EphrinB1 is essential in T-cell-T-cell co-operation during T-cell activation

Eph kinases are the largest family of receptor tyrosine kinases, and their ligands are ephrins (EFNs), which are also cell surface molecules. We have very limited knowledge about the expression and function of these kinases and their ligands in the immune system. In this study we investigated the effect of EFNB1 on mouse T-cells. EFNB1 and the Eph kinases it interacts with (collectively called EFNB1 receptors (EFNB1R)) were expressed on T-cells, B cells, and monocytes/macrophages. Some T-cells were double positive for EFNB1 and EFBB1R. Solid phase EFNB1 in the presence of suboptimal TCR ligation augmented T-cell responses in terms interferon-gamma secretion, proliferation, and cytotoxic T lymphocyte activity but not interleukin-2 production. After T-cell receptor (TCR) ligation, EFNB1R congregated to TCR caps, and then both of them translocated to raft caps. This provides a morphological basis for EFNB1R to enhance TCR signaling. Further downstream of the signaling pathway, EFNB1R stimulation led to increased LAT (linker for activation of T-cells) phosphorylation and p44/42 and p38 MAPK activation. Similar to CD28 costimulation, EFNB1R costimulation was insensitive to cyclosporin A inhibition. On the other hand, unlike the former, EFNB1R costimulation failed to activate Akt, which is essential in triggering interleukin-2 production. Our study suggests that EFNB1 is pivotal in T-cell-T-cell costimulation and in reducing T-cell response threshold to antigen stimulation.     

Mouse ephrinB3 augments T-cell signaling and responses to T-cell receptor ligation

Ephrins (EFN) are cell-surface ligands of Ephs, the largest family of cell-surface receptor tyrosine kinases. The function of EFNs in the immune system has not been well studied, although some EFNs and Ephs are expressed at high levels on certain leukocytes. We report here that EFNB3 and its receptors (collectively called EFNB3Rs, as EFNB3 binds to multiple EphBs) were expressed in peripheral T cells and monocytes/macrophages, with T cells being the dominant EFNB3+ and EFNB3R+ cell type. Solid-phase EFNB3-Fc in the presence of suboptimal anti-CD3 crosslinking enhanced T-cell responses in terms of proliferation, activation marker expression, interferon-gamma but not interleukin-2 production, and cytotoxic T-cell activity. EFNB3R costimulation in the presence of phorbol 12-myristate 13- acetate was insensitive to cyclosporin A, similar to CD28 costimulation, suggesting they might share a part of the signaling pathway. After crosslinking, T-cell receptor and EFNB3R congregated into aggregated rafts, and this provided a morphological basis for signaling pathways of T-cell receptor and EFNB3R to interact. Solid-phase EFNB3-Fc augmented p38 and p44/42 MAPK activation further downstream of the signaling pathway. These data suggest that EFNB3 is important in T-cell/T-cell and T-cell/antigen-presenting cell collaboration to enhance T-cell activation and function.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

Ephrin-B1 is critical in T-cell development

Eph kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. In this study, we investigated the role of EFNB1 and the Ephs it interacts with (collectively called EFNB1 receptors) in mouse T-cell development. In the thymus, CD8 single positive (SP) and CD4CD8 double positive (DP) cells expressed high levels of EFNB1 and EFNB1 receptors, whereas CD4 SP cells had moderate expression of both. Soluble EFNB1-Fc in fetal thymus organ culture caused significant subpopulation ratio skew, with increased CD4 SP and CD8 SP and decreased DP percentage, while the cellularity of the thymus remained constant. Moreover, in EFNB1-treated fetal thymus organ culture, CD117(+), CD25(+), DP, CD4 SP, and CD8 SP cells all had significantly enhanced proliferation history, according to bromodeoxyuridine uptake. In vitro culture of isolated thymocytes revealed that EFNB1-Fc on solid-phase protected thymocytes from anti-CD3-induced apoptosis, with concomitant augmentation of several antiapoptotic factors, particularly in CD4 SP and CD8 SP cells; on the other hand, soluble EFNB1-Fc promoted anti-CD3-induced apoptosis, as was the case in vivo. This study reveals that EFNB1 and EFNB1 receptors are critical in thymocyte development.     

Cross-linking of EphB6 resulting in signal transduction and apoptosis in Jurkat cells

Eph kinases are the largest family of receptor tyrosine kinases (RTK), and their ligands are cell surface molecules. The known functions of Eph kinases are mainly pattern formation in the CNS. Although several Eph kinases are expressed at high levels in hemopoietic cells and in the thymus, we have no knowledge of the functions of any Eph kinase in the immune system. In this study, we have demonstrated that an Eph kinase, EphB6, was expressed at high levels in Jurkat leukemic T cells. Co-cross-linking of EphB6 and CD3 led to an altered profile of lymphokine secretion along with proliferation inhibition of Jurkat cells. The cells subsequently underwent Fas-mediated apoptosis. Although EphB6 has no intrinsic kinase activity, its cross-linking triggered general protein tyrosine phosphorylation in Jurkat cells. EphB6 was found to associate with a number of molecules in the signaling pathways, notably Cbl. EphB6 cross-linking resulted in Cbl dephosphorylation and dissociation from Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1). Our results show that EphB6 has important functions in T cells, and it can transduce signals into the cells via proteins it associates with.     

Ephrin-A1 suppresses Th2 cell activation and provides a regulatory link to lung epithelial cells

Gene expression screening showed decreased ephrin-A1 expression in CD4+ T cells of asthma patients. Ephrin-A1 is the ligand of the Eph receptor family of tyrosine kinases, forming the largest family of receptor tyrosine kinases. Their immune regulatory properties are largely unknown. This study demonstrates significantly reduced ephrin-A1 expression in T cells of asthma patients using real time-PCR. Immunohistological analyses revealed strong ephrin-A1 expression in lung tissue and low expression in cortical areas of lymph nodes. It is absent in T cell/B cell areas of the spleen. Colocalization of ephrin-A1 and its receptors was found only in the lung, but not in lymphoid tissues. In vitro activation of T cells reduced ephrin-A1 at mRNA and protein levels. T cell proliferation, activation-induced, and IL-2-dependent cell death were inhibited by cross-linking ephrin-A1, and not by engagement of Eph receptors. However, anti-EphA1 receptor slightly enhances Ag-specific and polyclonal proliferation of PBMC cultures. Furthermore, activation-induced CD25 up-regulation was diminished by ephrin-A1 engagement. Ephrin-A1 engagement reduced IL-2 expression by 82% and IL-4 reduced it by 69%; the IFN-gamma expression remained unaffected. These results demonstrate that ephrin-A1 suppresses T cell activation and Th2 cytokine expression, while preventing activation-induced cell death. The reduced ephrin-A1 expression in asthma patients may reflect the increased frequency of activated T cells in peripheral blood. That the natural ligands of ephrin-A1 are most abundantly expressed in the lung may be relevant for Th2 cell regulation in asthma and Th2 cell generation by mucosal allergens.     

Activation-induced nonresponsiveness: a Th-dependent regulatory checkpoint in the CTL response.

CD8 T cells undergo autocrine IL-2-dependent proliferation upon TCR engagement and costimulation, but within 3-4 days, they become activation-induced nonresponsive (AINR) and display a split anergy. They can lyse targets and secrete IFN-gamma but they cannot produce IL-2 in response to TCR ligation and costimulation, due at least in part to an inability to up-regulate mitogen-activated protein kinases and IL-2 mRNA. Exogenous IL-2 can drive continued proliferation of AINR cells and nonresponsiveness is reversed within 1-2 days so that Ag-driven proliferation can resume. Mitogen-activated protein kinases and IL-2 mRNA can again be up-regulated, but "rewiring" has occurred so that these events no longer depend upon costimulation; TCR engagement is sufficient. Development of AINR appears to be a normal part of the differentiation program of CD8 T cells, providing a regulatory checkpoint to convert the initial helper-independent response to one that depends upon CD4 T cell help for continued expansion of the effector CTL. Once permission is given, in the form of IL-2, to pass this checkpoint, the CTL can make a prolonged response to persisting Ag in the absence of further CD4 T cell help.     

Tec kinase signaling in T cells is regulated by phosphatidylinositol 3-kinase and the Tec pleckstrin homology domain

Tec, the prototypical member of the Tec family of tyrosine kinases, is abundantly expressed in T cells and other hemopoietic cell types. Although the functions of Itk and Txk have recently been investigated, little is known about the role of Tec in T cells. Using antisense oligonucleotide treatment to deplete Tec protein from primary T cells, we demonstrate that Tec plays a role in TCR signaling leading to IL-2 gene induction. Interestingly, Tec kinases are the only known family of tyrosine kinases containing a pleckstrin homology (PH) domain. Using several PH domain mutants overexpressed in Jurkat T cells, we show that the Tec PH domain is required for Tec-mediated IL-2 gene induction and TCR-mediated Tec tyrosine phosphorylation. Furthermore, we show that Tec colocalizes with the TCR after TCR cross-linking, and that both the Tec PH and Src homology (SH) 2 domains play a role in this association. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, abolishes Tec-mediated IL-2 gene induction and Tec tyrosine phosphorylation, and partially suppresses Tec colocalization with the activated TCR. Thus, our data implicate the Tec kinase PH domain and phosphatidylinositol 3-kinase in Tec signaling downstream of the TCR.     

Induction of a Th1 response from Th2-polarized T cells by activated dendritic cells: dependence on TCR:peptide-MHC interaction, ICAM-1, IL-12, and IFN-gamma

Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of costimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this study, we demonstrate that the addition of activated DC to spleen cultures containing established Th2-polarized CD4(+) T cells was sufficient to suppress Th2 and induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite Ag specificity. The DC activator B7-DC cross-linking Ab (XAb) was >10,000-fold more efficient at inducing phenotype reversal than the TLR agonists CpG-oligodeoxynucleotide and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR:peptide-MHC complex, the expression of the costimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFN-gamma by the activated DC. The requirement for the costimulation/adhesion molecule SLAM (signaling lymphocytic activation molecule) was found to be quantitative. Thus, activation of DC, particularly by crosslinking B7-DC, can modulate well-established Th2 T cell responses in an Ag-specific manner. Because the regulation of mouse and human DC by B7-DC XAb overlaps in several significant ways, immune modulation with B7-DC XAb is a potential strategy for treating Th2-mediated diseases.     

Effective proliferation of human regulatory T cells requires a strong costimulatory CD28 signal that cannot be substituted by IL-2

The strength of immune repression by regulatory T (Treg) cells is thought to depend on the efficiency of Treg cell activation. The stimuli and their individual strength required to activate resting human Treg cells, however, have so far not been elucidated in detail. We reveal here that induction of proliferation of human CD4(+)C25(+) Treg cells requires an extraordinary strong CD28 costimulatory signal in addition to TCR/CD3 engagement. CD28 costimulation, noteworthy, cannot be substituted by IL-2 to induce proliferation of Treg cells, which is in contrast to CD4(+)CD25(-) T cells. IL-2, in contrast, prevents spontaneous apoptosis of Treg cells, but does not initiate their amplification. IL-2 and CD28 costimulation clearly exhibit disparate effects on Treg cells which are in contrast to those on CD4(+)CD25(-) T cells. Moreover, the prerequisites for Treg cell proliferation differ strikingly from those for effector T cells, implying a balanced orchestration in initiating and limiting a T cell immune response. In addition, data are of relevance for the design of therapeutic strategies involving IL-2 administration and CD28 costimulation.     

A regulatory role for CD37 in T cell proliferation

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37(-/-)) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37(-/-) mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37(-/-) T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37(-/-) T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56(Lck) kinase activity was increased in CD37(-/-) T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.     

CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation.

We investigate, here, the mechanism of the costimulatory signals for CD8 T cell activation and confirm that costimulation signals via CD28 do not appear to be required to initiate proliferation, but provide survival signals for CD8 T cells activated by TCR ligation. We show also that IL-6 and TNF-alpha can provide alternative costimulatory survival signals. IL-6 and TNF-alpha costimulate naive CD8 T cells cultured on plate-bound anti-CD3 in the absence of CD28 ligation. They act directly on sorted CD8-positive T cells. They also costimulate naive CD8 T cells from Rag-2-deficient mice, bearing transgenic TCRs for HY, which lack memory cells, a potential source of IL-2 secretion upon activation. IL-6 and TNF-alpha provide costimulation to naive CD8 T cells from CD28, IL-2, or IL-2Ralpha-deficient mice, and thus function in the absence of the B7-CD28 and IL-2 costimulatory pathways. The CD8 T cell generated via the anti-CD3 plus IL-6 and TNF-alpha pathway have effector function in that they express strong cytolytic activity on Ag-specific targets. They secrete only very small amounts of any of the cytokines tested upon restimulation with peptide-loaded APC. The ability of the naive CD8 T cells to respond to TCR ligation and costimulatory signals from IL-6 and TNF-alpha provides a novel pathway that can substitute for signals from CD4 helper cells or professional APC. This may be significant in the response to viral Ags, which can be potentially expressed on the surface of any class I MHC-expressing cell.     

Monocyte-driven activation-induced apoptotic cell death of human T-lymphotropic virus type I-infected T cells.

We attempted apoptotic cell death induction of T cells infected with human T lymphotropic virus type I (HTLV-I) which induces HTLV-I-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. T cells acutely infected and expressing HTLV-Igag Ags were killed by cross-linking their TCR with anti-CD3 mAb. Cells in apoptotic process were found by staining with annexin V. The apoptosis was not affected by costimulation through CD28 molecules and was resistant to ligation of Fas molecules. Whereas the virus-infected T cells expressed higher levels of HLA-DR, CD25, CD80, and CD86 Ags than apoptosis-resistant PHA-blasts, the T cell apoptosis was enhanced by addition of exogenous IL-2. Furthermore, in this apoptosis, monocytes played an important role because T cells infected in the absence of monocytes were resistant to the death signals. The apoptosis-sensitive T cells responded to TCR signaling more strongly by proliferating than those apoptosis-resistant cells. Monocytes weakly affected the expression levels of viral Ags on T cells. However, HTLV-I-infected monocytes primed T cells to die by subsequent TCR signaling. T cells primed with the monocytes, subsequently infected in the absence of monocytes, were killed by TCR signaling. These observations suggest that primed and infected T cells could be killed by activation-induced cell death.     

The poststimulation program of CD4 versus CD8 T cells (death versus activation-induced nonresponsiveness)

Both CD8 and CD4 T cells undergo autocrine IL-2-induced proliferation and clonal expansion following stimulation with Ag and costimulation. The CD8 T cell response is transient because the cells rapidly become activation-induced nonresponsive (AINR) and exhibit split anergy. In these cells, the capacity for IL-2 production is lost, but TCR-mediated IFN-gamma production and cytotoxicity are maintained. At this point, the CTL become dependent on IL-2 provided by CD4 Th cells for continued expansion. If IL-2 is available to support expansion for a brief period, AINR is reversed and the cells regain the ability to produce IL-2. In this study, we show that CD4 T cells do not become AINR, but instead are rendered susceptible to Fas-mediated activation-induced cell death following stimulation through TCR and CD28. Using z-VAD-fmk or anti-Fas ligand mAb to inhibit cell death, we demonstrate that previously activated CD4 T cells retain the ability to up-regulate c-Jun N-terminal kinase activity and IL-2 mRNA levels upon TCR engagement and no longer require costimulation. This rewiring of signaling pathways is similar to that seen following reversal of AINR in CD8 T cells. Thus, CD8 and CD4 T cells appear to use distinct mechanisms, AINR and activation-induced cell death, respectively, to limit excessive clonal expansion following a productive response, while permitting important effector functions to be expressed.     

TCR signals mediated by Src family kinases are essential for the survival of naive T cells

The role of TCR signals triggered by recognition of self MHCs in maintaining the survival of naive peripheral T cells remains controversial. Here we examine the role of the Src family kinases, p56(lck) (Lck) and p59(fyn) (Fyn), in the survival of naive T cells. We show that long term survival requires a combination of signals transduced by Src family kinases and signals through the IL-7R. In the absence of either one, naive T cells die slowly, but if both signals are removed, cell loss is greatly accelerated. The TCR signal can be mediated by either Fyn or Lck at wild-type levels of expression, but not by Lck alone if expressed suboptimally. The disappearance of T cells in the absence of Fyn and Lck was associated with a complete loss of TCRzeta-chain phosphorylation and down-regulation of CD5, both of which are also MHC contact dependent, indicating that the Src family kinases are critical for transducing a TCR-MHC survival signal.     

TNF type 2 receptor (p75) lowers the threshold of T cell activation.

T cell activation requires a threshold amount of TCR-mediated signals, an amount that is reduced by signals mediated through costimulatory molecules expressed on the T cell surface. Here the role of TNFR2 (p75) as a putative costimulatory receptor for T cell activation was examined. It was found that p75 deficiency in CD8(+) T cells increased the requirements for TCR agonist approximately 5-fold. Furthermore, p75(-/-) T cells display a marked reduction in the proliferative response to TCR agonist. This hypoproliferative response was associated with delayed kinetics of induction of the acute activation markers CD25 and CD69 as well as a marked decrease in the production of IL-2 and IFN-gamma. The net result is that very few cells are recruited into the dividing population. Interestingly, CD28 costimulation was only partially effective in rescuing the proliferative defect of p75(-/-)CD8(+) T cells. Thus, p75 provides an important costimulatory signal in addition to that provided by CD28 toward optimal T cell proliferation.     

Negative-feedback regulation of CD28 costimulation by a novel mitogen-activated protein kinase phosphatase, MKP6

TCR and CD28 costimulatory receptor-cooperative induction of T cell IL-2 secretion is dependent upon activation of mitogen-activated protein (MAP) kinases. Using yeast-hybrid technology, we cloned a novel CD28 cytoplasmic tail (CD28 CYT) interacting protein, MAP kinase phosphatase-6 (MKP6), which we demonstrate inactivates MAP kinases. Several lines of evidence indicate that MKP6 plays an important functional role in CD28 costimulatory signaling. First, in human peripheral blood T cells (PBT), expression of MKP6 is strongly up-regulated by CD28 costimulation. Second, transfer of dominant-negative MKP6 to PBT with the use of retroviruses primes PBT for the secretion of substantially larger quantities of IL-2, specifically in response to CD28 costimulation. A similar enhancement of IL-2 secretion is observed neither in response to TCR plus CD2 costimulatory receptor engagement nor in response to other mitogenic stimuli such as phorbol ester and ionomycin. Furthermore, this hypersensitivity to CD28 costimulation is associated with CD28-mediated hyperactivation of MAP kinases. Third, a retroviral transduced chimeric receptor with a CD28 CYT that is specifically unable to bind MKP6 costimulates considerably larger quantities of IL-2 from PBT than a similar transduced chimeric receptor that contains a wild-type CD28 CYT. Taken together, these results suggest that MKP6 functions as a novel negative-feedback regulator of CD28 costimulatory signaling that controls the activation of MAP kinases.     

Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking

Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.