scholarly journals The tyrosine phosphatase CD148 is excluded from the immunologic synapse and down-regulates prolonged T cell signaling

2003 ◽  
Vol 162 (4) ◽  
pp. 673-682 ◽  
Author(s):  
Joseph Lin ◽  
Arthur Weiss
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Clone ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Phosphatase Domain ◽  
T Cell Clone ◽  
Immunologic Synapse

CD148 is a receptor-like protein tyrosine phosphatase up-regulated on T cells after T cell receptor (TCR) stimulation. To examine the physiologic role of CD148 in TCR signaling, we used an inducible CD148-expressing Jurkat T cell clone. Expression of CD148 inhibits NFAT (nuclear factor of activated T cells) activation induced by soluble anti-TCR antibody, but not by antigen-presenting cells (APCs) loaded with staphylococcal enterotoxin superantigen (SAg) or immobilized anti-TCR antibody. Immunofluorescence microscopy revealed that the extracellular domain of CD148 mediates its exclusion from the immunologic synapse, sequestering it from potential substrates. Targeting of the CD148 phosphatase domain to the immunologic synapse potently inhibited NFAT activation by all means of triggering through the TCR. These data lead us to propose a model where CD148 function is regulated in part by exclusion from substrates in the immunologic synapse. Upon T cell–APC disengagement, CD148 can then access and dephosphorylate substrates to down-regulate prolongation of signaling.

scholarly journals Dynamic regulation of CD45 by tetraspanin CD53

2019 ◽  
Author(s):  
V. E. Dunlock ◽  
A. B. Arp ◽  
E. Jansen ◽  
S. Charrin ◽  
S. J. van Deventer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Tyrosine Phosphatase ◽  
Adaptive Immune Response ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Dynamic Regulation ◽  
And Function

AbstractT cells are central to the adaptive immune response, playing a role in both the direct and indirect killing of pathogens and transformed cells. The activation of T cells is the result of a complex signaling cascade, initiated at the T cell receptor (TCR), and ending with the induction of proliferation. CD45, a member of the protein tyrosine phosphatase family, is one of the most abundant membrane proteins on T cells and functions by regulating activation directly downstream of the TCR. As a result of alternative splicing, CD45 can be expressed in multiple isoforms, naive T cells express the CD45RA isoform, while activated T cells gain expression of CD45RO, which has been proposed to increase signaling. Though the importance of CD45 in TCR signaling, proliferation and cytokine production is well established, little is known about the regulation of CD45 activity. We discovered that the immune-specific tetraspanin CD53 directly affects the stability and function of CD45RO in T cells.We have identified CD53 as a T cell co-stimulatory molecule in primary human and murine cells. Furthermore, we have shown that the absence of CD53 leads to an altered CD45 isoform expression as a result of decreased CD45RO stability on the cell surface. This instability was accompanied by increased mobility as measured by FRAP.Together, this indicates that CD53 functions as a stabilizer of CD45RO, and therefore as a positive regulator of TCR signaling at the T cell surface. Our data provides novel insight into the role of tetraspanins in the regulation of immune signaling and may provide a new avenue for the regulation of T cell signaling.

A “non-fratricidal” αβ- T Cell Receptor That Targets Survivin Expressed By Hematological Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 141-141
Author(s):  
Caroline Arber ◽  
Xiang Feng ◽  
Harshal Abhyankar ◽  
Helen E. Heslop ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Hematological Malignancies ◽  
Cell Clone ◽  
Cell Receptor ◽  
Activated T Cells ◽  
T Cell Clone ◽  
Myeloid Leukemias

Abstract Survivin is broadly expressed by hematological malignancies as well as by solid tumors and may be a suitable target for T-cell immunotherapy. Previously, the utility of this target has been challenged by the occurrence of “fratricide” when T cells expressing a high avidity survivin-specific T cell receptor (TCR) killed each other because survivin epitopes can be presented by the T cells themselves (Leisegang M et al, J Clin Invest. 2010 Nov;120(11):3869-77). To overcome this obstacle, we used limiting dilution to isolate a new T-cell clone targeting the HLA-A*02-restricted survivin epitope ELT (survivin95-104) and its variant LML (survivin96-10497M) starting from autologous cultures, rather than from the allogeneic cultures previously used for this approach. In 51Chromium (Cr)-release assays, this T-cell clone, with nanomolar avidity, displayed specific killing against the survivin+HLA-A*02+ leukemia cells BV173 (39±16% specific lysis, E:T 40:1) and multiple myeloma cells U266 (20±7%) but not against HLA-A*02– HL-60 cells (2±2%). Furthermore, the colony formation of primary myeloid leukemias was inhibited (>50% reduction) while that of healthy bone marrow (BM) was unaffected. The TCR α- and β-chains were then cloned in an optimized retroviral vector that was used to transduce CD8+ T cells which then efficiently expressed the transgenic αβTCR (89±4%, n=6). As compared to non-transduced (NT) T cells, survivin-αβTCR+ T cells produced significant lysis of BV173 (46±14% vs 8±6%, E:T 20:1, n=12, p<0.001) and U266 (27±12% vs 14±6%, p=0.003) but not of HL-60 (14±7 vs 14±6 %, p=NS). Blocking the target cells with specific anti-MHC class I antibodies confirmed the HLA-restriction of TCR transgenic T cells. Importantly, transgenic cells recapitulated the function of the original clone by inhibiting colony formation (range 32-78% reduction, n=5) of primary myeloid leukemias while preserving normal clonogenic capacity of healthy BM or cord blood (n=5). When tested in vivo in a xenograft model of established systemic acute leukemia (FFLuc+BV173) using bioluminescent imaging, leukemia progression was significantly slower in mice treated with survivin-αβTCR+ versus NT T cells (40x106 ± 71x106 vs. 128 x 106 ± 176 x 106 photons/sec by day 28) (p=0.04) and survival improved (n=12/group, p=0.01). This effect was even more pronounced when T cells were transferred to mice with limited leukemia burden (bioluminescent signal by day 40: 8.1 x 106 ± 9 x 106 vs. 195 x 106 ± 85 x 106 photons/sec) (p=0.003, n=10/group). Overall survival was improved by day 80 (p<0.001) and 3/10 mice treated with TCR+ T cells completely cleared the leukemia. Crucially, the TCR cloned from our autologous culture system produced no fratricidal activity in 51Cr-release assays against HLA-A*02+ activated T cells (1±2%, E:T 20:1, n=7). Activated T cells were only killed by TCR+ T cells when they were also pulsed with survivin peptides (46±12% for LML, 68±14% for ELT, n=7). To elucidate at the molecular level why our “autologous” TCR had selective antitumor activity unlike the fratricidal activity of “allogeneic” TCRs (Leisegang M et al, J Clin Invest. 2010 Nov;120(11):3869-77), we modeled the structure of each TCR-peptide-HLA ternary complex using the Rosetta software. While the overall binding energies of TCR-peptide-HLA interfaces for both TCRs were similar, the “autologous” TCR showed a 48% higher binding energy contribution for the peptide as compared to the fratricidal TCR, whose interaction was primarily with the HLA molecule rather than with the survivin peptide in the HLA-binding groove. In conclusion, we have cloned a novel survivin-TCR with a highly epitope-specific binding mode that can be efficiently expressed in polyclonal T cells and provides antitumor activity in vitro and in vivo without affecting the survival of T cells or normal hematopoietic progenitors. Our results indicate that maximal recognition of the peptide presented in the HLA groove is critical for TCR selectivity. Disclosures: Heslop: Celgene: Patents & Royalties; Cell Medica: Patents & Royalties. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding. Savoldo:Celgene: Patents & Royalties, Research Funding.

scholarly journals Fyn-Binding Protein (Fyb)/Slp-76–Associated Protein (Slap), Ena/Vasodilator-Stimulated Phosphoprotein (Vasp) Proteins and the Arp2/3 Complex Link T Cell Receptor (Tcr) Signaling to the Actin Cytoskeleton

2000 ◽  
Vol 149 (1) ◽  
pp. 181-194 ◽  
Author(s):  
Matthias Krause ◽  
Antonio S. Sechi ◽  
Marlies Konradt ◽  
David Monner ◽  
Frank B. Gertler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Actin Cytoskeleton ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Signaling ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Antigen Presenting ◽  
Syndrome Protein

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76–associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3–coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.

scholarly journals An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells.

1994 ◽  
Vol 180 (3) ◽  
pp. 1171-1176 ◽  
Author(s):  
P Dellabona ◽  
E Padovan ◽  
G Casorati ◽  
M Brockhaus ◽  
A Lanzavecchia
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Clone ◽  
Cell Subset ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
T Cell Clone ◽  
Gene Usage ◽  
Beta Gene

The T cell receptor (TCR)-alpha/beta CD4-8- (double negative, DN) T cell subset is characterized by an oligoclonal repertoire and a restricted V gene usage. By immunizing mice with a DN T cell clone we generated two monoclonal antibodies (mAbs) against V alpha 24 and V beta 11, which have been reported to be preferentially expressed in DN T cells. Using these antibodies, we could investigate the expression and pairing of these V alpha and V beta gene products among different T cell subsets. V alpha 24 is rarely expressed among CD4+ and especially CD8+ T cells. In these cases it is rearranged to different J alpha segments, carries N nucleotides, and pairs with different V beta. Remarkably, V alpha 24 is frequently expressed among DN T cells and is always present as an invariant rearrangement with J alpha Q, without N region diversity. This invariant V alpha 24 chain is always paired to V beta 11. This unique V alpha 24-J alpha Q/V beta 11 TCR was found in expanded DN clones from all the individuals tested. These findings suggest that the frequent occurrence of cells carrying this invariant TCR is due to peripheral expansion of rare clones after recognition of a nonpolymorphic ligand.

scholarly journals Crucial Role for Nuclear Factor of Activated T Cells in T Cell Receptor-mediated Regulation of Human Interleukin-17

2004 ◽  
Vol 279 (50) ◽  
pp. 52762-52771 ◽  
Author(s):  
Xikui K. Liu ◽  
Xin Lin ◽  
Sarah L. Gaffen
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Promoter Activity ◽  
Cell Receptor ◽  
Cross Linking ◽  
Supporting Evidence ◽  
Nuclear Factor ◽  
Tcr Signaling ◽  
Activated T Cells

The biological activities of the inflammatory cytokine interleukin (IL)-17 have been widely studied. However, comparatively little is known about how IL-17 expression is controlled. Here, we examined the basis for transcriptional regulation of the human IL-17 gene. IL-17 secretion was induced in peripheral blood mononuclear cells following anti-CD3 cross-linking to activate the T cell receptor (TCR), and costimulatory signaling through CD28 strongly enhanced CD3-induced IL-17 production. To definecis-acting elements important for IL-17 gene regulation, we cloned 1.25 kb of genomic sequence upstream of the transcriptional start site. This putative promoter was active in Jurkat T cells following CD3 and CD28 cross-linking, and its activity was inhibited by cyclosporin A and MAPK inhibitors. The promoter was also active in Hut102 T cells, which we have shown to secrete IL-17 constitutively. Overexpression of nuclear factor of activated T cells (NFAT) or Ras enhanced IL-17 promoter activity, and studies in Jurkat lines deficient in specific TCR signaling pathways provided supporting evidence for a role for NFAT. To delineate the IL-17 minimal promoter, we created a series of 5′ truncations and identified a region between -232 and -159 that was sufficient for inducible promoter activity. Interestingly, two NFAT sites were located within this region, which bound to NFATc1 and NFATc2 in nuclear extracts from Hut102 and Jurkat cells. Moreover, mutations of these sites dramatically reduced both specific DNA binding and reporter gene activity, and chromatin immunoprecipitation assays showed occupancy of NFAT at this regionin vivo. Together, these data show that NFAT is the crucial sensor of TCR signaling in the IL-17 promoter.

scholarly journals Unique Clinical and Pathological Features in HLA-DRB1*0401–restricted MBP 111–129–specific Humanized TCR Transgenic Mice

2004 ◽  
Vol 200 (2) ◽  
pp. 223-234 ◽  
Author(s):  
Jacqueline A. Quandt ◽  
Mirza Baig ◽  
Karen Yao ◽  
Kazuyuki Kawamura ◽  
Jaebong Huh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cranial Nerve ◽  
Cell Clone ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Nerve Roots ◽  
Pathogenic Potential ◽  
Abnormal Gait ◽  
T Cell Clone

Amino acid residues 111–129 represent an immunodominant epitope of myelin basic protein (MBP) in humans with human leukocyte antigen (HLA)-DRB1*0401 allele(s). The MBP 111–129–specific T cell clone MS2-3C8 was repeatedly isolated from a patient with multiple sclerosis (MS), suggesting an involvement of MS2-3C8 T cells in the pathogenesis. To address the pathogenic potential of the MS2-3C8 T cell clone, we generated transgenic (Tg) mice expressing its T cell receptor and restriction element, HLA-DRB1*0401, to examine the pathogenic characteristics of MS2-3C8 Tg T cells by adoptive transfer into HLA-DRB1*0401 Tg mice. In addition to the ascending paralysis typical of experimental autoimmune encephalomyelitis, mice displayed dysphagia due to restriction in jaw and tongue movements and abnormal gait. In accordance with the clinical phenotype, infiltrates of MS2-3C8 Tg T cells and inflammatory lesions were predominantly located in the brainstem and the cranial nerve roots in addition to the spinal cord and spinal nerve roots. Together, these data suggest a pathogenic role of MBP-specific T cells in inflammatory demyelination within the brainstem and cranial nerve roots during the progression of MS. This notion may help to explain the clinical and pathological heterogeneity of MS.

Generating of Human CD4+ and CD8+ T Lymphocytes toward NY-ESO-1 by T Cell Receptor (TCR) Modification and Transduction for Adoptive TCR Gene-Transfer

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3533-3533
Author(s):  
Holger Krönig ◽  
Kathrin Hofer ◽  
Daniel Sommermeyer ◽  
Christian Peschel ◽  
Wolfgang Uckert ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
T Cell Receptor ◽  
Cell Clone ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
Cell Clones ◽  
T Cell Clone ◽  
T Cell Transfer

Abstract The Cancer Testis (CT) antigen NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in tumor patients and therefore it is a promising candidate antigen for successful adoptive T cell transfer. The aim of our studies is the transfer of autologous T cells re-directed towards CT antigens by T cell receptor (TCR) gene transfer. The first precondition for genetic transfer of CT-Ag-specific TCRs is the availability of tumor-reactive CD4+ and CD8+ T cell clones that express a CT-Ag-specific TCR. Therefore, we generated the autologous CD8+ T cell clone ThP2 through stimulating HLA-A2.1− PBMCs with autologous HLA-A2+DCs loaded with synthetic NY-ESO-1157–165. After two restimulations, FACS-sorting and cloning, the T cell line specifically recognized the NY-ESO-1157–165 peptide and also specifically lysed NY-ESO-1157–165 expressing tumor cells. In addition, we generated NY-ESO-1 specific T helper1 clones from HLA-DR1+ and HLA-DR4+ healthy donors by stimulation of CD4+ T cells with autologous dendritic cells (DC) pulsed with the NY-ESO-187–111 peptide. The specificity of CD4+ T helper cell clones was determined by proliferation assays and IFN gamma ELISPOT through screening with the NY-ESO-187–111 peptide. By limiting dilution of the NYESO- 1-specific T cell populations we succeeded to isolate CD4+ T cell clones, which recognized NY-ESO-1-pulsed target cells and DCs pulsed with NY-ESO-1 protein. The second precondition for TCR gene transfer is the availability of efficient vector systems. Using vectors based upon mouse myelo-proliferative sarcoma virus (MPSV), it was possible to achieve a high transgene expression in the TCR-transduced T cells. Therefore, we cloned the TCR of the HL-A2-restricted NY-ESO-1-specific CTL clone ThP2 in the retroviral vector and documented the correct expression of the TCR-chains using peptide/HLA-multimers following retroviral transduction of peripheral PBMCs. Moreover, the NY-ESO-1 specific lysis of HLA-A2+ NY-ESO-1+ tumor cell lines after transduction in primary T cells was as well effective as the primary T cell clone. Because the expression of naive transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks we modified the TCR of the ThP2 CTL clone by, murinisation, codon optimalization or by introducing cysteins into the constant regions. Afterwards we compared the expression efficiency of the three different modifications on naive T cells by tetramer-staining. We were able to show that codon optimalization leads to an increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The next step is the development of T cell transfer regiments, which are based on class-II-restricted TCR-transduced T cells.

scholarly journals Demonstration of functional CD40 in B-lineage acute lymphoblastic leukemia cells in response to T-cell CD40 ligand

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5162-5170 ◽  
Author(s):  
N Renard ◽  
M Lafage-Pochitaloff ◽  
I Durand ◽  
V Duvert ◽  
L Coignet ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Clone ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Activated T Cells ◽  
T Cell Clone ◽  
B Lineage

Because activated T cells were previously shown to induce proliferation of human normal B-cell precursors (BCP) via the CD40 pathway, we investigated the effects of T cells on leukemic blasts isolated from patients with B-lineage acute lymphoblastic leukemia (BCP-ALL). An anti- CD3 activated human CD4+ T-cell clone was found to induce significant call proliferation in four of nine BCP-ALL samples analyzed. In one of these cases, the T-cell effect was clearly dependent on interaction between CD40 and its ligand. Accordingly, a more thorough analysis was performed on this particular leukemia (case 461, adult early pre-B-ALL, mBCR+, Philadelphia+, i(9q)+). Thus, autologous CD4+ T cells isolated from the patient were also able to induce CD40-dependent proliferation of the leukemic blasts. Analysis of the phenotype after coculture showed that, among the CD19+ cells, a proportion gradually lost expression of CD10 and CD34, whereas some cells acquired CD23. In addition, and in contrast with normal BCP, activated T cells promoted maturation of a subset of the case 461 leukemic cells into surface IgM+ cells. The leukemic origin of the cycling and the maturing cells was assessed by the presence of i(9q), a chromosomal abnormality associated with this leukemia and evidenced by fluorescence in situ hybridization. Taken together, these results show that leukemic BCP can be activated via CD40 but that not all cases display detectable stimulation in response to T cells despite their expression of CD40. In addition, the present data suggest that CD4+ T cells could potentially play a role in the physiology of BCP-ALL.

scholarly journals CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

1994 ◽  
Vol 14 (12) ◽  
pp. 8078-8084 ◽  
Author(s):  
B B Niklinska ◽  
D Hou ◽  
C June ◽  
A M Weissman ◽  
J D Ashwell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phosphatase Activity ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Single Amino Acid ◽  
Tcr Signaling ◽  
Antigen Receptor Signaling ◽  
Membrane Anchoring

T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.

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