Consequence of the SLAM-SAP signaling pathway in innate-like and conventional lymphocytes

Signaling lymphocytic activation molecule (SLAM) family receptors mediate important regulatory signals in immune cells, as a result of their exquisite ability to associate with members of the SLAM-associated protein (SAP) family of adaptors. As discussed herein, recent findings show that the SLAM and SAP families carry out pivotal functions in innate-like and conventional lymphocytes. They are critically needed for the development of innate-like lymphocytes such as NKT cells. In addition, they influence several of the functions of conventional lymphocytes, including the ability of CD4(+) T cells to secrete certain cytokines and mediate B cell help; CD8(+) T cell proliferation and cytokine production; NK cell-mediated cytotoxicity; and B cell antibody production. These unique functional properties appear to be facilitated by the ability of SLAM-related receptors to serve as self-ligands during homotypic interactions between immune cells. The importance of the SLAM-SAP pathway in normal immunity is highlighted by the finding that SAP is mutated in humans suffering from the immunodeficiency X-linked lymphoproliferative disease.     

Altered Signaling Lymphocytic Activation Molecule (SLAM) Expression in HIV Infection and Redirection of HIV-Specific Responses via SLAM Triggering

Signaling lymphocytic activation molecule (SLAM) is a transmembrane lymphocytic receptor which gets rapidly upregulated following cell activation. SLAM engagement augments T cell expansion and interferon-gamma (IFN-gamma) production independently of CD28. SLAM signaling is regulated by the SLAM-associated protein. We evaluated the expression and function of SLAM on CD4(+) and CD8(+) lymphocytes in HIV-infected individuals with either recently acquired infection (Group A) or asymptomatic HIV infection (Group B) and in healthy controls (HC). Soluble antigen (HIV env peptides and tetanus toxoid)- and mitogen-stimulated proliferation and IFN-gamma and IL-10 production upon SLAM costimulation were also measured. Results showed that: (1) SLAM-expressing CD4(+) and CD8(+) lymphocytes diminish in group A patients compared to both group B patients and HC; (2) SLAM expression on CD4(+) lymphocytes is preferentially associated with the lack of CD7 on cell surface (CD4(+)CD7(-) produce IL-10 but not IFN-gamma); (3) SLAM engagement increases HIV env peptide-stimulated, but neither tetanus toxoid- nor PHA-stimulated proliferation of peripheral blood mononuclear cells (PBMC) in patients but not in HC; and (4) SLAM engagement augments IFN-gamma and reduces IL-10 production by env peptide-stimulated PBMC of HIV-infected individuals. These results demonstrate that early HIV infection results in an altered SLAM expression which correlates with a time-limited impairment of cell-mediated immunity. Furthermore, they show that triggering via SLAM potentiates HIV-specific proliferative responses with simultaneous downregulation of IL-10 and redirection of the response to TH0/TH1.     

Control of early stages in invariant natural killer T‐cell development

Natural killer T (NKT) cells develop in the thymus from the same precursors as conventional CD4(+) and CD8(+) αβ T cells, CD4(+) CD8(+) double-positive cells. In contrast to conventional αβT cells, which are selected by MHC-peptide complexes presented by thymic epithelial cells, invariant NKT cells are selected by lipid antigens presented by the non-polymorphic, MHC I-like molecule CD1d, present on the surface of other double-positive thymocytes, and require additional signals from the signalling lymphocytic-activation molecule (SLAM) family of receptors. In this review, we provide a discussion of recent findings that have modified our understanding of the NKT cell developmental programme, with an emphasis on events that affect the early stages of this process. This includes factors that control double-positive thymocyte lifespan, and therefore the ability to generate the canonical Vα rearrangements that characterize this lineage, as well as the signal transduction pathways engaged downstream of the T-cell receptor and SLAM molecules.     

Sialyl lewisx antigen-expressing human CD4+ T and CD8+ T cells as initial immune responders in memory phenotype subsets

Cytokine production by memory T cells in secondary immune responses has a critical role in host defenses. Previously, we had demonstrated that a unique antigen composed of sialyl lewis(x) (sLe(x)) was expressed on CD45RO(+) memory-phenotype subsets of human T cells. Here, we found that the sLe(x) antigen was up-regulated on CD45RA(+) na?ve human CD4(+) T and CD8(+) T cells by TCR stimulation. In addition, sLe(x) antigen-expressing CD4(+) T and CD8(+) T cells in human PBMCs were activated immediately by cytokine stimulations composed of IL-2 plus IL-12 or IL-15 in an antigen-independent manner. Moreover, the sLe(x)-positive human CD8(+) T cells significantly enhanced reverse antibody-dependent cellular cytotoxicity compared with a sLe(x)-negative population. These findings clearly indicate that sLe(x) antigen-expressing memory phenotype CD4(+) T and CD8(+) T cells contribute to early-stage immunity by providing a source of IFN-gamma and cytotoxicity, suggesting that they would be a key immunomodulator in host defenses.     

A subset of IL-10-producing gammadelta T cells protect the liver from Listeria-elicited, CD8(+) T cell-mediated injury

Although gammadelta T cells play a role in protecting tissues from pathogen-elicited damage to bacterial, viral and parasitic pathogens, the mechanisms involved in the damage and in the protection have not been clearly elucidated. This has been addressed using a murine model of listeriosis, which in mice lacking gammadelta T cells (TCRdelta(-/-)) is characterised by severe and extensive immune-mediated hepatic necrosis. We show that these hepatic lesions are caused by Listeria-elicited CD8(+) T cells secreting high levels of TNF-alpha that accumulate in the liver of Listeria-infected TCRdelta(-/-) mice. Using isolated populations of gammadelta T cells from wild-type and cytokine-deficient strains of mice to reconstitute TCRdelta(-/-) mice, the TCR variable gene 4 (Vgamma4)(+) subset of gammadelta T cells was shown to protect against liver injury. Hepatoprotection was dependent upon their ability to produce IL-10 after TCR-mediated interactions with Listeria-elicited macrophages and CD8(+) T cells. IL-10-producing Vgamma4(+) T cells also contribute to controlling CD8(+) T cell expansion and to regulating and reducing TNF-alpha secretion by activated CD8(+) T cells. This effect on TNF-alpha production was directly attributed to IL-10. These findings identify a novel mechanism by which pathogen-elicited CD8(+) T cells are regulated via interactions with, and activation of, IL-10-producing hepatoprotective gammadelta T cells.     

Functional characterization of OX40 expressed on human CD8+ T cells

In the present study, we investigated the expression of OX40 on human CD8(+) T cells with regard to expression induction, costimulatory function and possible involvement in cytotoxicity. Human CD8(+) T cells were purified from peripheral blood mononuclear cell (PBMC) of healthy donors and cocultured with allogeneic monocyte-derived dendritic cells. Flow cytometric analysis showed that expression of OX40 was induced on CD8(+) T cells within 1 day and increased to the maximum levels on day 3. An addition of anti-OX40 ligand (OX40L) mAb suppressed CD25 expression, proliferation and IFN-gamma production of CD8(+) T cells, suggesting that OX40 functions as a costimulatory molecule not only for CD4(+) T cells but also for CD8(+) T cells. In parallel, coculture of pre-activated CD8(+) T cells with OX40L-transfected murine epithelial cells (MMCE-OX40L) resulted in an increase in CD25 expression, proliferation and IFN-gamma producing cells, compared with that with the mock control (MMCE-mock). Finally, non-specific cytotoxic activity of preactivated CD8(+) T cells was examined using OKT3 hybridoma as target cells after coculture with these transfectants. Coculture with MMCE-OX40L induced slightly higher cytotoxicity of CD8(+) T cells than that with MMCE-mock. These results indicate that OX40 is induced transiently on CD8(+) T cells upon activation and its signals contribute to both clonal expansion and functional reinforcement.     

Signaling lymphocytic activation molecule (SLAM) is differentially expressed in human Th1 and Th2 cells

We have used a real-time quantitative RT-PCR technique (TaqMan, PE Biosystems) to identify genes that are differentially expressed by human polarised CD4(+) T cell subsets (Th1 or Th2). The goal was to test the feasibility of the detection method in profiling the expression of a set of marker genes important for Th1 and Th2 differentiation. We demonstrate that in polarised human Th1 cells signaling lymphocytic activation molecule (SLAM), a member of the immunoglobulin superfamily, is expressed at 7-25-fold higher levels than in Th2 cells. Along with SLAM, expression of the IL-12 receptor chain beta 2 (IL-12R beta 2) and the IFN-gamma receptor chain beta (IFN-gamma R beta) proved to be useful molecular markers indicating the state of T cell polarisation, as previously reported. Treatment with IL-12 increased SLAM mRNA expression in T cells by 3-4-fold, whereas a number of other cytokines including PDGF-BB, IFN-alpha A, IFN-alpha A/D, IFN-beta, IFN-gamma or IL-9 had no effect. Stimulating T cells by co-ligating CD3 and CD28 increased SLAM protein surface expression in both Th1 and Th2 cells. In conclusion, real-time RT-PCR detection was found to be an accurate, sensitive and highly reproducible method for fast profiling of mRNA expression in Th1 and Th2 cell subsets.     

Molecular and cellular pathogenesis of X-linked lymphoproliferative disease

Summary: X‐linked lymphoproliferative disease (XLP) is an inherited immune defect caused by mutations in the Src homology 2 domain‐containing gene 1A, which encodes the adapter protein, signaling lymphocytic activation molecule (SLAM)‐associated protein (SAP). SAP is expressed in T cells, natural killer (NK) cells, and NKT cells, where it binds to the cytoplasmic domain of the surface receptor SLAM (CD150) and the related receptors, 2B4 (CD244), CD84, Ly9 (CD229), NK‐T‐B‐antigen, and CD2‐like receptor‐activating cytotoxic T cells. SAP also binds to the Src family tyrosine kinase Fyn and recruits it to SLAM, which leads to the generation of downstream phosphotyrosine signals. While the roles of the SLAM family receptors are only beginning to be understood, experiments suggest that these molecules regulate important aspects of lymphocyte function, such as proliferation, cytokine secretion, cytotoxicity, and antibody production. Thus, in XLP patients who lack functional SAP, the SLAM family receptors may not signal properly. This property likely contributes to the phenotypes of XLP, including fulminant infectious mononucleosis, lymphoma, and hypogammaglobulinemia. Further studies of SAP and the SLAM family receptors will provide insights into XLP and elucidate the signaling events regulating lymphocyte ontogeny and function.     

A co-stimulatory molecule on activated T cells,H4/ICOS,delivers specific signals in T(h) cells and regulates their responses

We examined the co-stimulatory activity of H4/ICOS on murine activated CD4(+) T cells and found that the cross-linking of H4/ICOS enhanced their proliferation, in addition to raising IFN-gamma, IL-4 and IL-10 production to levels comparable to those induced by CD28. However, IL-2 production was only marginally co-stimulated by H4/ICOS. This distinct pattern of lymphokine production appears to be induced by a specific intracellular signaling event. Compared with CD28, H4/ICOS dominantly elicited the Akt pathway via phosphatidylinositol 3-kinase. In addition, mitogen-activated protein kinase family kinases were activated in different ways by CD28 and H4/ICOS. The strong phosphorylation of p46 c-Jun N-terminal kinase was observed upon CD28 co-stimulation, but was less potently induced by H4/ICOS. The strain diversity in the induction of H4/ICOS was recognized. The expression of H4/ICOS on BALB/c activated CD4(+) T cells was >6-fold higher compared with C57BL/6 activated CD4(+) T cells. Furthermore, BALB/c activated CD4(+) T cells exhibited more T(h)2-deviated lymphokine production as compared with C57BL/6 activated CD4(+) T cells and signaling through H4/ICOS during the primary stimulation of naive CD4(+) T cells promoted the generation of T(h)2 cells. Thus, the difference in H4/ICOS expression on activated CD4(+) T cells, which is regulated among the mouse strains, may also regulate the polarization of T(h) cells.     

Induction of NK1.1(+) alpha beta TCR(+) T cells by bypassing TCR signals in ZAP-70 deficient mice

The mechanism of development of a unique subset of T cells, thymic NK1.1(+) alpha beta T cells, has been poorly understood. We found that the development of thymic NK1.1(+) alpha beta T cells was defective in mice deficient in ZAP-70. Instead, an accumulation of NK1.1(+) TCR beta(-) NK-like population was detected in the thymus and spleen of the ZAP-70 deficient (ZAP -/-) mouse. In the present report, we examined whether biochemical treatments that replace TCR-mediated positive selection signals could restore the generation of thymic NK1.1(+) alpha beta T cells in ZAP -/- mice using the thymus organ culture. We found that a higher concentration of phorbol ester (PMA) than that required for CD4(+) T cell generation and ionomycin induced the generation of NK1.1(+) alpha beta T cells. Phenotypic analysis of the induced NK1.1(+) alpha beta T cell population suggested that these cells expressed CD8 but not CD4 molecules, which is a different characteristic from ordinary thymic NK1.1(+) alpha beta T cells. These results suggest that differential signaling is required for the generation of mainstream T cells and thymic NK1.1(+) alpha beta T cells.     

Elevated rate of Thelper1 (T(H)1) lymphocytes and serum IFN-gamma levels in psoriatic patients

Several disorders are known to be associated with altered Thelper1/Thelper2 (T(H)1/T(H)2) cytokine balance. Psoriasis is characterized by increased systemic and local production of T(H)1 and pro-inflammatory cytokines. Furthermore recent data indicate the dominant presence of T(H)1 lymphocytes in the circulation and T(H)1 and Tcytotoxic1 (T(C)1) cells in lesional skin of psoriatic patients. In order to assess the systemic T(H)1/T(H)2 imbalance in psoriasis most of the studies so far tested isolated peripheral mononuclear cells. As a new approach we applied a whole blood flow cytometric assay to determine the rate of circulating T(H)1/T(H)2 and T(C)1/Tcytotoxic2 (T(C)2) lymphocytes based on their intracellular IFN-gamma, IL-4 and IL-10 expression. Besides, serum levels of these cytokines were determined in healthy controls and psoriatic patients by commercial ELISAs. In psoriatic patients we found significantly (P<0.02) increased rates of CD4(+)/IFN-gamma(+) lymphocytes (30.3+/-8.8%) while the percent of CD4(+)/IL-4(+) cells (0.37+/-0.31%) were significantly (P<0.03) lower compared to healthy controls (CD4(+)/IFN-gamma(+): 20.1+/-7.3% and CD4(+)/IL-4(+): 0.78+/-0.44%). The IL-10-positive CD4(+) and CD8(+) cells also had higher rate in psoriasis, but the difference between patients and controls was not significant, similarly to the rate of CD8(+)/IFN-gamma(+) and CD8(+)/IL-4(+) lymphocytes. Beside cellular expression, serum IFN-gamma levels were also significantly higher (control: 4.9+/-6.4 pg/ml; psoriatic patients: 35.9+/-47.0 pg/ml; P<0.05). Our results provide further evidence for an altered T(H)1/T(H)2 balance in psoriasis measured in non-separated whole blood T cells.     

SAP regulates T(H)2 differentiation and PKC-theta-mediated activation of NF-kappaB1

XLP is caused by mutations affecting SAP, an adaptor that recruits Fyn to SLAM family receptors. SAP-deficient mice recapitulate features of XLP, including increased T cell activation and decreased humoral responses post-infection. SAP-deficient T cells also show increased TCR-induced IFN-gamma and decreased T(H)2 cytokine production. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca(2+) mobilization and ERK activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation, and nuclear NF-kappaB1/p50 levels. Similar defects were observed in Fyn-deficient cells. SLAM engagement amplified PKC-theta recruitment in wt but not SAP- or Fyn-deficient cells, arguing that a SAP/Fyn-mediated pathway enhances PKC-theta/NF-kappaB1 activation and suggesting a role for this pathway in T(H)2 regulation.