Anti-HLA-A2 antibody-enhancement of peptide association with HLA-A2 as detected by cytotoxic T lymphocytes

Most cytotoxic T lymphocytes (CTL) not only recognize epitopes of viral or other foreign proteins in association with class I major histocompatibility complex (MHC) molecules, but also recognize target cells sensitized with short synthetic peptides representing the epitopes. There is increasing evidence that these synthetic peptides associate with the class I molecule both at the cell surface and intracellularly. We have now investigated the effect of a monoclonal antibody specific for HLA-A2 and HLA-B17 (B57/58) molecules (antibody MA2.1)3 on the sensitization of target cells with peptide for lysis by HLA-A2-restricted CTL. Previously, anti-HLA class I monoclonal antibodies have been shown to inhibit the recognition of target cells, infected with influenza A virus, by virus-specific CTL. We find, however, that target cells treated with MA2.1 antibody can be sensitized with peptide for CTL lysis much more rapidly than untreated cells, or at greater than 100-fold lower peptide concentration than that required for sensitization of untreated cells. This implies that the antibody, which is believed to bind to one side of the peptide-binding groove, directly affects the binding of peptide to the HLA-A2 molecule at the cell surface.     

Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody

The specificity of cytotoxic T lymphocytes (Tc) cells is conferred by an antigen-specific receptor, Ti, which in humans is physically associated with an invariant cell-surface glycoprotein, T3. Monoclonal antibodies specific for either T3 and Ti are able to elicit a variety of T-cell responses such as lymphokine production, mitogenesis and cytotoxicity. For example, human Tc cells lyse anti-T3-expressing hybridoma cells, but not cells of other specificity, presumably because anti-T3 on the hybridoma cells binds to T3 on the Tc cells and triggers lysis. Here, we have adapted approaches used in a different cytotoxic effector system, antibody-dependent cellular cytotoxicity (ADCC), to alter the specificity of Tc cell. Studies of ADCC showed that heteroaggregates containing anti-Fc receptor (Fc gamma R) antibody cross-linked to a second antibody bind to Fc gamma R on ADCC effectors and cause them to kill target cells bearing antigen recognized by the second antibody. The present studies use anti-T3-containing heteroaggregates to re-target human Tc cells to cells for which we have appropriate antibodies, including xenogeneic tumour cells and chicken erythrocytes. These results extend previous observations on the role of T3 in triggering cytotoxicity and suggest that effector cell re-targeting could be used for in vivo treatment of neoplasms and other pathogens that express distinctive surface antigens.     

Monoclonal antibodies demonstrate protection of polymorphonuclear leukocytes against complement attack

Studies on erythrocytes have shown that the formation of the membrane attack complex on a cell surface inevitably results in lysis. However, it is known that nucleated cells are much more difficult to kill with complement, although the molecular basis of this resistance has never been established. We have shown that a very early intracellular event, occurring within seconds of formation of the attack complex in the membrane, is a rise in cytoplasmic Ca2+, which can activate cell responses without cell death 5,6. Here we report the use of a monoclonal antibody to the terminal complement component C9, quantified by 125I and visualized by fluorescein, to demonstrate a protection mechanism in polymorphonuclear leukocytes (PMNs) attacked by complement, involving removal of the attack complex by vesiculation. Concomitantly, there is a Ca2+-dependent activation of reactive oxygen metabolite production without cell lysis. These findings have important implications in the evolutionary and pathological significance of the terminal components of the complement pathway.     

MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells.

The molecular basis of target cell recognition by CD3- natural killer (NK) cells is poorly understood, despite the ability of NK cells to lyse specific tumour cells. In general, target cell major histocompatibility complex (MHC) class I antigen expression correlates with resistance to NK cell-mediated lysis, possibly because NK cell-surface molecules engage MHC class I antigens and consequently deliver inhibitory signals. Natural killer cell allospecificity involves the MHC class I peptide-binding cleft, and further understanding of this allospecificity should provide insight into the molecular mechanisms of NK cell recognition. The Ly-49 cell surface molecular mechanisms of NK cell recognition. The Ly-49 cell surface molecule is expressed by 20% of CD3- NK cells in C57BL/6 mice (H-2b). Here we show that C57BL/6-derived, interleukin-2-activated NK cells expressing Ly-49 do not lyse target cells displaying H-2d or H-2k despite efficient spontaneous lysis by Ly-49- effector cells. This preferential resistance correlates with expression of target cell MHC class I antigens. Transfection and expression of H-2Dd, but not H-2Kd or H-2Ld, renders a susceptible target (H-2b) resistant to Ly-49+ effector cells. The transfected resistance is abrogated by monoclonal antibodies directed against Ly-49 or the alpha 1/alpha 2 domains of H-2Dd, suggesting that Ly-49 specifically interacts with the peptide-binding domains of the MHC class I alloantigen, H-2Dd. Inasmuch as Ly-49+ effector cells cannot be stimulated to lyse H-2Dd targets, our results indicate that NK cells may possess inhibitory receptors that specifically recognize MHC class I antigens.     

Expression of cell-surface HLA-DR, HLA-ABC and glycophorin during erythroid differentiation

The unexpected discovery that Ia-like (HLA-DR) antigens in humans were present on blast cells from acute myeloblastic leukaemia led to the finding that normal granulocytic progenitors, in contrast to their mature descendents, also expressed HLA-DR antigens. Thus, anti-Ia sera stain a proportion of myeloblasts in normal bone marrow, inhibit myeloid progenitor (CFU-GM) colony formation in the presence of complement and can be used to label and separate CFU-GM on a fluorescence-activated cell sorter (FACS). Winchester et al. subsequently reported that erythroid progenitors (BFU-E and CFU-E) were also inhibited or killed by anti-Ia (p28,37) and complement. These observations raised the possibility that HLA-DR (or presumptive I-region equivalent) products might have a regulatory role in early haematopoiesis. We have now analysed HLA-DR and HLA-ABC antigen expression on normal erythroid progenitors using monoclonal antibodies to non-polymorphic determinants and fluorescence-activated cell sorting. In parallel experiments, we tested a monoclonal antibody to glycophorin, a well defined erythroid-specific cell-surface membrane glycoprotein. We report that HLA-DR, HLA-ABC and glycophorin are all expressed at various stages during erythroid differentiation.     

Rejection of transplantable AKR leukaemia cells following MHC DNA-mediated cell transformation

Major histocompatibility complex (MHC) class I molecules can function as specific target antigens in T-cell-mediated cytotoxity. In addition, T cells can kill target cells through non-MHC antigens, for example, virally infected cells, if the target and effector cells express the same MHC class I antigens. Consequently, quantitative and/or qualitative variations in the expression of the H-2/HLA antigens on the target cells could interfere with MHC-restricted immune reactions. We have reported that the AKR leukaemia cell line K36.16, a subline of K36 (ref. 3), on which the H-2Kk antigen cannot be detected, is resistant to T-cell lysis and grows very easily in AKR mice. Other AKR tumour cell lines, like 369, which have a relatively large amount of H-2Kk on their surface, are easily killed by T cells in vitro and require a much larger inoculum to grow in vivo. Monoclonal antibodies against H-2Kk, but not against H-2Dk, prevented the killing by T cells. This suggests that some tumour cells grow in vivo because tumour-associated antigen(s) cannot be recognized efficiently by the host's immune system, due to the absence of MHC molecules which would function as restriction elements for T-cell cytotoxicity. We have tested this hypothesis by introducing the H-2Kk gene into the H-2Kk-deficient AKR tumour cell line K36.16 and have now demonstrated directly the biological relevance of H-2Kk antigen expression in the regulation of the in vivo growth of this tumour cell line.     

Two antigen-independent adhesion pathways used by human cytotoxic T-cell clones

Cell-cell adhesion is essential for many immunological functions, including interaction of cytotoxic T lymphocytes (CTLs) with their targets. We have explored CTL-target interactions using well-characterized cloned human CTLs. Conjugate formation between these CTLs and many antigen-negative targets is almost as efficient as with specific target cells, but does not lead to target-cell lysis. Thus, on specific target cells, adhesion by antigen-independent pathways may occur concurrently with or precede antigen recognition. The molecules LFA-1, CD2 (T11, LFA-2) and LFA-3 have been shown to be involved in human CTL conjugation with and lysis of specific target cells. Here we describe monoclonal antibody inhibition studies using individual monoclonal antibodies and mixes which demonstrate (1) that LFA-1, CD2 and LFA-3 are involved in antigen-independent conjugate formation; and (2) suggest that CD2 and LFA-3 are involved in one pathway and LFA-1 in another. We confirmed the existence of distinct pathways by the demonstration that LFA-1-dependent adhesion requires divalent cations and is temperature-sensitive whereas CD2- and LFA-3-dependent adhesion does not require divalent cations and is temperature-insensitive. Together with previous data, our studies suggest that CD2 on the effector interacts with LFA-3 as its ligand on targets.     

Monoclonal antibody production by receptor-mediated electrically induced cell fusion

Fusion of myeloma cells and B lymphocytes to form hybridomas which produce monoclonal antibodies has been a major advance, but the poor efficiency and randomness of viral or polyethylene glycol fusion techniques generally gives poor yields of specific, high affinity antibodies. High voltage electrical fields with dielectrophoresis to ensure cell alignment can fuse a limited number of cells under direct microscopic examination, but it is not possible to identify B-cells destined to secrete relevant antibodies. However, B-cells express, on their surface, antigen receptor immunoglobulins of the same antigenic specificity as the secreted antibodies. Binding of antigen to surface immunoglobulins stimulates proliferation and differentiation of B-cells into plasma cells. Here we report the use of the selective, high affinity interaction of antigen with surface immunoglobulins on B-cells to facilitate a close adherence to myeloma cells. The antigen, covalently conjugated to avidin, binds to the surface immunoglobulins on B-cells. This B-cell-antigen-avidin complex binds to biotin covalently attached to the surface of myeloma cells. An intense electric field across a bulk cell suspension then produces selective fusion of cells in contact, that is, of myeloma cells with B-cells which make the appropriate antibody. We have used this technique with several antigens, and all resultant hybridomas secrete appropriate antibodies with very high affinity.     

Monoclonal antibody and ligand binding sites of the T cell erythrocyte receptor (CD2)

The human T cell erythrocyte receptor (CD2 antigen) allows thymocytes and mature T cells to adhere to thymic epithelium and target cells through a cell surface protein, LFA-3 (refs 1-6). Monoclonal antibodies recognizing CD2 can either block adhesion or, in certain combinations, induce an antigen-independent T cell activation. We have identified the binding sites for 16 monoclonal antibodies against CD2 by a rapid and generally applicable mutational analysis. The binding sites fall in three discrete regions: antibodies that participate in activation and block erythrocyte adhesion bind to the first region; antibodies that block adhesion bind to the second region; and antibodies that participate in activation but do not block adhesion bind to the third region. A large number of mutations selected for loss of antibody reactivity in the first two regions also weaken the CD2-LFA-3 interaction. Good agreement was observed between mutational lesions blocking LFA-3 binding and lesions blocking binding by activating antibodies, which supports the view that such antibodies induce T cell activation by mimicking the effect of LFA-3 binding. CD2 sequences that participate in LFA-3 binding correspond to immunoglobulin variable region hypervariable sequences when the homologous domains are aligned.     

Monoclonal antibodies identify a cell-surface antigen associated with an activated cellular oncogene

A variety of antigens have been identified on the surface of the malignant cell. However, identical antigens are often found on non-malignant cells of the same or different histological origin, or of a different stage of embryonic development. Many of these tumour-associated antigens appear to be only incidentally expressed on neoplastic cells. Clearly, it would be of great interest to identify cell-surface antigens whose expression is associated specifically with the transformed state and linked directly with the mechanisms responsible for transformation. The detection of activated cellular oncogenes in human and animal cancer cells by the technique of DNA transfection has allowed the isolation of genetic elements which are thought to have a critical role in malignancy. Here, in an effort to identify cell-surface antigens associated with the neoplastic process, we have generated hybridomas which secrete monoclonal antibodies that react specifically with cell-surface determinants found on NIH 3T3 cells transformed by transfection with a group of rat neuroblastoma oncogenes. These antibodies bind to and immunoprecipitate a phosphoprotein of relative molecular mass 185,000 (185 K) from a DNA donor rat neuroblastoma and 13 independent rat neuroblastoma DNA transfectants. There was no antibody reactivity with normal NIH 3T3 cells or with NIH 3T3 cells transformed by various other agents.     

Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.     

HLA-DR products are a subset of human Ia antigens

Human Ia antigens are polymorphic cell-surface sialoglycoproteins which have restricted tissue distribution. They are bimolecular complexes of 34,000 (alpha) and 28,000 (beta) molecular weight and most of the polymorphism is found in the smaller polypeptides. They are involved in the initiation of immune responses and particular Ia antigens are associated with increased susceptibility to certain diseases. They are also the major barrier to human allogeneic tissue transplantation. Whereas serological analysis and mixed lymphocyte typing have defined three polymorphic families of Ia antigens, HLA-DR, -DC and -SB, protein sequencing results and studies with monoclonal antibodies indicate that the complexity is much greater. Thus the HLA-DR and DC specificities as defined by alloantisera, could represent groups of antigens which are controlled by HLA genes in linkage disequilibrium. Here, we have used a monoclonal antibody specific for HLA-DR2 to show that this determinant is carried by molecules which are distinct from those of the DC series and which represent 30% of the Ia antigens expressed on the cell surface of an HLA homozygous line PGF.     

Immunization to a syngeneic sarcoma by a monoclonal auto-anti-idiotypic antibody

Idiotypic networks regulate the immune response to a variety of antigens. Antibodies generated against other antibodies, called anti-idiotypic antibodies, can themselves mimic antigen and elicit a specific immune response. They have been shown to induce delayed-type hypersensitivity (DTH) to model antigens in the mouse. As anti-idiotypic antibodies are thought to be involved in the response to tumour-associated antigens we tested whether injection of monoclonal antibodies derived from mice hyperimmunized to a syngeneic, chemically induced sarcoma could mimic antigen and induce DTH to the sarcoma in naive mice. One of the monoclonal antibodies, 4.72, primed BALB/c mice for DTH to the sarcoma but not for DTH to another sarcoma or to sheep erythrocytes. Antibody 4.72 did not induce DTH in mice of immunoglobulin allotype congeneic strains nor did it bind to the sarcoma cells. As antibodies specific for this sarcoma have not been detected, we do not know whether idiotype on immunoglobulin molecules is recognized by antibody 4.72. However, as the response induced by antibody 4.72 was both antigen-specific and allotype-restricted, analogous to those induced by anti-idiotypic antibodies in other systems, we propose that antibody 4.72 is an anti-idiotypic antibody.     

Precursor and effector phenotypes of activated human T lymphocytes

In mice, thymus-derived lymphocytes are differentiated into functional subclasses by their cell surface antigens. The Ly 1 determinants are present on T cells with a helper function, whereas Ly 2 and Ly 3 antigens are expressed on the surface of lymphocytes with suppressor or cytotoxic functions. In man also, T-cell subsets have been identified using allo- and heteroimmune sera and, more recently, using monoclonal antibodies, which seem to identify helper and suppressor or cytotoxic subpopulations. The major histocompatibility system (MHS)-encoded Ia antigens belong to several polymorphic families of membrane associated glycoproteins originally found on B lymphocytes; however, they have also been shown to be markers for suppressor T cells in mice. Recent studies have shown that in both mouse and man, T cells activated by a mixed lymphocyte reaction or by mitogens become Ia+. Furthermore, some human T lymphoid cells, either freshly isolated from peripheral blood or after in vitro activation by lectins or alloantigens, possess suppressor properties. We report here the phenotype of a T suppressor-cell subpopulation which was induced in long-term culture of lymphoid cells after activation with phytohaemagglutinin (PHA). Our results suggest that a subset of T cells was progressively expanded over a period of 8 days in culture and that, with the expression on the surface of these cells of 'Ia-like' antigens, they acquired the capacity to suppress the proliferative response of syngeneic or allogeneic lymphocytes to alloantigens or mitogens.     

Localization of the binding site for the human high-affinity Fc receptor on IgG

A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen.     

H-2-restricted cytolytic T cells specific for HLA can recognize a synthetic HLA peptide

It is generally accepted that T lymphocytes recognize antigens in the context of molecules encoded by genes in the major histocompatibility complex (MHC). MHC class II-restricted T cells usually recognize degraded or denatured rather than native forms of antigen on the surface of class II-bearing antigen presenting cells. It has recently been shown that short synthetic peptides corresponding to mapped antigenic sites of the influenza nucleoprotein (NP) can render uninfected target cells susceptible to lysis by NP-specific class I-restricted cytolytic T cells (CTL). These and earlier experiments that showed specific recognition of NP deletion mutant transfectants suggest that class I-restricted recognition might also involve processed antigenic fragments. One important issue arising from these studies is whether the model applies not only to viral proteins that are expressed internally (such as NP) but also to antigens normally expressed as integral membrane proteins at the cell surface. We have recently isolated class I-restricted mouse CTL clones that recognize class I gene products of the human MHC (HLA) as antigens in mouse cell HLA-transfectants. Here we show that these anti-HLA CTL can lyse HLA-negative syngeneic mouse cells in the presence of a synthetic HLA peptide. These results suggest that the model applies generally.     

A late-differentiation antigen associated with the helper inducer function of human T cells

T lymphocytes possessing helper function produce soluble factors that greatly augment B-cell proliferation and differentiation into antibody-secreting cells. In humans the subset of T lymphocytes bearing the T4 surface antigen comprises most of the cells that display helper activity and recognize class II antigens of the major histocompatibility complex (MHC), while the subset bearing the T8 antigen comprises T cells recognizing class I MHC antigens and exhibiting cytotoxic or suppressor function. Monoclonal antibodies to T4 or T8 greatly inhibit the cognitive and effector function of cells with the corresponding phenotype. This function/phenotype correlation is not absolute, however, for there are many examples of T8-positive clones that recognize MHC class II antigens and have helper activity, as well as of T4-positive clones with suppressor or cytotoxic function. Recently a family of cell-surface neoantigens, which might be relevant to T-cell function and which are present on activated but not on resting T lymphocytes, has been identified in mouse and humans using monoclonal antibodies. Some of these antibodies block the cytolytic activity of alloreactive T-cell clones, suggesting the possible involvement of such molecules in the activation of cytotoxic T-cell clones or in the lytic process itself. We now describe a similar late-differentiation antigen (LDA1) that is expressed by human T lymphocytes only following activation and is recognized by a monoclonal antibody that inhibits the antibody-inducing helper function of T lymphocytes.     

Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1

Cell surface molecules have been implicated in cell interactions which underlie formation of the nervous system. The analysis of the functional properties of such molecules has profited from the combined use of antibodies and cell culture systems. It has been suggested that the interplay between these molecules modulates cell-to-cell interaction at critical developmental stages. In the mouse, N-CAM and L1 antigen have been shown to mediate Ca2+-independent adhesion among neural cells. N-CAM plays a role in fasciculation of neurites and formation of neuromuscular junction. L1 is apparently not involved in synaptogenesis, but in migration of granule cell neurones in the developing mouse cerebellar cortex. The two antigens are distinct molecular and functional entities which act synergistically in aggregation of neuroblastoma and early postnatal cerebellar cells. In view of a certain similarity in function between the two groups of molecules, it was not surprising to find that structural similarities are detectable by the monoclonal antibody L2. We show here that a carbohydrate moiety recognized by L2 and HNK-1 monoclonal antibodies, is present in mouse N-CAM and L1. The L2 epitope appears on all major neural cell types but not all N-CAM molecules express it. This heterogeneity points to a previously undetected molecular diversity which may have functional implications for modulating cell adhesion during development.     

A novel epitope of the LFA-1 antigen which can distinguish killer effector and suppressor cells in human CD8 cells

The CD4 subset of cells displays helper/inducer activity and recognizes class II antigens of the major histocompatibility complex (MHC), while the CD8 subset recognizes class I MHC antigens and exhibits cytotoxic or suppressor function. Considerable functional as well as corresponding phenotypic heterogeneity exists within the two major T cell subsets. Although the CD8+ population contains pre-cytotoxic, cytotoxic, pre-suppressor and suppressor effector T cells, these distinctions still rest largely on the use of functional assays. Attempts have been made to define the CD8+ precursor of the killer cell with new monoclonal antibodies. But more precise phenotypic distinctions between the functional subpopulations within CD8+ cells will be needed. We have now developed a monoclonal antibody, anti-S6F1 which can distinguish killer effector and suppressor effector cells in CD8 lymphocyte populations. The cell-surface structure defined by this antibody comprises two glycoproteins with relative molecular mass (Mr) 180K and 95K respectively. Also sequential immunoprecipitation studies and two dimensional gel electrophoresis indicate that anti-S6F1 recognizes a novel epitope on the LFA-1 antigen.     

Functional modifications of cytotoxic T-lymphocyte T200 glycoprotein recognized by monoclonal antibodies

Plasma membrane glycoproteins of cytotoxic T lymphocytes (CTLs) are involved in the binding to and subsequent destruction of appropriate target cells. The electrophoretic profile of surface proteins of mature CTLs, particularly those of high relative molecular mass (Mr), is markedly different from that of naive peripheral T cells or non-cytolytic T cells, suggesting the possible involvement of these molecules in the activation of CTLs and/or in the lytic process itself. By generating monoclonal antibodies to cell-surface proteins of CTL clones, we have now detected CTL-specific modifications in one of these high-Mr membrane proteins, T200. Although forms of T200 are found on a wide variety of cell types, the neoantigenic determinants recognized by our antibodies are present exclusively on activated T cells and in high concentrations only on CTLs. Furthermore, the expression of the modifications recognized by our antibodies is influenced by soluble factors and also seems to have functional significance, as monoclonal antibodies specific for these novel epitopes block cytolytic activity.