Molecular and functional characterization of mouse signaling lymphocytic activation molecule (SLAM): differential expression and responsiveness in Th1 and Th2 cells

Optimal T cell activation and expansion require engagement of the TCR plus costimulatory signals delivered through accessory molecules. SLAM (signaling lymphocytic activation molecule), a 70-kDa costimulatory molecule belonging to the Ig superfamily, was defined as a human cell surface molecule that mediated CD28-independent proliferation of human T cells and IFN-gamma production by human Th1 and Th2 clones. In this study, we describe the cloning of mouse SLAM and the production of mAb against it which reveal its expression on primary mouse T and B cells. Mouse SLAM is expressed on highly polarized Th1 and Th2 populations, and is maintained on Th1, but not on Th2 clones. Anti-mouse SLAM mAb augmented IFN-gamma production by Th1 cells and Th1 clones stimulated through the TCR, but did not induce IFN-gamma production by Th2 cells, nor their production of IL-4 or their proliferation. Mouse SLAM is a 75-kDa glycoprotein that upon tyrosine phosphorylation associates with the src homology 2-domain-containing protein tyrosine phosphatase SHP-2, but not SHP-1. Mouse SLAM also associates with the recently described human SLAM-associated protein. These studies may provide new insights into the regulation of Th1 responses.     

CD28 is not required for c-Jun N-terminal kinase activation in T cells.

Studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for activation of c-Jun N-terminal kinase (JNK), suggesting that JNK activity may mediate the costimulatory function of CD28. To examine the role of JNK signaling in CD28 costimulation in normal T cells, murine T cell clones and CD28(+/+) or CD28(-/-) TCR transgenic T cells were used. Although ligation with anti-CD28 mAb augmented JNK activation in Th1 and Th2 clones stimulated with low concentrations of anti-CD3 mAb, higher concentrations of anti-CD3 mAb alone were sufficient for JNK activation even in the absence of anti-CD28. JNK activity was comparably induced in both CD28(+/+) and CD28(-/-) 2C/recombinase-activating gene 2(RAG2)(-/-) T cells stimulated with anti-CD3 mAb alone, and with L(d)/peptide dimers, a direct alphabeta TCR ligand. Moreover, JNK activation was also detected in 2C/RAG2(-/-) T cells stimulated with P815 cells that express the relevant alloantigen L(d) whether or not B7-1 was coexpressed. However, IL-2 production by both Th1 clones and CD28(+/+) 2C/RAG2(-/-) T cells was detected only upon TCR and CD28 coengagement. Thus, CD28 coligation is not necessary, and stimulation through the TCR is sufficient, for JNK activation in normal murine T cells. The concept that JNK mediates the costimulatory function of CD28 needs to be reconsidered.     

Induction of competence to respond to IL-4 by CD4+ T helper type 1 cells requires costimulation.

Rested murine CD4+ Th1 clones do not produce IL-4, but have previously been shown to be capable of responding to IL-4 if they are first activated with Ag and APC. In this study, we have examined the activation requirements for induction of competence to respond to IL-4 in these clones. TCR occupancy alone (given either as chemically fixed APC and Ag, anti-CD3, Con A, or ionomycin and PMA) was inadequate, but the addition of a source of costimulation to any of these stimuli resulted in complete induction of competence to respond to IL-4. Pretreatment of the Th1 clones with TCR occupancy alone induced an anergic state from which subsequent full stimulation with Ag and APC failed to give IL-4 responsiveness. Pretreatment of the cells with IL-2 alone was an inadequate signal to induce IL-4 responsiveness and only a partial response was obtained when TCR occupancy was combined with IL-2. Addition of anti-IL-2 and anti-IL-2R antibodies during full activation with APC and Ag gave a 50% inhibition of competence induction. These results demonstrate that costimulation, in addition to its role in IL-2 production, is an important second signal for inducing T cells to become competent to respond to IL-4.     

IL-2 secretion and T cell clonal anergy are induced by distinct biochemical pathways.

In Th1 clones, TCR occupancy together with a costimulatory signal from APC results in IL-2 production. TCR occupancy alone results in unresponsiveness (anergy) to antigenic stimulation, a phenomenon that may be important for self-tolerance in vivo. Inasmuch as inositol phosphate production occurs during the induction of anergy other biochemical signals must be necessary for IL-2 production. Here we assess the role of tyrosine-specific protein kinases using the specific inhibitor, genistein. IL-2 secretion and responsiveness were very dependent on tyrosine-specific protein kinase activation and could be completely blocked under conditions where inositol phosphate generation occurred normally. Although anergy induction could also be blocked by inhibition of tyrosine-specific protein kinase activation this probably occurred indirectly via inhibition of inositol phospholipid hydrolysis. The differential susceptibility of IL-2 secretion and anergy induction to inhibition by genistein indicates that positive and negative outcomes of TCR occupancy may be mediated by distinct biochemical pathways.     

TCR-dependent cell response is modulated by the timing of CD43 engagement

Binding of Ag by the Ag receptor in combination with other stimuli provided by costimulatory receptors triggers the expansion and differentiation of T lymphocytes. However, it is unclear whether the time when costimulatory molecules interact with their counterreceptors with regards to Ag recognition leads to different T cell responses. Provided that the coreceptor molecule CD43 is a very abundant molecule evenly distributed on the membrane of T cell surface protruding 45 nm from the cell, we hypothesized that CD43 is one of the first molecules that interacts with the APC and thus modulates TCR activation. We show that engaging CD43 before or simultaneously with the TCR inhibited Lck-Src homology 2 domain containing phosphatase-1 interaction, preventing the onset of a negative feedback loop on TCR signals, favoring high levels of IL-2, cell proliferation, and secretion of proinflammatory cytokines and chemokines. In contrast, the intracellular signals resulting of engaging the TCR before CD43 were insufficient to induce IL-2 production and cell proliferation. Interestingly, when stimulated through the TCR and CD28, cells proliferated vigorously, independent of the order with which molecules were engaged. These results indicate that CD43 induces a signaling cascade that prolongs the duration of TCR signaling and support the temporal summation model for T cell activation. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune T cell signal quality, and ultimately immune function.     

Clonal anergy is induced in vitro by T cell receptor occupancy in the absence of proliferation.

Murine Th1 clones that receive signals through their TCR in the absence of APC-derived co-stimulatory signals do not produce IL-2 and instead become anergic, i.e., they are subsequently unable to produce IL-2 in response to Ag and normal APC. The critical cellular event required to prevent the induction of this anergic state appears to be T cell proliferation. Anergy was induced when T cell clones were stimulated under conditions where both TCR occupancy and costimulatory signals were provided but where proliferation in response to the IL-2 produced was prevented. Once induced, anergy could be reversed if the T cells were allowed to undergo multiple rounds of cell division. These results show that anergy is induced as a consequence of TCR occupancy in the absence of cell division; this can be achieved either by limiting IL-2 production because of deficient provision of co-stimulatory signals or by preventing response to IL-2.     

The processing and presentation of the self-antigen hemoglobin. Self-reactivity can be limited by antigen availability and costimulator expression.

APC do not distinguish between self- and foreign proteins. Previous studies from our laboratory demonstrated that most endogenous host APC constitutively processed and presented the self-Ag, hemoglobin (Hb), as detected by the Hb-specific T cell hybridoma, YO1.6. We have now examined APC in organs known to be involved in RBC degradation (liver Kupffer cells and splenic small resting B cells) for the presence of Hb/Ia complexes and for the expression of the costimulation necessary to trigger proliferation of T cell clones. We detected Hb/Ia complexes not only on splenic small resting B cells, but also on liver Kupffer cells. Interestingly, complexes were not present on lymph node small resting B cells. Splenic small resting B cells expressed costimulatory activity and efficiently stimulated the Th2 clones only. The opposite pattern was observed with liver Kupffer cells, which expressed costimulatory activity for Th1 clones only. However, if costimulatory activity was provided for the Th2 clones (IL-1 beta) and Th1 clones (allogenic spleen cells), the clones did proliferate in response to Kupffer cells and small resting B cells, respectively. In this report we have demonstrated that 1) endogenously formed self Hb/Ia complexes are expressed on splenic small resting B cells and liver Kupffer cells but not on lymph node small resting B cells and 2) these APC are also able to limit the expression of costimulatory activity for Th2 and Th1 T cell clones. Thus, endogenous APC not only constitutively process and present the self-Ag Hb, but also limit expression of the costimulatory activity necessary to trigger T cell proliferation against a self-Ag. The constitutive processing and presentation of self-Ag, as well as the regulation of costimulatory activity on APC, is likely an important feature of the maintenance of self-tolerance.     

Dual effects of Sprouty1 on TCR signaling depending on the differentiation state of the T cell

Sprouty (Spry) is known to be a negative feedback inhibitor of growth factor receptor signaling through inhibition of the Ras/MAPK pathway. Several groups, however, have reported a positive role for Spry involving sequestration of the inhibitory protein c-Cbl. Thus, Spry may have various functions in the regulation of receptor-mediated signaling depending on the context. In the immune system, the function of Spry is unknown. In this study, we investigated the role of Spry1 in T cell activation. Spry1, among the four mammalian homologs, was specifically induced by TCR signaling of CD4(+) murine T cells. In fully differentiated Th1 clones, overexpressed Spry1 inhibited TCR signaling and decreased IL-2 production while reducing expression with specific siRNA transfection had the opposite effect, increasing IL-2 production. In contrast, in naive T cells, Spry1 overexpression enhanced TCR signaling, and increased proliferation and IL-2 production, while siRNA transfection again had the opposite effect, reducing IL-2 production following activation. The enhancing effect in naive cells was abrogated by preactivation of the T cells with Ag and APC, indicating that the history of exposure to Ag is correlated with a hierarchy of T cell responsiveness to Spry1. Furthermore, both the NF-AT and MAPK pathways were influenced by Spry1, implying a different molecular mechanism from that for growth factor receptor signaling. Thus, Spry1 uses a novel mechanism to bring about differential effects on TCR signaling through the same receptor, depending on the differentiation state of the T cell.     

Intercellular adhesion molecule-2, a second counter-receptor for CD11a/CD18 (leukocyte function-associated antigen-1), provides a costimulatory signal for T-cell receptor-initiated activation of human T cells.

Activation of T cells often requires both activation signals delivered by ligation of the TCR and those resulting from costimulatory interactions between certain T cell surface accessory molecules and their respective counter-receptors on APC. CD11a/CD18 complex on T cells modulate the activation of T cells by interacting with its counter-receptors intracellular adhesion molecule (ICAM-1) (CD54) and/or ICAM-2 on the surface of APC. The costimulatory ability of ICAM-1 has been demonstrated. Using a soluble ICAM-2 Ig fusion protein (receptor globulin, Rg) we demonstrate the costimulatory effect of ICAM-2 during the activation of CD4+ T cells. When coimmobilized with anti-TCR-1 mAb ICAM-2 Rg induced vigorous proliferative response of CD4+ T cells. This costimulatory effect of ICAM-2 was dependent on its coimmobilization with mAb directed at the CD3/TCR complex but not those directed at CD2 or CD28. Both resting as well as Ag-primed CD4+ T cells responded to the costimulatory effects of ICAM-2. The addition of mAb directed at the CD11a or CD18 molecules almost completely inhibited the responses to ICAM-2 Rg. These results are consistent with the role of CD11a/CD18 complex as a receptor for ICAM-2 mediating its costimulatory effects. Stimulation of T cells with coimmobilized anti-TCR-1 and ICAM-2 resulted in the induction of IL-2R (CD25), and anti-Tac (CD25) mAb inhibited this response suggesting the contribution of endogenously synthesized IL-2 during this stimulation. These results demonstrate that like its homologue ICAM-1, ICAM-2 also exerts a strong costimulatory effect during the TCR-initiated activation of T cells. The costimulatory effects generated by the CD11a/CD18:ICAM-2 interaction may be critical during the initiation of T cell activation by ICAM-1low APC.     

Counter-antigen presentation: fibroblasts produce cytokines by signalling through HLA class II molecules without inducing T-cell proliferation

Fibroblasts are known to express histocompatibility leukocyte antigen DR (HLA-DR) molecules on their cell surface upon stimulation with interferon gamma (IFN- gamma), while the exact roles of HLA-DR on fibroblasts remain undetermined. To understand the role of HLA-DR molecules on fibroblasts, we examined whether: (1) fibroblasts act as antigen presenting cells (APC) which activate helper T (Th) cells; and/or (2) fibroblasts are activated via HLA-II molecules by making a T-cell receptor (TCR)-peptide-major histocompatibility complex (MHC) complex. We used Th(0) clone HT8.3, which recognizes an antigenic peptide (Ag53 p141-161) in the context of DRB1*1501, as well as IFN - gamma - treated and irradiated periodontal ligament fibroblasts (PDL) expressing DRB1*1501 molecules. When peptide-pulsed fibroblasts were co-incubated with HT8.3 treated by the protein synthesis inhibitor emetine, peptide-induced de novo expression of lymphokines and cell-surface molecules on T cells can be neglected. The antigen presenting capacity of these fibroblasts was evaluated by examining the proliferative responses of Th cells. Possible activation of fibroblasts by stimulation via HLA-DR molecules was evaluated by quantitating secreted cytokines in the supernatants after 18-h culture with or without anti-HLA-DR monoclonal antibody (mAb) or emetine-treated HT8.3. Indeed, Th cells did not show proliferative responses when peptide-pulsed PDL were used as APC, whereas PDL produced larger amounts of interleukin (IL) 6, IL-8, monocyte chemoattractant protein 1 (MCP-1) and regulated upon activation, normal T expressed and secreted (RANTES) compared with controls, when cultured with anti-HLA-DR mAb or emetine-treated HT8.3. These findings suggest that HLA-DR expressed on fibroblasts do not present antigens to induce T-cell proliferation, but may act as receptor molecules that transmit signals into fibroblasts, based on DR-peptide-TCR interaction, resulting in the secretion of several cytokine species.     

T cell activation through TCR/-CD3 complex. IL-2 production of T cell clones stimulated with anti-CD3 without cross-linkage.

To elucidate the Th cell activation mechanism through the TCR/CD3 complex, we examined the reactivity of T cell clones to soluble monovalent and divalent anti-CD3 without accessory cells or costimulatory factor. All T cell clones tested produced IL-2 in response to monovalent anti-CD3, although reactivity to divalent anti-CD3 was variable depending upon clones. IL-2 production of T cell clones induced by monovalent anti-CD3 was suppressed by cross-linking of the antibody with anti-hamster IgG. IL-2 mRNA expression and the increment of intracellular Ca2+ concentration were consistent with the IL-2 production. When T cell clones were stimulated with monovalent anti-CD3, they increased in size, although divalent anti-CD3 stimulation did not affect their size irrespective of their IL-2 production. These results indicate that monovalent anti-CD3 is more efficient than divalent anti-CD3 in induction of IL-2 production and that the cross-linkage of the TCR/CD3 complex is not necessarily required for the T cell clone activation.     

Purified HLA class II peptide complexes can induce adherence and activation of peptide-specific human T cell clones.

An initial event in T cell activation is the specific adherence of T cells via their T cell receptor to the MHC peptide complex. We have studied this adherence by incubating T cells with preformed HLA DR4Dw4 peptide complexes attached to a solid support. Adherence of sodium 51Cr-labeled T cell clones specific for the influenza hemagglutinin peptide, HA 307-319, was maximal after 15 min and was specific for the HLA DR4Dw4-HA 307-319 complex. The binding was temperature dependent and could be blocked with azide or protein kinase C inhibitors, indicating that for adherence the T cells need to be metabolically active and have a functioning protein kinase C pathway. The adherence could be blocked with CD4- or CD3-reactive murine mAb, suggesting that the TCR and CD4 molecules work in concert to induce strong adherence to the HLA DR4Dw4-HA 307-319 complex. A subsequent event in T cell activation is proliferation, which is thought to need additional proteins such as IL-1 or other adhesion molecules. MHC peptide complexes coated on microtiter plates also induced proliferation in the human T cell clones. Removal of any monocytes by treatment of human T cell clones with anti-CD14 in conjunction with C, followed by purification over a nylon wool column, did not abrogate proliferation. After prolonged culture of the T cell clones in plates coated with peptide-pulsed HLA DR4Dw4 in the presence of IL-2, the T cell clones continued to proliferate in response to peptide. These results suggest that human T cell clones do not require a second signal from a monocyte or other APC to proliferate.     

Rapid identification of antigenic T-cell epitopes by extracellular acidification rate signals

We used a silicon-based biosensor, a microphysiometer, to measure real-time extracellular acidification rate signals associated with T lymphocyte responses to peptide ligands interacting with the T-cell receptor (TCR). We compared these effector responses with those of interferon-gamma (IFN-gamma) production, and T-cell proliferation. Within minutes, major histocompatibility complex (MHC)-bound peptides on antigen-presenting cells (APCs) engaged the TCR to increase acidification rates of the extracellular media was measured by microphysiometer. We exposed two myelin peptide-specific human T-cell clones, MSF132E11 (DRB1*1501 restricted) and TOM3A6 (DRB5*0101 restricted), to truncated analogues of the parent MBP 84-102 peptide, in the presence of MHC restricted human antigen-presenting cells, and measured the extracellular acidification rate signal changes, IFN-gamma production and T-cell proliferation. The core epitopes recognized by these clones were identified by microphysiometer and found to be MBP 88-100 and MBP 91-100, respectively. These epitopes were identical to those identified by the IFN-gamma and proliferation assays. We conclude that measurement of real-time extracellular acidification rate signals by the microphysiometer may facilitate rapid identification of human T-cell epitopes involved in immune disorders and the development of specific T-cell antagonists.     

Threshold signaling of human Th0 cells in activation and anergy: modulation of effector function by altered TCR ligand

Molecular interactions between TCR and its natural ligand, in the presence of costimulatory signals, elicit T cell effector functions, whereas subtle changes in the structure of antigenic peptides may induce only selected T cell effector function including anergy. In this study, we have investigated the immunological activity of an altered TCR ligand (p 2, 28-40A34,36) derived from the immunodominant T cell epitope of the group 2 allergen of house dust mite, in which residues at positions 34 and 36 were substituted by alanine. Elevated IFN-gamma synthesis was induced by equimolar concentrations of the analogue compared with native peptide (p 2, 28-40) and was paralleled by increased down-regulation of cell surface CD3. IL-5 and IL-10 production exhibit the same sensitivity to both peptides, implying that the induction of T cell effector functions are not all proportional to TCR occupancy. Both native peptide and the analogue bound to MHC class II (DRB1*1101) molecules with similar affinities. Furthermore, p 2, 28-40A34,36 induced T cell anergy at lower concentrations than native peptide. During the induction of anergy, TGF-beta production was comparable for both peptides, whereas IL-10 secretion was markedly increased but more so in response to p 2, 28-40A34,36. Membrane expression of costimulatory ligands CD80 and CD86 was similar for native peptide and p 2, 28-40A34,36 and increased in activation, whereas only CD86 was elevated during anergy. The modulation of T cell effector function with altered TCR ligands may have practical applications in reprogramming allergic inflammatory responses through the induction of T cell anergy and/or the promotion of Th1 cytokines.     

Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling

BACKGROUND: The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. RESULTS: Here we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. CONCLUSIONS: The Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.     

Antigen presentation by resting B cells. Effectiveness at inducing T cell proliferation is determined by costimulatory signals, not T cell receptor occupancy

Resting B cells stimulated the proliferation of two T cell clones much less efficiently than T cell-depleted low-density APC. In contrast, low-density cells and resting B cells stimulated the clones to produce similar levels of inositol phosphates, a rapid biochemical event dependent only on occupancy of the TCR. The inefficient stimulation of T cell proliferation by resting B cell APC was dramatically improved by the addition of allogeneic low-density accessory cells incapable of being recognized by the TCR on the responding T cells. The results are most consistent with a model where low-density and resting B cell APC display similar amounts of Ag/Ia molecule complexes capable of being recognized by the TCR on the responding T cells but differ in the provision of costimulatory signals that, together with TCR occupancy, are required for IL-2 production.     

CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells.

CD4+ T cells require two signals to produce maximal amounts of IL-2, i.e., TCR occupancy and an unidentified APC-derived costimulus. Here we show that this costimulatory signal can be delivered by the T cell molecule CD28. An agonistic anti-CD28 mAb, but not IL-1 and/or IL-6, stimulated T cell proliferation by tetanus toxoid-specific T cells cultured with Ag-pulsed, costimulation-deficient APC. Furthermore, the ability of B cell tumor lines to provide costimulatory signals to purified T cells correlated well with expression of the CD28 ligand B7/BB-1. Finally, like anti-CD28 mAb, autologous human APC appeared to stimulate a cyclosporine A-resistant pathway of T cell activation. Together, these results suggest that the two signals required for IL-2 production by CD4+ T cells can be transduced by the TCR and CD28.     

CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation.

Untransformed CD4(+) Th1 cells stimulated with Ag and APC demonstrated a dependence on B7- and CD28-mediated costimulatory signals for the expression and function of AP-1 proteins. The induction of transactivation by the c-fos gene regulator Elk-1 mirrored this requirement for TCR and CD28 signal integration. c-Jun N-terminal kinase (JNK) (but not extracellular signal-regulated kinase or p38) protein kinase activity was similarly inhibited by neutralizing anti-B7 mAbs. Blockade of JNK protein kinase activity with SB 202190 prevented both Elk-1 transactivation and c-Fos induction. These results identify a unique role for B7 costimulatory molecules and CD28 in the activation of JNK during Ag stimulation in Th1 cells, and suggest that JNK regulates Elk-1 transactivation at the c-fos gene to promote the formation of AP-1 complexes important to IL-2 gene expression.