CD28 co-stimulation restores T cell responsiveness in NOD mice by overcoming deficiencies in Rac-1/p38 mitogen-activated protein kinase signaling and IL-2 and IL-4 gene transcription.

Previously, we reported that T cell hyporesponsiveness induced by TCR ligation is causal to autoimmune diabetes in NOD mice. Neonatal CD28 co-stimulation reverses T cell hyporesponsiveness and protects NOD mice from diabetes by an IL-4-mediated mechanism, indicating that a deficiency in TCR signaling may be overcome by CD28/B7-2 co-stimulation in NOD T cells. To investigate which co-stimulation-induced signaling events mediate this protection, we analyzed the activity of Ras, Rac-1, mitogen-activated protein kinases (MAPK) and several transcription factors in TCR-activated NOD T cells in the presence or absence of CD28 co-stimulation. We show that CD28 co-stimulation restores normal TCR-induced activation of Rac-1 and p38 MAPK in NOD T cells. Deficiencies in TCR-induced nuclear expression of activating protein (AP)-1 binding proteins as well as activation of AP-1 and NF-AT in the IL-2 and IL-4 P1 promoters are also corrected by CD28 co-stimulation. Thus, CD28 co-stimulation reverses NOD T cell hyporesponsiveness by restoring TCR signaling leading to the activation of AP-1 and NF-AT during IL-2 and IL-4 gene transcription. Our findings provide additional evidence that CD28 co-stimulation amplifies signals delivered by the TCR and further explain the mechanism by which CD28 co-stimulation may protect against autoimmune diabetes.

Regulation of fas ligand expression during activation-induced cell death in T cells by p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.

Activation-induced cell death (AICD) is a mechanism of peripheral T cell tolerance that depends upon an interaction between Fas and Fas ligand (FasL). Although c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) may be involved in apoptosis in various cell types, the mode of regulation of FasL expression during AICD in T cells by these two MAPKs is incompletely understood. To investigate the regulatory roles of these two MAPKs, we analyzed the kinetics of TCR-induced p38 MAPK and JNK activity and their regulation of FasL expression and AICD. We report that both JNK and p38 MAPK regulate AICD in T cells. Our data suggest a novel model of T cell AICD in which p38 MAPK acts early to initiate FasL expression and the Fas-mediated activation of caspases. Subsequently, caspases stimulate JNK to further upregulate FasL expression. Thus, p38 MAPK and downstream JNK converge to regulate FasL expression at different times after T cell receptor stimulation to elicit maximum AICD.

ZAP-70 is essential for the T cell antigen receptor-induced plasma membrane targeting of SOS and Vav in T cells.

Translocation of the SOS and Vav GDP/GTP exchange factors proximal to Ras and Rac GTPases localized in the plasma membrane glycolipid-enriched microdomains is a pivotal step required for T cell antigen receptor-induced T cell activation. Here we demonstrate that the T cell antigen receptor zeta-chain-associated ZAP-70 kinase and T cell antigen receptor zeta-chain immunoreceptor tyrosine-based activation motifs are essential for the membrane recruitment of SOS and Vav. Plasma membrane targeting of SOS or Vav begins with the assembly of ZAP-70 with Grb-2 and SOS. The subsequent tyrosine phosphorylation of LAT (linker for activation of T cell) by ZAP-70 leads to a shift in equilibrium from the ZAP-70.Grb-2.SOS(Vav) complex to the (Vav)SOS.Grb-2.LAT complex. This shift results in the targeting of SOS and Vav into glycolipid-enriched microdomains and initiation of the Ras and Rac signaling cascades involved in T cell activation, proliferation, and cytokine production.

TCR and CD28 are coupled via ZAP-70 to the activation of the Vav/Rac-1-/PAK-1/p38 MAPK signaling pathway.

CD28 costimulation amplifies TCR-dependent signaling in activated T cells, however, the biochemical mechanism(s) by which this occurs is not precisely understood. The small GTPase Rac-1 controls the catalytic activity of the mitogen-activated protein kinases (MAPKs) and cell cycle progression through G1. Rac-1 activation requires the phospho-tyrosine (p-Tyr)-dependent recruitment of the Vav GDP releasing factor (GRF) to the plasma membrane and assembly of GTPase/GRF complexes, an event critical for Ag receptor-triggered T cell activation. Here, we show that TCR/CD28 costimulation synergistically induces Rac-1 GDP/GTP exchange. Our findings, obtained by using ZAP-70-negative Jurkat T cells, indicate that CD28 costimulation augments TCR-mediated T cell activation by increasing the ZAP-70-mediated Tyr phosphorylation of Vav. This event regulates the Rac-1-associated GTP/GDP exchange activity of Vav and downstream pathway(s) leading to PAK-1 and p38 MAPK activation. CD28 amplifies TCR-induced ZAP-70 activity and association of Vav with ZAP-70 and linker for activation of T cells (LAT). These results favor a model in which ZAP-70 regulates the intersection of the TCR and CD28 signaling pathways, which elicits the coupling of TCR and CD28 to the Rac-1, PAK-1, and p38 MAPK effector molecules.

p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells.

Optimal T cell activation requires two signals, one generated by TCR and another by the CD28 costimulatory receptor. In this study, we investigated the regulation of costimulation-induced mitogen-activated protein kinase (MAPK) activation in primary mouse T cells. In contrast to that reported for human Jurkat T cells, we found that p38 MAPK, but not Jun NH2-terminal kinase (JNK), is weakly activated upon stimulation with either anti-CD3 or anti-CD28 in murine thymocytes and splenic T cells. However, p38 MAPK is activated strongly and synergistically by either CD3/CD28 coligation or PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3/CD28-mediated signaling. Activation of p38 MAPK correlates closely with the stimulation of T cell proliferation. In contrast, PMA-induced JNK activation is inhibited by Ca2+ ionophore. T cell proliferation and production of IL-2, IL-4, and IFN-gamma induced by both CD3 and CD3/CD28 ligation and the nuclear expression of the c-Jun and ATF-2 proteins are each blocked by the p38 MAPK inhibitor SB203580. Our findings demonstrate that p38 MAPK 1) plays an important role in signal integration during costimulation of primary mouse T cells, 2) may be involved in the induction of c-Jun activation and augmentation of AP-1 transcriptional activity, and 3) regulates whether T cells enter a state of functional unresponsiveness.

T-cell anergy and altered T-cell receptor signaling: effects on autoimmune disease.

Immunological self-tolerance can be acquired by several mechanisms, including the induction of anergy in autoreactive T cells. In this sense, anergy is predictably advantageous for the immune system. Here, Konstantin Salojin and colleagues present an alternative view that the induction of anergy in regulatory T cells may be harmful to the host and elicit autoimmune disease.

TCR alpha beta chains associate with the plasma membrane independently of CD3 and TCR zeta chains in murine primary T cells.

The TCR is a multisubunit complex composed of the clonotypic alpha/beta disulfide-linked heterodimer and noncovalently linked invariant CD3 gamma epsilon and CD3 delta epsilon and TCR zeta chains. Recent studies demonstrate that the surface expression of CD3 components can occur independently of the clonotypic TCR complexes in both thymocytes and splenic T cells. In this study, we report that free noncovalently associated TCR alpha beta heterodimers that exist independently of CD3 and TCR zeta chains are expressed on the cell surface of immature thymocytes and peripheral T cells, but not of T cell lines and T cell hybridomas. This suggests that the regulation of surface expression of TCR alpha beta heterodimers differs between primary T cells and T cell lines or T cell hybridomas. The isolation and biochemical characterization of surface clonotype-independent CD3 complexes and free membrane-associated TCR alpha beta complexes may provide a structural basis for the quantitative difference in amount of T cell proliferation stimulated by anti-CD3 epsilon and anti-TCR beta.

Sequestration of CD4-associated Lck from the TCR complex may elicit T cell hyporesponsiveness in nonobese diabetic mice.

The Lck protein tyrosine kinase associates noncovalently with the cytoplasmic domain of CD4. Upon ligand engagement of the TCR, CD4-associated Lck is rapidly activated and recruited to the TCR complex. Coupling of this complex to an intracellular signaling pathway may result in T cell proliferation. Previously, we reported that thymocytes from nonobese diabetic (NOD) mice (> or = 6 wk of age) exhibit a proliferative hyporesponsiveness after TCR stimulation, which is associated with defective TCR-mediated signaling along the protein kinase C/Ras/mitogen-activated protein kinase pathway of T cell activation. Here, we investigated whether differential association of Lck with TCR or CD4 mediates the control of NOD thymocyte hyporesponsiveness. We demonstrate that less CD4-associated Lck is recruited to the TCR in activated NOD thymocytes than in control thymocytes. This CD4-mediated sequestration of Lck from the TCR correlates with the increased binding of CD4-associated Lck through its Src homology 2 domain to free TCRzeta and CD3gamma epsilon chains on the plasma membrane. Sequestration of Lck by CD4 does not occur in activated thymocytes from 3-wk-old NOD mice and is only apparent in thymocytes from NOD mice >5 to 6 wk of age. This diminished recruitment of CD4-associated Lck to the TCR is not mediated by an increase in the amount of CD8-associated Lck. Thus, impaired recruitment of CD4-associated Lck to the TCR complex may represent an early event that results in deficient coupling of the TCR complex to downstream signaling events and gives rise to NOD thymocyte hyporesponsiveness.

Impaired plasma membrane targeting of Grb2-murine son of sevenless (mSOS) complex and differential activation of the Fyn-T cell receptor (TCR)-zeta-Cbl pathway mediate T cell hyporesponsiveness in autoimmune nonobese diabetic mice.

Nonobese diabetic (NOD) mouse thymocytes are hyporesponsive to T cell antigen receptor (TCR)-mediated stimulation of proliferation, and this T cell hyporesponsiveness may be causal to the onset of autoimmune diabetes in NOD mice. We previously showed that TCR-induced NOD T cell hyporesponsiveness is associated with a block in Ras activation and defective signaling along the PKC/Ras/MAPK pathway. Here, we report that several sequential changes in TCR-proximal signaling events may mediate this block in Ras activation. We demonstrate that NOD T cell hyporesponsiveness is associated with the (a) enhanced TCR-beta-associated Fyn kinase activity and the differential activation of the Fyn-TCR-zeta-Cbl pathway, which may account for the impaired recruitment of ZAP70 to membrane-bound TCR-zeta; (b) relative inability of the murine son of sevenless (mSOS) Ras GDP releasing factor activity to translocate from the cytoplasm to the plasma membrane; and (c) exclusion of mSOS and PLC-gamma1 from the TCR-zeta-associated Grb2/pp36-38/ZAP70 signaling complex. Our data suggest that altered tyrosine phosphorylation and targeting of the Grb2/pp36-38/ZAP70 complex to the plasma membrane and cytoskeleton and the deficient association of mSOS with this Grb2-containing complex may block the downstream activation of Ras and Ras-mediated amplification of TCR/CD3-mediated signals in hyporesponsive NOD T cells. These findings implicate mSOS as an important mediator of downregulation of Ras signaling in hyporesponsive NOD T cells.

Anti-endothelial cell antibody, thrombomodulin, and von Willebrand factor in idiopathic inflammatory myopathies.

Sera from 19 patients with idiopathic inflammatory myopathy (IIM) were examined for the presence of anti-endothelial cell antibodies (AECA) by an immunoglobulin G-specific cellular enzyme-linked immunosorbent assay. The mean binding index of AECA was found to be 37.7% +/- 26.5% for the patients, compared with a mean of 7.2% +/- 2.7% for normal controls (P < 0.04). Levels of thrombomodulin, von Willebrand factor antigen, and serum creatine kinase were also shown to be augmented. Interestingly, positive correlations between AECA on the one hand and Raynaud's phenomenon and interstitial lung disease on the other were demonstrated. Given that the pathogenesis of IIM remains uncertain, these findings may be of importance.

Antiendothelial cell antibodies: useful markers of systemic sclerosis.

BACKGROUND: Systemic sclerosis (SS) encompasses a wide spectrum of clinical presentations. Antiendothelial cell antibodies (AECA) in patients with primary Raynaud's phenomenon (PRP), limited SS (lSSc), or diffuse SS (dSSc) may help to determine the long-term prognosis of the disease. METHODS: Twenty-seven normal controls, 13 patients with PRP, 36 with lSSc, and 31 with dSSc were included in the study. Sera were examined for the presence of AECA, using a cellular enzyme-linked immunosorbent assay (ELISA). Angiotensin-converting enzyme (ACE) activity, plasma von Willebrand factor antigen (vWfAg), and thrombomodulin (Tm) concentrations were also evaluated. The medical records of 50 of the lSSc and dSSc patients were reviewed and the organ system involvement noted. RESULTS: Antiendothelial cell antibodies were present in 3 patients with PRP, 16 patients with lSSc, and 26 patients with dSSc. These autoantibodies were mainly of the IgG isotype. There was no difference in ACE activity between patients and controls. In contrast, vWfAg and Tm concentrations were higher in patients with PRP relative to controls, and higher in patients with lSSc compared with those with PRP. The presence of AECA was associated with digital scars and ulcers (P < 0.004 and P < 0.003, respectively), severe RP (P < 0.01), grade 3 tortuosity of vessels (P < 0.0004), and lung involvement (P < 0.02). CONCLUSION: The significant trend for AECA to increase with disease severity across the three groups of patients studies suggests that the AECA test can identify subsets of SSc with differing prognoses.

Anti-endothelial cell antibodies in patients with various forms of vasculitis.

OBJECTIVE: To determine the prevalence of anti-endothelial cell antibodies (AECA) in various forms of vasculitis and to evaluate their relationships with markers of endothelial cell (EC) injury such as thrombomodulin (TM), von Willebrand factor antigen (vWf) and protein S. METHODS: A total of 167 disease-associated sera, from 79 patients with large- or medium-sized (group I) and 88 with small-sized vessel vasculitis (group II), were examined for the presence of AECA using a cellular enzyme-linked immunosorbent assay (ELISA). These were evaluated before and after incubation with epithelial cells. EC plus epithelial cell (eC) extracts were fractionated and blotted with selected sera, and EC plus mononuclear cell extracts were dotted and blotted with lupus sera. Soluble TM, vWf and protein S levels were measured by ELISA. RESULTS: The binding of antibodies to eC was significant in group II sera (p < 0.01) but not in group I sera, so that the remaining EC-specific activity was significantly higher (p < 0.001) in the latter group than in the former. Eight antigenic specificities appeared to be specific for EC, whereas three were shared with eC. Despite absorption, the sera remained as reactive with EC and MNC as before. Taking the patient group as a whole, the levels of serum TM correlated with the titers of IgG, IgM and IgA AECA. CONCLUSION: EC-specific activity is more often encountered in group I than in group II patients. At present, the explanation for the distinct AECA specificities in these disease associated sera is not clear.