T Cell Receptor Down-modulation Research Articles

Crk adaptor proteins regulate CD3ζ chain phosphorylation and TCR/CD3 down-modulation in activated T cells

T cell receptor (TCR) recognition of a peptide antigen in the context of MHC molecules initiates positive and negative cascades that regulate T cell activation, proliferation and differentiation, and culminate in the acquisition of effector T cell functions. These processes are a prerequisite for the induction of specific T cell-mediated adaptive immune responses. A key event in the activation of TCR-coupled signaling pathways is the phosphorylation of tyrosine residues within the cytoplasmic tails of the CD3 subunits, predominantly CD3ζ. These transiently formed phosphotyrosyl epitopes serve as docking sites for SH2-domain containing effector molecules, predominantly the ZAP70 protein tyrosine kinase, which is critical for signal propagation. We found that CrkI and CrkII adaptor proteins also interact with CD3ζ in TCR activated-, but not in resting-, T cells. Crk binding to CD3ζ was independent of ZAP70 and also occurred in ZAP70-deficient T cells. Binding was mediated by Crk-SH2 domain interaction with phosphotyrosine-containing motifs on CD3ζ, via a direct physical interaction, as demonstrated by Far-Western blot. CrkII binding to CD3ζ could also be demonstrated in a heterologous system, where coexpression of a catalytically active Lck was used to phosphorylate the CD3ζ chain. TCR activation-induced Crk binding to CD3ζ resulted in increased and prolonged phosphorylation of CD3ζ, as well as ZAP70 and LAT, suggesting a positive role for CrkI/II binding to CD3ζ in regulation of TCR-coupled signaling pathways. Furthermore, Crk-dependent increased phosphorylation of CD3ζ coincided with inhibition of TCR downmodulation, supporting a positive role for Crk adaptor proteins in TCR-mediated signal amplification.

Regulation of T Cell Receptor Signaling by DENND1B in TH2 Cells and Allergic Disease

The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.

PD‐L1 co‐stimulation contributes to ligand‐induced T cell receptor down‐modulation on CD8 + T cells

T cell receptor (TCR) down-modulation after antigen presentation is a fundamental process that regulates TCR signal transduction. Current understanding of this process is that intrinsic TCR/CD28 signal transduction leads to TCR down-modulation. Here, we show that the interaction between programmed cell death 1 ligand 1 (PD-L1) on dendritic cells (DCs) and programmed death 1 (PD-1) on CD8 T cells contributes to ligand-induced TCR down-modulation. We provide evidence that this occurs via Casitas B-lymphoma (Cbl)-b E3 ubiquitin ligase up-regulation in CD8 T cells. Interference with PD-L1/PD-1 signalling markedly inhibits TCR down-modulation leading to hyper-activated, proliferative CD8 T cells as assessed in vitro and in vivo in an arthritis model. PD-L1 silencing accelerates anti-tumour immune responses and strongly potentiates DC anti-tumour capacities, when combined with mitogen-activated kinase (MAPK) modulators that promote DC activation.

Dual T cell receptor T cells with two defined specificities mediate tumor suppression via both receptors.

Grafting T cells with new antigen specificity by T cell receptor (TCR) gene transfer could greatly facilitate adoptive T cell immunotherapy. Little is known about how two TCR on one T cell influence each other. Among other reasons, this is often due to the fact that only one TCR specificity is known. We have genetically generated murine dual TCR T cells (OT-I/P14), specific for ovalbumin(ova257) and lymphocyte choriomeningitis virus glycoprotein (gp33). These cells retain both specificities and can be stimulated by either antigenic peptide to proliferate and produce IFN-gamma. Even though one TCR (P14) is expressed at reduced levels on dual TCR T cells, the peptide sensitivity of these cells is similar to that of single TCR T cells of the same specificity. TCR down-modulation on dual TCR T cells depends primarily on binding of the specific ligand. Adoptively transferred dual TCR T cells suppress the growth of both B16-ova and B16-gp33 melanoma cells, regardless of the peptide used for in vitro activation. Taken together, despite a certain dominance of expression between two TCR on the same T cell, this need not necessarily have functional consequences.

T cell development and T cell responses in mice with mutations affecting tyrosines 292 or 315 of the ZAP-70 protein tyrosine kinase.

After stimulation of the T cell receptor (TCR), the tyrosine residues 292 and 315 in interdomain B of the protein tyrosine kinase ZAP-70 become phosphorylated and plausibly function as docking sites for Cbl and Vav1, respectively. The two latter proteins have been suggested to serve as substrates for ZAP-70 and to fine-tune its function. To address the role of these residues in T cell development and in the function of primary T cells, we have generated mice that express ZAP-70 molecules with Tyr to Phe substitution at position 292 (Y292F) or 315 (Y315F). When analyzed in a sensitized TCR transgenic background, the ZAP-70 Y315F mutation reduced the rate of positive selection and delayed the occurrence of negative selection. Furthermore, this mutation unexpectedly affected the constitutive levels of the CD3-zeta p21 phosphoisoform. Conversely, the ZAP-70 Y292F mutation upregulated proximal events in TCR signaling and allowed more T cells to produce interleukin 2 and interferon gamma in response to a given dose of antigen. The observation that ZAP-70 Y292F T cells have a slower rate of ligand-induced TCR downmodulation suggests that Y292 is likely involved in regulating the duration activated TCR reside at the cell surface. Furthermore, we showed that Y292 and Y315 are dispensable for the TCR-induced tyrosine phosphorylation of Cbl and Vav1, respectively. Therefore, other molecules present in the TCR signaling cassette act as additional adaptors for Cbl and Vav1. The present in vivo analyses extend previous data based on transformed T cell lines and suggest that residue Y292 plays a role in attenuation of TCR signaling, whereas residue Y315 enhances ZAP-70 function.

Mechanisms of immunomodulation by glatiramer acetate.

To define the mechanism of action of glatiramer acetate (GA; formerly known as copolymer-1) as an immunomodulatory treatment for MS. The proposed mechanisms of action of GA include 1) functional inhibition of myelin-reactive T cells by human leukocyte antigen (HLA) blocking, 2) T-cell receptor (TCR) antagonism, and 3) induction of T helper 2 (Th2) immunomodulatory cells. In this report, the authors examined the effects of GA on the functional activation of human T-cell clones (TCC) specific for myelin basic protein (MBP) and for foreign antigens. Several questions were addressed: Is the inhibitory effect of GA specific for autoantigens? Is it mediated by blocking the interaction between peptide and HLA molecule? Is GA a partial agonist or TCR antagonist, or does it induce anergy? Does it induce Th2 modulatory T cells? The effects of GA on antigen-induced activation of human TCC specific for MBP, influenza virus hemagglutinin, and Borrelia burgdorferi were studied by proliferation and cytokine measurements, TCR downmodulation, and anergy assays. GA-specific TCC were generated in vitro from the peripheral blood of patients and healthy controls by limiting dilution. GA more strongly inhibited the proliferation of MBP, as compared with foreign antigen-specific TCC; in some MBP-specific TCC, the production of Th1-type cytokines was preferentially inhibited. In addition to HLA competition, the induction of anergy, but not direct TCR antagonism, was observed. Numerous GA-specific TCC were generated from the peripheral blood of both MS patients and normal controls, and a fraction of these showed a Th2 phenotype. This study confirms a preferential inhibitory effect of GA on autoreactive TCC. With respect to cellular mechanisms, although HLA competition appears to play the most important role in functional inhibition in vitro, a direct effect on the TCR may be involved at least in some autoreactive T cells as shown by anergy induction. Although not confirmed at the clonal level, it is demonstrated further that GA induces T cells that crossreact with myelin proteins. GA-specific, Th2-modulatory cells may play an important role in mediating the effect of the drug in vivo.

T-cell activation and receptor downmodulation precede deletion induced by mucosally administered antigen.

The fate of antigen-specific T cells was characterized in myelin basic protein (MBP) T-cell receptor (TCR) transgenic (Tg) mice after oral administration of MBP. Peripheral Th cells are immediately activated in vivo, as indicated by upregulation of CD69 and increased cytokine responses (Th1 and Th2). Concurrently, surface TCR expression diminishes and internal TCR levels increase. When challenged for experimental autoimmune encephalomyelitis during TCR downmodulation, Tg mice are protected from disease. To characterize Th cells at later times after antigen feeding, it was necessary to prevent thymic release of naive Tg cells. Therefore, adult Tg mice were thymectomized before treatment. TCR expression returns in thymectomized Tg mice 3 days after MBP feeding and then ultimately declines in conjunction with MBP-specific proliferation and cytokine responses (Th1-type and Th2-type). The decline correlates with an increase in apoptosis. Collectively, these results demonstrate that a high dose of fed antigen induces early T-cell activation and TCR downmodulation, followed by an intermediate stage of anergy and subsequent deletion.

Partial T cell activation with an altered superantigenic ligand.

T cells have the capacity to respond to ligands as full, weak, partial or null agonists, or indeed as antagonists. In the present paper, it is reported that staphylococcal enterotoxin B (SEB) mutated in a T cell receptor (TCR) contact site (SEBDelta61Y) behaves as an altered ligand for a T cell clone (AC20) that expresses the Vbeta17 TCR. The T cells were partially activated by SEBDelta61Y, as shown by TCR down-modulation and up-regulation of the IL-2 receptor. However, these cells did not secrete IL-2, IL-3, IL-4 or IFN-gamma, nor did they proliferate. Analysis of intracellular protein tyrosine phosphorylation after cellular activation provided further evidence that SEBDelta61Y could transduce a signal via the Vbeta17 TCR. The events following receptor ligation were clearly different when the T cells were stimulated with SEB or SEBDelta61Y, manifested as both quantitatively and qualitatively different patterns of phosphorylation of intracellular substrates. In contrast, only quantitative differences were apparent when a transfectant expressing the same alpha/beta TCR was stimulated with the different superantigens. Together, these results provide the first demonstration that altered TCR ligands are not restricted to peptides substituted at secondary TCR contact residues. Rather, an altered superantigenic ligand mutated in the TCR binding site can behave as a partial agonist.

High frequency of T cell clonal expansions in primary human melanoma. Involvement of a dominant clonotype in autologous tumor recognition.

It was previously found that primary melenomas from HLA-A2-matched patients display an increased expression of a few T cell receptor (TCR) variable-region beta-chain transcripts (BV) compared with autologous peripheral blood lymphocytes (PBL) and uninvolved skin. In order to see whether expansions of clonal/oligoclonal subsets of T cells occurred, complementarity-determining region 3 (CDR3) of BV transcripts overexpressed in the neoplastic infiltrate were cloned and sequenced. Dominant rearrangements were found for BV14, which were commonly increased in the neoplastic lesions of all analysed HLA-A2 melanoma patients, as well as for other overexpressed BV gene families, but none of them could be identified among autologous PBL. No identical CDR3 sequences could be detected in the dominant BV14 rearrangements obtained from the different patients. In one patient a single clonally expanded SLSGTGVNEQF CDR3 clonotype accounted for the entire BV14 relative frequency of expression (24%) in tumor-infiltrating lymphocytes (TIL). Two independent mixed lymphocyte/tumor cultures (MLTC) could be successfully established from TIL of the patient and were found to exert HLA-class-I-restricted cytotoxicity for the autologous melanoma line. BV14 T cells that constituted from 22% to 32% of all T cells present in both MLTC lines, as assessed by flow cytometry, all displayed the same CDR3 clonotype found in vivo and could be shown, by TCR down-modulation experiments, to be involved in autologous tumor recognition. These results support the hypothesis of a tumor-antigen-driven origin of clonally amplified T cells present at high frequency in the in situ neoplastic infiltrate.

Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR down-modulation.

The Nef protein of simian immunodeficiency virus (SIV) is dispensable for replication in established T-cell lines but essential for high level virus replication in the adult host, though the mechanism by which Nef contributes to this has remained unclear. We demonstrate here that SIV Nef binds to the zeta chain of the T-cell receptor (TCR). SIV Nef proteins that interact with TCR zeta in a yeast two-hybrid system also reduce T-cell surface expression levels of TCR alphabeta, CD3 and CD4. These findings are the first demonstration that Nef can bind directly to a component of the TCR-CD3 complex and modulate its surface expression.

The minimal number of antigen-major histocompatibility complex class II complexes required for activation of naive and primed T cells.

Although previous studies have shown that 50-200 antigen-major histocompatibility complex complexes (Ag-MHC) are sufficient to stimulate significant secretion of interleukin (IL)-2 from MHC class II-restricted T cell hybridomas, there have been no studies of this nature on more physiologically relevant T cell populations. In this study we have analyzed the ligand requirements for stimulation of responses from naive and previously primed T cells derived from T cell receptor (TCR)-transgenic animals whose TCR is specific for the pigeon cytochrome c (PCC) 88-104 peptide presented by I-Ek. Primed T cells were as sensitive as the previously reported T cell hybridomas, requiring about 100 Ag-MHC complexes to synthesize readily detectable quantities of IL-2, whereas naive T cells required 15 times more ligand to produce equivalent quantities of IL-2. Similarly, primed T cells required about 40 Ag-MHC complexes to produce a significant proliferative response, whereas naive T cells required about 400 complexes. In contrast to these results, naive and primed T cells showed similar ligand requirements when early events in the T cell activation pathway were analyzed; i.e. TCR down-modulation, CD69 and CD25 expression, and blast transformation. A further analysis of IL-2 and IL-2R expression indicated: 1) The first synthesis of IL-2 was detected at the same ligand concentration in both primed and naive T cells, but primed T cells made much more IL-2 as the ligand concentrations increased; 2) primed T cells expressed about fivefold more IL-2 receptor (R) than naive T cells, despite the fact that the antigen dose-response curves with respect to the percentage of cells expressing IL-2R were identical. These results suggest that naive and primed T cells have the same threshold with respect to the number of Ag-MHC complexes required to initiate T cell activation, but that due to the inefficient expression of IL-2 and IL-2R, engagement of more complexes is needed to enable naive T cells to synthesize the necessary amounts of these two molecules to allow T cells to go through a complete cycle of replication.

Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogeneity for individual cytokine responses of CD4+ T cells.

T cell receptor (TCR) recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4(+) cell population. Low concentrations of TCR ligand elicit only interferon-gamma (IFN-gamma) production. Increasing ligand recruits more cells into the IFN-gamma+ pool, increases IFN-gamma produced per cell, and also elicits IL-2, but only from cells already making IFN-gamma. Most cells producing only IFN-gamma show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-gamma synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-gamma/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-gamma and not IL-2, and the amount of IFN-gamma exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.

Control of thymus-independent intestinal intraepithelial lymphocytes by beta 2-microglobulin.

Murine intestinal intraepithelial lymphocytes (i-IEL) comprise thymus-dependent cells such as T cell receptor (TcR) alpha/beta CD8 alpha/beta+ i-IEL, as well as thymus-independent ones such as TcR alpha/beta CD8 alpha/alpha+ and TcR gamma/delta CD8 alpha/alpha+ i-IEL. Whilst the development of the CD8 alpha/beta expressing i-IEL is strictly contingent on major histocompatibility complex (MHC) class I surface expression, that of CD8 alpha/alpha i-IEL appears largely MHC class I independent. We have used beta 2-microglobulin (beta 2m)-/- mutant mice lacking surface-expressed MHC class I and TcR alpha/beta CD8 alpha/beta+ i-IEL to analyze the potential impact of MHC class I on regional activation of thymus-independent i-IEL. To analyze the role of TcR gamma/delta i-IEL in regional cell interactions, these mice were treated with the anti-TcR gamma/delta mAb, GL3. Whilst numbers of TcR alpha/beta CD8 alpha/alpha i-IEL were markedly reduced in beta 2m-/- mice, those of TcR gamma/delta i-IEL were elevated. Administration of GL3 in vivo caused TcR down-modulation and functional inactivation of TcR gamma/delta i-IEL in beta 2m+/- mice. In contrast, TcR expression and functional activities of TcR gamma/delta i-IEL from beta 2m-/- mice were not impaired by GL3 treatment. The TcR alpha/beta CD8 beta- i-IEL from beta 2m-/- mice were expanded and functionally activated as a consequence of TcR gamma/delta engagement. The TcR gamma/delta i-IEL and TcR alpha/beta CD8 alpha/alpha+ i-IEL from athymic nu/nu mice which express MHC class I, but lack TcR alpha/beta CD8 alpha/beta+ i-IEL, responded to TcR gamma/delta engagement as those from the beta 2m+/- controls. In addition, the TcR gamma/delta i-IEL from TcR beta-/- and TCR beta+/- mutants were equally affected by GL3. We conclude that the absence of beta 2m renders TcR gamma/delta i-IEL resistant to TcR-mediated inactivation and promotes activation of TcR alpha/beta CD8 beta- i-IEL. The activation of TcR gamma/delta i-IEL seems to be directly controlled by beta 2m/MHC class I expression and independent from TcR alpha/beta CD8 beta+ i-IEL. Regulation of self-reactive thymus-independent i-IEL through beta 2m/ MHC class I may contribute to control of autoreactive immune responses in the intestine.