Small cell lung carcinomas express shared and private tumor antigens presented by HLA-A1 or HLA-A2.

Tumor-derived peptides presented by MHC class I molecules are targets for tumor rejection by CD8+ CTLs. MHC-restricted CD8+ CTLs are required also for the identification and characterization of tumor antigens that will be useful for immune therapy. For many human solid tumors, however, tumor antigens remain undefined because of the difficulty of generating MHC-restricted, tumor-specific CTLs required for their analysis. CD8+ CTL responses are modulated by CD4+ helper T cells and by antigen-presenting cells. In this study, highly purified CD8+ T cells were mixed with tumor cells in primary cultures in the absence of any other cells to reduce the complexity of CTL generation. Tumor cells were transfected with HLA-A1 or HLA-A2 and used to stimulate partly matched HLA-A1- or HLA-A2-positive CD8+ T cells. Partial MHC class I matching of tumor and CD8+ T cells and omission of other cells in primary culture was highly effective in generating MHC class I-restricted CTL to poorly immunogenic small cell lung carcinomas (SCLCs). Cytotoxicity was further enhanced by cotransfection of tumor cells with B7.1 (CD80). ICAM-1 (CD54) was not as effective as costimulation. SCLC cells presented tumor-specific peptides with HLA-A1 and HLA-A2 and were lysed by A1- or A2-restricted CD8+ CTLs. A1- and A2-restricted CD8+ CTLs detected shared tumor antigens on unrelated SCLC tumor lines in addition to private antigens. The use of direct antigen presentation by MHC class I-transfected tumors to MHC class I-matched CD8+ T cells is an effective way to generate MHC class I-restricted CTLs toward poorly immunogenic tumors in vitro, permitting the molecular identification of their tumor antigens.

Perforin/Fas-ligand double deficiency is associated with macrophage expansion and severe pancreatitis.

We report that perforin/Fas-ligand double-deficient mice die early of severe pancreatitis. Female mice, in addition, are infertile and suffer from hysterosalpingitis. Tissue destruction is accompanied by infiltration with Mac-1 (CD11b)-positive monocytes/macrophages, Mac-1-positive T cells, and expansion of CD8+ T cells. In vivo inactivation of monocytes/macrophages by carrageenan reverses disease progression and restores fertility of female mice. Perforin/Fas-ligand double-deficient CD4+ or CD8+ CTL are unable to lyse cognate-activated macrophages, and therefore are unable to mediate negative feedback regulation by lysis of APCs, thereby preventing further T cell activation. These studies demonstrate a novel role for perforin in homeostatic regulation of the immune response.

Consequences of Fas-ligand and perforin expression by colon T cells in a mouse model of inflammatory bowel disease.

BACKGROUND & AIMS: We describe a type of colitis that develops after transplantation of nonallogeneic wt bone marrow cells into T cell- and natural killer cell-deficient Tg26 mice (BM-->Tg26). In these animals, severe wasting and inflammation of the colon correlates with the expansion of mucosal T lymphocytes that displays cytotoxic activity. The aims of this study were to determine the relative contribution of perforin and Fas ligand (Fas-L) expression to the cytotoxic action of these T cells and to examine the influence of each pathway in this model of colitis. METHODS: Colonic T cells were tested for their ability to mediate Fas- and perforin-dependent killing in redirected cytotoxicity assays. Bone marrow cells from donor mice lacking either Fas-L (gld mice) or perforin (PFPnull mice) or both molecules were used to reconstitute Tg26 mice. RESULTS: Colon cytotoxic T lymphocyte displayed both Fas- and perforin-dependent killing. Deficiency in perforin, but not Fas-L, resulted in reduced incidence of wasting and, to a lesser extent, severe colitis in BM-->Tg26 animals. CONCLUSIONS: Colon T cells from BM-->Tg26 mice express both perforin and Fas-L. Although neither pathway is critical in the development of colitis, perforin does have a measurable influence on disease in the BM-->Tg26 colitis model.

CD80 (B7.1) and CD54 (intracellular adhesion molecule-1) induce target cell susceptibility to promiscuous cytotoxic T cell lysis.

Overexpression of B7 and intracellular adhesion molecule-1 (ICAM-1) on syngeneic cells triggered MHC-unrestricted lysis by MHC-restricted CD8+ CTL without TCR/CD3 engagement. Both CD28/B7 and LFA-1/ICAM-1 interactions were required for MHC-nonrestricted cytotoxicity. Cytotoxicity was measured in 4-h and 16- to 18-h cytotoxicity assays. B7-ICAM-1 overexpression triggered perforin- and Fas ligand-mediated lysis, while TNF-alpha was not elicited in MHC-unrestricted cytotoxicity. These results suggest that target cells may elicit MHC-unrestricted lysis from CTL through up-regulation of membrane adherence and costimulatory receptors.

Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.

Bcl-2 protects against Fas-based but not perforin-based T cell-mediated cytolysis.

Fas ligand and perforin are the two key effector mechanisms in T cell-mediated cytotoxicity. These molecules mediate cytolysis of target cells by membrane damage and apoptosis. bcl-2 is known to protect cells against apoptosis induced by many stimuli including growth factor removal. However bcl-2's effect on Fas ligand and perforin-induced lysis has not been studied extensively. We investigated the effect of overexpression of bcl-2 alone, Fas alone or their combined overexpression on lysis of a commonly used target, P815, by perforin-sufficient, Fas ligand-sufficient and perforin-deficient or Fas ligand-deficient, allospecific cytotoxic T lymphocytes (CTL). Wild-type P815 are susceptible to lysis by perforin-sufficient CTL, regardless of the presence or absence (gld) of Fas ligand, but are poorly lysed by perforin-deficient CTL. Fas transfection of P815 makes target cells highly susceptible to lysis by both perforin-sufficient and -deficient CTL, indicating the presence of the Fas ligand-mediated cytotoxicity on both types of CTL. Co-transfection of P815-fas with bcl-2 abolishes their increased susceptibility to Fas-mediated lysis, even in the face of Fas overexpression on the cell membrane. The protective effect of bcl-2 against cell lysis is evident with perforin-deficient CTL as effector cells or when perforin activity is eliminated by the absence of extracellular calcium in perforin-sufficient CTL. bcl-2 overexpression by P815, however, does not protect against CTL lysis by the perforin pathway, regardless of Fas overexpression, as demonstrated by fas ligand mutated gld and wild-type perforin-sufficient CTL. Therefore bcl-2 can protect P815 target cells against Fas-mediated lysis when triggered by the Fas ligand on CTL, but not against perforin-mediated lysis.

Scoliosis in familial dysautonomia. Operative treatment.

The results of operative treatment of scoliosis were reviewed for twenty-two patients (ten boys and twelve girls) who had familial dysautonomia, an autosomal recessive disorder affecting primarily Ashkenazi Jews. The indication for operative intervention was progressive kyphoscoliosis to 45 degrees or more in a skeletally immature patient for whom bracing had failed. The mean age at the time of the operation was fifteen years and five months (range, eight years and two months to nineteen years). Seventeen patients had a thoracic curve with a mean preoperative Cobb angle of 69 degrees (range, 47 to 112 degrees), and five patients had a double major curve with a mean preoperative Cobb angle of 71 degrees (range, 42 to 87 degrees) for the cephalad curves and 60 degrees (range, 45 to 72 degrees) for the caudad curves. Twenty patients had a rigid kyphosis; in fourteen, the apex was at the seventh thoracic vertebra or more cephalad. Two patients had a lordoscoliosis. The mean preoperative kyphosis was 64 degrees (range, 12 to 110 degrees) in the thirteen patients who had a thoracic curve and for whom information regarding kyphosis was available, and it was 70 degrees (range, 54 to 84 degrees) in the five patients who had a double major curve. Postoperior spinal arthrodesis and instrumentation was performed in all patients. Two patients had an anterior arthrodesis as well because of the severity and rigidity of the curve. Allograft bone was used in eighteen patients. Postoperatively, all patients were managed with a body cast or with a custom-molded thoracolumbar brace.(ABSTRACT TRUNCATED AT 250 WORDS)

T cell adhesion to extracellular matrix molecules secreted by endothelial cells cultured on a substrate of type IV collagen.

T cell emigrating from the bloodstream into lymphoid organs or sites of inflammation in the connective tissue must adhere to, and traverse, the subendothelial basement membrane (BM). The goal of the current investigation was to develop a method to study the adhesion of T cells to endothelial cell (EC)-derived extracellular matrix (ECM) as a model for the interaction of T cells with the subendothelial BM in vivo. To be certain that we were truly measuring T cell adhesion to ECM molecules secreted by the EC, it was necessary to culture the EC on a substrate to which T cells could not attach. Non-tissue culture-treated microtiter plate wells which had been coated with type IV collagen (tIVC), a major constituent of BM in vivo, were found to be suitable for this purpose since EC, but very few T cells, adhered to such wells. After incubating the EC on a substrate of tIVC in non-treated wells for a period of 48 h, the EC were gently removed from their underlying ECM and T cell adhesion to that ECM was examined. Using this system, it was observed that approximately 15-40% of human peripheral blood T cells specifically adhered to ECM molecules produced by the EC. This method should be useful as a model for the interactions of T cells and other leukocytes with the vascular BM in vivo.

Biosynthetic maturation of an ascites tumor cell surface sialomucin. Evidence for O-glycosylation of cell surface glycoprotein by the addition of new oligosaccharides during recycling.

Previous biosynthetic studies of the ascites 13762 rat mammary adenocarcinoma cell surface sialomucin ASGP-1 (ascites sialoglycoprotein-1) showed that it is synthesized initially as a poorly glycosylated immature form, which is converted to a larger premature form (t1/2 30 min) and more slowly to the mature glycoprotein (t1/2 greater than 4 h). In the present study O-glycosylation of ASGP-1 polypeptide is shown to occur in two phases: an early phase complete in less than 30 min, which corresponds to the synthesis of the premature form, and a later phase that continues for hours and corresponds to the synthesis of the mature form. Pulse-chase labeling studies indicate that 95% of the ASGP-1 has moved to the cell surface in 2 h. Since transit to the cell surface is faster than the slow phase of addition of new oligosaccharides, some new oligosaccharides must be added after ASGP-1 has reached the cell surface. Initiation of new oligosaccharides on cell surface ASGP-1 was demonstrated directly using a biotinylation procedure to identify cell surface molecules. Glucosamine labeling of biotinylated ASGP-1 was shown to occur on galactosamine residues, which are linked to the polypeptide, establishing the addition of new oligosaccharides to the cell surface molecules. Finally, resialylation studies indicate that ASGP-1 rapidly recycles through a sialylating compartment. From these results we propose that ASGP-1 reaches the cell surface in an incompletely glycosylated state and that additional oligosaccharides are added to the glycoprotein in a second process involving recycling.

Interferon-gamma increases susceptibility of murine pancreatic beta cells to lysis by allogeneic cytotoxic T lymphocytes.

The selective loss of insulin-producing pancreatic beta cells which occurs in IDDM has been postulated to result from lysis by beta cell-specific cytotoxic T lymphocytes (CTL). CTL typically recognise antigen in the context of MHC class I molecules, which are normally present at low levels on beta cells. However, hyperexpression of class I antigens on islet cells has been observed in the early stages of beta cell destruction in IDDM. Since interferon-gamma (IFN-gamma) is known to increase class I expression on a number of cell types, we have investigated the responses of murine beta cells to this cytokine under various conditions. Two color immunostaining followed by FACS analysis showed that on average, only 14.9 +/- 3.1% of cultured beta cells were class I positive. However, a majority of beta cells could be induced to express class I after 24 hours of IFN-gamma treatment, and maximal induction (80-90% positive) occurred after 48 hours. Importantly, increased class I expression on beta cells could be achieved with very low concentrations of IFN-gamma (1-10 U/ml). Expression of class II MHC was never detected under any of the conditions employed to up-regulate class I. Interestingly, although islet cells were only moderately susceptible to lysis by allospecific CTL, this susceptibility was markedly enhanced by prior exposure of the islets to IFN-gamma. Taken together, these results suggest that beta cells are extremely susceptible to up-regulation of class I MHC molecules by IFN-gamma, and that this property may render these cells particularly susceptible to lysis by autologous class I-restricted CTL. Since enhanced expression of class I frequently accompanies inflammatory responses and viral infections, this property of beta cells may account in part for their selective destruction in IDDM.

Biosynthesis of a tumor cell surface sialomucin. Maturation and effects of monensin.

The major cell surface glycoprotein (ascites sialoglycoprotein-1 (ASGP-1] of ascites 13762 rat mammary tumor cells is a large (Mr greater than 500,000), highly glycosylated sialomucin which is present in great abundance (greater than 0.5% of total cell protein). Thus, these tumors provide a useful system for investigating the biosynthesis of O-glycosylated glycoproteins. Previous studies in this system have demonstrated that initiation of O-linked oligosaccharides occurs throughout most of the transit period of ASGP-1 from the endoplasmic reticulum to the cell surface. By pulse-chase threonine labeling and precipitation with peanut agglutinin, ASGP-1 is first observed as an immature lightly glycosylated form (Mr approximately 200,000) which is converted to a more mature, more heavily glycosylated form (designated the premature or P form) with a half-time of about 30 min. The P form is then more gradually converted into the mature ASGP-1. Analysis of glucosamine-labeled oligosaccharitols obtained from the immature form showed primarily unsialylated derivatives consisting of the structures of the size of the tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc and smaller, whereas the mature form showed a mixture of sialylated and unsialylated structures. Desialylation of glucosamine-labeled mature form resulted in a glycoprotein intermediate in size between the immature and mature forms, indicating that the size change with maturation is not solely due to sialylation. Treatment of the cells with 10(-6) M monensin significantly reduced the conversion of immature to mature form without inhibiting initiation of O-linked oligosaccharides and without preventing sialylation. Analysis of oligosaccharitols obtained from ASGP-1 of monensin-treated cells showed that the major oligosaccharides are trisaccharide GlcNAc beta 1,6(Gal beta 1,3)GalNAc and sialylated trisaccharide GlcNAc beta 1,6(NeuAc alpha 2,3-Gal-beta 1,3) GalNAc. These results suggest that monensin specifically disrupts the compartment of the biosynthetic pathway which adds most of the beta 1,4-Gal to the oligosaccharides of ASGP-1 and that this compartment is separate from the primary site of sialylation.

Temporal aspects of O-glycosylation and cell surface expression of ascites sialoglycoprotein-1, the major cell surface sialomucin of 13762 mammary ascites tumor cells.

We have investigated the biosynthesis and cell surface expression of the major cell surface sialomucin (ascites sialoglycoprotein-1 (ASGP-1] of 13762 rat mammary ascites tumor cells by pulse or pulse-chase metabolic labeling combined with precipitation with peanut agglutinin and alkaline borohydride elimination or proteolytic fragmentation. The minimum time for initial glycosylation was estimated from the time required for the protein to acquire the ability to bind to peanut agglutinin to be less than 5 min. Moreover, when cells were labeled with threonine for 5 min and the ASGP-1 isolated by peanut agglutinin precipitation, 3% of the labeled threonine could be converted to 2-aminobutyric acid by alkaline borohydride elimination of the carbohydrate, indicating that at least 3% of the threonines of ASGP-1 are O-glycosylated within 5 min of polypeptide synthesis. The minimum time between the final glycosylation reactions in the cell and appearance of ASGP-1 at the cell surface was determined by trypsinizing galactose- or glucosamine-labeled cells at timed intervals after labeling to occur within 5-10 min of labeling. Both labeled glucosamine and galactosamine appeared in ASGP-1 fragments within 5 min, but the amount of labeled galactosamine was less than the amount of labeled glucosamine until after 20 min, when the 1:1 equilibrium ratio was reached. The half-time for appearance of glucosamine-labeled ASGP-1 at the cell surface was found to be greater than 4 h. The minimum time required from synthesis of the ASGP-1 polypeptide to appearance at the cell surface was determined by leucine labeling and proteolysis to be 70-80 min. These combined studies suggest a continuum of O-linked oligosaccharide initiation events extending over most of the period of ASGP-1 biosynthesis and transit from the endoplasmic reticulum to the cell surface.

Structural and functional aspects of tumor cell sialomucins.

Sialomucins are abundant on the surfaces of certain ascites tumor cells and have been implicated in the escape of tumors from immune destruction and metastasis. They are large, highly glycosylated glycoproteins which are rich in serine and threonine and have a variety of 0-linked oligosaccharides. The sialomucin (ASGP-1) or 13762 rat mammary adenocarcinoma ascites cells represents more than 0.5% of the total cell protein and can be isolated from cell membranes by centrifugation in 4 M guanidine hydrochloride-cesium chloride. ASGP-1 can also be isolated from membranes or cells by nonionic detergent extraction as a 1:1 complex with a second glycoprotein ASGP-2. Studies with the fluorescent lectins peanut agglutinin, which binds ASGP-1, and Concanavalin A, which binds ASGP-2, indicate that the glycoproteins are present at the cell surface as a complex. ASGP-1 is shed into cell culture medium or ascites fluid, apparently by a proteolytic cleavage mechanism. 13762 ascites cells grown in culture or as solid tumors lose their ASGP-1. The sialomucin reappears with extensive passage of the tumor cells in ascites form. Studies on the biosynthesis of ASGP-1 indicate that carbohydrate is being added over nearly the entire period of transit of ASGP-1 from the site of polypeptide synthesis to the plasma membrane. The negatively charged, rod-like structure of the sialomucins suggests that they may play a role in inhibiting recognition or binding processes necessary for the immune destruction of these tumor cells.

Spotted fever group rickettsiae in Dermacentor variabilis from Cape Cod, Massachusetts.

Spotted fever group (SFG) rickettsiae were identified by indirect immunofluorescent antibody tests in 1.1% of 6,956 specimens of Dermacentor variabilis collected on Cape Cod, Massachusetts. Forty-one of 45 rickettsial isolates were serotyped as Rickettsia montana, one as Rickettsia rickettsii, and three were unidentified. Studies of canines and humans with clinical evidence of spotted fever, and of healthy dogs with antibody to SFG rickettsiae, indicated that exposure of the canine population to R. montana was common but that R. montana was not involved in the etiology of spotted fever.

Natural infection of dogs on Cape Cod with Rickettsia rickettsii.

Four isolates of rickettsiae from sick dogs on Cape Cod, Mass., were serologically identical to isolates of Rickettsia rickettsii from human patients with Rocky Mountain spotted fever. The antigenic analysis used the indirect fluorescent-antibody test and antisera prepared in mice to each of the isolates and to reference strains of R. rickettsii and Rickettsia montana. Serological responses of infected dogs were specific for R. rickettsii, although antibodies to R. montana were also detected in the sera of most of the canines.

An indirect immunofluorescent test for human antibodies to tetanus toxoid using an insoluble toxoid as antigen.

An indirect fluorescent antibody (FA) test for detection of human antibodies to tetanus toxoid is described using an ethylchloroformate-prepared polymer of tetanus toxoid as the particulate slide test antigen. Titres of the FA test were compared with those obtained with the toxin neutralization (TN) test in mice. No antisera were FA-positive at less than 0-0025 AU/ml. Positive correlation of the FA test with the TN test was 50% between 0-0025 and 0-01 antitoxin units/ml (AU/ml) and 100% between 0-02 and 160 AU/ml. In general, an increase in FA titres correlated with an increase in TN titres beginning at about 0-08-0-16 AU/ml.