The partial isolation of subcellular MHC products which are recognized by alloimmune T lymphocytes

H-2 antigens from three murine tumor-cell lines (YAC, EL-4 and P815) were solubilized with triton X-100 and separated by SDS-polyacrylamide gel electrophoresis. Proteins were eluted from gel slices and assayed for inhibition by a target-effector binding (TBC) assay. Fractions inhibiting alloimmune T cells also contained serologically detectable H-2 molecules as judged by a complement-dependent microcytotoxicity-inhibition assay. Fractions containing Moloney cell-surface antigen (MCSA), gp 71, or p30 did not inhibit alloimmune TBC. H-2 antigens were selective for T cells since they failed to inhibit binding of natural killer (NK) cells to NK-sensitive targets, whereas, in the reciprocal experiment, the NK target antigens inhibited NK cells but not alloimmune T cell binding to intact targets. Cross inhibition tests in the T-cell system revealed that the H-2 molecules maintained their specific antigenic structure. These observations suggest that it will be possible to characterize further those parts of the subcellular H-2 preparations reacting with distinct killer T-cell clones.     

Mapping of epitopes recognized by alloreactive cytotoxic T lymphocytes using inhibition by MHC peptides

To identify epitopes recognized by alloreactive CTL we have examined H-2Kb-specific CTL for their recognition of synthetic peptides with sequences derived from the native Kb class I molecule. Consecutive nested peptides spanning the immunogenic alpha 1 and alpha 2 domains of Kb were tested for their capacity to inhibit CTL clones in their recognition of cells expressing the native Kb molecule. Inhibition by these peptides was found to be an extremely rare event. One peptide (Kb.111-122) did inhibit recognition by one particular CTL clone, clone 13. Upon further investigation it was observed that clone 13 also recognized peptide Kb.111-122 when presented in the context of the syngeneic MHC molecule, Kd. Considering that residues 111 to 122 are located at the base of the antigen groove, and clone 13 is able to recognize Kb.111-122 when presented by syngeneic target cells, we suggest that inhibition of this CTL clone may be due to MHC restricted, self-presentation of peptide rather than to direct binding of free peptide to the TCR. Taken together, these results suggest inhibition of allospecific CTL by MHC peptides is a rare event at least for Kb recognition. Furthermore, they demonstrate the need for caution when interpreting inhibition by peptide as evidence for recognition by the TCR of the corresponding region on the native molecule.     

Mechanisms for generating cell membrane antigens that are recognized by cytotoxic T lymphocytes

Studies with many viruses have revealed that viral specific protein synthesis is an obligatory step in generating antigens on target cells for antiviral cytotoxic T lymphocytes. This has been most clearly demonstrated with DI particles, virions that are structurally complete but lack infectious RNA. Adsorption of such particles onto target cell membranes does not render these cells susceptible to lytic attack by antiviral effector cells, unless some viral protein synthesis transpires. However, some viruses, such as Sendai virus, circumvent the requirement for viral protein synthesis via fusion of the viral envelope with the target cell membrane, a process mediated by a specialized fusion protein. Once inserted into the lipid bilayer, it is likely that viral components and self H-2 noncovalently associate so that the complex can be recognized by antiviral cytotoxic T cells. This idea is supported by the demonstration that viral proteins and H-2 containing membrane proteins, incorporated into reconstituted membrane vesicles or liposomes are recognized by cytotoxic T cells. These data further show that native rather than altered viral and H-2 molecules are the moieties recognized. Associations between antigen and H-2 have been detected by a variety of techniques and in some cases are not random but selective; that is, viral antigens perferentially associate with some H-2 alleles and not others. In summary, these findings indicate that although viral antigens are present in the mature virions, these components are not recognized by antiviral killer cells until integrated into the plasma membrane. This may be achieved either through direct fusion of the viral envelope with the target cell or following viral protein synthesis and insertion of viral antigens into the plasma membrane.     

Helper T lymphocytes which recognize the MHC class I alloantigens in vivo are CD4+CD8-1

To determine the CD4 or CD8 phenotype of the Th lymphocyte which recognizes in vivo the MHC class I alloantigens, B10 recombinant mice were treated with anti-CD8 or anti-CD4 mAb and immunized with lymphoid cells from donors differing in the K or D region of the MHC. Alloantibodies were evaluated by a 51Cr-release assay or by indirect immunofluorescence. The production of IgG anti-Dd and anti-Kk alloantibodies was increased by the deletion of the CD8+ and absent in mice depleted of the CD4+ subset. These experiments indicate that the helper influence elicited by the recognition of a MHC class I alloantigen in vivo is due to cells of the CD4+CD8- phenotype.     

The isolation of cloned cDNA sequences which are differentially expressed in human lymphocytes and fibroblasts.

Poly(A)+ RNA populations derived from normal lymphocytes and fibroblasts have been compared by hybridising each RNA to cDNA derived from the other RNA population. This indicated that approximately 75% of the sequences were common to both, and that these were present at different concentrations in the two cell types. The two RNA populations were further compared by hybridising them to a cDNA recombinant library derived from lymphocyte poly(A)+ RNA. This allowed the identification of clones containing sequences which are abundant in lymphocyte poly(A)+ RNA but absent or rare in fibroblast poly(A)+ RNA. A direct estimation of the abundance of five of these sequences in lymphocyte cDNA demonstrated that clones can be detected by such a procedure if they represent 0.2% or greater of the original cDNA population.     

Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys.

Infection of macaque monkeys with the simian immunodeficiency virus of macaques (SIVmac) results in disease similar to human AIDS. Therefore, the macaque monkey is proving to be an important model for testing the effectiveness of various AIDS vaccine approaches. A detailed analysis of the cellular immune responses is necessary for the evaluation of candidate vaccines. However, this has not been possible in macaques, due, in part, to the unknown nature of the MHC molecules that restrict their T lymphocytes. In our report we demonstrate that a particular MHC class I molecule involved in the rhesus monkey's effector T lymphocyte response to SIVmac is expressed at a high frequency in a colony of rhesus monkeys. SIVmac-infected monkeys that express this MHC class I molecule all develop CTL that are restricted by that molecule and recognize an identical nine amino acid epitope of the SIVmac gag protein. This MHC class I molecule has been defined as an HLA-A homolog by cDNA cloning and sequencing. It has also been expressed in an MHC class I-deficient cell line to demonstrate directly the cloned molecule's capacity to bind and present peptide Ag to CTL. These studies illustrate that AIDS virus-specific CTL can be characterized in detail in the rhesus monkey and lay the foundation for exploring novel approaches to AIDS virus vaccination in this species.     

MHC allele-specific molecular features determine peptide/HLA-A2 conformations that are recognized by HLA-A2-restricted T cell receptors

The structures of alphabeta TCRs bound to complexes of class I MHC molecules and peptide show that the TCRs make multiple contacts with the alpha1 and alpha2 helixes of the MHC. Previously we have shown that the A6 TCR in complex with the HLA-A2/Tax peptide has 15 contact sites on HLA-A2. Single amino acid mutagenesis of these contact sites demonstrated that mutation of only three amino acids clustered on the alpha1 helix (R65, K66, A69) disrupted recognition by the A6 TCR. In the present study we have asked whether TCRs that recognize four other peptides presented by HLA-A2 interact with the MHC in identical, similar, or different patterns as the A6 TCR. Mutants K66A and Q155A had the highest frequency of negative effects on lysis. A subset of peptide-specific CTL also selectively recognized mutants K66A or Q155A in the absence of exogenous cognate peptides, indicating that these mutations affected the presentation of endogenous peptide/HLA-A2 complexes. These findings suggest that most HLA-A2-restricted TCRs recognize surfaces on the HLA-A2/peptide complex that are dependent upon the side chains of K66 and Q155 in the central portion of the peptide binding groove. Crystallographic structures of several peptide/HLA-A2 structures have shown that the side chains of these critical amino acids that make contact with the A6 TCR also contact the bound peptide. Collectively, our results indicate that the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs as well as influence the binding and presentation of the bound peptides.     

Identification of MHC class II-restricted tumor antigens recognized by CD4+ T cells

CD4+ T cells play a central role in orchestrating host immune responses against cancer as well as autoimmune and infectious diseases. Identification of major histocompatibility complex (MHC) class II-restricted helper T peptides is important for development of effective vaccines. The lack of effective methods to identify such T-cell peptides is a major hurdle in the use of antigen-specific CD4+ T cells in cancer vaccines. Here we describe a genetic targeting expression system for cloning genes encoding for MHC class II-restricted tumor antigens recognized by tumor-reactive CD4+ T cells. Helper T peptides are subsequently identified by using synthetic peptides to test their ability to stimulate CD4+ T cells.     

Mouse T lymphocytes proliferative responses specific for human MHC products in mouse anti-human xenogeneic MLR

It has been reported that human T cells recognize the polymorphism of murine Ia antigens in the human anti-mouse xenogeneic mixed lymphocyte reactions (MLR). In this study, murine T cell recognition of human Class II antigens of the major histocompatibility complex (MHC) was analyzed in mouse anti-human xenogeneic MLR responses. The xenoreactive murine T cell proliferative response was blocked by adding anti-HLA-DR monoclonal antibody to the xenogeneic MLR culture. The specificity of xenoreactive murine T cells was examined with regard to the secondary and tertiary xenogeneic MLR system. The xenoreactive murine T cells were restimulated by distinct human stimulator cells that had no shared HLA antigens with the stimulator used in the primary MLR. The data presented here show that the murine xenoreactive T cells recognize the shared determinant(s) of HLA-DR antigen on non-T, non-B stimulator cells. The xenoreactive murine T cell proliferative responses were mediated by Thy-1+, Lyt-1+, and Lyt-2- cells. Furthermore, the xenoreactive T cell responses required Ia+ cells, and Ia antigen on accessory cells plays a crucial role in eliciting the xenoreactive responses against human stimulator cells, while Ia+ accessory cells in the responding cell population are not essential for the elicitation of allogeneic MLR responses, as reported previously.     

A naturally occurring peptide recognized by alloreactive CD8+ cytotoxic T lymphocytes in association with a class I MHC protein.

The antigenic structures that initiate T cell responses to foreign (allogeneic) cells have long attracted considerable interest. We have purified and sequenced a peptide from mouse spleen that is recognized in association with the class I MHC protein H-2Ld by 2C, an alloreactive CD8+ T cell clone. The peptide (LSP-FPFDL) greatly enhances the susceptibility of Ld+ cells to lysis by 2C, and this activity is completely blocked by a clonotypic antibody against the 2C T cell receptor. Thus, this study characterizes the naturally occurring peptide moiety of an MHC-I/peptide complex recognized by alloreactive CD8+ T cells. The peptide, which occurs in the thymus of MHC-disparate mice, can be used to study T cell development in mice expressing transgenes for the 2C T cell receptor.     

Retrovirus antigens recognized by cytolytic T lymphocytes activate tumor rejection in vivo

We have initiated the molecular definition of the antigens recognized by Gross MuLV-specific cytolytic T lymphocytes on the surface of Gross MuLV-induced tumor cells. A panel of target cells was obtained by the double transfection and expression of a retrovirus gene and a foreign H-2 gene in recipient mouse fibroblasts. Our results show that class I H-2 transplantation antigens have a directive influence in determining the antigenicity of proteins encoded by the gag and env MuLV genes. Genes not linked to H-2 influence the intensity and the specificity of the cytolytic T lymphocyte response to Gross MuLV-induced tumors. Finally, MuLV-induced antigens expressed by transfected fibroblasts induce tumor immunity and lead to accelerated tumor rejection in vivo.     

The reovirus nonstructural protein sigma1NS is recognized by murine cytotoxic T lymphocytes.

The cytotoxic T-lymphocyte (CTL) response in reovirus-infected C3H mice was investigated by using reovirus-vaccinia virus recombinants. Results of cytotoxicity assays indicated that the nonstructural protein sigma1NS elicited a significant CTL response. Experiments with sigma1NS-specific CTL lines showed that both strain-specific and cross-reactive epitopes exist in the sigma1NS protein.     

Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor

Single amino acid substitutions at nine different positions on the H-2Kb molecules from in vitro-mutagenized, immunologically altered, somatic cell variants were correlated with their patterns of recognition by monoclonal antibodies (MAbs) and allogeneic cytotoxic T lymphocyte (CTL) clones. While MAbs were found to detect spatially discrete, domain-specific sites, CTLs interacted simultaneously with multiple residues on the alpha 1 and alpha 2 domains of the Kb molecule. The computer graphic three-dimensional Kb model structure showed that, of the seven CTL-specific residues analyzed, six residues were located on the alpha-helical regions of the two domains. Every CTL clone was found to interact with a distinct pattern of residues composed of a specific subset of the CTL-specific residues.     

Structural identity between HLA-A2 antigens differentially recognized by alloreactive cytotoxic T lymphocytes

Alloreactive CTL raised against HLA-A2 Ag often display heterogeneous recognition of HLA-A2+ target cells. This heterogeneity has been found to reflect structural polymorphism among the corresponding target Ag, thus defining HLA-A2 subtypes. A previous study (van der Poel et al. 1986. Human Immunol. 16:247) established the existence of a new HLA-A2.4 variant, A2-SCHU, that was distinguished from A*0206 (A2.4a) by HLA-A2-specific alloreactive CTL. The same CTL subdivided HLA-A2.1 Ag into two subgroups. In the present study, the molecular basis of this heterogeneity has been examined by double-label comparative peptide mapping analysis of differentially recognized A2.1 and A2.4 Ag. In addition, we have determined the complete sequence of polymerase chain reaction-amplified full length cDNA from A2-SCHU. The results show that: 1) A2-SCHU is indistinguishable from A*0206 by peptide mapping; 2) the cDNA sequence of A2-SCHU is identical to that of A*0206; and 3) two differentially recognized A2.1 Ag are both indistinguishable from A*0201 by comparative peptide mapping. These results indicate that differential recognition by alloreactive CTL can occur among structurally identical class I HLA Ag and suggest that allorecognition by such CTL may involve corecognition of endogenous peptides, presumably derived from polymorphic proteins.     

The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides

A proportion of cytotoxic T lymphocytes (CTL) responding to infection by influenza recognize target cells that express the viral nucleoprotein. Recent work showed that CTL can recognize short overlapping regions of large nucleoprotein fragments expressed in transfected L cells. This led to the suggestion that CTL recognize segmental epitopes of denatured or degraded proteins in a similar way to helper T cells. One corollary of this idea is that CTL should recognize appropriate short peptides on the target cell surface. We demonstrate that the epitopes of nucleoprotein recognized by CTL in association with class I molecules of the major histocompatibility complex in both mouse and man can be defined with short synthetic peptides derived from the nucleoprotein sequence.     

The diversity of T cell receptors specific for self MHC gene products

Cytolytic and helper T cells exhibit, in addition to their specificity for foreign antigen, a restriction specificity for self MHC gene products. The present study was designed to assess the degree of diversity within the repertoire of receptors that are involved in T cell recognition of self MHC gene products. For this purpose, we generated a series of murine cytolytic T lymphocyte (CTL) clones specific for a hapten antigen and restricted to the self MHC gene product H-2Kb. An analysis of the hapten fine specificity of these clones by using hapten analogues revealed the presence of substantial diversity within the repertoire of CTL receptors specific for the hapten. The degree of diversity within the repertoire of self H-2 recognition structures on these clones was assessed by testing clones on panels of syngeneic, congenic H-2K disparate, and H-2Kb mutant target cells bearing varying amounts of antigen. A striking degree of heterogeneity in H-2K recognition was found among these H-2Kb restricted CTL. We estimate that there are probably a minimum of 65 different patterns of H-2K recognition among these clones. Our results suggest a high degree of diversity exists within the repertoire of self MHC recognition structures on antigen-specific T cells restricted to a single self MHC gene product.     

Affinity-purified antigen-specific products produced by T cells share epitopes recognized by heterologous antisera raised against several different antigen-specific products from T cells

Heterologous antisera to murine or rat T-cell antigen-binding molecules (T-ABM) were raised in rabbits or sheep. The T-ABM used for immunization were purified by affinity for antigen and did not bear known immunoglobulin isotypes. T-ABM and anti-T-ABM were raised in three separate laboratories. Antisera to T-ABM were exchanged and tested for binding to T-ABM in three separate laboratories. Thus antisera to at least three distinct T-ABM were tested directly for binding to T-ABM or by adsorption of biological activity. Rabbit antisera to murine trinitrophenol (TNP)-specific T-ABM or rat AgB-specific T-ABM bound both murine or rat T-ABM, indicating evolutionary conservation of T-ABM. Similar results were found with sheep antisera to murine T-ABM. In addition, all heterologous anti-T-ABM antisera used bound murine T-ABM specific for TNP, 4-hydroxy-3-nitrophenyl acetate (NP), SRBC, or T-cell membrane proteins with similar structure. Thus, there is a commonality of antigenic determinants between various T-ABM and T-cell membrane homologues which may be T-cell surface receptors for foreign antigen.