Inhibition of alloreactive cytotoxic T lymphocytes by peptides from the alpha 2 domain of HLA-A2

Class I major histocompatibility complex (MHC) molecules function in the recognition of antigens by cytotoxic T lymphocytes (CTL). Although this biological role is firmly established and much has been learnt about their structure and polymorphic variation, little is known of the regions of class I molecules that are involved in functional interactions with components of the T-cell surface. Here we show that peptides derived from residues 98-113 of the alpha 2 domain of HLA-A2 specifically inhibit the recognition of target cells by many HLA-A2-specific CTL. In addition to identifying a region that is probably involved in binding the T-cell receptor these results raise the possibility that alloreactive CTL may recognize degraded fragments of class I histocompatibility antigens.     

Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2

Both human and murine cytotoxic T cells (CTL) elicited in response to infection with influenza A viruses have been shown to be specific for internal viral proteins, such as the matrix and nucleoprotein. Individual CTL epitopes have been identified in the nucleoprotein by successfully substituting short synthetic peptides for the intact virus in the preparation of target cells in cytotoxicity assays. The defined peptide epitopes have each been recognized by CTL in association with individual class I major histocompatibility complex (MHC) proteins, H-2Db, H-2Kk, H-2Kd (Taylor, P. et al., unpublished data) and HLA-B37. A logical strategy to investigate the molecular details of the interaction between antigen and MHC class I proteins would be to define an epitope recognized by the MHC class I molecule HLA-A2. This is because the amino-acid sequence is known, several variants of A2 have been characterized and the protein has been purified and crystallized. Here we describe a peptide derived from the influenza matrix protein that is recognized by human CTL in association with the HLA-A2 molecule.     

A minor transplantation antigen detected by MHC-restricted cytotoxic T lymphocytes during graft-versus-host disease

Transplantation of bone marrow can give rise to graft-versus-host disease when donor T lymphocytes, mismatched with the host for major histocompatability (MHC) antigens, become sensitized and attack host tissues. However, graft-versus-host disease can also arise between donor and host with compatible MHC antigens but mismatched for a minor histocompatability antigen. We report here on the occurrence of severe acute graft-versus-host disease in a male patient with acute myeloid leukaemia who had received bone marrow matched for MHC (HLA) antigens from his sister. Strong cytotoxicity of the posttransplantation (that is, donor) lymphocytes against the patient's pretransplantation lymphocytes was found. Thus, the transplanted lymphocytes differed in a non-HLA antigen from the patient. The possible role of this strong cytotoxic minor histocompatability antigen in the development of graft-versus-host disease in man is being evaluated. Furthermore, with the use of cytotoxic T-cell lines, derived from the patient's 6 day effector cells, we are now able to type for it before grafting.     

Recognition of H-2 domains by cytotoxic T lymphocytes

The polymorphic major histocompatibility antigens (H-2) have a crucial role in the activation of antigen-specific T lymphocytes. Thus, H-2 antigens are not only recognized by allogenic lymphocytes leading to generation of cytotoxic T lymphocytes (CTLs), but it has also been demonstrated that in syngeneic systems most T cells are only able to recognize foreign antigens in conjunction with their own MHC (major histocompatibility complex) antigens. This phenomenon, termed H-2 restriction, may be the key to our understanding to the biological function of MHC antigens. It is not clear whether recognition by T cells of H-2 on a molecular level is confined to particular domains on the H-2 molecule, nor whether the same polymorphic H-2 sites, which are characterized by antibodies, are recognized by allogeneic as well as by H-2 restricted syngeneic CTLs. Previous findings indicate the existence of at least two major polymorphic domains on the H-2Kk molecule as defined by antibodies. Here we show the existence of CTLs with specifity for these polymorphic domains, and the preferential recognition of a particular domain by both alloreactive as well as H-2 restricted CTLs.     

HLA-A2 peptides can regulate cytolysis by human allogeneic T lymphocytes

The class-I and class-II molecules encoded by the major histocompatibility complex (MHC) are homologous proteins which allow cytotoxic and helper T cells to recognize foreign antigens. Recent studies have shown that the form of the antigen recognized by T cells is generally not a native protein but rather a short peptide fragment and that class-II molecules specifically bind antigenic peptides. Furthermore, the three-dimensional structure of the human MHC class-I molecule, HLA-A2, is consistent with a peptide-binding function for MHC class-I molecules. An outstanding question concerns the molecular nature and involvement of MHC-bound peptides in antigens recognized by alloreactive T cells. In this study the effects of peptides derived from HLA-A2 on cytolysis of alloreactive cytotoxic T cells (TC) cells are presented. Peptides can inhibit lysis by binding to the T cell or sensitize to lysis by binding an HLA-A2-related class-I molecule (HLA-Aw69) on the target cell. Thus, allospecific TC cells can recognize HLA-derived peptides in the context of the MHC.     

Restricted recognition of beta 2-microglobulin by cytotoxic T lymphocytes

Recognition of foreign antigen by cytotoxic T lymphocytes (CTL) is restricted by class I major histocompatibility complex (MHC) products. Class I heavy chains (relative molecular mass (Mr) 45,000-48,000) are reversibly and noncovalently associated with beta 2-microglobulin (beta 2M, Mr = 12,000). Cells expressing human or murine class I heavy chains can exchange their native beta 2M for exogenously added free beta 2M, which is present in serum. Two allelic forms of beta 2M exist among the common laboratory mouse strains, beta 2M-A and beta 2M-B, which are represented in BALB and C57BL mice, respectively. The two forms differ at a single amino acid at position 85, the gene (beta 2m) is located on chromosome 2 linked to a minor histocompatibility (H) region, H-3. It has been proposed that one of the H-3 loci is identical with beta 2m, and that CTL raised across certain H-3 incompatibilities are actually specific for beta 2M. Here we describe CTL raised in such a combination which recognize endogenous as well as exogenous beta 2M-B in the context of H-2Kb. This represents a unique case of CTL recognition, as CTL usually recognize antigens inserted into the membrane, and it is the first molecular identification of the product of a minor H locus.     

Peptide-dependent recognition of H-2Kb by alloreactive cytotoxic T lymphocytes

Antigen-specific T lymphocytes appear to recognize foreign antigens in the form of peptide fragments presented within the antigen-binding groove of class I or class II molecules encoded by the major histocompatibility complex (MHC). Alloreactive T cells also show specificity for MHC molecules, and various reports suggest that residues of the MHC molecules constitute at least part of the ligand to which alloreactive T-cell receptors bind. The X-ray crystal structure of the human MHC class I molecule, HLA-A2, has provided evidence to strengthen the argument that MHC-bound self-peptide might also contribute to such recognition. We now provide direct evidence for this, showing that at least some alloreactive cytotoxic T lymphocyte clones recognize peptide fragments derived from cytoplasmic proteins. We reasoned that if self-peptides were involved in allorecognition, then the sequence of some of these peptides could vary between species, resulting in species-restricted distribution of the relevant ligand(s). Several alloreactive cytotoxic T lymphocyte clones specific for H-2Kb, expressed by the murine cell line EL4, did not lyse a human-cell transfectant expressing the H-2Kb molecule (Jurkat-Kb cells). However, these clones were able to lyse Jurkat-Kb cells sensitized by preincubation with an EL4 cytoplasmic extract cleaved by cyanogen bromide. The sensitizing activity from this extract was destroyed by protease and appeared to be due to a peptide consisting of 10 to 15 amino acids.     

A novel serine esterase expressed by cytotoxic T lymphocytes

Cytotoxic T (Tc) lymphocytes recognize and lyse target cells and are thought to serve as an important defence against viral infections and possibly against neoplasms. The nature of the receptors responsible for antigen recognition by these cells is becoming clearer, but the molecular mechanisms responsible for their cytolytic activity remain largely unknown. The possibility that proteases are involved in this process has been suggested by the effects of certain inhibitors. Here we demonstrate that clones of murine Tc cells possess considerable trypsin-like esterase activity when assayed by a sensitive colorimetric assay. This activity was blocked completely by two serine esterase inhibitors, diisopropylfluorophosphoridate (DFP) and phenylmethylsulphonyl fluoride (PMSF), but not by N alpha-tosyl lysyl chloromethyl ketone (TLCK). The use of 3H-DFP as an affinity-labelling reagent demonstrated that the esterase activity resides in a protein of relative molecular mass (Mr) 28,000 (28K). A wide variety of other lymphocytes, including those from thymus, spleen and lymph node, established lines of B cells and noncytotoxic T cells, and clones of T helper cells, had about 300-fold less esterase activity than the Tc-cell clones and far smaller amounts of the DFP-reactive 28K protein. However, in thymocytes the esterase activity increased 20-50-fold and the 28K protein became more prominent 4 days after these cells had been stimulated in vitro to generate Tc cells.     

Acquisition of specific cytotoxic activity by human T4+ T lymphocytes in culture

Mature human T lymphocytes can be separated by monoclonal antibodies OKT4 and OKT8 according to their surface phenotypes into T4+T8- and T4-T8+ subsets. From short-term experiments using bulk cultures, the helper/inducer function has been assigned to the T4+T8- subset and the cytotoxic/suppressor function to the T4-T8+ subset. Thus if T lymphocytes are separated after stimulation in a mixed lymphocyte reaction (MLR), the entire cytotoxic activity is found in the T4-T8+ fraction whereas the T4+T8- fraction shows no detectable cytotoxicity. If, however, T lymphocytes are cloned after MLR and grown in long-term culture, a surprisingly large fraction of T4+ T lymphocyte clones (TLC) shows cytotoxic activity. Here we report that T4+ TLC can acquire specific cytotoxicity during in vitro cultivation.     

Generation of F1 hybrid cytotoxic T lymphocytes specific for self H-2

Cytotoxic T lymphocytes specific for self H-2 antigens are generated by murine F1 hybrid (H-2 heterozygous) spleen cells cultured with irradiated parental (H-2 homozygous) splenocytes. The effectors bind to heterozygous and homozygous cells bearing the appropriate H-2 alleles but only lyse homozygous targets. Autoreactivity for membrane-bound molecules of normal cells may be a mechanism for regulating cellular interactions.     

AIDS virus-specific cytotoxic T lymphocytes in lung disorders

Human immunodeficiency virus (HIV) is implicated in the development of AIDS (acquired immune deficiency syndrome). HIV infection leads to the generation of HIV-specific thymus-derived (T) lymphocytes in humans and apes. We describe an experimental system permitting the quantitative and systematic analysis of HIV-specific cytotoxic T lymphocytes (CTL). Functional, HIV-specific CTL are obtained by broncho-alveolar lavage (BAL) from the lungs of seropositive patients with lymphocytic alveolitis. These alveolar CTL: (1) recognize and kill HIV-infected alveolar macrophages in vitro under autologous, but not heterologous, conditions; (2) correspond to standard CTL as they express the CD3 and CD8 surface markers, but not the CD4 marker; and (3) are restricted by class I HLA transplantation antigens in their cytotoxic activities. We propose the hypothesis that interactions between HIV-specific CTL and infected macrophages induce major inflammatory reactions in seropositive patients.     

In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine

Cytotoxic T lymphocytes (CTL) constitute an essential part of the immune response against viral infections. Such CTL recognize peptides derived from viral proteins together with major histocompatibility complex (MHC) class I molecules on the surface of infected cells, and usually require in vivo priming with infectious virus. Here we report that synthetic viral peptides covalently linked to tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can efficiently prime influenza-virus-specific CTL in vivo. These lipopeptides are able to induce the same high-affinity CTL as does the infectious virus. Our data are not only relevant to vaccine development, but also have a bearing on basic immune processes leading to the transition of virgin T cells to activated effector cells in vivo, and to antigen presentation by MHC class I molecules.     

Signal requirements in the step-wise functional maturation of cytotoxic T lymphocytes

The generation of effector cytotoxic T lymphocytes from resting precursors proceeds through a series of steps in a pathway that, in aggregate, involves both proliferation and development of cytotoxicity. To understand the relationship of the various signals (mitogens and/or lymphokines) that bring about progress along this pathway, it is desirable to define a series of 'minimal signals', each of which stimulates the cell to proceed to a further stage in this process of differentiation. Stimulation of lymphocytes with two different monoclonal antibodies directed against the CD2 surface molecule induces a proliferative response; we report here that both CD4+ and CD8+ cells proliferate in response to such a stimulus but do not develop cytotoxicity. Addition of recombinant gamma-interferon (rIFN-gamma) or recombinant IL-2 (rIL-2) to the activated cells leads to acquisition of cytotoxic status by the CD8+ cells but not the CD4+ cells. The availability, in addition to precursors and effectors, of an apparently intermediate stage in the form of proliferating CD8+ cells that are non-cytotoxic should facilitate both cellular and molecular studies of this maturation pathway; the differences between CD4+ and CD8+ cells in development of cytotoxicity under these experimental conditions is a valuable model for understanding differentiation of T lymphocytes.     

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.     

Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes.

Potent virus-specific cytotoxic T lymphocyte (CTL) responses elicited by candidate AIDS vaccines have recently been shown to control viral replication and prevent clinical disease progression after pathogenic viral challenges in rhesus monkeys. Here we show that viral escape from CTL recognition can result in the eventual failure of this partial immune protection. Viral mutations that escape from CTL recognition have been previously described in humans infected with human immunodeficiency virus (HIV) and monkeys infected with simian immunodeficiency virus (SIV). In a cohort of rhesus monkeys that were vaccinated and subsequently infected with a pathogenic hybrid simian-human immunodeficiency virus (SHIV), the frequency of viral sequence mutations within CTL epitopes correlated with the level of viral replication. A single nucleotide mutation within an immunodominant Gag CTL epitope in an animal with undetectable plasma viral RNA resulted in viral escape from CTLs, a burst of viral replication, clinical disease progression, and death from AIDS-related complications. These data indicate that viral escape from CTL recognition may be a major limitation of the CTL-based AIDS vaccines that are likely to be administered to large human populations over the next several years.     

T-cell-mediated association of peptide antigen and major histocompatibility complex protein detected by energy transfer in an evanescent wave-field

Helper T cells recognize foreign antigen displayed on antigenpresenting cells which also express self-molecules of the major histocompatibility complex (MHC). A single T-cell receptor mediates recognition of both MHC and foreign antigen. A proposed ternary complex between T-cell receptor, foreign antigen and MHC antigen has not yet been demonstrated (see ref. 1 for review). Here, we show that a fluorescein-labelled synthetic peptide, together with Texas red-labelled class II MHC antigen, I-Ad, stimulates the production of interleukin-2 by a peptide-specific I-Ad-restricted T-cell hybridoma when reconstituted in a lipid membrane on a glass substrate. Under the same conditions, resonance-energy transfer from donor peptide to acceptor I-A can be stimulated in an evanescent wave-field only in the presence of the specific T-hybrid. Our results show that the T cell stabilizes an association between peptide antigen and class II MHC protein to within a distance of about 40 A.