Surface programmable activation receptor (SPAR) cell platform for the rapid detection of melanoma-specific biomarkers.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14263-e14263
Author(s):  
Joel Lwande ◽  
Joseph D Kittle ◽  
M. Russell Williams ◽  
Shengwen Liang ◽  
Kyle McQuaid ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
Target Detection ◽  
Cell Activation ◽  
Cell Receptor ◽  
Jurkat Cells ◽  
Cell Surface Expression ◽  
Specific Cell ◽  
Luminescent Signal

e14263 Background: We have previously demonstrated that SPAR modified T cells effectively detect bacterial and viral pathogens. SPAR cells are engineered to express a modified T cell receptor (TCR) capable of antibody-directed signal transduction and respond with a Ca2+-mediated production of luminescent signal upon target detection. Here we describe the ability of SPAR cells to rapidly detect the cell surface expression of melanoma biomarkers without a requirement for extensive sample preparation. Methods: SPAR cells expressing both an engineered T cell receptor and the luminescent reporter protein aequorin were developed via the transfection of Jurkat cells with the aequorin expression vector pEF1-Aeq and an engineered TCR complex composed of mouse FcγRI fused to the CD3ζ subunit. The mFcγRI-CD3ζ receptor binds to the Fc region of full-length mouse IgG2 antibodies: SPAR cells are programmed for target detection via the addition of target-specific antibody. The ability of programmed SPAR cells to accurately detect melanoma-specific cell surface biomarkers was evaluated using whole cells from cultured mouse (B16-F10) and human (SK-Mel-28) melanoma cell lines. SPAR cells were programmed by incubation with murine antibodies directed toward the melanoma biomarkers CD133 or TRP1, then mixed with melanoma cells or K562 control cells and evaluated for signal generation. Results: SPAR cells programmed with anti-CD133 or anti-TRP1 antibody produced luminescent signal within minutes when combined with human SK-Mel-28 cells or mouse B16-F10 cells, respectively, known to abundantly express the appropriate biomarker. No signal was generated when programmed SPAR cells were incubated with K562 cells. Further studies also document cytokine production following receptor engagement. Conclusions: SPAR cells can be programmed for the rapid and specific detection of known cell surface cancer biomarkers. The Ca2+-dependent production of luminescent signal and cytokine release in response to TCR engagement suggests SPAR cell activation. Thus, in addition to biomarker detection the SPAR system may ultimately provide predictive insights into the potency of antibody-directed cell therapy.

Protein Phosphatase 2A Isotypes Regulate Cell Surface Expression of the T Cell Receptor

2001 ◽  
Vol 18 (1) ◽  
pp. 24-33 ◽  
Author(s):  
Jens Peter H. Lauritsen ◽  
Charlotte Menné ◽  
Jesper Kastrup ◽  
Jes Dietrich ◽  
Carsten Geisler
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cell Receptor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Phosphatase 2A

scholarly journals Downregulation of the T-Cell Receptor Complex and Impairment of T-Cell Activation by Human Herpesvirus 6 U24 Protein

2007 ◽  
Vol 82 (2) ◽  
pp. 602-608 ◽  
Author(s):  
Brian M. Sullivan ◽  
Laurent Coscoy
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Human Herpesvirus ◽  
Cell Receptor ◽  
Immune Recognition ◽  
Human Herpesvirus 6 ◽  
Herpesvirus 6

ABSTRACT We have performed a screen aimed at identifying human herpesvirus 6 (HHV-6)-encoded proteins that modulate immune recognition. Here we show that the U24 protein encoded by HHV-6 variant A downregulates cell surface expression of the T-cell receptor (TCR)/CD3 complex, a complex essential to T-cell activation and the generation of an immune adaptive response. In the presence of U24, the TCR/CD3 complex is endocytosed but is not recycled back to the plasma membrane. Instead, it accumulates in early and late endosomes. Interestingly, whereas CD3 downregulation from the cell surface is normally associated with T-cell activation, U24 downregulates CD3 independently of T-cell activation. Moreover, we found that U24-expressing T cells are resistant to activation by antigen-presenting cells. HHV-6 has evolved a unique mechanism of inhibition of T-cell activation that may impair the establishment of an adaptive immune response. Furthermore, lymphocyte activation creates an environment favorable to the reactivation and replication of lymphotropic herpesviruses. Thus, by inhibiting T-cell activation, HHV-6 might limit its reactivation and thus minimize immune recognition.

Defective synthesis or association of T-cell receptor chains underlies loss of surface T-cell receptor–CD3 expression in enteropathy-associated T-cell lymphoma

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5103-5110 ◽  
Author(s):  
Jennifer M. L. Tjon ◽  
Wieke H. M. Verbeek ◽  
Yvonne M. C. Kooy-Winkelaar ◽  
Binh H. Nguyen ◽  
Arno R. van der Slik ◽  
...  
Keyword(s):  
Celiac Disease ◽  
T Cell ◽  
Cell Surface ◽  
Cell Line ◽  
T Cell Receptor ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
Intraepithelial Lymphocytes ◽  
T Cell Lymphoma ◽  
Cell Surface Expression

Abstract Enteropathy-associated T-cell lymphoma, an often fatal complication of celiac disease, can result from expansion of aberrant intraepithelial lymphocytes in refractory celiac disease type II (RCD II). Aberrant intraepithelial lymphocytes and lymphoma cells are intracellularly CD3ϵ+ but lack expression of the T-cell receptor (TCR)–CD3 complex on the cell surface. It is unknown what causes the loss of TCR-CD3 expression. We report the isolation of a cell line from an RCD II patient with the characteristic phenotype of enteropathy-associated T-cell lymphoma. We demonstrate that in this cell line the TCR-α and -β chains as well as the CD3γ, CD3δ, CD3ϵ, and ζ-chains are present intracellularly and that assembly of the CD3γϵ, CD3δϵ, and ζζ-dimers is normal. However, dimerization of the TCR chains and proper assembly of the TCR-CD3 complex are defective. On introduction of exogenous TCR-β chains, but not of TCR-α chains, assembly and functional cell surface expression of the TCR-CD3 complex were restored. Defective synthesis of both TCR chains was found to underlie loss of TCR expression in similar cell lines isolated from 2 additional patients. (Pre)malignant transformation in RCD II thus correlates with defective synthesis or defective association of the TCR chains, resulting in loss of surface TCR-CD3 expression.

Engagement of the CD4 molecule influences cell surface expression of the T-cell receptor on thymocytes

Nature ◽  
1988 ◽  
Vol 336 (6194) ◽  
pp. 76-79 ◽  
Author(s):  
Susan A. McCarthy ◽  
Ada M. Kruisbeek ◽  
Ingeborg K. Uppenkamp ◽  
Susan O. Sharrow ◽  
Alfred Singer
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Surface Expression ◽  
Cell Surface Expression

scholarly journals Synergistic T cell activation via the physiological ligands for CD2 and the T cell receptor.

1988 ◽  
Vol 168 (3) ◽  
pp. 1145-1156 ◽  
Author(s):  
B E Bierer ◽  
A Peterson ◽  
J C Gorga ◽  
S H Herrmann ◽  
S J Burakoff
Keyword(s):  
T Cells ◽  
Cell Adhesion ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Cytoplasmic Domain ◽  
Hla Dr

T cells may be activated either by the antigen-specific T cell receptor (TCR)-CD3 complex or the cell surface receptor CD2. A natural ligand for CD2 has been found to be lymphocyte function-associated antigen 3 (LFA-3), a widely distributed cell surface glycoprotein. To investigate the interaction of these two pathways, we have expressed the cDNA encoding the human CD2 molecule in a murine T cell hybridoma that produces IL-2 in response to HLA-DR antigens. Expression of the CD2 molecule markedly enhances IL-2 production in response to LFA-3+ antigen-bearing stimulator cells, and this stimulation is inhibited by anti-CD2 and anti-LFA-3 mAb. To further define the role of LFA-3 in antigen-dependent T cell activation, we have studied the ability of the purified ligands of CD2 and the TCR to stimulate the hybridoma. Neither liposomes containing purified HLA-DR antigens nor liposomes containing purified LFA-3 were able to stimulate the parent or the CD2+ hybridoma. However, liposomes containing both purified LFA-3 and HLA-DR, the physiological ligands for CD2 and the TCR, respectively, stimulate IL-2 production by the CD2+ but not the parent hybridoma, suggesting that complementary interactions between the TCR-CD3 complex and the CD2 pathway may regulate lymphocyte activation. To determine whether the CD2/LFA-3 interaction participates in cell-cell adhesion and provides an activation signal, we have constructed a cytoplasmic deletion mutant of CD2, CD2 delta B, in which the COOH-terminal 100 amino acids of CD2 have been replaced with a serine. Hybridomas expressing the CD2 delta B molecule were examined. Deletion of the cytoplasmic domain of CD2 did not alter binding of LFA-3 but eliminated the ability of CD2 to increase the response of the hybridoma to liposomes containing both HLA-DR and LFA-3, demonstrating that adhesion of LFA-3 to CD2 alone was insufficient for activation, and that the cytoplasmic domain was required for LFA-3 stimulation through the CD2 molecule. T cells may be activated by purified LFA-3 binding to CD2 and the TCR interacting with its ligand, and these signals appear to be synergistic for the T cell. These results suggest that the CD2/LFA-3 interaction not only plays a role in cell-cell adhesion but provides a stimulatory signal for T cell activation.

scholarly journals The Transmembrane Adaptor Protein Trim Regulates T Cell Receptor (Tcr) Expression and Tcr-Mediated Signaling via an Association with the Tcr ζ Chain

2001 ◽  
Vol 193 (11) ◽  
pp. 1269-1284 ◽  
Author(s):  
Henning Kirchgessner ◽  
Jes Dietrich ◽  
Jeanette Scherer ◽  
Pia Isomäki ◽  
Vladimir Korinek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Jurkat T Cells ◽  
Integral Component

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-ζ chains and to a lesser extent via the CD3-ε/γ heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca2+ mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

scholarly journals Allelic Exclusion in pTα-deficient Mice: No Evidence for Cell Surface Expression of Two T Cell Receptor (TCR)-β Chains, but Less Efficient Inhibition of Endogeneous Vβ→ (D)Jβ Rearrangements in the Presence of a Functional TCR-β Transgene

1997 ◽  
Vol 186 (5) ◽  
pp. 767-775 ◽  
Author(s):  
Anna Krotkova ◽  
Harald von Boehmer ◽  
Hans Jörg Fehling
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Allelic Exclusion ◽  
Thymocyte Development ◽  
Β Chain ◽  
Deficient Mice

Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-β chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-β chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-β rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-β rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-α (pTα) chain, a core component of the pre-TCR. Staining of CD3+ thymocytes and lymph node cells with antibodies specific for Vβ6 or Vβ8 and a pool of antibodies specific for most other Vβ elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTα-deficient mice expressing a functionally rearranged TCR-β transgene. Interestingly, although the transgenic TCR-β chain significantly influenced thymocyte development even in the absence of pTα, it was not able to inhibit fully endogeneous TCR-β rearrangements either in total thymocytes or in sorted CD25+ pre-T cells of pTα−/− mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.

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