Thirty-six views of T-cell recognition

While much is known about the signalling pathways within lymphocytes that are triggered during activation, much less is known about how the various cell surface molecules on T cells initiate these events. To address this, we have focused on the primary interaction that drives T-cell activation, namely the binding of a particular T-cell receptor (TCR) to peptide-MHC ligands, and find a close correlation between biological activity and off-rate; that is, the most stimulatory TCR ligands have the slowest dissociation rates. In general, TCRs from multiple histocompatibility complex (MHC) class-II-restricted T cells have half-lives of 1-11s at 25 degrees C, a much narrower range than found with antibodies and suggesting a strong selection for an optimum dissociation rate. TCR ligands with even faster dissociation rates tend to be antagonists. To observe the effects of these different ligands in their physiological setting, we made gene fusions of various molecules with green fluorescent protein (GFP), transfected them into the relevant lymphocytes, and observed their movements during T-cell recognition using multicolour video microscopy. We find that clustering of CD3zeta-GFP and CD4-GFP on the Tcell occurs concomitantly or slightly before the first rise in calcium by the T cell, and that various GFP-labelled molecules on the B-cell side cluster shortly thereafter (ICAM-1, class II MHC, CD48), apparently driven byT-cell molecules. Most of this movement towards the interface is mediated by signals through the co-stimulatory receptors, CD28 and LFA-1, and involves myosin motors and the cortical actin cytoskeleton. Thus, we have proposed that the principal mechanism by which co-stimulation enhances T-cell responsiveness is by increasing the local density of T-cell activation molecules, their ligands and their attendant signalling apparatus. In collaboration with Michael Dustin and colleagues, we have also found that the formation and stability of the TCR-peptide-MHC cluster at the centre of the interaction cap between T and B cells is highly dependent on the dissociation rate of the TCR and its ligand. Thus, we are able to link this kinetic parameter to the formation of a cell surface structure that is linked to and probably causal with respect to T-cell activation.     

Effect of glutaraldehyde treatment of human melanoma cells on the expression of melanoma-associated antigens

Summary Human melanoma cells were treated with different concentrations of glutaraldehyde, and retention of serological reactivity with antisera against melanoma-associated antigens, HLA antigen, and 2-microglobulin was assessed by quantitative absorption analysis in mixed hemadsorption microassays. Glutaraldehyde concentrations of 0.025% or greater significantly impaired binding to melanoma cells of antibody against melanoma-associated antigens. At a concentration of 0.0025% antibody binding was not decreased although plating efficiency was reduced to less than 1%. Glutaraldehyde concentrations of 0.25% or greater significantly reduced binding to the same melanoma cells of antisera against HLA antigen and 2-microglobulin. Glutaraldehyde treatment (up to 2.5%) of HT-29 colon carcinoma cells failed to reduce reactivity of antisera against CEA and blood group A isoantigen, which are present on these cells. These studies indicate that the effect of glutaraldehyde treatment of cells on retention of surface antigens is critically dependent on the concentration of glutaraldehyde used and the type of antigens involved. Abbreviations used in this paper: MAA, melanoma-associated antigens; GA, glutaraldehyde; FCS, fetal calf serum; RPMI, Roswell Park Memorial Institute; 2M, 2-microglobulin; CEA, carcinoembryonic antigen; PBS, phosphate-buffered saline; NGP, normal glycoprotein cross-reacting with CEA; SRBC, sheep red blood cells     

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.     

Immunochemical analysis of the modulation of human melanoma-associated antigens by DNA recombinant immune interferon

By utilizing the human melanoma cell line Colo 38, a panel of monoclonal antibodies, and a combination of serologic and immunochemical assays, the effect of recombinant immune interferon (IFN-gamma) on the synthesis, expression, and shedding of a cytoplasmic melanoma-associated antigen (MAA) and of the membrane-bound high m.w. MAA (HMW-MAA), 115K MAA, and 100K MAA has been investigated. IFN-gamma increased the synthesis and shedding of the cytoplasmic MAA, but reduced the synthesis and cell surface expression of the HMW-MAA and of the 100K MAA. The cell surface expression of the 115K MAA on IFN-gamma-treated melanoma cells was reduced, although its synthesis was not markedly changed. The effects were dose-dependent and were related to the incubation time of cells with IFN-gamma. Among the three membrane-bound MAA analyzed, the 100K MAA was the most susceptible to modulation by IFN-gamma. The effects of IFN-gamma preparations are not mediated by contaminants in IFN-gamma preparations because removal of IFN-gamma by affinity chromatography on anti-IFN-gamma monoclonal antibodies abolished its modulating activity. The effects of IFN-gamma on the cytoplasmic MAA are similar to those of leukocyte and fibroblast interferons, whereas those on the membrane-bound MAA are significantly different. The potential implications of the marked changes in the antigenic profile of melanoma cells treated with IFN-gamma are discussed in view of the changes in the immunogenicity of IFN-gamma-treated melanoma cells.     

Distinct classes of human T-cell activation antigens

The characterization of three groups of antigens expressed by activated human T lymphocytes and detected by monoclonal antibodies is reported. Antigens defined by OKT19, OKT21, and OKT22 do not appear on in vitro activated T cells until increases in DNA synthesis become apparent and are not detected on most Interleukin 2 (IL-2)-independent cell lines and normal peripheral blood lymphocytes, monocytes, and granulocytes. Cell surface molecules reactive with the monoclonal antibodies OKT23 and OKT24 are displayed prior to any notable increase in DNA synthesis and are present on IL-2 independent cell lines, irrespective of lineage. T23 and T24 do not appear on peripheral blood cells and their distribution more closely resembles that of the T9 antigen (the receptor for transferrin) than antigens of the other groups. The third group of antigens, T14 and T20, have been classified as "early" antigens relative to DNA synthesis. They are expressed by distinct populations of normal lymphoid cells as well as by some IL-2-independent cell lines. Display of each group of activation antigens on T lymphocytes can be induced by either phytohemagglutinin, purified protein derivative from tuberculin, or allogeneic non-T cells, is not restricted to the OKT4+ or OKT8+ subsets, and is predominant on cells exhibiting the light-scattering properties of blast cells. The relative lack of expression of these antigens among normal peripheral blood cells make them attractive candidates for identifying changes in the status of immune activation.     

Immunity to melanoma in mice immunized with transfected allogeneic mouse fibroblasts expressing melanoma-associated antigens

Summary Transfection of genomic DNA from B16 mouse melanoma into LM(TK^–) fibroblasts led to the generation of several clones of transfected cells that strongly expressed B 16 melanoma-associated antigens (MAA). The transfected cells retained their H-2^k markers and served as allogeneic cells with expressive MAA in C57BL/6 mice, syngeneic with the melanoma. The cells were capable of eliciting primary anti-B16 immune responses in vitro in spleen cells from C57BL/6 mice. Immunization of C57BL/6 mice with the transfected cells led to the generation of anti-B16 cytotoxic activity in spleen cells, and C57BL/6 mice immunized with the MAA-positive transfected cells were partially resistant to a lethal challenge with B16 melanoma cells. Under similar conditions, B16 cells were nonimmunogenic. Therefore, transfected allogeneic LM(TK^–) fibroblast cells expressing MAA served as more potent anti-melanoma immunogens than the parental B16 tumor cells themselves.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells: II. Sensitization against melanoma-associated antigens

Peripheral blood lymphoid cells from patients with malignant melanoma can be sensitized on allogeneic or autochthonous melanoma monolayers. Peak cytotoxicity occurred after 5 days of sensitization. Sensitization appeared to be directed against melanoma-associated antigens, as judged by the pattern of cytotoxic reactivity. Sensitized cells were cytotoxic against autochthonous or allogeneic melanoma cells, but not against autochthonous fibroblasts or allogeneic tumor cells of different histologic types. Sensitization of responder lymphoid cells from melanoma patients on allogeneic melanoma cells usually resulted in more pronounced cytotoxicity against autochthonous melanoma target cells than did sensitization on autochthonous melanoma monolayers. These results indicate that cell cultures of human malignant melanoma contain tumor-associated antigens which can sensitize human peripheral blood lymphoid cells in vitro. These results also support the concept that there are cross-reactive tumor-associated antigens in human malignant melanomas.     

The immune recognition of malaria antigens

Owing to the demonstration that the immune response of inbred mice to some defined malaria antigens is influenced by the major histocompatibility complex (MHC), and the finding that only a minority of individuals living in malaria-endemic areas appear to recognize such antigens, there are fears that synthetic subunit malaria vaccines will be poorly immunogenic in a substantial proportion of the target population. Such fears have been reinforced by the results of the first two human malaria vaccine trials. In this review Eleanor Riley, Olle Olerup and Marita Troye-Blomberg summarize the experimental evidence for MHC-related genetic restriction of malaria immunity and discuss some alternative explanations for nonresponsiveness in populations living in malaria-endemic areas.     

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.     

Cross-reactive T-cell antigens among mammalian species.

Fluoresceinated heteroantisera prepared against T cells of rats, monkeys, and humans were reacted withthymus and spleen cells from 11 selected species. These reagents recognized cross-reacting T cell antigen(s) among rodent species (mouse, rat, guinea pig, and hamster) and among primate species (monkey and humans). With one exception, the cross-reactivity was restricted to a phylogenetic order. All three antisera required relatively few absorptions to achieve T cell specificity for related species when compared to absorption requirements for the isologous species. Differentiation antigens within a phylogenetic order thus appear to be more homologous than other cell surface constituents on T cells.     

Expression of T-cell differentiation antigens on activated Thymocytes

The expression of Thy 1, TL, Lyt1, and Lyt2 antigens on resting and proliferating thymocytes activated by concanavalin A in the presence of interleukin 2 has been studied by conventional complement-dependent cytotoxicity assay. The predominant population of resting thymocytes has a TL⁺Lytl⁺Lyt2⁺ phenotype while the predominant population of proliferating thymocytes has a TL — Lytl⁺Lyt2⁺ phenotype. Using several separation procedures such as agglutination by peanut lectin, BSA density gradient centrifugation, and pretreatment with high dilutions of anti-H-2 serum it was impossible to obtain a 100% pure population of TL⁺Lytl⁺Lyt2⁺ cells, suggesting that the population of resting immature thymocytes contains small subpopulations of phenotypically differentiated cells. The population of proliferating thymocytes is also phenotypically heterogenous and contains cells bearing all phenotypes that were described for different stages of T-cell differentiation, including TL⁺Lytl⁺Lyt2^? and TL⁺Lytl^?Lyt2⁺ with the following approximate frequency: TL⁺Lytl⁺Lyt2⁺—27%, TL⁺Lytl⁺Lyt2^?—8%, TL⁺Lyt1^?Lyt2⁺—4%, TL^?Lytl⁺Lyt2⁺—45%, TL^?Lyt1⁺Lyt2^?—13%, TL^?Lyt1^?Lyt2⁺—3%.     

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.     

CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.