Serine esterase in cytolytic T lymphocytes

The mechanisms that enable cytotoxic T lymphocytes (Tc cells) to destroy target cells are only vaguely understood. However, recent studies have identified in Tc cells and natural killer cells cytoplasmic granules that contain perforin, a cytolytic protein that resembles the ninth component of complement (C9). Antigen-specific lysis of target cells, traditionally ascribed solely to Tc cells, has now also been demonstrated in some T-helper cell (Th cell) lines, referred to here as T helper-killer or Th/c cells. We recently found a novel serine esterase that is present at greatly elevated levels in cloned murine Tc cell lines and one Th/c cell line, but not in two non-cytolytic Th cell lines. These findings suggest that the serine esterase is involved in cytolytic activity and that a variety of effector cells share a common cytolytic mechanism. To explore the role of the serine esterase in this process, we have been studying additional properties of the enzyme in murine T cells. We show here that it is a membrane-associated, disulphide-linked dimer, it has trypsin-like properties but is not a general protease, in density gradient centrifugation it sediments with perforin, it is secreted by Tc cells during their cytolytic attack on target cells, and antiserum to Tc-cell serine esterase reacts with the enzyme in Th/c cells.     

人穿孔素羧基端肽段的表达纯化与活性鉴定

穿孔素,即成孔蛋白（pore forming protein,PFP),其溶细胞作用与免疫调节和自身免疫病以及其它多种疾病过程中的免疫性病理损伤相关。为得到足够量的PFP建立与之相关的免疫学研究手段用于基础和临床研究,在已克隆人PFP cDNA的基础上,用基因工程方法表达了人PFP C端124个氨基酸肽段（hPFP-C),并通过谷胱甘肽琼脂糖新和层析获得纯化的GST/hPFP-C融合蛋白,经凝血酶酶切和再次北极和层析去除GST部分,得到了纯化的hPFP-C蛋白。纯化的hPFP-C蛋白与兔红细胞共育,呈现钙依赖的溶血活性。     

Phosphorylcholine acts as a Ca2+-dependent receptor molecule for lymphocyte perforin

Large granular lymphocytes and cytolytic T-lymphocytes (CTL) contain numerous cytoplasmic granules thought to be responsible, at least in part, for the cytolytic activity of these effector cells. Isolated granules are lytic for a variety of target cells and the granule proteins are specifically released upon target-cell interaction. Major proteins in mouse CTL granules are a family of seven serine proteases designated granzymes A to G, and a pore-forming protein called perforin (cytolysin). Purified perforin is cytolytic in the presence of Ca2+ and shows ultrastructural, immunological and amino-acid sequence similarities to complement component C9. Despite these similarities, perforin and C9 are clearly distinct in their mode of target-cell recognition. Whereas C9 insertion is absolutely dependent on a receptor moiety assembled from the complement proteins C5b, C6, C7, and C8 on the target-cell membrane, no requirement for a receptor molecule has been reported for perforin. Here, we demonstrate that phosphorylcholine acts as a specific, Ca2+-dependent receptor molecule for perforin.     

Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis

Cytolysis mediated by complement or cytolytic lymphocytes results in the formation of morphology similar lesions in the target membrane. These lesions, formed by the polymerization of C9 or perforin respectively, contribute the major killing action by causing osmotic lysis of the target cell. Following the suggestion of Mayer that the mechanisms of humoral and cell-mediated cytotoxicity might be related, studies into the morphology of the membrane lesions formed, and the proteins responsible for causing the lesions, have shown several similarities. While the lesion caused by natural and T-killer cells is a little larger than that caused by complement, its overall shape is similar and in both cases the cylindrical pore is formed by polymerization of a monomeric subunit, C9 (relative molecular mass, Mr = 71,000) for complement, and perforin (Mr = 66,000) for cell-mediated cytotoxicity. C9 has an absolute requirement for a receptor in the target membrane formed by the earlier membrane attack complex components, C5b, C6, C7 and C8 (ref. 8). For perforin, polymerization in a target membrane requires no receptor, specificity being derived from the specific recognition between killer and target cell. Both proteins can be made to polymerize in vitro by the addition of divalent cations (Zn2+ for C9 (ref. 16) and Ca2+ for perforin) and the resultant complexes closely resemble their physiological counterparts. Antibodies raised against lymphocyte-killed targets have also been shown to cross-react with complement proteins, but the antigenically related proteins were not determined in these studies. We show here using purified proteins that perforin, C9 and complexes involving C7 and C8 share a common antigenic determinant which is probably involved in polymerization.     

Herpesvirus-transformed cytotoxic T-cell lines

Investigations of cellular cytotoxicity of the immune system are hampered by the lack of continuously growing, transformed cell lines which express a cytotoxic potential. Here we describe cytotoxic cell lines from the cotton-topped marmoset monkey, transformed by Herpesvirus Ateles (HVA) or Herpesvirus Saimiri (HVS), which can kill certain target cells in a short-term in vitro test. HVA/HVS-transformed cells have earlier been classified as belonging to the T-cell lineage in contrast to Epstein-Barr virus (EBV)-transformed cells derived from B lymphocytes. We suggest that the HVA/HVS-transformed killer cell lines described here represent an effector population resembling, or corresponding to, marmoset natural killer (NK) cells and that they may be used to define the cytolytic mechanism involved in cellular cytotoxicity and possibly also effector cell receptors and target cell antigens, as well as regulatory mechanisms of general biological interest.     

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.     

The structural basis for membrane binding and pore formation by lymphocyte perforin

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca(2+)-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.     

Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells.

CD8-positive T cells protect the body against viral pathogens by two important mechanisms: production of antiviral cytokines and lysis of infected cells. Cytokine production can have both local and systemic consequences, whereas cytolytic activity is limited to infected cells that are in direct contact with T cells. Here we analyse activated CD8-positive T cells from mice infected with lymphocytic choriomeningitis virus and find that cytokines are not produced ex vivo in the absence of peptide stimulation, but that they are rapidly generated after T cells encounter viral peptides bound to the major histocompatibility complex. Remarkably, cytokine production ceases immediately upon dissociation of the T cells from their targets and resumes when antigenic contact is restored. In contrast to the 'on/off/on' cycling of cytokines, the pore-forming cytotoxic protein perforin is constitutively maintained. Our results indicate that there is differential expression of effector molecules according to whether the antiviral product is secreted (like cytokines) or stored inside the cell (like perforin). The ability to turn cytokines on and off while maintaining intracellular stores of perforin shows the versatility of the cellular immune response and provides a mechanism for maintaining effective immune surveillance while reducing systemic immunopathology.     

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.     

Aged murine killer T-cell clones acquire specific cytotoxicity for P815 mastocytoma cells

T-cell clones that grow continuously in tissue culture have become a major tool for studying the properties of T lymphocytes. It is therefore important to know to what extent such clones resemble their normal counterparts. Several reports have appeared recently which demonstrate that long-term T-cell lines may lose the specificity for which they were initially selected and acquire cytotoxic activity to a variety of targets, typical of the activity displayed by natural killer cells. We now report a number of instances in which murine cytotoxic T-cell clones have lost their original specific cytotoxic activity but have acquired strong specific cytotoxic activity for P815 mastocytoma target cells. Loss of the original specificity was usually observed after continuous in vitro cultivation for more than 6 months. We propose that this novel type of cytotoxicity should be called aged killer activity.     

Structure and function of human perforin

Perforin (P1) is a cytolytic protein with similarity to complement component C9. P1 has been described as a unique component of murine cytolytic T-cell and rat natural killer cell granules Previous studies indicated that human granules and P1 differed from murine granules and P1 in that they appeared to be cytolytically less active and lacked the haemolytic activity characteristic of P1. It has been suggested that P1, like C9, is under the control of the homologous restriction factor. Here we determine the primary structure of human P1, re-examine its functional properties, and address the question of homologous restriction.     

Recombinant interleukin-2 directly augments the cytotoxicity of human monocytes

Interleukin-2 (IL-2), originally described as a growth factor required for sustained proliferation of T cells in vitro is a glycoprotein hormone of known structure which appears to be important for the generation of immune responses in vivo. As well as T lymphocytes, B lymphocytes and large granular lymphocytes with natural killer activity (NK cells) can also respond to IL-2. The action of IL-2 seemed to be limited specifically to lymphocytes, however, and the term 'T-lymphocytotrophic hormone' was used. Here we provide evidence that human monocytes display a substantially increased cytotoxic activity as a direct and rapid response to human recombinant IL-2 but not to human recombinant glycosylated interferon-gamma (IFN-gamma) or lipopolysaccharide. Our results reveal a previously unknown function of IL-2 and suggest its possible involvement in monocyte-T cell interactions.     

Unique structure of murine interleukin-2 as deduced from cloned cDNAs

Interleukin-2 (IL-2) is a lymphokine originally described as a humoral factor required for the continued proliferation of activated T-cell clones. It also seems to be involved in the mitogenic response of thymocytes, in augmenting natural killer cell activity, in the generation of cytotoxic T cells and in the induction of other lymphokines such as gamma-interferon and a B-cell growth factor (BCGF-1). More recently, there has been evidence for the involvement of IL-2 per se in the stimulation of B-cell growth (ref. 10 and T. Kishimoto and J. Vilcek, personal communications). We have reported previously the cloning and expression of a human IL-2 complementary DNA. The cDNA encodes biologically active IL-2 which would consist of 153 amino acids, including a signal sequence. Because so much of the work on IL-2 has been done in the human and mouse, we sought to obtain cDNA encoding murine IL-2, and we now report the cloning, expression and sequence analysis of murine IL-2 cDNAs. The longest cDNA insert encodes a polypeptide of 169 amino acids, containing unique repeats of a CAG sequence which would encode 12 consecutive glutamine residues within the active IL-2 molecule.     

A new immunodeficiency disorder in humans involving NK cells

Immunodeficiency disorders have provided much information on the development and interaction of the various B and T lymphoid components in the immune system of man. As the lymphoid system becomes increasingly divided into functional subsets of cells it will be important to find immunodeficiencies affecting newly discovered cell types. Natural killer (NK) cells are a recently described but ill-defined subpopulation of lymphocytes which is thought to play an important part in surveillance against tumour development. Mice homozygous for the beige gene were found to have a selective deficiency in NK function and were more susceptible to transplantation of syngeneic tumours as predicted. We report here that patients carrying the analogous, autosomal recessive Chediak-Higashi (CH) gene have a profound defect in their ability to spontaneously lyse various tumour cells in vitro by either antibody-dependent or independent mechanisms. Since other cell-mediated cytolytic functions were relatively normal, these results suggest that the beige or Chediak-Higashi gene in both man and mouse controls NK function.     

Gene rearrangement in cells with natural killer activity and expression of the beta-chain of the T-cell antigen receptor

The mammalian host defence system can be divided broadly into adaptive and non-adaptive immunity. Adaptive immunity is acquired and is mediated by B and T lymphocytes. Non-adaptive immunity is mediated in part by a small subclass of heterogeneous peripheral blood mononuclear cells. This population, termed null cells, consists of haematopoietic precursors and cells mediating natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC). NK cells are a class of non-adherent, non-phagocytic, rapidly cytotoxic lymphocytes which can efficiently lyse a wide variety of tumour cells, virally infected cells and immature cell types of normal origin. Despite the broad range of targets, only a limited number of specificities are thought to be involved in target-cell recognition. Morphologically, NK cells are large granular lymphocytes, but they have been shown to exhibit cell-surface markers characteristic of both T cells and monocytes, raising doubt over their lineage. The recent cloning of the beta-chain of the T-cell antigen receptor has now allowed us to investigate whether some NK cells are T-cell-related. We have examined rearrangement and expression of the beta-chain of the T-cell receptor in cloned murine NK cell lines and fresh murine NK cell populations, and our results support the hypothesis that a subpopulation of NK cells is related to T cells and provide basis for examining whether some NK activity is mediated by a small number of T-cell receptors.     

Mediation of mouse natural cytotoxic activity by tumour necrosis factor

Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.     

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.     

Precise prediction of a dominant class I MHC-restricted epitope of Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive bacterium which grows in the cytoplasm of eukaryotic cells and can cause severe disease in immunocompromised individuals. In murine systems CD8+ T lymphocytes have been shown to be important effectors of acquired protective immunity against L. monocytogenes. Class I MHC-restricted CD8+ cytotoxic T lymphocytes (CTL), which lyse J774 macrophage-like targets infected with L. monocytogenes, are induced following in vivo injection of live organisms. Natural peptide epitopes derived from L. monocytogenes can be acid-extracted from heavily infected BALB/c spleens and detected by CTL. A CTL clone, B9, derived from a (BALB/c x C57BL/6)F1 (H-2dxb) mouse, recognizes one of these natural epitopes in an H-2Kd-restricted fashion. B9 also recognizes P815 (H-2d) mastocytoma cells transfected with the listeriolysin gene. To identify the region of the listeriolysin recognized by CTL we used the H-2Kd peptide-binding motif described by Rammensee and colleagues to synthesize 11 nonamer peptides. One of these peptides, listeriolysin 91-99, was recognized very efficiently by B9. This represents the first identified class I MHC-restricted epitope of bacteria and demonstrates the utility of the allele-specific motif for predicting CTL epitopes.