Phenotypic analysis of CTLA-4 and CD28 expression during transient peptide-induced T cell activation in vivo.
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The T cell co-stimulatory receptors CD28 and CTLA-4 appear to have opposite effects on T cell activation, mediating augmentation and inhibition of T cell responses respectively. Since these two receptors use the same ligands, CD80 (B7-1) and CD86 (B7-2), the co-ordinate timing of CD28 and CTLA-4 expression has a major impact on the regulation of immune responses. While the kinetics of co-stimulatory molecules have been established for T cell stimulation in vitro, little is known about CD28 and CTLA-4 expression in response to T cell activation in vivo. In this study we have investigated the kinetics of CD28 and CTLA-4 expression upon CD4(+) T cell activation in response to soluble peptide in vivo. Using mice transgenic for a T cell receptor specific for the I-Au-restricted N-terminal peptide of myelin basic protein MBP Ac1-9, we show maximal up-regulation of both CD28 and CTLA-4 2 days after peptide administration. CTLA-4 expression correlated positively with early activation markers on the same cells and was high on blast cells. Administration of peptide analogs with higher affinity for I-Au MHC class II revealed a higher increase in CTLA-4 than in CD28 expression in response to improved TCR ligation. Further, a small population of CD4(+) T cells expressing CTLA-4, CD25 and CD45RBlow was identified in mice that had not been treated with specific peptide. The implications of these observations for immune regulation are discussed. (+info)
Cell spreading distinguishes the mechanism of augmentation of T cell activation by integrin-associated protein/CD47 and CD28.
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Integrin-associated protein (IAP/CD47) is a 50 kDa transmembrane protein initially defined as a regulator of beta3 integrin-mediated functions in neutrophils. IAP also can synergize with the TCR in T cell activation independent of beta3 integrins. To analyze the mechanism for IAP synergy with TCR, we expressed in Jurkat cells a chimeric molecule, consisting of the CD16 extracellular domain, the CD7 transmembrane domain and the TCR zeta chain cytoplasmic tail (CD16-7-zeta), which on its own is unable to induce IL-2 production. Ligation of IAP acted in synergy with TCR to induce IL-2 transcription and synthesis, but failed to synergize with the signal generated by CD16-7-zeta, while CD28 was a potent co-stimulator with both TCR and CD16-7-zeta. The failure of IAP to activate Jurkat together with CD16-7-zeta correlated with a lack of c-Jun N-terminal kinase, but not extracellular-signal-regulated kinase activation. Jurkat adhesion to anti-IAP, but not anti-CD28, induced cell spreading and the same domains of IAP required for augmentation of T cell activation were required to induce cell spreading. IAP synergy with TCR signaling likely results from its ability to stimulate adhesion to a ligand-expressing surface or antigen-presenting cell (APC), rather than from initiation of a novel signaling cascade. We conclude that a major role for ligation of IAP in T cell activation is to enhance the efficiency of TCR signaling by causing T cells to spread on an APC or surface. (+info)
Generation of antigen-specific cytotoxic T lymphocytes and regulation of cytokine production takes place in the absence of CD3zeta.
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The TCR-associated protein CD3zeta plays a major role in regulating the state of responsiveness to peptide-MHC complexes on the surface of antigen-presenting cells. In this paper the requirement of CD3zeta in the generation of cytotoxic T cells was compared with its requirement in cytokine gene activation in two mutant mice: ZKO mice with a disrupted CD3zeta gene and ZTG mice in which a truncated CD3zeta segment was expressed as a transgene on the ZKO background. Upon infection of ZTG mice with lymphocytic choriomeningitis virus (LCMV), antigen-specific cytotoxic T lymphocyte (CTL) responses were detected, identical to responses in wild-type mice. In addition, antigen-specific CTL responses to allogeneic class I and class II MHC in ZTG animals were indistinguishable from those in wild-type animals. However, CTL responses to the same major antigens were not detectable in ZKO mice. We conclude that the signal transduction pathways leading to CTL development and cytokine production can be triggered through TCR in the absence of functional CD3zeta, provided the remainder of the TCR-CD3 complex is expressed at high levels on the cell surface. Surprisingly, IFN-gamma production in response to LCMV followed the same kinetics in ZKO, ZTG and wild-type mice. However, in vitro studies showed that cytokine production in general was abnormally regulated in T lymphocytes from ZKO mice, in contrast to ZTG T cells. Taken together, these studies support the hypothesis that development of CTL can take place in the absence of functional CD3zeta. However, CTL development requires stronger TCR-initiated signal transduction events than induction of cytokine genes. (+info)
Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naive and memory T cells.
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The question of whether enhanced memory T cell responses are simply due to an increased frequency of specific cells or also to an improved response at the single cell level is widely debated. In this study, we analyzed T cell receptor (TCR) transgenic memory T cells and bona fide memory T cells isolated from virally infected normal mice using the tetramer technology. We found that memory T cells are qualitatively different from naive T cells due to a developmentally regulated rearrangement of the topology of the signaling machinery. In naive cytotoxic T cells, only a few CD8 molecules are associated with Lck and the kinase is homogeneously distributed inside the cell. However, in vivo priming of naive T cells induces the targeting of Lck to the CD8 coreceptor in the cell membrane and the consequent organization of a more efficient TCR signaling machinery in effector and memory cells. (+info)
Generation of CD28- cells from long-term-stimulated CD8+CD28+ T cells: a possible mechanism accounting for the increased number of CD8+CD28- T cells in HIV-1-infected patients.
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According to CD28 molecule expression, CD8+ T cells can be classed as CD28bright, CD28dim, and CD28-. The CD28dim T cells were found to derive from mitogenic stimulated CD28-T cells but also from CD28bright T cells through a mechanism of CD28 down-modulation. Moreover, after prolonged in vitro interleukin-2 stimulation, clonal CD28bright, cells showed a CD28dim expression before further evolution to a stable CD28-phenotype. This loss was concomitant with the disappearance of CD28 mRNA. A study of the cytokine production pattern revealed that CD28dim and CD28- T cell clones produced similar levels of type 1 and type 2 cytokines, which differed from those produced by the CD28bright T cell clones. A high percentage of CD28dim and CD28- cells, with similarities in their cytokine production pattern, were found in the blood samples of HIV-infected patients, as compared to healthy donors. The CD28 down-modulation may account for the increased number of CD8+CD28- T cells in HIV-infected patients. (+info)
SLP-76 and Vav function in separate, but overlapping pathways to augment interleukin-2 promoter activity.
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SLP-76 and Vav, two hematopoietic cell specific molecules, are critical for T cell development and activation. Following T cell antigen receptor stimulation, SLP-76 and Vav both undergo tyrosine phosphorylation and associate with each other via the SH2 domain of Vav and phosphorylated tyrosines of SLP-76. Furthermore, SLP-76 and Vav have a synergistic effect on interleukin (IL)-2 promoter activity in T cells. In this report, we show that two tyrosines, Tyr-113 and Tyr-128, of SLP-76 are required for its binding to Vav, both in vitro and in intact cells. Surprisingly, we find also that the interaction between SLP-76 and Vav is not required for their cooperation in augmenting IL-2 promoter activity, as the two molecules appear to function in different signaling pathways upstream of IL-2 gene expression. Overexpression of SLP-76 in the Jurkat T cell line potentiates the activities of both nuclear factor of activated T cells and AP-1 transcription factors. In contrast, overexpression of Vav leads to enhanced nuclear factor of activated T cells activity without affecting AP-1. Additionally, overexpression of Vav, but not SLP-76, augments CD28-induced IL-2 promoter activity. These findings suggest that the synergy between SLP-76 and Vav in regulating IL-2 gene expression reflects the cooperation between different signaling pathways. (+info)
Functional integrity of the CD28 co-stimulatory pathway in T lymphocytes from elderly subjects.
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INTRODUCTION: the antigen CD28, expressed in most T cells, has co-stimulatory properties and plays a pivotal role in clonal T cell anergy mechanisms. METHODS: we have compared proliferative T cell responses after anti-CD3 or in phorbol myristate acetate activation with concomitant CD28 signal in peripheral blood mononuclear cells from healthy donors aged over 65 [elderly donors; ED] and young healthy donors (YD); mean age 30+/-2.7 years). RESULTS: no proliferative responses were observed in ED and YD with anti-CD28 monoclonal antibody alone. These responses both were defective in ED, particularly after anti-CD3 monoclonal antibody stimulus (7604 compared with 12,438 c.p.m. in YD, P=0.001) and were corrected when anti-CD28 monoclonal antibody was added to the culture (17,216 vs 18,536, not significant). Functional integrity of the CD28 co-stimulatory pathway was demonstrated by analysis of CD25 expression, interleukin-2 secretion and interleukin-2 gene expression on T cells from ED and YD. Age-associated phenotypic T cell changes were not crucial for an adequate CD28 response. CONCLUSION: these experiments demonstrate the integrity of the CD28 pathway in elderly people, and suggest that ageing does not affect different T cell activation pathways equally. (+info)
Opposing roles of CD28:B7 and CTLA-4:B7 pathways in regulating in vivo alloresponses in murine recipients of MHC disparate T cells.
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Blockade with B7 antagonists interferes with CD28:B7 and CTLA-4:B7 interactions, which may have opposing effects. We have examined the roles of CD28:B7 and CTLA-4:B7 on in vivo alloresponses. A critical role of B7:CD28 was demonstrated by markedly compromised expansion of CD28-deficient T cells and diminished graft-versus-host disease lethality of limited numbers of purified CD4+ or CD8+ T cells. When high numbers of T cells were infused, the requirement for CD28:B7 interaction was lessened. In lethally irradiated recipients, anti-CTLA-4 mAb enhanced in vivo donor T cell expansion, but did not affect, on a per cell basis, anti-host proliferative or CTL responses of donor T cells. Graft-versus-host lethality was accelerated by anti-CTLA-4 mAb infusion given early post-bone marrow transplantation (BMT), mostly in a CD28-dependent fashion. Donor T cells obtained from anti-CTLA-4 mAb-treated recipients were skewed toward a Th2 phenotype. Enhanced T cell expansion in mAb-treated recipients was strikingly advantageous in the graft-versus-leukemia effects of delayed donor lymphocyte infusion. In two different systems, anti-CTLA-4 mAb enhanced the rejection of allogeneic T cell-depleted marrow infused into sublethally irradiated recipients. We conclude that blockade of the selective CD28-B7 interactions early post-BMT, which preserve CTLA-4:B7 interactions, would be preferable to blocking both pathways. For later post-BMT, the selective blockade of CTLA-4:B7 interactions provides a potent and previously unidentified means for augmenting the GVL effect of delayed donor lymphocyte infusion. (+info)