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.     

Homology of perforin to the ninth component of complement (C9)

Perforin is one of the cytolytic factors present in the cytoplasmic granules of mouse cytotoxic T lymphocytes and natural killer cells. We have determined the sequence of the N-terminal amino acids of perforin purified from a mouse natural killer cell line, and, by using oligonucleotide probes corresponding to the amino acid residues, we have identified a complementary DNA encoding perforin from the cDNA library of a mouse cytotoxic T lymphocyte clone. As predicted from the functional similarities between perforin and the ninth component of the serum cytolytic system, complement (C9) (refs 4-8), the deduced primary structure of perforin has homology with C9 at their respective functionally conserved regions. We find that perforin is only expressed in killer cell lines, and not in helper T lymphocytes or other tumour cells tested. Thus we have provided direct molecular evidence that a killer-cell-specific protein evolutionally linked to C9 is involved in cell-mediated cytolysis.     

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.     

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.     

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。     

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.     

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.     

Murine cells expressing an HLA molecule are specifically lysed by HLA-restricted antiviral human T cells

Class I HLA (histocompatibility locus antigen) molecules are the targets of allospecific cytolytic T lymphocytes (CTL) in graft rejection, and constitute the restricting elements necessary for the interaction between antiviral CTL and virus-infected cells. Cells expressing only one HLA in the absence of other human molecules would provide a remarkable model for studying the function of these molecules. However, HLA+ murine cells transfected with human genes are generally not lysed by allospecific human CTL, and this is ascribed to insufficient HLA expression, lack of human beta 2-microglobulin, alteration of HLA molecules or absence of receptors for human T8 or LFA1 molecules in murine cells. Here we report, for the first time, the specific lysis of virus-infected HLA+ murine cells by HLA-restricted antiviral human CTL. Therefore, these murine cells constitute an excellent model for studying the role of HLA molecules.     

Cytotoxic T lymphocyte mediated lysis without release of serine esterase

Cloned cytotoxic T lymphocytes (CTL) can lyse target cells in a Ca2+-dependent or Ca2+-independent fashion, depending on the target cell used. We have used these Ca2+-free assay conditions to determine whether there is serine esterase release in the absence of extracellular Ca2+. We find that under conditions where there is significant target cell killing in the absence of Ca2+, there is no detectable serine esterase release. To the extent that serine esterase and perforin-like molecules reside in the same granules, these results also suggest that target cell killing may occur in the absence of degranulation. Our results provide an example of target cell killing without serine esterase release, and the first indication that lysis and degranulation can be functionally dissociated.     

Detection of refolding conformers of complement protein C9 during insertion into membranes

Human complement protein C9 is a hydrophilic serum glycoprotein responsible for efficient expression of the cytotoxic and cytolytic functions of complement. It assembles on the surface of a target cell together with C5, C6, C7 and C8 to form the membrane attack complex (MAC) and therefore has to change structure to become an integral membrane protein. As the protein assumes a stable structure in an aqueous environment, the question arises as to how it can enter the hydrophobic interior of a membrane. During MAC assembly C9 polymerizes into a circular structure, termed poly(C9) (ref. 8), which is responsible for the cylindrical electron microscopic appearance of the MAC. The suggestion has been made that C9 must at least partly unfold in order to enter a membrane and also that polymerization of the molecule is intimately linked to insertion and cytotoxicity. The extent of unfolding and the mechanism of polymerization are not understood, nor is it known precisely which parts of the molecule participate in the proposed structural changes. We have been able to capture refolding C9 conformers during membrane insertion with the help of sequence-specific anti-peptide antibodies. Some of these antibodies inhibit C9-mediated haemolysis but not C9 polymerization, while others have the opposite effect. This suggests that the two processes are independent.     

Pleiotropic loss of activation pathways in a T-cell receptor alpha-chain deletion variant of a cytolytic T-cell clone

Untransformed T-cell clones maintained in culture are dependent on signals transmitted through their antigen receptors (Ti; alpha and beta chains associated with the CD3 molecules) for growth and effector function. For cytolytic T cells (CTL), Ti stimulation also activates the killing machinery and induces synthesis of gamma interferon (IFN-gamma) messenger RNA and IFN-gamma secretion. The Thy-1 molecule, expressed on all murine cells of the T-cell lineage, has been suggested to function in transmembrane signalling, based on the ability of some anti-Thy-1 monoclonal antibodies (mAb) to activate T cells. Recently, it was suggested that Thy-1 could function as a signal-transduction molecule when expressed in B-cell lymphomas after transfection of the gene, leading to speculation that the molecule was part of an activation pathway independent of the Ti/CD3 structures. Here we report the immunoselection of a variant CTL clone which has lost expression of mRNA for the alpha-chain of the Ti. The Ti- variant was defective in lectin-mediated activation whether measured by increase in intracytoplasmic Ca2+, CTL effector function or IFN-gamma synthesis. The variant, which expressed normal levels of Thy-1, was also unresponsive to Thy-1 mAb activation as measured by IFN-gamma secretion, whereas it responded to calcium ionophore plus phorbol ester. These results indicate that in a non-transformed, functional mature T-cell, Thy-1 mediated signalling is not an alternative to, but might depend on elements associated with the Ti/CD3-mediated T-cell activation pathway.     

Specificity of peptide presentation by a set of hybrid mouse class I MHC molecules

The class I molecules of the major histocompatibility complex (H-2 in mouse, HLA in man) are membrane proteins composed of a polymorphic heavy chain associated with beta-2-microglobulin. Recent studies suggest that class I molecules present peptides derived from processed antigens to the receptor of cytolytic T cells. In particular, in the H-2d haplotype, synthetic HLA peptides can be recognized on Kd-bearing target cells by Kd-restricted cytolytic T cells specific for HLA. Here we analyse the specificity of presentation of two HLA peptides by a set of chimaeric Kd/Dd molecules to four different cytolytic T-cell clones. We identify two distinct regions within the second external (alpha 2) domain of Kd that contribute to its specificity as a restriction element. Our results indicate that the binding of an immunogenic peptide by a class I molecule is not always sufficient for its recognition by the T-cell antigen receptor. This suggests that the major histocompatibility complex restriction element either interacts with the T-cell antigen receptor or induces the recognized conformation of the peptide.     

Cytotoxic T lymphocytes and glucocorticoids activate an endogenous suicide process in target cells

Cytotoxic T lymphocytes (CTL) induce a cytolytic process in target cells which, like the glucocorticoid-mediated cytolysis of immature thymocytes, effects a rapid and characteristic degradation of chromosomal DNA. I have explored the possibility that these two lethal processes share a common pathway by studying the susceptibility of glucocorticoid-resistant mutants to CTL-mediated killing. Here, I report that an unusual thymoma mutant, which has normal hormone receptor activity, is resistant to both glucocorticoids and CTL. The failure to be killed by CTL is not due to an inability of this 'deathless' mutant to be recognized. Further, a single-step reversion can restore sensitivity to both glucocorticoids and CTL. The genetic locus thus identified may reveal one element of an endogenous suicide pathway that can be triggered by different effectors. Unlike complement-mediated lysis, the processes of glucocorticoid- and CTL-mediated cytolysis seem to require that target cells be active in their own death.     

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.     

Role of intracellular calcium mobilization in the regulation of protein kinase C-mediated membrane processes

Phorbol esters are potent tumour-promoting agents that exert pleiotropic effects on cells. Among these are the control of growth, stimulation of release of stored bioactive constituents and regulation of growth-factor surface receptors. Phorbol esters bind to and activate protein kinase C, leading to the phosphorylation of specific protein substrates presumed to be necessary for eliciting the full response. Strong evidence exists that specific binding of tumour promoter occurs at the membrane level in intact cells, resulting in activation of protein kinase C. Recent evidence concerning the release of bioactive constituents from platelets and neutrophils has linked agonist-induced protein kinase C activation and Ca2+ mobilization in a synergistic mechanism. Here we present a novel model of synergism between Ca2+ and phorbol esters that leads to transferrin receptor phosphorylation and down-regulation in HL-60 human leukaemic cells. Raising intracellular Ca2+, although ineffective by itself, increases the potency and rate of action of phorbol ester for activating protein kinase C and mediating transferrin receptor phosphorylation and down-regulation. We propose a molecular model in which increased intracellular Ca2+ recruits protein kinase C to the plasma membrane, thus "priming' the system for activation by phorbol ester.     

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.     

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.     

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.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.