Cross-reactivity in T-cell antigen recognition

The molecular interactions between the T-cell receptor (TCR) and peptide-MHC (pMHC) have been elucidated in recent years. Nevertheless, the fact that binding of only slightly different ligands by a TCR, or ligation of the same pMHC at different developmental stages of the T cell, can have opposing consequences, continues to pose intellectual challenges. Kinetic proofreading models, which have at their core the dissociation rates of pMHC from the TCR, are best suited to account for these observations. However, T cells can be triggered by peptides with often minimal homology to the primary immunogenic peptide. This cross-reactivity of the TCR is manifest at several levels, from positive selection of immature thymocytes to homeostasis and antigen-cross- reactive immune responses of mature peripheral T cells. The implications of the high cross-reactivity of T-cell antigen recognition for self-tolerance and T-cell memory are discussed.     

Models for MHC-restricted T-cell antigen recognition

Current models for T-cell recognition of foreign antigen depict the T-cell receptor as having a single antibody-like combining site which binds a complex of MHC and antigen. An alternative hypothesis is presented here; it is proposed that the first domains of the MHC function as inverted V-like regions to complement the TcR V-regions in creating antigen binding sites.     

Multimolecular associations of the T-cell antigen receptor

T cells are activated when the T-cell receptor for antigen (TCR) interacts with an antigenic peptide bound to a major histocompatibility complex (MHC) molecule on the surface of another cell. It is often assumed that T-cell activation is induced by the crosslinking of TCRs. In this article, Albertus Beyers, Louise Spruyt and Alan Williams argue that this mechanism is not generally applicable. They hypothesize that the key event in T-cell activation is the formation of multimolecular complexes consisting of the TCR and several other polypeptides, including CD4 or CD8, CD2, CD5 and the associated tyrosine kinases p59(fyn) and p56(lck).     

T-cell antigen receptor (TCR) transmembrane peptides

Cell surface membranes are generally considered as inert and hydrophobic providing a stable physical barrier that anchor proteins and maintain cellular homeostasis between the intra- and the extra-cellular environment. The integral proteins that transverse membranes do so once or multiple times and can function alone or as part of a larger complex. Far from being inert, there is a multiplicity of biophysical factors that drive protein-protein and protein-lipid interactions within membranes that are being increasingly recognised as very important for cellular function. Unravelling these “hot-spots” on the contact surface of transmembrane (TM) proteins and targeting peptides to these sites to interrupt the cohesive interaction between the proteins provides both an enormous challenge and a huge therapeutic potential that as yet remains unrecognized. Indeed, with biopharmaceutical research on the rise, TM peptides may prove a useful innovation. Using the T-cell antigen receptor (TCR) as a model system of multi-subunits interacting at the TM via electrostatic charges the potential for peptides as therapeutic agents to interfere with normal immune responses is discussed. The principles of such can be extended to other similar receptor systems including those involved in cancer or infection.     

The T-cell receptor mediating restrictive recognition of antigen

Four facts characterize restrictive recognition of antigen. First, in large measure, allele-specific determinants on R are recognized when R is functioning either as a restricting element (RL) or as an allo-target (or even xeno-target) (RF). Second, there is a high frequency of virgin antigen-responsive t cells with alloreactivity, i.e. anti-RF. Third, there is a strict relationship between the class of effector function and the class of RL recognized (restrictive recognition of antigen, XF) but a relaxed relationship between class of effector function and class of RF recognized (alloreactivity). Fourth, the effector T cell functions anti-RL-dependently when XF is the target (restrictive recognition of antigen) and anti-RL-independently when RF is the target (alloreactivity). From these facts are derived the following conclusions. The T cell uses a dual recognitive, single receptor (Model I, Figure 1). A single germ-line VT locus specifying anti-allele-specific recognition of species R encodes both the anti-R and the anti-X combining sites. A "learning" process (occurring in the thymus) is required to establish the restriction specificity (anti-RL) as well as the effector function/class of RL relationship. The repertoire is derived by somatic mutation of all germ-line VT genes specifying anti-RF (Model IA, Table 3 and Figure 9). Given Model IA (Table 3 and Figure 9), we can account further for the existence of an extensive polymorphism of R and minimal polygeneism, for the high frequency of crossreactivity between anti-XF and RF, and for the physiology and genetics of cell-cell communication in immune responsiveness.     

Signal transduction through the T-cell antigen receptor.

The mechanism by which ligand binding to the T-cell antigen receptor triggers the T-cell activation program has long been one of the most fascinating questions in lymphocyte biology. Here, we review recent insights into the transmembrane signaling functions of this multisubunit receptor complex.     

T-cell antigen receptor assembly and cell surface expression is not affected by treatment with T-cell antigen receptor-alpha chain transmembrane Peptide

A synthetic peptide termed core peptide (CP), which corresponds to a specific sequence of the TCR-alpha chain transmembrane domain, is known to inhibit IL-2 production in antigen stimulated T-cells. The molecular mechanism of the TCR inhibition is not known. This study examined the effects of CP on TCR subunit assembly and TCR cell surface expression in vitro. Co-transfection experiments between TCR-alpha and CD3-delta using COS-7 cells, and the interaction between TCR-alpha and the CD3 proteins in a T-cell line (2B4) were analysed after incubation with CP or its conjugates. Results indicate that CP co-precipitates with CD3-delta and CD3-epsilon in vitro, without any effect on TCR-alpha/CD3-delta dimerisation or TCR multisubunit assembly and cell surface expression.     

Modulation of T-cell antigen receptor on lymphocyte membrane

A mouse monoclonal antibody, Mo. Ab. 108.45, detecting cell-surface determinants associated with the T-cell receptor for alloantigen was produced by immunizing mice with an alloreactive human T-cell clone and fusing the splenocytes with the NS₁ plasmocytoma. This Mo. Ab. (1) reacts with the immunizing T-cell clone but not with autologous or allogeneic lymphocytes, lymphoblasts or monocytes; (2) stimulates the proliferation of the immunizing T cells in the absence of the alloantigen; (3) inhibits the response to the specific stimulator; and (4) precipitates a disulfied linked heterodimer composed of two distinct glycoproteins of molecular weights 40 000 and 46 000. The receptor molecule detected by Mo. Ab. 108.45 modulates on the surface of the cells, reaching the highest levels 5 days following exposure to the specific stimulator. The receptor-associated molecule detected by Mo. Ab. 108.45 was expressed by T-cell clones obtained independently in two different mixed lymphocyte cultures between the same responder and stimulator.     

Biosynthesis and processing of murine T-cell antigen receptor

The antigen-specific receptor of C6VL T-lymphoma cells is a disulfide-linked heterodimer composed of 39 kd alpha chain and a 41 kd beta chain, both of which exhibit charge microheterogeneity. Pulse-chase labeling experiments indicate that epitopes reactive with the anti-receptor xenoantiserum #8177 were detectable by 2 min, while the clonotypic epitope reactive with monoclonal antibody 124-40 was not detectable until 10 min. Digestion with endoglycosidases H and F revealed that both subunits have at least three N-linked oligosaccharide side chains. The deglycosylated alpha and beta subunits were 27 and 32 kd, respectively. These data suggest that the dimeric receptor is formed shortly after translation, followed by extensive glycosylation. Emergence of the C6VL clonotypic epitope, and perhaps the antigen binding site, may therefore be dependent on post-assembly events.     

A novel predictive algorithm to personalize autologous T-cell harvest for chimeric antigen receptor T-cell manufacture

Background aimsThe most widely accepted starting materials for chimeric antigen receptor T-cell manufacture are autologous CD3+ T cells obtained via the process of leukapheresis, also known as T-cell harvest. As this treatment modality gains momentum and apheresis units struggle to meet demand for harvest slots, strategies to streamline this critical step are warranted.MethodsThis retrospective review of 262 T-cell harvests, with a control cohort of healthy donors, analyzed the parameters impacting CD3+ T-cell yield in adults with B-cell malignancies. The overall aim was to design a novel predictive algorithm to guide the required processed blood volume (PBV) (L) on the apheresis machine to achieve a specific CD3+ target yield.ResultsFactors associated with CD3+ T-cell yield on multivariate analysis included peripheral blood CD3+ count (natural log, ×10⁹/L), hematocrit (HCT) and PBV with coefficients of 0.86 (95% confidence interval [CI], 0.80–0.92, P < 0.001), 1.30 (95% CI, 0.51–2.08, P = 0.001) and 0.09 (95% CI, 0.07–0.11, P < 0.001), respectively. The authors’ model, incorporating CD3+ cell count, HCT and PBV (L), with an adjusted R² of 0.87 and root-mean-square error of 0.26 in the training dataset, was highly predictive of CD3+ cell yield in the testing dataset. An online application to estimate PBV using this algorithm can be accessed at https://cd3yield.shinyapps.io/cd3yield/.ConclusionsThe authors propose a transferrable model that incorporates clinical and laboratory variables accessible pre-harvest for use across the field of T-cell therapy. Pending further validation, such a model may be used to generate an individual leukapheresis plan and streamline the process of cell harvest, a well-recognized bottleneck in the industry.     

The T-cell receptor as analyzed by functional T-cell lines specific to a synthetic polypeptide antigen

For a better understanding of the molecular nature of the antigen-specific T-cell recognition system, continuous T-cell lines specific to the synthetic polypeptide antigen poly(Tyr,Glu)-poly(DLAla)--poly(Lys) [(T,G)–A--L] were established from C3H.SW (high-responder) activated T-cells, cloned, and characterized. These lines and their derived clones are also constitutive secretors of antigen-specific T-cell replacing helper factors. The secreted T-cell helper factor was shown to possess MHC determinants as well as V-region determinants, or more specifically, idiotypic determinants that are cross-reactive with those expressed on (T,G)–A--L-specific antibodies of the same mouse strain. Using the fluorescence-activated cell sorter (FACS II) and individual C57BL/6 anti-idiotypic sera produced against (T,G)–A--L-specific antibodies of C3H.SW origin, we have demonstrated the expression of the cross-reactive idiotypic markers on the monoclonal helper T-cells. Attempts were made to purify the active fraction of the T-cell factors secreted by the (T,G)–A--L continuous helper lines. Gel analysis of the twice affinity-purified eluate of a (T,G)–A--L column revealed the existence of iodinated bands with molecular weight of 17,000 and 15,000, in addition to a diffuse band of high molecular weight. The specific helper activity of the factors was associated with a 65–75% ammonium sulfate precipitate. Gel electrophoresis of the latter fraction, as well as of an eluate of a (T,G)–A--L–Sepharose column indicated that a high-molecular-weight (< 67,000) and a low-molecular-weight (15,000–17,000) fraction contained the biological activity of the factor. Similar results were obtained following chromatography of the factor on Sephadex G-100 columns. The two fractions were shown to be synthesized by the T-cell lines, as indicated by internal labeling experiments using ³⁵S-methionine. Thus, it is suggested that a fraction of an apparent molecular weight of 15,000–17,000 preserves both the antigen specificity and the helper activity of the factor produced by the (T,G)–A--L-specific T-cell lines.     

Mutants in signal transduction through the T-cell antigen receptor

Mutants of an untransformed helper T-cell clone have been derived by chemical mutagenesis followed by selection for cells incapable of proliferating in response to antigen or anti-CD3. The selection was designed to enrich cells bearing mutations distal to the T-cell antigen receptor. The mutants express normal levels of functional T-cell receptors but are uncoupled from cellular responses, including gene induction, lymphokine secretion, proliferation, and phosphatidylinositol turnover. Responses to phorbol ester plus calcium ionophore and to interleukin-2 are unimpaired. Responses to antigen were restored by fusion with a T-cell receptor-negative thymoma, making the mutants valuable for investigating the mechanisms that couple T-cell receptor stimulation to the induction of second messengers and subsequent physiologic responses.     

Induction of differential T-cell epitope by plain- and liposome-coupled antigen

The T-cell receptors of CD4(+) T lymphocytes recognize immunogenic peptide sequences bound within the groove of MHC class II molecules, and the peptides that bind to these molecules are known to share common structural motifs. For example, OVA(323-339), an I-A(d)-binding peptide, involves a motif of the I-A(d) peptide-binding groove. In the present study, OVA peptides of up to 26-mer were sequentially synthesized and screened, and two additional I-A(d) binding OVA peptides, OVA(20-43) and OVA(264-286), were found to stimulate CD4(+) T cells of OVA-immune BALB/c mice. OVA(20-43) involved structural motifs of the I-A(d) peptide-binding groove, while OVA(264-286) did not. The ability of these three I-A(d) binding OVA peptides to induce antigen-specific cytokine production was compared among CD4(+) T cells of mice immunized either with alum-adsorbed OVA (OVA-alum) or OVA chemically coupled to the surface of liposome (OVA-liposome). CD4(+) T cells of mice immunized with OVA-alum produced more cytokines when stimulated with OVA(264-286) than with OVA(323-339), while CD4(+) T cells of mice immunized with OVA-liposome conjugates produced more cytokines when stimulated with OVA(323-339) than with OVA(264-286). OVA(20-43) induced production of comparable levels of cytokines in mice immunized either with OVA-alum or OVA-liposome. Confocal laser scanning microscopic analysis demonstrated that chemically coupled OVA and liposomes were colocalized in APCs until OVA received processing. Three-dimensional structural analysis demonstrated that both OVA(264-286) and OVA(323-339) were present on the surface of OVA, but OVA(20-43) was not. These results suggested that the chemical coupling of OVA to liposome affected antigen processing in APCs and thus resulted in the induction of differential T-cell epitopes as compared with those induced by plain OVA.     

Molecular genotyping of human T-cell antigen receptor variable gene segments

The gene complex encoding the chain of the T-cell antigen receptor (Tcr) in man was previously reported to contain a restriction fragment length polymorphism (RFLP) involving a single Bgl II site adjacent to the second constant region gene. This RFLP allowed assignment of Tcr genotypes in certain human families. In the present study, two different RFLP in a V gene family were detected using the murine probe V8.1 in genomic DNA samples digested with the restriction endonucleases Hind III and Bam HI. Use of these RFLP to mark the V gene complex allowed complete haplotype assignment in four of seven families studied and provided support for linkage of the V gene complex to the constant region genes. Different combinations of the C and two V region markers can result in eight possible distinct haplotypes. The observation of all but one of the eight possible haplotypes in parents of the families studied suggests that recombination events occur between the C and V region and among members of the V region subfamily marked by the V8.1 probe. These markers can be used for mapping studies of the V gene complex in man and will allow an appraisal of possible associations between Tcr genes and disease susceptibility.Abbreviations used in this paper: Tcr T-cell antigen receptor - RFLP restriction fragment length polymorphism - C2 second Tcr constant region gene - V Variable - C constant - J joining - D diversity     

Mouse T-cell antigen rt6.1 has thiol-dependent NAD glycohydrolase activity

Mouse Rt6.1 and Rt6.2, homologues of rat T-cell RT6 antigens, catalyze arginine-specific ADP-ribosylation. Without an added ADP-ribose acceptor, Rt6.2 shows NAD glycohydrolase (NADase) activity. However, Rt6.1 has been reported to be primarily an ADP-ribosyltransferase, but not an NADase. In the present study, we obtained evidence that recombinant Rt6.1 catalyzes NAD glycohydrolysis but only in the presence of DTT. The NADase activity of Rt6.1 observed in the presence of DTT was completely inhibited by N-ethylmaleimide (NEM). Native Rt6.1 antigen, immunoprecipitated from BALB/c mouse splenocytes with polyclonal antibodies generated against recombinant RT6.1, also exhibited NADase activity in the presence of DTT. Compared with Rt6.2, Rt6.1 has two extra cysteine residues at positions 80 and 201. When Cys-80 and Cys-201 in Rt6.1 were replaced with the corresponding residues of Rt6.2, serine and phenylalanine, respectively, Rt6.1 catalyzed the NADase reaction even in the absence of DTT. Conversely, replacing Ser-80 and Phe-201 in Rt6.2 with cysteines, as in Rt6.1, converted the thiol-independent Rt6.2 NADase to a thiol-dependent enzyme. Kinetic study of the NADase reaction revealed that the affinity of Rt6.1 for NAD and the rate of catalysis increased in the presence of DTT. Moreover, the NADase activity of Rt6.1 expressed on COS-7 cells was stimulated by culture supernatant from activated mouse macrophages, even in the absence of DTT. From these observations, we conclude that t!he Rt6.1 antigen has thiol-dependent NADase activity, and that Cys-80 and Cys-201 confer thiol sensitivity to Rt6.1 NADase. Our results also suggest that upon the interaction of T-cells expressing Rt6.1 with activated macrophages, the NADase activity of the antigen will be stimulated.     

Role of HLA-DR antigen on T-cell activation in visceral leishmaniasis

Ability of peripheral blood monocytes in association with HLA-DR molecules to support T-cell activation in response to soluble Leishmania donovani antigen was investigated. Adherent cells were stained with monoclonal antibodies. The increased number of cells with DR expression was more efficient in presenting L. donovani antigen to sensitized T-cells. The results suggest that quantitative variation in monocytes with expression of DR molecules, correlates with their ability to support T-cell response to L. donovani antigen, in vitro, as assessed by migration inhibition factor (MIF). However, it is not clear whether this is due to only HLA-DR antigen on the surface or whether other factors are involved.