance. Cytokine...

BackgroundEzrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored.Methodology/Principal FindingsWe characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin−/− mice likely arise as a consequence of nutritional stress.Conclusions/SignificanceWe conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.

The adaptor protein Src homology 2 (SH2) domain containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adaptor proteins that function together to activate second messengers required for TCR signal propagation. The N-terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with the adhesion- and degranulation-promoting adaptor protein (ADAP) and hematopoietic progenitor kinase 1 (HPK1). Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production and phosphorylation of protein kinase D (PKD) and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells.