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C F Ware

Publications and source records attributed to C F Ware.

At least 91 records · Page 5Linked to original sources
PubMed1988

Inhibition of cytotoxic T lymphocyte and natural killer cell-mediated lysis by O,S,S,-trimethyl phosphorodithioate is at an early postrecognition step.

O,S,S,-Trimethyl phosphorodithioate (OSS-TMP), an organophosphate esterase inhibitor, has been shown to block the effector phase of the cytolytic reaction mediated by murine and human cytotoxic T lymphocytes (CTL) and human natural killer cells. The murine interleukin 2-dependent CTLL-1 (anti-Iad) clone was used to determine the phase of the cytolytic pathway inhibited by OSS-TMP. Pretreatment of the CTL or target cell with OSS-TMP was not effective at blocking lysis; however, inhibition of lysis was achieved if the reaction was carried out in the continuous presence of OSS-TMP (IC50 = 55 microM) or when CTL-target conjugates were performed and incubated with OSS-TMP (IC50 = 640 microM). Two structural analogues of OSS-TMP were unable to inhibit CTL-mediated lysis. In contrast to OSS-TMP, N-alpha-p-tosyl-L-lysine chloromethylketone required only a 5-min preincubation with the CTL to inhibit lysis. OSS-TMP did not block recognition-adhesion step(s) of the reaction since the ability to form conjugates was not impaired; however, the lytic efficiency of individual CTL-target pairs were blocked. OSS-TMP did not appear to be an inhibitor of the major granule-associated protease that cleaves the substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzylester. Ca2+ pulse and kinetic experiments indicated that the OSS-TMP-sensitive site was at a pre-Ca2+-dependent phase but after recognition-adhesion. Human CTL and natural killer cell activity was also inhibited by OSS-TMP, suggesting the presence of a common site of action among these cytolytic systems. The results indicate that OSS-TMP may be a useful reagent in characterizing the early post-recognition events in the cytolytic pathway of CTL and natural killer effector cells.

Animals↗
PubMed1988

Effects of acute administration of O,S,S-trimethyl phosphorodithioate on the generation of cellular and humoral immune responses following in vitro stimulation.

The time course of immune modulation induced by acute treatment with O,S,S-trimethyl phosphorodithioate (OSS-TMP), an impurity in technical formulations of malathion, was examined in female C57BL/6 mice. The immune parameters studied included the generation of cytotoxic T lymphocytes (CTL) to alloantigen (H-2 incompatible) and antibody secreting cells to sheep red blood cells, proliferative response to the mitogens, and interleukin-2 (IL-2) production. Acute administration of the non-toxic doses of OSS-TMP, i.e. 20 or 40 mg/kg, led to an elevation in the generation of a CTL response on day 1 or 7, respectively. At 20 mg/kg OSS-TMP, the antibody response was elevated at day 3. However, at a dose of 40 mg/kg OSS-TMP, the antibody response was suppressed at day 1 following treatment. Following acute administration of 60 or 80 mg/kg OSS-TMP, the generation of an antibody and CTL responses was suppressed at all time points tested with 1 exception. One day following treatment at a dose of 60 mg/kg OSS-TMP, there was no change in the CTL response. At day 7 following treatment, the mitogenic responses to lipopolysaccharide and phytohemagglutinin were elevated at all doses of OSS-TMP administered. At this time point, however, the proliferative response to Concanavalin A was elevated in a dose dependent manner. IL-2 production was suppressed following acute administration of 60 or 80 mg/kg OSS-TMP at all time points tested and at all doses tested on day 5 following treatment. These data indicate that OSS-TMP, unlike its congener, O,O,S-trimethyl phosphorothioate, enhances the generation of humoral and cell mediated immune responses of C57BL/6 mice following administration of non-toxic doses.

Animals↗
PubMed1988

The relationship of receptor occupancy to the kinetics of cell death mediated by tumor necrosis factor.

An analysis of the relationship between TNF receptor occupancy and the kinetics of cell death has been conducted in the sensitive L929.10 murine fibrosarcoma cell line. The receptor affinity and number, measured by competitive and saturation binding assays with human 125I-TNF, is consistent with a single class of binding sites having a Kd = 1.5 nM and 7,000 sites/cell. Human lymphotoxin competed equally well with TNF for binding to the receptor. Determination of the kinetics of L929.10 cell death as a function of TNF concentration revealed three sequential phases to the cytolytic process: An induction phase, that lasted 4 to 9 hrs after the addition of TNF, in which no cell death occurred; the burst phase in which cells died at a rate of 12 to 14% per hr; and the third phase, termed the slow lytic phase, was characterized by a rate of lysis of 1 to 2% per hr. Two parameters, the length of the induction phase and the percentage of cells killed in the burst phase, were dependent on TNF receptor occupancy. Both of these receptor dependent processes were maximized at 10% receptor occupancy. Half maximal lysis of the L929.10 clone was achieved at 10 pM or 0.67% receptor occupancy. In contrast, the rates of cell death during the burst phase and the slow lytic phase were independent of the concentration of TNF. Mitomycin C, an antitumor drug that alkylates and crosslinks DNA, enhanced the sensitivity of the L929.10 cells to TNF mediated lysis by modulating both of the TNF receptor dependent processes and by doubling the (apparent) rate of cell death (22 to 25%/hr) during the burst phase. These results suggest a direct relationship exists between the strength of the TNF-receptor signal and nuclear events, perhaps those associated with processes involved in DNA replication or cell division, during the cytotoxic reaction induced by these inflammatory cytokines.

Animals↗
PubMed1987

Resistance to the cytolytic action of lymphotoxin and tumor necrosis factor coincides with the presence of gap junctions uniting target cells.

The resistance of target cells to the cytolytic action of lymphotoxin (LT) and recombinant tumor necrosis factor (rTNF) has been investigated by using clonally derived cell lines with defined gap junction-mediated, intercellular communication properties. Gap junction-competent Chinese hamster ovary cells are normally insensitive to the action of LT/TNF. However, treatment with 12-o-tetradecanoylphorbol-13-acetate, which promotes the loss of gap junctions, or culturing at low cell density to reduce intercellular contacts, significantly increased their sensitivity to LT/TNF. The LT/TNF-sensitive murine CL-1D and L929 cell lines, which in normal culture conditions are unable to form gap junctions, were not changed in their susceptibility to LT/TNF after treatment with phorbol ester or low culture density. However, the formation of gap junctions by CL-1D can be promoted by treatment with 8-bromo-cyclic adenosine monophosphate (1 mM), and this treatment completely suppressed the ability of LT and rTNF to kill CL-1D. Additionally, the LA25-normal rat kidney cell line, which is infected with a temperature-sensitive mutant of Rous sarcoma virus (LA25), is gap junction-competent and resistant to the effects of LT at the restrictive temperature (39 degrees C). However, when shifted to the permissive temperature (33 degrees C), LA25-normal rat kidney cells express the pp60v-src viral gene product, lose their ability to form gap junctions, and become sensitive to the lytic activity of LT. The results demonstrate that the expression of the retroviral pp60v-src, a tyrosine protein kinase, is sufficient to render cells susceptible to the lytic effects of LT and rTNF. Collectively, these experiments demonstrate a strong correlation between the resistance of target cells to the action of LT/TNF and their ability to cooperate metabolically through gap junctions. The results do not completely exclude the possibility that other mechanisms, such as LT receptor modulation, are also occurring under these experimental conditions. These data also suggest that a possible physiologic function of the stable cytotoxic lymphokines is to induce cytolysis/cytostasis of cells that have lost gap junctional contact, such as those in the process of mitosis or metastasis that have separated from the main tissue mass.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Effects of O,S,S-trimethyl phosphorodithioate on immune function.

The effect of acute administration of 20-80 mg/kg O,S,S-trimethyl phosphorodithioate (OSS-TMP) to C57BL/6 female mice on the murine immune system was determined. The parameters examined to evaluate overt toxicity of the compound included body weight, plasma cholinesterase levels, splenic nucleated cell number and thymic weight and nucleated cell number. Acute administration of 60 or 80 mg/kg OSS-TMP led to a 75 or 63% decrease, respectively, in plasma cholinesterase levels and a decrease in thymic size. At a dose of 80 mg/kg OSS-TMP, the animals also exhibited some lethargy and body weight loss. Below 60 mg/kg OSS-TMP, no overt toxic manifestations were observed. These studies were carried further to determine the effect of OSS-TMP on the generation of in vivo primary and in vitro secondary cellular and humoral immune responses. At nontoxic doses of the compound, i.e. 20 and 40 mg/kg OSS-TMP, the in vivo generation of a primary cytotoxic T lymphocyte (CTL) response to alloantigen was significantly elevated, but this response was unaffected following restimulation of the splenocytes by alloantigen in vitro. The generation of an in vivo primary and in vitro secondary humoral responses to sheep red blood cells (SRBC) was elevated following a single dose of 40 mg/kg OSS-TMP. Administration of toxic doses of OSS-TMP, i.e. 60 and 80 mg/kg, did not alter the ability of splenocytes to generate a primary or secondary CTL response, but suppressed the generation of humoral immune responses. These results differ significantly from those observed in a similar system following acute administration of a structural analog, O,O,S-trimethyl phosphorothioate which was previously shown to have potent immunosuppressive activity at nontoxic doses.

Animals↗
PubMed1987

Purification and characterization of a pectin lyase produced by Pseudomonas fluorescens W51.

A pectin lyase (PNL; EC 4.2.2.10) was isolated from culture filtrates of Pseudomonas fluorescens W51 and purified to apparent homogeneity. The enzyme catalyzed a random eliminative cleavage of pectin but not sodium polypectate, and it macerated plant tissue. The Mr of the PNL on sodium dodecyl sulfate-polyacrylamide gels was 32,000 +/- 1,000, and the isoelectric point was 9.4 as determined by isoelectric focusing. The enzyme was constitutively produced, since the highest yields were obtained when glycerol was used as a sole carbon source, and addition of pectin to the medium did not increase its production. Antibodies against purified PNL reacted in Western blots (immunoblots) with a pectate lyase (PLb) produced by Erwinia chrysanthemi EC16. The PNL appeared to be the only factor secreted into the culture medium by P. fluorescens W51 which macerated plant tissue and is probably involved in the soft rot disease caused by the bacterium.

Animals↗
PubMed1986

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. II. A novel CTL-produced cytotoxin that is antigenically distinct from tumor necrosis factor and alpha-lymphotoxin.

We have cloned lines of IL 2-dependent human T cells derived from alloantigen, soluble antigen (tetanus toxoid), mitogen, or IL 2-stimulated peripheral blood lymphocytes and characterized their surface marker expression and cytolytic activity. The surface phenotype and cytolytic function was compared with the ability of these T cell clones to release cytotoxic lymphokines in response to mitogenic lectins. The cytotoxins released by these CTL clones were detected on the murine L929 target cells in a 16-hr assay. All of the T cell clones, whether stimulated by HLA alloantigens, tetanus toxoid, or mitogens, exhibited killer cell activity and the capacity to secrete a soluble cytotoxin(s). Specific polyclonal antisera to recombinant human tumor necrosis factor (rTNF) and human alpha-lymphotoxin (alpha LT) were unable to neutralize the cytotoxic activity released by most of these CTL clones. These results indicate that human CTL produce a novel antigenic form(s) of cytotoxin that we have termed CTL-toxin. Supernatants from several CTL clones yielded a cytotoxic activity that was partially neutralized (10 to 40%) by saturating levels of anti-TNF (but not anti-alpha LT) indicating that human CTL may be capable of producing a TNF-like molecule. Only two out of 60 CTL clones studied thus far produced a cytotoxic activity that was partially neutralized by anti-alpha LT (20 to 40%). Collectively, these results suggest that although both the CD4 and the CD8 subpopulations of human cytotoxic T cells may be capable of releasing several types of cytotoxins in response to mitogenic signals, the predominant cytotoxin is distinct from alpha LT and TNF.

Antibodies, Monoclonal↗
PubMed1986

A rat monoclonal antibody which interacts with mammalian ornithine decarboxylase at an epitope involved in phosphorylation.

Ornithine decarboxylase was purified from androgen-treated mouse kidney to homogeneity and high specific activity. The purified enzyme was utilized for production and screening of rat monoclonal and polyclonal antibodies. A rat monoclonal antibody was isolated which was capable of immunoprecipitation of native mouse kidney ornithine decarboxylase activity or the [3H]difluoromethylornithine-inactivated enzyme. Phosphorylation of mouse ornithine decarboxylase by casein kinase-II prior to immunoprecipitation led to complete loss of the epitope recognized by the monoclonal antibody but did not alter recognition by polyclonal antibody. Mammalian ornithine decarboxylase activity obtained from several species, in crude or partially purified extracts, was subjected to quantitative immunoprecipitation with monoclonal and polyclonal antibody. Polyclonal antibody immunoprecipitated all of the ornithine decarboxylase activity from every extract tested, while monoclonal antibody was capable of only limited immunoprecipitation (60-80%). Due to the inability of the monoclonal antibody to recognize ornithine decarboxylase phosphorylated in vitro by casein kinase-II and the partial immunoprecipitation of ornithine decarboxylase activity from cell extracts, a portion of the ornithine decarboxylase molecule population must exist in a phosphorylated state. This immunological evidence further confirms existing data that the enzyme exists in at least two distinct forms.

Androgens↗
PubMed1986

The human LT system. XI. Identification of LT and "TNF-like" forms from stimulated natural killers, specific and nonspecific cytotoxic human T cells in vitro.

These studies demonstrate that specific and nonspecific cytolytic human T cells can release LT forms in vitro. Nonspecific cytolytic T cells were derived from IL 2-dependent cultures initiated by allogeneic mixed lymphocyte reaction (AMLR). Specific cytolytic T cells (CTL) were derived from IL 2-dependent T cell clones, initiated by mixed lymphocyte reaction and specific for class II antigens. alpha-LT was the major lytic component released by these cells in IL 2-dependent cultures. However, on Con A stimulation or contact with target cells, both AMLR and CTL effectors release a new LT form. The new LT form released by AMLR and CTL effectors appear similar, for they both elute from gel filtration at 60,000 to 70,000 m.w. and migrate as a single peak with an Rf of 0.4 on 7% native PAGE tube gels. Moreover, testing these materials on a panel of target cells in vitro indicates that they are both nonspecific, and lyses NK-resistant target cells in vitro. Additional studies revealed that in vitro lytic activity of this form(s) is not affected by either anti-alpha-LT serum or a monoclonal reagent which inactivates macrophage cell toxins (MCT) and tumor necrosis factor (TNF). However, when these two immunologic reagents are tested together, activity is totally neutralized. Thus, this LT form expresses antigens in common with alpha-LT, MCT, and TNF. Finally, studies with NK-CF and NK-LT forms revealed that they were also completely neutralized with a mixture of anti-LT and monoclonal anti-TNF antibody. These data suggest that certain macrophage- and lymphocyte-derived cell toxins are interrelated.

Cells, Cultured↗
PubMed1986

Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase.

Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme.

Journal Article↗
PubMed1986

Human T cell hybridomas producing cytotoxic lymphokines: induction of lymphotoxin release and killer cell activity by anti-CD3 monoclonal antibody or lectins and phorbol ester.

Human T cell hybrids were constructed between lectin-activated peripheral blood lymphocytes and CEM.TET1, a variant of the CEM T lymphoblastoid cell line. Hybrids were screened for their ability to produce cytotoxic lymphokines and interleukin-2 (IL-2) following lectin activation. Five hybrids were identified that released significant levels (greater than 75 units/ml) of cytotoxic activity that was detected on the murine L929 target cells; three hybrids were identified that produced IL-2. Cell surface phenotype of the T cell hybrids did not correlate with their ability to produce these lymphokines. The II-23 hybrid (CD3+;4+) produced an inducible cytotoxic activity that was fully neutralized by anti-lymphotoxin antibodies. The release of lymphotoxin (LT) rapidly attained maximum levels 12 hrs to 24 hrs after lectin activation; IL-2 production was maximum at 48 hrs. The induction of LT and IL-2 release required similar membrane stimulating agents. The phorbol ester, phorbol 12-myristate 13-acetate (PMA) in combination with phytohemagglutinin or concanavalin A, were required for maximal release of LT and IL-2. Anti-CD3 monoclonal antibodies coupled to agarose, but not anti-T200-agarose, induced the release of high levels of LT and IL-2. Soluble anti-CD3 was not sufficient to induce IL-2 or LT release from the II-23 hybrid; however, soluble anti-CD3 combined with PMA was a strong stimulus for lymphotoxin and IL-2 release. The II-23 hybrid also functioned as a cytotoxic T cell line in a 51Chromium release assay. Induction of killer cell function required similar perturbation of Ti/CD3 complex. This human T cell hybrid line offers an inducible model system for the simultaneous study of the molecular events regulating the production of growth inhibitory/cytotoxic (LT) and growth promoting (IL-2) lymphokines and cytotoxic T cell function.

Antibodies, Monoclonal↗
PubMed1985

Human, rat or mouse hybridomas secrete high levels of monoclonal antibodies following transplantation into mice with severe combined immunodeficiency disease (SCID).

Mice with severe combined immunodeficiency disease (SCID) have been investigated for their ability to grow xenogenic hybridomas of mouse, rat and human origin. Two rat X mouse hybridoma lines (187.1.10 and 3B9) and 1 mouse X mouse hybridoma (2D9) grown in pristane-treated SCID mice as ascites tumors showed a 100-200-fold increase in monoclonal antibody levels over the amount produced in vitro with a total yield up to 0.5 g of antibody per animal. A human X human hybridoma, CLL-11-D1, exhibited a 1000-fold increase in human immunoglobulin levels in ascites (1.3 mg/ml) as compared to that obtained in tissue culture. Analyses of the antibody protein in the SCID ascites produced by these hybridomas using protein electrophoresis, SDS polyacrylamide gel electrophoresis and high resolution isoelectric focusing indicated the antibodies were monoclonal and free from any contaminating immunoglobulins. Yields of monoclonal antibodies of over 90% purity could be obtained from the ascites by a single ammonium sulfate precipitation step. This study indicates that SCID mice provide several significant advantages over other in vivo methods for the production of pure monoclonal antibodies of human, rat, or mouse origin.

Animals↗
PubMed1985

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. I. Cytotoxins produced by antigen-specific and natural killer-like CTL are dissimilar to classical lymphotoxins.

Several cloned lines of IL 2-dependent human T cells derived from alloantigen, mitogen, or IL 2-stimulated peripheral blood lymphocytes were examined for their surface marker expression, cytolytic activity in a 51Cr-release assay, and capacity to release cytotoxic lymphokines. Thirty cell lines exhibiting either antigen-specific natural killer cell activity or lectin-dependent killer cell function, which expressed either the CD4 or CD8 surface differentiation markers, were capable of producing cytotoxin(s) in response to the lectins phytohemagglutinin and concanavalin A. Cytotoxin activity was detected on the murine L929 target cell in a 16-hr cytotoxicity assay. In contrast, several nonlytic T cell tumor lines failed to produce a soluble cytotoxin. Antibodies capable of neutralizing human alpha-lymphotoxin were completely ineffective in inhibiting the cytotoxin(s) produced by any of the cytotoxic T lymphocytes (CTL) cell lines. Comparative gel filtration and HPLC hydrophobic chromatography of alpha-lymphotoxin and CTL toxin produced by the CTL-830.B2 clone revealed significant differences in their elution profiles. The CTL-produced toxin and alpha-lymphotoxin exhibited similar kinetics of lysis of the L929 target cells, with 50% target cell lysis occurring at 10 hr. These data indicate human CTL produce a cytotoxin(s) antigenically distinct from alpha-lymphotoxin and imply that human cytolytic effector T cells are not the cellular source for the production of human alpha-lymphotoxin. The relationship of alpha-lymphotoxin and CTL toxin production was investigated in unseparated peripheral blood mononuclear cells stimulated with lectins or IL 2 for 1 and 5 days. Anti-alpha-lymphotoxin antibodies were capable of neutralizing only 30 to 50% of the cytotoxic activity in 24-hr supernatants. Cytotoxic activity in supernatants harvested after 120 hr stimulation with PHA or Con A was neutralized 70 to 100%, whereas the toxin(s) released from IL 2-stimulated lymphocytes was only neutralized 30%. These data suggest the observed heterogeneity of cytotoxic lymphokines produced by unseparated mononuclear cells depends in part on the subpopulations of effector cells responding to a given stimulus and the capacity of different subpopulations to produce distinct cytotoxins.

Cell Separation↗
PubMed1984

A rat anti-mouse kappa chain specific monoclonal antibody, 187.1.10: purification, immunochemical properties and its utility as a general second-antibody reagent.

A rat IgG1 monoclonal antibody, produced by hybridoma 187.1.10, exhibits specificity for mouse immunoglobulins containing kappa light chains (Yelton et al., 1981). The 187.1.10 hybridoma cell line secreted upwards of 200 micrograms/ml of monoclonal antibody in tissue culture and the secreted product was purified in a single step by antigen-immunoadsorbent affinity chromatography. The homogeneity of the purified 187.1.10 protein was determined by isoelectrofocusing and SDS gel electrophoresis. Equilibrium binding analyses of the radioiodinated 187.1.10 antibody indicated a strong interaction with its antigen of KA = 2 X 10(9) l/mole. The 187.1.10 antibody did not readily bind to Staph. aureus protein A unless it was complexed with antigen. The binding of immune complexes of 187.1.10 to protein A was shown to be dependent on the Fc region of the antigen. The utility of the 187.1.10 monoclonal antibody as a general second antibody reagent for studying mouse immunoglobulins was demonstrated in a rapid solid phase immunoprecipitation assay to detect and analyze radioiodinated membrane proteins of a human cytotoxic T cell line.

Animals↗
PubMed1984

Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines.

A rapid colorimetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colorimetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH -5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10-50-fold more sensitive than the ESH -5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.

Animals↗
PubMed1983

Human lymphocyte function associated antigen-1 (LFA-1): identification of multiple antigenic epitopes and their relationship to CTL-mediated cytotoxicity.

Human lymphocyte function-associated antigen (LFA)-1, a heterodimeric lymphocyte surface glycoprotein of 177,000 and 95,000 relative molecular weight has been implicated to function in the cytotoxic T lymphocyte (CTL) effector mechanism. Seven mouse hybridoma lines producing monoclonal antibodies (MAb) reactive with this structure were studied. Three unique and 3 partially over-lapping epitopes on human LFA-1 were defined by competitive cross inhibition binding assays using biosynthetically labeled anti-LFA-1 MAb. In contrast, of five rat antimouse LFA-1 MAb, all five recognized a common or shared epitope. An HLA-B7 specific human CTL line expressed 1.1 X 10(5) LFA-1 sites per cell with a direct saturation binding assay. Human CTL expressed two to four times more LFA-1 than peripheral blood lymphocytes or B and T lymphoblastoid cell lines. Titration of each of the anti-LFA-1 MAb in a 51chromium release cytolytic assay revealed quantitative differences in the ability of the different anti-LFA-1 MAb to block cytolysis indicating distinct functional and antigenic epitopes exist on the human LFA-1 molecule. Anti-LFA-1 MAb reversibly inhibited the CTL reaction by slowing the initial rate of cytolysis. These results suggest anti-LFA-1 MAb inhibit CTL function by specific blockade of a functionally relevant molecule.

Animals↗
PubMed1983

Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line.

HLA-A2 specific human cytotoxic T lymphocytes (CTL) cell lines have been developed using T cell growth factor and coculture of peripheral blood lymphocytes with selected allogeneic target cell lines. The CTL-8 line showed specificity for human leukocyte antigens (HLA)-A2 bearing target cells after 5 weeks in culture when tested against a panel of 14 lymphoblastoid cell lines in a 51Chromium (51Cr) release assay. Purified anti-human leukocyte antigens (HLA) monoclonal antibodies W6/32 and PA2.1 inhibited cytolysis by 85% and 60%, respectively. The CTL-8 line lysed non-HLA-A2 target cells in the presence of lectins concanavalin A (Con A) or phytohemagglutinin-P lectin (PHA-P) indicating the specificity of cytolysis was not due to nonspecific resistance of target cells to the CTL-lytic mechanism. The T5-1 HLA-A2 mutant cell series were tested as targets for the CTL-8 line. Cell clones 8.18.1, 8.21.1 and 8.6.1, which express altered HLA-A2 molecules as determined by their decreased reactivity with allospecific monoclonal antibodies, were lysed by the CTL-8 line as efficiently as the T5-1 wild type. These cell lines also acted as efficient cold target competitors for a normal HLA-A2 target cell. The 8.14.1 cell clone expressed a lower amount of HLA-A2 alloantigen and showed a corresponding decreased reactivity with CTL-8 in direct cytolytic and cold target competitive inhibition assays. In contrast, the M7 and DK1 HLA-A2 variant cell lines, which express normal HLA-A2 serological determinants, were inefficiently lysed by CTL-8 and did not act as competitive inhibitors of normal HLA-A2 target cells. These results support the concept that the alloantigenic determinant(s) recognized by T cells and antibodies occur at separate regions on the HLA-A2 molecule.

Animals↗