Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity

Natural killer (NK) cells attack many tumour cell lines, and are thought to have a critical role in anti-tumour immunity; however, the interaction between NK cells and tumour targets is poorly understood. The stimulatory lectin-like NKG2D receptor is expressed by NK cells, activated CD8+ T cells and by activated macrophages in mice. Several distinct cell-surface ligands that are related to class I major histocompatibility complex molecules have been identified, some of which are expressed at high levels by tumour cells but not by normal cells in adults. However, no direct evidence links the expression of these 'induced self' ligands with tumour cell rejection. Here we demonstrate that ectopic expression of the murine NKG2D ligands Rae1beta or H60 in several tumour cell lines results in potent rejection of the tumour cells by syngeneic mice. Rejection is mediated by NK cells and/or CD8+ T cells. The ligand-expressing tumour cells induce potent priming of cytotoxic T cells and sensitization of NK cells in vivo. Mice that are exposed to live or irradiated tumour cells expressing Rae1 or H60 are specifically immune to subsequent challenge with tumour cells that lack NKG2D ligands, suggesting application of the ligands in the design of tumour vaccines.     

Characterization of murine cytolytic-helper hybrid T cell clones

L3T4, Lyt-2 and the T-cell receptor for antigen are cell-surface molecules involved in antigen specific T cell activation. We have constructed functional murine cytolytic-helper T-cell hybrid clones to study the link between expression of cell-surface molecules and specific cell function. Three of the clones express two antigen receptors and both Lyt-2 and L3T4, normally expressed on mutually exclusive subsets of mature T lymphocytes. The pattern of lymphokines produced by the hybrid cells in response to antigen was not controlled by the specific antigen receptor; both T-cell growth factor, produced only by the helper T-cell partner, and gamma-interferon, produced only by the cytolytic T-cell partner, were secreted when either antigen receptor was stimulated. However, cytolytic activity appeared to be restricted to the recognition of antigen by the T-cell receptor of the cytolytic partner. Thus cytolysis appears to be rightly linked to the antigen receptor of the cytolytic parent but lymphokine release is not tightly linked.     

Induction and kinetics of natural killer cells in humans following interferon therapy

Natural killer (NK) cells are non-B, non-T lymphocytes that effect spontaneous cytolysis of both virus-infected and neoplastically transformed target cells. These NK lymphocytes have been detected in several species including man. Interferon is a primary regulator of natural killer activity. Because NK cells have been implicated in the regulation of tumour cell expression and can be induced by interferon in murine models, we have studied patients receiving large doses of interferon to determine (1) whether interferon could induce NK lymphocytes in the peripheral blood of man, and (2) whether there are characteristic kinetics for the appearance, disappearance and reactivation of NK lymphocytes following interferon therapy. We report here the activation of human NK cells by the systemic inoculation of human subjects with interferon. Five patients received interferon as therapy for non-Hodgkin's lymphoma. All showed a marked increase in NK cell activity 12--24 h after inoculation. Peak NK activity occurred 18 h after introducing interferon, and thereafter declined rapidly but remained above pre-interferon levels. Induced NK activity occurred with reintroduction of interferon but at lower levels of activity and with different kinetics.     

Activation of cytolytic T lymphocyte and natural killer cell function through the T11 sheep erythrocyte binding protein

The T11 sheep erythrocyte binding glycoprotein [relative molecular mass (Mr)50,000(50K)] is expressed throughout human T-lymphocyte ontogeny and appears to play an important physiological role in T-cell activation. Thus, the treatment of T cells with certain monoclonal anti-T11 antibodies results in antigen-independent polyclonal T-cell activation as assessed by proliferation and lymphokine secretion. In addition, the majority of thymocytes that have not yet acquired the T3-Ti antigen/major histocompatibility complex (MHC) receptor can be activated to express interleukin-2 (IL-2) receptors through this T11 structure. We show here that the triggering of cytolytic T (Tc) cells via T11 causes an antigen-independent activation of the cytolytic mechanism as evidenced by the induction of nonspecific cytolytic activity. Furthermore, T11+T3-Ti- natural killer (NK) cell clones can also be induced to lyse NK-cell-resistant targets by treatment with anti-T11 monoclonal antibodies directed at defined T11 epitopes. These results indicate that T11 triggering can activate cytotoxic lymphocytes to express their functional programmes in the absence of specific antigen recognition via the T3-Ti complex and provide further evidence for the notion that certain NK cells and T lymphocytes are related.     

Isolation and characterization of a cDNA clone encoding the murine homologue of the human 20K T3/T-cell receptor glycoprotein

The antigen receptor on the surface of human T lymphocytes, which consists of a heterodimer of relative molecular mass (Mr) 90,000 (90K) (alpha- and beta-chains), is associated with the T3 antigen (gamma = 25K, delta = 20K and epsilon = 20K). A working model for the mode of action of the T3/T-cell receptor complex is that the clonotypic alpha- and beta-chains are involved in the recognition and binding of antigen in the context of polymorphic major histocompatibility complex (MHC) gene products on the surface of target cells. Antigen binding by the clonotypic receptor probably results in conformational changes in this structure which are recognized by and subsequently trigger the associated T3 complex to transmit signals into the cell, resulting in a proliferative response. The similarity in structure between murine and human clonotypic antigen receptors suggests that such a mechanism of recognition and activation also exists in mouse T lymphocytes, but so far there has been no evidence for the existence of a murine T3 complex. Here we demonstrate the existence of a T3 delta-chain mRNA in murine T lymphocytes. Our sequence data strongly suggest that this mouse mRNA codes for a complete T3 delta polypeptide chain and reveal some interesting properties of the protein.     

Identification of antigen receptor-associated structures on murine T cells

The specific antigen receptor on human and murine T lymphocytes is a heterodimer of relative molecular mass (Mr) 80,000-90,000 (80-90K) composed of two 40-50K disulphide-linked glycoprotein subunits. Peptide map analysis of the alpha- and beta-chains of receptor isolated from distinct tumour cell lines suggests the presence of both constant and variable regions. Unlike the antigen receptor on B lymphocytes (that is, surface immunoglobulin), the human T-cell antigen receptor seems to be non-covalently associated with another invariant structure recognized by monoclonal antibodies to the cell-surface antigens T3 and Leu 4 (refs 4, 5, 9, 12). Meuer et al. have demonstrated comodulation of the T3 structure and T-cell antigen receptor using anti-clonotypic and anti-T3 monoclonal antibodies. Furthermore, immunoprecipitation with anti-T3 weakly co-precipitates a small amount of the 80-90K heterodimer in certain conditions. The murine homologue of the Leu 4/T3 structure has not been identified, although Gunter et al. have suggested that Thy-1 may be the counterpart of Leu 4/T3 (ref. 13). Here we describe a Leu 4/T3-like structure, distinct from Thy-1, associated with the T-cell receptor of a murine T-lymphoma cell line.     

Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes

The vast majority of mature T lymphocytes in the peripheral blood and lymphoid organs use the CD3-associated alpha, beta T-cell receptor (TCR) heterodimer for antigen recognition. A second class of TCRs consists of disulphide-linked gamma and delta proteins that are also CD3-associated. A subset of early CD3+ fetal and adult CD4- 8- thymocytes express gamma, delta TCRs before alpha, beta TCRs are detectable. In addition, a minor (1-5%) subpopulation of peripheral T lymphocytes, and some spleen cells from nude mice express gamma, delta TCRs. Notably, dendritic epidermal cells have also been shown to express gamma, delta TCRs. All of these populations lack CD4 and CD8 molecules. We now report that most mature T cells residing in the murine intestinal epithelium express CD3-associated TCRs composed of gamma-chains disulphide-linked to a protein resembling the delta-chain. The striking feature of these intraepithelial lymphocytes (IEL) was that they were exclusively CD4-8+. In addition, approximately half of CD3-bearing IEL lacked detectable Thy-1 on the cell surface, which is unprecedented for murine T cells. In contrast to other CD8+ peripheral T cells, freshly isolated IEL could be induced to display cytolytic activity by engaging the CD3 molecule, indicating that activation had occurred in vivo. Thus, CD8+ IEL are a phenotypically diverse and anatomically restricted population of lymphocytes that use gamma-chain containing heterodimers for antigen recognition.     

CD2-mediated adhesion facilitates T lymphocyte antigen recognition function

The CD2 T lymphocyte-surface glycoprotein serves to mediate adhesion between T lymphocytes and their cognate cellular partners which express the specific ligand LFA-3. In addition, CD2 by itself or in conjunction with T-cell receptor stimulation, transduces signals resulting in T-lymphocyte activation. One or both of these functions seems to be physiologically important, given that certain anti-CD2 monoclonal antibodies block T-cell activation and that antigen-responsive memory T cells express a high level of CD2 relative to virgin T cells, which are largely antigen-unresponsive. Nevertheless, the contribution of the individual CD2 functions in T-cell responses has not been independently examined. To this end, human CD2 complementary DNAs encoding an intact LFA-3-binding adhesion domain, but lacking a functional cytoplasmic signal transduction element (CD2trans-), were introduced into an ovalbumin-specific, I-Ad restricted murine T-cell hybridoma. The antigen-specific response of T hybridoma cells expressing human CD2trans- protein was enhanced up to 400% when the human LFA-3 ligand was introduced into the I-Ad expressing murine antigen-presenting cells. In contrast, no augmentation was observed if human LFA-3 was absent or expressed on a third-party cell lacking the I-Ad restriction element. These results directly demonstrate the functional significance of adhesion events mediated between CD2 on the antigen-responsive T lymphocyte and LFA-3 on the presenting cell in optimizing antigen-specific T-cell activation.     

A T3-like protein complex associated with the antigen receptor on murine T cells

Antigen recognition by human T lymphocytes and initiation of T-cell activation are mediated by a group of integral membrane proteins, the T-cell antigen receptor (TCR) and the T3 complex. The polypeptides which comprise T3 (a gamma-chain of relative molecular mass (Mr) 25,000 (25K), and delta and epsilon chains of 20K each) are physically associated with the TCR chains. Surface expression of the complex requires the presence of all the component T3 and TCR proteins. In contrast to the human system, murine T3 has not been identified using antibodies. Here we describe a murine T3-like protein complex. It appears to be more complicated than human T3, containing three monomeric glycoproteins (21-28K), two of which have N-linked carbohydrate side chains and a novel family of TCR-associated homo- and heterodimers. The 28K protein is identified as the murine T3 delta-chain. The 21K protein is phosphorylated on cell activation with concanavalin A (Con A).     

Thy-1-mediated T-cell activation requires co-expression of CD3/Ti complex

In addition to monoclonal antibodies against the CD3 (T3)-T-cell antigen receptor (CD3/Ti) complex, several other monoclonals directed towards distinct cell surface structures on human (CD2 (T11) and Tp44) and murine (Thy-1, TAP, and Ly-6) T lymphocytes are capable of activating T cells. It has been proposed that such structures may function as alternative pathways of stimulation. To examine directly whether any relationship exists between Thy-1-dependent activation phenomena and T-cell activation mediated through the CD3/Ti complex, we have transfected several CD3/Ti- variants of the human T-cell line Jurkat with the murine Thy-1.2 gene. Our data indicate that in CD3/Ti-, Thy-1.2+ transfectants, monoclonal antibodies against Thy-1.2 can induce a rise in cytoplasmic free calcium ([Ca2+]i), but fail to stimulate interleukin-2 (IL-2) production. The only defect in these variant cell lines responsible for the inability to produce IL-2 in response to Thy-1 stimulation was in the expression of the CD3/Ti complex, because replacement of defective Ti alpha- or beta-chain genes reconstributed both surface expression of CD3/Ti and responsiveness to Thy-1 in the IL-2 production assay.     

Co-association of CD3 zeta with a receptor (CD16) for IgG Fc on human natural killer cells

Natural killer (NK) cells are a subset of lymphocytes that mediate major histocompatibility complex (MHC)-nonrestricted cytotoxicity against tumours and virus-infected cells and secrete numerous cytokines on activation. NK cells are distinct from mature T lymphocytes, because they do not rearrange or productively transcribe T-cell receptor alpha-, beta-, gamma- or delta-chain genes and do not express the CD3 gamma- or delta-subunits. But recent studies indicate that NK cells do express CD3 zeta, co-associated with other membrane proteins. Here we report that CD16, the receptor for the Fc (constant) region of IgG, specifically associates with the CD3 zeta homodimer on the membrane of human NK cells, and that co-transfection with CD3 zeta complementary DNA permits expression of a transmembrane-linked CD16 complex on COS-7 cells. These findings indicate that CD3 zeta can co-associate with membrane receptors of diverse cell types and function as a common structure for signal transduction.     

Human large granular lymphocytes are potent producers of interleukin-1

Natural killer (NK) activity against tumour and virus-infected target cells is shown by a subpopulation of peripheral blood mononuclear leukocytes with the morphological features of large granular lymphocytes (LGL). The lineage of human LGL is still controversial, as they display surface markers of both T lymphocytes and myelomonocytic cells. LGL have recently been reported to produce lymphokines such as interleukin-2 (IL-2) and alpha- as well as gamma-interferons, functions associated mainly with T cells. To determine whether cytokines associated with other cell lineages are also produced by LGL, we examined whether they might produce a myelomonocyte -associated cytokine such as interleukin-1 (IL-1). IL-1 is a 12-18,000 molecular weight (MW) lymphokine produced by a variety of cell types such as monocytes, keratinocytes and a human dendritic cell line, which plays a crucial role in immunoregulation and inflammation. Moreover, IL-1 has recently been reported to act synergistically with IL-2 and interferons in boosting LGL-mediated NK activity. We now show that a subset of highly purified human LGL with NK activity can be stimulated to secrete a soluble factor with the biochemical and biological characteristics of human IL-1.     

Natural killer cells act as rheostats modulating antiviral T cells

Antiviral T cells are thought to regulate whether hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections result in viral control, asymptomatic persistence or severe disease, although the reasons for these different outcomes remain unclear. Recent genetic evidence, however, has indicated a correlation between certain natural killer (NK)-cell receptors and progression of both HIV and HCV infection, implying that NK cells have a role in these T-cell-associated diseases. Although direct NK-cell-mediated lysis of virus-infected cells may contribute to antiviral defence during some virus infections--especially murine cytomegalovirus (MCMV) infections in mice and perhaps HIV in humans--NK cells have also been suspected of having immunoregulatory functions. For instance, NK cells may indirectly regulate T-cell responses by lysing MCMV-infected antigen-presenting cells. In contrast to MCMV, lymphocytic choriomeningitis virus (LCMV) infection in mice seems to be resistant to any direct antiviral effects of NK cells. Here we examine the roles of NK cells in regulating T-cell-dependent viral persistence and immunopathology in mice infected with LCMV, an established model for HIV and HCV infections in humans. We describe a three-way interaction, whereby activated NK cells cytolytically eliminate activated CD4 T cells that affect CD8 T-cell function and exhaustion. At high virus doses, NK cells prevented fatal pathology while enabling T-cell exhaustion and viral persistence, but at medium doses NK cells paradoxically facilitated lethal T-cell-mediated pathology. Thus, NK cells can act as rheostats, regulating CD4 T-cell-mediated support for the antiviral CD8 T cells that control viral pathogenesis and persistence.     

Positive selection of CD4+ T cells mediated by MHC class II-bearing stromal cell in the thymic cortex

T lymphocytes differentiate in the thymus, where functionally immature, CD4+CD8+ (double positive) thymocytes develop into functionally mature CD4+ helper cells and CD8+ cytotoxic (single positive) T cells. The thymus is the site where self-reactive T cells are negatively selected (clonally deleted) and where T cells with the capacity to recognize foreign antigens in association with self-proteins encoded by the major histocompatibility complex (MHC) are positively selected. The net result of these developmental pathways is a T-cell repertoire that is both self-tolerant and self-restricted. One unresolved issue is the identity of the thymic stromal cells that mediate the negative and positive selection of the T-cell repertoire. Previous work has pointed to a bone-marrow-derived macrophage or dendritic cell as the inducer of tolerance, whereas a radiation-resistant, deoxyguanosine-resistant thymic cell seems to mediate the positive selection of self-MHC restricted T cells. Thymic stromal cells in the cortex interact with the T-cell antigen receptor on thymocytes. Using several strains of transgenic mice that express the class II MHC molecule I-E in specific regions of the thymus, we show directly that the positive selection of T cells is mediated by an I-E-bearing cell in the thymic cortex.     

Intestinal intraepithelial lymphocytes are a distinct set of gamma delta T cells

Lymphocytes are most reliably subdivided on the basis of their receptors for antigen at the cell surface. Three subtypes of lymphocytes are well defined: B cells that bear surface immunoglobulin and make antibody, CD4+T cells with CD3 alpha beta receptors specific for antigen associated with class II major histocompatibility complex molecules, and CD8+T cells with CD3 alpha beta receptors specific for antigen associated with class I MHC molecules. These T cells are responsible for known forms of cell-mediated immunity. The discovery of a third rearranging T-cell specific gene called gamma (refs 1 and 2) has revealed the presence of a new class of T cells bearing a new receptor type, CD3 gamma delta (refs 3-7). To date, neither the function nor the specificity of cells bearing this receptor has been determined. Because gamma delta T cells are the main lymphocyte of epidermis, it was proposed that such cells could be important in surveillance of all epithelia. We have isolated intraepithelial lymphocytes from murine small intestine, and shown that they predominantly or exclusively express CD3 gamma delta receptors. Unlike the epidermal lymphocytes, these cells also express CD8, and they use a different V lambda gene to form their receptor. This strongly suggests that gamma delta T cells home in a very specific manner to epithelia, where they presumably mediate their function.     

Production of immune interferon by murine T-cell clones from long-term cultures

Production of leukocyte interferon (IFN-alpha) and fibroblast interferon (IFN-beta) can be induced by a variety of agents but immune interferon, IFN-gamma, is only obtained when lymphoid cells are stimulated by specific antigens, allo-antigens or T-cell mitogens. Moreover, in bulk cultures, only small quantities of IFN-gamma are produced. The type of cell producing IFN-gamma has not been unambiguously defined and so we set out to determine whether a pure T-cell population could produce it, exploiting the knowledge that T cells can be maintained indefinitely in tissue culture by the addition of T-cell growth factors. Although not all T cells can found long-term cultures of this kind, cultures of antigen-specific helper, suppressor and killer T cells have been obtained in this way. We now describe the production of substantial amounts of INF-gamma when some (but not all) murine T-cell clones derived from such cultures are stimulated by either concanavalin A (Con A) or phytohaemagglutinin (PHA).     

Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand

Natural killer (NK) cell function is regulated by NK receptors that interact with MHC class I (MHC-I) molecules on target cells. The murine NK receptor Ly49A inhibits NK cell activity by interacting with H-2D(d) through its C-type-lectin-like NK receptor domain. Here we report the crystal structure of the complex between the Ly49A NK receptor domain and unglycosylated H-2D(d). The Ly49A dimer interacts extensively with two H-2D(d) molecules at distinct sites. At one interface, a single Ly49A subunit contacts one side of the MHC-I peptide-binding platform, presenting an open cavity towards the conserved glycosylation site on the H-2D(d) alpha2 domain. At a second, larger interface, the Ly49A dimer binds in a region overlapping the CD8-binding site. The smaller interface probably represents the interaction between Ly49A on the NK cell and MHC-I on the target cell, whereas the larger one suggests an interaction between Ly49A and MHC-I on the NK cell itself. Both Ly49A binding sites on MHC-I are spatially distinct from that of the T-cell receptor.     

A new subunit of the human T-cell antigen receptor complex

The T-cell antigen receptor binds antigen in association with a cell surface molecule encoded by the major histocompatibility complex (MHC). MHC restricted recognition of antigen by this receptor leads to the complex pattern of programmed gene expression that characterizes T-cell activation. The eventual understanding of human T-cell function will require the complete elucidation of the structure of the human T-cell antigen receptor. On human T cells, clonally determined, disulphide-linked alpha and beta chains of the receptor are non-covalently and stoichiometrically associated with three additional polypeptides known as the T3 complex. These receptor subunits are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20K (gamma and delta) and a non-glycosylated 20K protein (epsilon). Our studies of murine T cells show that the mouse T-cell antigen receptor consists of at least seven distinct polypeptide chains. In addition to clonotypic alpha and beta chains, the murine complex consists of glycoproteins of 26K and 21K and endoglycosaminidase F (endo F)-insensitive polypeptides of 25K, 21K and 16K. The latter, which we have termed zeta (zeta), exists as a homodimer within the complex. The 26K component (gp26) has been shown to be the murine analogue of the human delta chain. Other cross species homologies remain to be established, however none of the described human receptor components appear similar to the murine zeta polypeptide. We report here the use of an antiserum raised against the murine zeta subunit to identify a previously unrecognized component of the human T-cell antigen receptor. This human protein is T-cell specific and biochemically similar to the murine zeta polypeptide.     

Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES.

An important process in the immune response is the migration of different populations of lymphocytes at the proper time to sites of antigenic challenge. Although several chemoattractants are known for broad classes of lymphocytes, such as T and B cells, the process by which lymphocytes of specific subsets, such as helper, cytotoxic or memory T cells, migrate to the appropriate sites remains obscure. Interleukin-8 is a chemoattractant for T cells and neutrophils and is a member of a superfamily of soluble molecules related by a conserved motif containing four cysteine residues. IL-8 and related molecules, including platelet factor 4, constitute the C-X-C class of the superfamily and a group of cytokines produced by haematopoietic cells constitute the RANTES/sis or C-C class. The roles of most of these molecules are not well known, although murine MIP-1 alpha of the C-C branch is a specific inhibitor of haematopoietic stem cell proliferation and some members of the C-X-C branch are neutrophil-targeted inflammatory agents. Here we report that the RANTES protein of the C-C class causes the selective migration of human blood monocytes and of T lymphocytes expressing the cell surface antigens CD4 and UCHL1. CD4+/UCHL1+T cells are thought to be prestimulated or primed helper T cells involved in memory T cell function. The preferential attraction of T-cell subsets by specific cytokines could in part explain how lymphocytes are targeted, and may provide insight into the workings of T cell memory.