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H Spits

Publications and source records attributed to H Spits.

At least 163 records · Page 9Linked to original sources
PubMed1985

The role of T8 in the cytotoxic activity of cloned cytotoxic T lymphocyte lines specific for class II and class I major histocompatibility complex antigens.

It is reported here that most cytotoxic T lymphocytes (CTL), which recognize class I major histocompatibility complex (MHC) loci, express the T cell differentiation antigen T8. However, a minority of T8+ CTL clones was found to recognize class II MHC antigens. To test the hypothesis that T8 is involved only in T cell recognition of class I MHC antigens, we studied the role of T8 in the cytotoxic activity of class II MHC-specific CTL. Monoclonal antibodies specific for T8 blocked the activity of most class I MHC-specific CTL clones but did not affect the activity of class II MHC-specific CTL clones. Moreover, a mild trypsin treatment of the clones, which removed and T8 determinant, affected the activity of class I MHC but not that of class II MHC-specific CTL clones. These findings indicate that the class II-specific MHC CTL clones described here did not require T8 for their cytolytic activity. The activity of one T8+ class I MHC-specific (HLA-B27) CTL clone (HG-61) against the B cell line JY, which was used to raise this CTL clone, was not blocked by trypsin treatment of this clone. However, the activity of CTL clone HG-61 against target cells different from JY but carrying the appropriate HLA specificity was blocked by anti-T8 antibodies and trypsin treatment. The implications of these findings for the hypothesis that T8 is involved only in the activity of CTL with a relatively low avidity for class I MHC antigens are discussed.

Antibodies, Monoclonal↗
PubMed1985

Induction of nonspecific cytotoxicity by monoclonal anti-T3 antibodies.

The effects of monoclonal anti-T3 antibodies on the effector phase of cytotoxic T lymphocytes (CTL) were studied with respect to antigen-specific and antigen-nonspecific lysis of different target cells. Anti-T3 antibodies inhibited the antigen-specific lysis by CTL generated in mixed lymphocyte cultures (MLC), but they concomitantly augmented the nonspecific killing of third-party cells such as the cell lines Daudi, Raji, and K562. This nonspecific cytotoxicity was induced by various anti-T3 antibodies, whereas antibodies reactive with other antigens expressed on the cytotoxic effector cells lacked any such activity. Anti-T3 antibodies induced nonspecific cytotoxicity only when activated T cells, obtained by primary MLC, by repeated restimulation, or after cloning, were used. The antibodies had no effect on unstimulated peripheral T lymphocytes or thymocytes. The inhibition of the antigen-specific lysis and the induction of nonspecific lysis by anti-T3 was dose dependent, and both effects occurred at the same concentration range of anti-T3. F(ab')2 fragments of anti-T3 inhibited the specific lysis but were not able to induce cytotoxic activity, indicating that this induction is an Fc-dependent process. When different target cells were tested, only Fc receptor-positive cells were susceptible for this nonspecific cytotoxicity. Thus, anti-T3 antibodies have a dual effect on effector CTL: they inhibit antigen-specific lysis and concomitantly induce nonspecific lysis in an Fc-dependent way.

Antibodies, Monoclonal↗
PubMed1985

Polymorphisms within the HLA-DRw6 haplotype. I. Restriction fragment length variation and its correlation with serology.

The Dw6/DRw6 complex, one of the MHC class II specificities that can be defined by cellular techniques and by serology, probably has one or more immunoregulatory functions. To obtain information on the molecular structure of the DRw6 region, we studied several DRw6 homozygous cell lines, of which three were of consanguineous origin. DNA-DNA hybridization comprised the use of seven restriction enzymes in combination with three DR beta cDNA probes. The obtained results were compared with similar analyses of an HLA homozygous cell panel, expressing DR1-w8 specificities. This comparison indicated that in DRw6 homozygous individuals the coding potential for DR beta chains resembles closely that of all other DR specificities, thus identifying DRw6 as a regular DR region. In addition, we found a restriction fragment length pattern unique for DRw6, indicating the possibility to type for DRw6 by DNA-DNA hybridization. Comparisons within the DRw6 cell panel revealed the occurrence of several HLA class II DNA subtypes. These subdivisions partly correlated with serologically obtained reaction patterns. No correlation, however, could be observed between the different DNA subtypes and cellular reaction patterns as obtained by MLC and T cell cytotoxicity.

Antigen-Antibody Reactions↗
PubMed1984

Recognition of HLA-A2 by cytotoxic T lymphocytes after DNA transfer into human and murine cells.

A gene coding for the major histocompatibility antigen HLA-A2 was transferred into human HLA-A2 negative M1 cells and murine L cells. Following transfection, these cells expressed molecules at the cell surface that are biochemically indistinguishable from HLA-A2 antigens on the human cell line JY from which the HLA-A2 gene was isolated. The M1A2 cells were recognized and lysed by a cytolytic T-cell clone specific for HLA-A2. The transfected L cells which express HLA-A2 in association with human beta 2-microglobulin were not lysed by this T-cell clone. The specific cytolysis of M1A2 cells could be inhibited by monoclonal antibodies to HLA-A2, and monoclonal antibodies to T3, T8, and LFA-1 on cytotoxic T lymphocytes. These results suggest that killing by allospecific T cells requires HLA-A2 antigens as well as other species-specific structures on the target cell surface.

Animals↗
PubMed1984

Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones.

A serum-free lymphocyte culture medium is described in which serum is replaced by bovine serum albumin, transferrin, insulin, ethanolamine and a mixture of saturated and unsaturated fatty acids (linoleic acid, oleic acid and palmitic acid). In this serum-free medium proliferative and cytotoxic responses induced in mixed lymphocyte culture were comparable with those obtained in medium containing serum. Antigen-specific cytotoxic and helper T cells were isolated and could be propagated in serum-free medium without loss of function.

Blood Physiological Phenomena↗
PubMed1984

A cloned human T cell line cytotoxic for autologous and allogeneic B lymphoma cells.

A human cytotoxic T cell clone (MWS-14) with auto-tumor reactivity was established in serum-free medium in a mixed tumor cell culture by repetitive stimulation with fresh autologous lymphoma cells. This clone and its subclones are of the T3+ T4+ T8- phenotype. They were strongly cytotoxic for the autologous lymphoma cells, whereas autologous PHA blasts were not killed. Analysis of the specificity of MWS-14, MWS-14-30, and MWS-14-34 indicated that these CTL clones were cytotoxic for 7/7 allogeneic lymphoma cells, whereas only 3/23 of normal and non-lymphoma cells were lysed. Blocking studies with monoclonal antibodies directed at MHC class I and class II antigens showed that this preferential, anti-lymphoma reactivity was not directed at HLA determinants. The anti-lymphoma activity is not due to an aspecific susceptibility of the lymphoma cells to lysis. In contrast to CTL clones specific for HLA antigens present on the lymphoma cells, T3 and T4 were not involved in the cytotoxic reaction of MWS-14 against the autologous lymphoma cells. The reactivity of this clone could be blocked by a monoclonal antibody directed at leukocyte function-associated antigen. It can be concluded from these results that these T4+ CTL clones recognize a determinant, which is preferentially expressed on autologous and allogeneic lymphoma cells.

Aged↗
PubMed1984

HLA-DC antigens can serve as recognition elements for human cytotoxic T lymphocytes.

The specificity of four cytotoxic T lymphocyte (CTL) clones which recognize class II major histocompatibility complex (MHC) antigens was analyzed. All clones recognized antigens associated with the serologically defined HLA-DRw6 specificity. The activity of two of these clones, JR-2-2 and JR-2-10, could be inhibited by a monoclonal antibody Q 5/13 specific for a monomorphic determinant present on HLA-DR. In contrast, the activity of the two other CTL clones, JR-2-19 and JR-2-26, was not blocked by Q 5/13, but by a new monoclonal reagent, SPV-L3. This latter monoclonal antibody precipitated a two-chain structure of 28 kDa and 33 kDa and reacts with a monomorphic determinant. The molecular weight of the polypeptides precipitated with SPV-L3 was slightly less than those precipitated with a HLA-DR-specific monoclonal reagent. In addition two-dimensional gel electrophoresis showed that the antigen precipitated by SPV-L3 differed in charge from those precipitated with the anti-HLA-DR antibody. These results indicate that SPV-L3 recognizes a class II MHC product different from HLA-DR. This observation was confirmed by partial amino acid sequence analysis of the two chains which revealed that the molecule precipitated by SPV-L3 is homologous to HLA-DC/DS molecules. Therefore this report provides the first evidence that human cytotoxic T cells can recognize HLA-DC/DS antigens.

Amino Acid Sequence↗
PubMed1984

Both Fc receptors and lymphocyte-function-associated antigen 1 on human T gamma lymphocytes are required for antibody-dependent cellular cytotoxicity (killer cell activity).

A monoclonal antibody, designated CLB-LFA-1/1, directed to the human lymphocyte-function-associated antigen 1 (LFA-1) was raised by immunization of mice with the peripheral blood lymphocytes of a T gamma lymphocytosis patient. The monoclonal antibody was selected by inhibition of the natural killer cell and the antibody-dependent killer cell activity of the patient's T gamma lymphocytes. In addition, the monoclonal antibody was shown to inhibit the cytotoxic activity of T cell clones specific for either class I or class II HLA molecules. The antigen recognized by CLB-LFA-1/1 consisted of three polypeptide chains with molecular weights of 180 000 (alpha), 155 000 and 94 000 (beta). The antibody reacted with T cells, B cells, monocytes and granulocytes, and stained normal T gamma cells and T gamma cells of patients with T gamma lymphocytosis two- to threefold stronger than normal T cells. It was shown that LFA-1 and the Fc receptor on T gamma cells did not comodulate and it is therefore concluded that Fc receptors and LFA-1 are independent membrane structures, both required for the killer cell activity of T gamma cells.

Antibodies, Monoclonal↗
PubMed1984

A cloned cytotoxic T-lymphocyte (CTL) line recognizing a subtype of HLA B27.

The lymphoblastoid cell-line JY (HLA-A2,2;B7,7;C-; DR4,w6) was used to stimulate T cells from donor HG (HLA-A2,w23; B40,w44; Cw4; DRw6,7). Cloned CTL line were obtained by limiting dilution after tertiary stimulation. Strong cytotoxic activity on stimulator cells was found with all CTL clones obtained. One of the clones (HG-31 recognized a subtype of the HLA-B7 antigen. In this paper, we describe another long-term cloned CTL line (HG-61). This line, when tested on a panel of 107 target cells from unrelated individuals, recognized a subtype of HLA-B27 (B27 "K"). There was no significant association with any other HLA antigen. The cloned CTLs were T8+ and their cytotoxic activity could be blocked by the monoclonal antibody W6/32 which recognizes a framework determinant on HLA-A, -B, and -C molecules. In families, reactivity with cells of the CTL line (HG-61) segregated with HLA. It is concluded that the CTL line interacts with an antigenic determinant shared between the HLA-B7 antigen and JY and the subtype of HLA-B27 (B27 "K"), or detects products of a gene closely linked to HLA-B, not revealed by present-day serology.

Antibodies, Monoclonal↗
PubMed1984

Deficiency of a leukocyte surface glycoprotein (LFA-1) in two patients with Mo1 deficiency. Effects of cell activation on Mo1/LFA-1 surface expression in normal and deficient leukocytes.

Mo1, a phagocyte surface glycoprotein heterodimer, is involved in a number of phagocyte adhesion functions such as binding and ingestion of serum-opsonized particles, zymosan-induced degranulation, and superoxide generation. Deficiency of this antigen in humans has been associated with increased susceptibility to recurrent bacterial infections. The beta subunit of Mo1 is shared by another surface glycoprotein named LFA-1, which is involved in lymphocyte proliferation, cytolytic T cell, and natural killing activities. Two unrelated patients with Mo1 deficiency were found to be deficient in LFA-1 as well as in the common beta subunit. Investigation of lymphocyte functions in these two patients revealed normal mixed leukocyte culture-generated cytolytic T cell and natural killing activities and significantly reduced proliferative response to phytohemagglutinin. LFA-1-deficient cells also proliferated in response to soluble antigen and different alloantigens. These responses were partially blocked by anti-LFA-1 antibody. Whereas LFA-1 was undetectable by immunofluorescence and immunoprecipitation on the patients' resting T cells, significantly reduced (approximately 5% of normal) but detectable amounts of the heterodimeric LFA-1 antigen were found on mitogen and alloantigen-activated T cells. On granulocytes, Mo1 surface expression was also dependent on the state of cellular activation. The amount of surface Mo1 present on resting normal granulocytes increased by 3-10-fold following exposure to stimuli that induced degranulation, suggesting the presence of a major intracellular pool for this antigen. Analysis of subcellular fractions from granulocytes showed that intracellular Mo1 is located primarily in the specific granule fraction. Activated granulocytes had little or no increase in their surface expression of LFA-1 antigen. Deficient granulocytes had significantly increased numbers of Mo1 antigen expressed on the surface following stimulation with calcium ionophore (1 microM). However, the amount expressed continued to be significantly reduced compared with normal cells. Quantitation of surface Mo1 on granulocytes exposed to calcium ionophore (1 microM) showed that both parents in one family but only the mother in the other family had significantly reduced levels of Mo1, suggesting heterogeneity in the inheritance of this disorder. Whereas LFA-1 deficiency on lymphocytes was associated with normal alloantigen-induced cytolytic T cell and natural killing activities in these two patients, functions which were in part dependent on small amounts of detectable LFA-1 antigen, the Mo1 deficiency state led to significant defects in phagocyte adhesion functions. Hence, the clinical symptoms associated with this combined deficiency state reflect a more profound phagocyte than lymphocyte disorder.

Animals↗
PubMed1984

Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity.

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated. The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones. In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined. The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes.

It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.

Amino Acid Sequence↗
PubMed1984

Coexpression of the human HLA-A2 or HLA-B7 heavy chain gene and human beta 2-microglobulin gene in L cells.

L cells expressing human HLA-A2 or HLA-B7 class I antigen heavy chains are not recognized by human cytotoxic T lymphocytes directed at HLA-A2 or HLA-B7 antigens. To test whether the absence of human beta 2-m was the cause of the lack of recognition by the human cytotoxic T lymphocytes, coexpression of the human beta 2-m gene and the HLA-A2 or HLA-B7 heavy chain in L cells ("double transfectants") was obtained. In addition, L cells expressing HLA-A2 or HLA-B7 antigens in association with human beta 2-m were obtained by an exchange reaction, in which human beta 2-m from serum replaced the endogenous murine beta 2-m. Both types of transfectant cells were used in 51Cr-release assays and cold target inhibition assays for human cytotoxic T cell clones which were directed at HLA-A2 or HLA-B7. Neither human CTL clones nor a mixture of CTL specific for HLA-A2 and HLA-B7 were able to recognize these cells. Several alternative explanations for these observations are discussed.

Animals↗
PubMed1983

Comparison of target antigens of monoclonal reagents OKT5, OKT8, and Leu2A, which inhibit effector function of human cytotoxic T lymphocytes.

The OKT8 antigen, which is present on a subset of thymocytes and peripheral blood T lymphocytes, was examined utilizing a variety of biochemical techniques. It is shown to be a glycoprotein of 34 kD molecular weight which contains a hydrophobic region. Further, under nonreducing conditions, dimers of 76 kD and 67 kD are found. These species are most likely generated by differences in internal disulfide bonding. When two previously described antigens, OKT5 and Leu2A, were compared with OKT8, no structural differences could be found among the three antigens. Evidence is presented, however, which indicates that the three monoclonals recognize different epitopes on the molecule. Functional analysis on cytotoxic T lymphocyte (CTL) clones confirmed the biochemical findings concerning the identity of the three proteins.

Antibodies, Monoclonal↗
PubMed1983

Target antigen of monoclonal reagent S5.7: comparison with T3 antigen.

S5.7 recognizes a 20 kD cell surface protein which is present on T lymphocytes. S5.7 binds to a nonglycosylated protein, which can be labeled by cell-surface radioiodination and by a hydrophobic reagent [125I]-iodo-5-naphthyl-1-azide (INA). As the T-lymphocyte-specific T3 complex was found to contain a nonglycosylated 20 kD species, and since this 20 kD T3 form can be labeled preferentially by INA, a comparison between T3 and S5.7 was made. Isoelectric focusing experiments showed, however, that the two proteins are different. Moreover, the S5.7 monoclonal antibody does not block CML, is not mitogenic, reacts with immature cells of several hemopoietic lineages, and differs in that respect from anti-T3 monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1983

Characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned CTL lines.

Supernatants of hybridomas from fused spleen cells of mice immunized with either a T4+ or a T8+ CTL clone, were screened for their ability to inhibit the cytotoxic activity of the T4+ and the T8+ CTL clone. Eight monoclonal antibodies (MAbs) with blocking activity were obtained and a preliminary characterization of these antibodies was carried out. The MAbs SPV-L1 and SPV-L5 were found to react with thymocytes and all peripheral blood leukocytes. SPV-L1 and SPV-L5 precipitated a molecular complex consisting of two noncovalently bound chains of molecular weights of 95 and 160 kD indicating that they recognize the human equivalents of the recently described leukocyte function associated (LFA-1) antigens. The SPV-L1 and SPV-L5 antibodies inhibited the reactivity of six CTL clones tested in this study. In addition, SPV-L1 and L5 blocked the lectin dependent cellular cytotoxicity mediated by these CTL clones. Natural killer cell activity mediated by fresh peripheral blood lymphocytes and activated killer cell activity generated in MLC both measured against K562 was also inhibited by SPV-L1 and SPV-L5. Two other antibodies, SPV-L3 and SPV-L4 blocked strongly the cytotoxic activity of two T4+ CTL clones, whereas the reactivity of the three T8+ CTL clones and one T4+ CTL clone was not or only moderately inhibited. These antibodies recognized an Ia antigen as judged from tissue distribution and immunoprecipitation studies. However, these latter studies also suggest that SPV-L3 and SPV-L4 recognize HLA-DC rather than HLA-DR antigens. Finally, three antibodies (SPV-T3a, SPV-T3b, SPV-T3c) were obtained directed against the T3 molecular complex and one MAb (SPV-T8) was found to react with T8. The anti-T3 reagents were shown to be mitogenic for peripheral blood lymphocytes, with the exception of SPV-T3a. The results presented here indicate that the antigens recognized by SPV-L1 and SPV-L5 are involved in various cytotoxic reactions. In contrast, SPV-L3, SPV-T3 and SPV-T8 only seem to play a role in antigen specific cytotoxic reactions.

Animals↗