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PubMed1994

The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency.

Stimulation of antigen receptors of lymphocytes triggers a transitory release of Ca2+ from internal stores and the opening of a transmembrane Ca2+ conductive pathway. The latter underlies the sustained increase of intracellular free calcium concentration, and it seems to be a key event in the Ca(2+)-dependent biochemical cascade leading to T cell proliferation. Alternatively, pharmacological depletion of internal stores by itself activates Ca2+ influx. This has led to the hypothesis that antigen-triggered Ca2+ influx is secondary to Ca2+ release from internal stores. However, the precise relationship between antigen and Ca2+ release-activated Ca2+ currents remains unclear, particularly since neither of them has been electrophysiologically recorded in normal lymphocytes. Using the whole-cell and the perforated configurations of the patch clamp technique on peripheral blood lymphocytes, we found that a low amplitude Ca(2+)-selective current was triggered when intracellular stores were depleted by stimuli such as the intracellular perfusion of inositol triphosphate or thapsigargin and the extracellular perfusion of ionomycin. A similar current was elicited by the cross-linking of the T cell receptor-CD3 complex. This current displayed an inward rectification below 0 mV and was completely blocked by the divalent cation Cd2+. It was very selective for Ca2+ over Na+ and insensitive to changes in chloride concentration. The physiological relevance of this conductance was investigated with the analysis of abnormal Ca2+ signaling in lymphocytes from a patient suffering from a primary immunodeficiency associated with a defective T cell proliferation. Using fura-2 video imaging, an absence of Ca2+ influx was established in the patient's lymphocytes, whereas the Ca2+ release from internal stores was normal. This was the case whether cells were stimulated physiologically through their antigen receptors or with store depleting pharmacological agents. Most importantly, no Ca(2+)-selective current was elicited in these cells. Our data strongly suggest that the Ca2+ release-activated current underlies the sustained Ca2+ influx during antigenic stimulation and that it plays a key role in the immune function.

Biological Transport↗
PubMed1994

Severe combined immunodeficient mice engrafted with human splenocytes have functional human T cells and reject human allografts.

Previous studies have shown that human hemopoietic cells can be adoptively transferred into immunodeficient C.B-17 scid/scid (SCID) mice that lack autologous T and B lymphocytes, to generate chimeric animals. The future development of novel immunomodulatory drugs in transplantation will depend increasingly on experimental animal models to investigate the properties of the agents on human cells before starting clinical trials. However, in previous models of SCID mice engrafted with human PBLs, human T cells have been found either to be in an unresponsive state, unable to respond to mitogenic stimulations in vitro, or to mediate skin graft rejection only when HLA-primed in vivo before their adoptive transfer into SCID mice. In addition, T cells and other leukocyte subsets engraft quite poorly in the lymphoid tissues of the animals. In an attempt to develop a useful model for transplantation research, we have inoculated SCID mice with fresh human splenocytes from cadaveric organ donors (hu-Spl-SCID mice). In this model, various leukocyte subsets engraft effectively in different lymphoid compartments. In addition, human T cells retain their proliferative responses to mitogens and to alloantigens when tested 3 wk after engraftment into SCID mice. Finally, mice engrafted with unprimed human spleen cells acutely reject human foreskin allografts. Treatment of hu-Spl-SCID mice with OKT3, an immunosuppressive mAb directed against the human CD3 complex associated with the TCR, prevents the rejection of most human skin allografts, indicating a major role for human T cells in this phenomenon. Thus, this hu-Spl-SCID model may be useful for the study of immunosuppressive therapies in a preclinical in vivo setting.

Animals↗
PubMed1993

T-cell recognition of ovarian cancer.

BACKGROUND: The existence of a tumor-specific T-cell immune response to human malignant melanoma has been well documented. In contrast, the existence of tumor-specific cytotoxic T lymphocyte to ovarian cancer remains controversial despite the abundant lymphocytic infiltrates in the malignant ascites and solid tumor of these patients. METHODS: Tumor-associated lymphocytes (TAL) from the malignant ascites and tumor-infiltrating lymphocytes (TIL) from the solid tumors were isolated from six untreated patients with ovarian cancer. TAL and TIL were grown with initial anti-cluster of differentiation of T cells (CD3), low-dose interleukin-2, and tumor stimulation. T-cell lines were analyzed in functional studies. RESULTS: At 5 weeks, TAL and TIL from five of six patients were > 50% CD8+, and one of six was > 70% CD4+. In all five pairs of CD8 positive cultures, both TAL and TIL exhibited high levels of tumor-specific cytotoxicity for ascite and solid tumor, respectively. T-cell recognition of tumor was mediated through the T-cell receptor-CD3 complex and was human leukocyte antigen class I restricted. TAL and TIL lysed autologous ascitic tumor equally well; however, TAL-mediated tumoricidal activity against autologous solid tumor was consistently and significantly poorer than TIL-mediated killing. CONCLUSIONS: Tumor-specific cytotoxic T lymphocytes can be expanded from both TAL and TIL. However, TAL do not kill solid tumor as efficiently as TIL. This suggests the requirement of TIL, or a combination of TIL and TAL, for effective immunotherapy.

Adult↗
PubMed1996

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide-38 inhibit IL-10 production in murine T lymphocytes.

Vasoactive intestinal peptide (VIP), a neuropeptide present in the peptidergic innervation of lymphoid organs and expressed in thymocytes and peripheral lymphocytes has been previously reported to modulate cytokine expression in T lymphocytes. In this study, we investigated the effects of VIP and of the structurally related neuropeptide PACAP-38 on the expression of IL-10 in murine lymphocyte cultures. Both neuropeptides inhibit IL-10 production by spleen cells or thymocytes activated via the TCR-associated CD3 complex in a similar dose-response manner. The inhibition is specific, presumably mediated through the VIP-R1, and maximum inhibitory levels are achieved within the first 5 to 15 min of exposure to VIP or PACAP-38. CD4+ T cells function as direct cellular targets for the two neuropeptides. The fact that VIP, PACAP-38, and forskolin, all known cAMP inducers, also inhibit IL-10 production, suggests the participation of cAMP in signal transduction. VIP and PACAP-38 regulate transcriptional expression of IL-10, since IL-10 steady state mRNA levels are significantly reduced by treatment with the two neuropeptides. These results expand the range of neuroendocrine-regulated cytokines and support the idea that neuropeptides such as VIP and PACAP, which are released or produced in the local lymphoid microenvironment and specifically modulate the expression of various cytokines, may participate in the intricate cytokine network controlling local immune responses.

Animals↗
PubMed1996

Thymic and extrathymic T cell development.

The majority of T cells located in peripheral blood, spleen and lymph nodes are dependent on the thymus for proper differentiation and function. Only a minority of T lymphocytes located in these lymphoid organs is thymic-independent. In contrast, a large number of thymus-independent T cells is present in the gut epithelium. This review deals with phenotypic and functional characteristics of T cell development, summarizes the knowledge on the cytokine requirement in this process and describes positive and negative selection. The differences between thymic-dependent and thymic-independent T cells are emphasized, including selection processes, CD4-CD8 expression and the composition of the CD3 complex.

Animals↗
PubMed1998

A mucosal intranet: intestinal epithelial cells down-regulate intraepithelial, but not peripheral, T lymphocytes.

Epithelial cells and lymphocytes, including gammadelta and alphabeta T cells, in the gastrointestinal tract epithelium represent a major host defense intranet that is incompletely understood. Cell-to-cell interactions between intraepithelial lymphocytes (IELs) and intestinal epithelial cells (IECs) comprise this intranet, and we have assessed the role of IECs in the regulation of gammadelta and alphabeta T cell responses. When highly purified CD3+ IEL T cells were stimulated via the TCR-CD3 complex, high proliferative responses and cytokine synthesis were induced. However, the addition of viable IECs or purified IEC membranes (mIEC) down-regulated T cell proliferative and cytokine responses. Further, the inhibitory effect of mIEC was not restored by antibodies to TGF-beta, CD1d, E-cadherin, or MHC class I or II. This inhibitory effect was noted for both gammadelta and alphabeta T cell subsets from IELs, and mRNA levels were reduced for both Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-5) cytokines in gammadelta and alphabeta IELs. In contrast, a purified membrane fraction obtained from thymocytes did not inhibit IEL proliferative responses. Further, mIEC did not inhibit splenic alphabeta T cell proliferative responses. These findings show that cell-to-cell interactions between intraepithelial gammadelta and alphabeta T cells and IECs occur via cell surface molecules, suggesting an intranet to prevent potential inflammatory responses at the intestinal mucosal surface.

Animals↗
PubMed1999

Profound enhancement of T cell activation mediated by the interaction between the TCR and the D3 domain of CD4.

CD4 plays an important role in the activation and development of CD4+ T cells. This is mediated via its bivalent interaction with MHC class II molecules and the TCR:CD3 complex through p56lck. Recent studies have implicated a third site of interaction between the membrane-proximal extracellular domains of CD4 and the TCR. Due to these multiple interactions, direct evidence for the functional importance of this extracellular association has remained elusive. Furthermore, the residues that mediate this interaction are unknown. In this study, we analyzed the function of 61 CD4 mutants. Alanine substitution of just 2 residues, either Q114/F182 or F182/F201, which are partially buried and located close to the D2/D3 interface, completely abrogated CD4 function. Direct evidence for the functional importance of TCR:CD4.D3 interaction was obtained using an anti-CD3fos:anti-CD4jun-bispecific Ab. Surprisingly, it induced strong T cell activation in hybridomas transfected with cytoplasmic-tailless CD4, despite the lack of association with either p56lck or MHC class II molecules. However, this effect was completely abrogated with the CD4 mutants Q114A/F182A or F182A/F201A. These data demonstrate that TCR:CD4.D3 interaction can have a profound effect on T cell activation and obviates the need for receptor oligomerization.

Amino Acid Sequence↗
PubMed1991

Expression and function of a variant T cell receptor complex lacking CD3-gamma.

A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface-radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3-epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma.

Antibodies, Monoclonal↗
PubMed1997

Signalling through either the p38 or ERK mitogen-activated protein (MAP) kinase pathway is obligatory for phorbol ester and T cell receptor complex (TCR-CD3)-stimulated phosphorylation of initiation factor (eIF) 4E in Jurkat T cells.

Initiation factor (elF) 4E plays a key role in the regulation of translation. Its activity is modulated both by phosphorylation and by its association with an inhibitory protein, 4E-BP1, which precludes its interaction with eIF4G. Although increased eIF4E phosphorylation has been correlated with the activation of protein synthesis in T cells, the kinase(s) and/or phosphatase(s) involved have not been characterised. There is evidence for phosphorylation of eIF4E mediated by both protein kinase C-dependent and -independent signalling pathways. In these studies, I show that activation of protein kinase C with phorbol ester, stimulation via the T cell receptor complex with the monoclonal antibody OKT3 and cellular stresses increase the phosphorylation of eIF4E in Jurkat T cells. In contrast to published data, inhibition of either the ERK MAP kinase or p38 MAP kinase signalling pathways does not affect the PMA- or OKT3-stimulated increase in eIF4E phosphorylation. However, simultaneous inhibition of both of these pathways with selective inhibitors is required to completely abrogate the enhanced phosphorylation of eIF4E. These data show that in Jurkat cells, protein kinase C modulates the phosphorylation status of eIF4E indirectly via the ERK and/or p38 MAP kinase signalling pathways.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Altered functional responsiveness of thymocyte subsets from CD3delta-deficient mice to TCR-CD3 engagement.

CD3delta-deficient (delta degrees) mice are defective in alphabeta T cell development. Here we explore the capacity of TCR-CD3 signaling complexes expressed on delta degrees thymocytes to mediate the following functional outcomes in response to antibody cross-linking: (i) the transition from the CD4-CD8- to CD4+CD8+ stage, (ii) the transition from the CD4+CD8+ to CD4+CD8- or CD4-CD8+ stages and (iii) the induction of apoptosis. We provide evidence that CD3deltaepsilon complexes are dispensable for mediating the anti-CD3-mediated CD4-CD8- to CD4+CD8+ transition. On the other hand, CD3delta is critical at the CD4+CD8+ stage. We demonstrate that CD4+CD8+ thymocytes from delta degrees mice, unlike delta degrees CD4-CD8- thymocytes and wild-type CD4+CD8+ thymocytes, require prolonged or consecutive stimuli to elicit functional responses. Depending on the nature of the secondary stimulus, delta degrees thymocytes can be induced to undergo apoptosis or preferential maturation to the CD4-CD8+ stage. Taken together these results indicate that the signaling capacity of the TCR-CD3 complex is noticeably altered in the absence of CD3delta. The essential role of CD3delta at the CD4+CD8+ stage of development correlates with the onset of TCRalpha rearrangement, consistent with a critical structural and/or functional relationship between CD3delta and TCRalpha.

Animals↗
PubMed1990

Selective signal transduction through the CD3 or CD2 complex is required for class II MHC expression by human T cells.

Ag-dependent activation of human T cells results in high level expression of class II MHC molecules. As part of this process, Ag recognition by TCR generates a series of second signals including protein kinase C, tyrosine kinase, and Ca2+ mobilization. To investigate the role of these second messengers in class II MHC expression, purified T cells were first stimulated by PMA, ionomycin, OKT3 accompanied by IL-2, or the mitogenic anti-CD2 antibodies T112 and T113 and were then stained with FITC-conjugated anti-class I and -class II MHC antibodies for analysis by flow cytometry. OKT3 and IL-2 induced optimal expression of HLA-DR (DR) on 70% of T cells with high density. Despite their high mitogenicity, induction of class II MHC expression by PMA, even with co-stimulation by ionomycin, was reduced to less than 20% of T cells, with an intensity 50-fold lower than in OKT3/IL-2-stimulated T cells. Furthermore, PMA inhibited class II MHC expression by OKT3/IL-2-stimulated T cells in a dose-dependent manner and additional stimuli, such as IL-1, IL-4, IFN-gamma, TCR cross-linkers, or monocytes, did not restore class II MHC expression by PMA-activated T cells. DR beta mRNA analysis showed that the low induction of class II molecules by PMA extends to the transcriptional level. Interestingly, anti-T112 and anti-T113 induced not only proliferation of T cells but also DR expression on more than 90% of T cells. These results indicate that transduction of a specific signal, probably selective phosphorylation of the CD3 molecule, contributes to class II MHC induction in the process of T cell activation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Characterization of human peripheral lymphocytes expressing the CD3-gamma/delta complex with anti-receptor monoclonal antibodies.

Three mAb, anti-Ti gamma A, anti-TCR delta 1, and anti-delta TCS1, have been developed against the CD3-associated gamma/delta molecular complex. One of this antibody anti-Ti gamma A is specific for an epitope encoded by the V9 gamma-gene. The two others react with the delta-chain but their fine epitopic specificity has not been characterized previously. In the present study, we have compared the surface expression of these three antigenic determinants on 27 cloned and 5 polyclonal CD3+ TCR gamma/delta + cell lines derived from human peripheral blood of 13 distinct individuals. It was found that all CD3+ TCR alpha/beta- clones and polyclonal cell lines tested were recognized by anti-TCR delta 1. In contrast, only a fraction of both clones and cell lines reacted with either anti-Ti gamma A or anti-delta TCS1 mAb. In fact reactivity of the latter reagents was found to be mutually exclusive on the cell panel. Northern blot analysis of RNA extracted from a series representative clones showed a positive correlation between the surface expression of the delta TCS1 epitope and the transcription of the V-delta gene isolated from the IDP2 cell line. These data support the view that anti-TCR delta 1 can be used to positively define the entire TCR gamma/delta+ fraction. Moreover, the reciprocal reactivity of anti-delta TCS1 and anti-Ti gamma A on cultured cell lines suggests that these reagents should delineate in human peripheral blood distinct, essentially non-overlapping, subsets. Taken together, the present results indicate that the complementary use of these three antibodies will be helpful to further characterize the TCR gamma/delta + peripheral lymphocyte fraction.

Adult↗
PubMed2000

Competition of peptide-MHC class I tetrameric complexes with anti-CD3 provides evidence for specificity of peptide binding to the TCR complex.

BACKGROUND: Major histocompatibility complex (MHC)-peptide tetrameric complexes (tetramers) are valuable tools for detecting and characterizing peptide-specific T cells. Because the frequency of these cells is generally very low, it may be difficult to discriminate between nonspecific and specific tetramer binding. METHODS: A four-color flow cytometric assay that simultaneously measures tetramer, CD3, CD8, and CD14 was used to investigate the sensitivity and specificity of MHC class I tetramer staining. This was accomplished by using the influenza virus matrix protein peptide, GILGFVFTL (FLU), as a model recall antigen and the human immunodeficiency virus (HIV) reverse transcriptase peptide, ILKEPVHGV (HIV), as a model novel antigen. Peripheral blood mononuclear cells (PBMC) from 31 HLA-A2.1(+) and 10 HLA-A2.1(-) healthy individuals were stained with the tetramers. RESULTS: The lower limit of detection was established at approximately 1/8,000. In HLA-A2(+) PMBC, frequencies of tetramer-positive CD8(+) T cells were log normally distributed and were high for FLU (1/910) but low for HIV (1/6,067). A novel competition assay, in which tetramer binding was shown to diminish subsequent staining with anti-CD3 antibody, was used to confirm the specificity of tetramer binding to the T-cell receptor (TCR) complex. The competition assay was validated by evaluating several anti-CD3 antibodies and showing that in PBMC from HLA-A2(-) subjects, spurious tetramer-positive events (1/20,000) failed to compete with CD3 binding. For the "recall" FLU tetramer, the degree of competition was proportional to the frequency, suggesting a selection of high avidity cells. Although CD3 competition was also highly correlated with the intensity of tetramer staining, competition allowed the identification of false positive cases with relatively high tetramer staining intensity. CONCLUSION: The data indicate that competition of CD3 binding allows confirmation of the specificity of tetramer binding to the TCR, extending the usefulness of tetramers in the frequency analysis of peptide-specific T lymphocytes.

Amino Acid Sequence↗
PubMed1995

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells.

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.

Amino Acid Sequence↗
PubMed2004

Crystal structure of the human T cell receptor CD3 epsilon gamma heterodimer complexed to the therapeutic mAb OKT3.

The CD3 epsilon gamma heterodimer is essential for expression and function of the T cell receptor. The crystal structure of the human CD3 epsilon gamma heterodimer is described to 2.1-A resolution complexed with OKT3, a therapeutic mAb that not only activates and tolerizes mature T cells but also induces regulatory T cells. The mode of CD3 epsilon gamma dimerization provides a general structural basis for CD3 assembly and maps candidate T cell antigen receptor docking sites, including a duplicated linear region rich in acidic residues that is unique to human CD3 epsilon. OKT3 binds to an atypically small area of CD3 epsilon and has a low affinity for the isolated CD3 epsilon gamma heterodimer. The structure of the OKT3/CD3 epsilon gamma complex has implications for T cell signaling and therapeutic design.

Amino Acid Sequence↗
PubMed1989

Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain.

The TCR/CD3 complex is a multimeric protein complex composed of a minimum of seven transmembrane chains (TCR alpha beta-CD3 gamma delta epsilon zeta 2). Whereas earlier studies have demonstrated that both the TCR-alpha and -beta chains are required for the cell surface expression of the TCR/CD3 complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat was investigated. The results indicate that: 1) CD3-zeta is required for the cell surface expression of the TCR/CD3 complex; 2) the pentameric form (TCR alpha beta-CD3 gamma delta epsilon) of the TCR/CD3 complex and single TCR chains associated with CD3 (TCR alpha-CD3 gamma delta epsilon and TCR beta-CD3 gamma delta epsilon) are produced in the endoplasmic reticulum in the absence of CD3-zeta; 3) the CD3-zeta does not associate with TCR alpha-CD3 gamma delta epsilon or TCR beta-CD3 gamma delta epsilon complexes; 4) CD3-zeta associate with the pentameric form of the TCR/CD3 complex in the endoplasmic reticulum to form the heptameric complex (TCR alpha beta-CD3 gamma delta epsilon----TCR alpha beta-CD3 gamma delta epsilon 2); and 5) CD3-zeta is required for the export of the TCR/CD3 complex from the endoplasmic reticulum to the Golgi apparatus for subsequent processing.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

A conformational epitope expressed upon association of CD3-epsilon with either CD3-delta or CD3-gamma is the main target for recognition by anti-CD3 monoclonal antibodies.

We have performed immunofluorescence analysis of COS cells transfected with human CD3 genes and detergent permeabilized to define the specificity of several anti-CD3 antibodies. We have found that the mAb OKT3, WT31, UCHT1, and Leu-4 did not stain COS cells singly transfected with the CD3-epsilon chain. However, these antibodies very strongly stained COS cells doubly transfected with a combination of CD3-epsilon plus either CD3-gamma or CD3-delta. By contrast, the antibodies SP34 and APA 1/1, which were raised against isolated SDS-denatured CD3-epsilon protein, gave a strong staining of COS cells singly transfected with CD3-epsilon as well as of the double transfectans. The recognition by this panel of anti-CD3 antibodies of CD3-gamma/epsilon and CD3-delta/epsilon complexes and not of CD3-epsilon alone was assessed by immunoprecipitation. These findings suggest that the most widely used mAb specific for the CD3 complex recognize conformational epitopes on CD3-epsilon, which are expressed when this chain is bound to either CD3-gamma or CD3-delta. It should also be highlighted that antibody WT31 clearly recognizes the CD3 moiety of the TCR/CD3 complex.

Antibodies, Monoclonal↗