JY Cells Research Articles

Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells.

The genes that code for the human major histocompatibility class I antigens, HLA-A2 and HLA-B7, were introduced into human, monkey, and mouse cell lines by cotransfection with suitable biochemical markers and the fluorescence-activated cell sorter was used to identify and/or select stable cell populations expressing high surface levels of these antigens. Levels of expression obtained were similar to those observed for endogenous HLA antigens on various human cell lines and were 25-80% of those observed on the human B-lymphoblastoid cell line JY. Serologically defined HLA-A2 and HLA-B7 polymorphic determinants remained intact on all transfected recipient cells analyzed. Cloned human allospecific cytotoxic T lymphocytes (CTL) specific for HLA-A2 or HLA-B7 were capable of lysing appropriate HLA-transfected human cells with comparable efficiency to JY cell lysis. Two of 10 CTL clones lysed appropriate monkey cell transfectants with approximately equal to 20% the efficiency of human cell transfectants. No specific lysis of any HLA-transfected mouse cell lines, including a B cell lymphoma, was observed despite comparable levels of surface antigen expression or after induction of higher levels by mouse gamma-interferon. Furthermore, L cells expressing human beta 2-microglobulin in addition to HLA-A2 or -B7 were not lysed by these CTL. Thus, an additional species-specific component may be involved in lysis by allogeneic CTL--possibly related to the function(s) of other surface proteins on target cells.

High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

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)

Recognition by xenogeneic cytotoxic T lymphocytes of cells expressing HLA-A2 or HLA-B7 after DNA-mediated gene transfer.

Murine L cells expressing HLA-A2 or -B7 antigens were isolated after cotransformation of thymidine kinase-negative cells with the herpes simplex virus thymidine kinase gene and the genomic clones containing either the HLA-A2 or -B7 genes. Monoclonal antibody binding analyses demonstrated the stable cell surface expression of HLA antigens by these cells at levels of up to 40% of the amount expressed by the human B lymphoblastoid cell line, JY. The HLA-A2 and -B7 antigens expressed by the L cells retained all of the antibody-defined, heavy-chain-associated antigenic determinants but lacked those determinants associated with human beta 2-microglobulin. These HLA transformants were capable of functioning as targets for monoclonal cytotoxic T lymphocytes (CTL) that specifically recognize the HLA-B7 or -A2 antigens expressed by JY cells. However, the efficiency of lysis, relative to the JY cell line, was 50-99% for individual CTL. In addition, not all of these CTL were capable of lysing the appropriate transformants. Because the antigens appear by serological criteria to be structurally intact and expressed at high levels, these results suggest that the complementation of the HLA heavy chains with mouse, rather than human, beta 2-microglobulin may alter the antigenic determinants that are important for CTL recognition.

NH2-terminal sequence of the ? and ? chains of human DC-1 antigen isolated from the JY cell line. Homology with murine I-A molecules

The DC-1 antigen has been isolated from the JY cell line (DR4, w6). The amino terminal sequences of its alpha and beta chains are both reported and are homologous to the murine I-A antigens. The JY and previously reported LB DC-1 alpha chain sequences appear to be variants of the DC alpha chains reported by other authors. The JY DC-1 beta chain sequence appears to be identical with that deduced from a beta chain cDNA clone and thus identifies this clone. The JY and LB DC-1 beta-chains are clearly different since the latter has a blocked amino terminus.

A permanent human cytotoxic T-cell line with high killing capacity against a lymphoblastoid B-cell line shows preference for HLA A, B target antigens and lacks spontaneous cytotoxic activity

The optimal conditions for the generation of highly cytotoxic human T lymphocytes (CTL) in vitro against a lymphoblastoid B-cell line (JY) in primary and secondary mixed lymphocyte cultures (MLC) were investigated. Variation of the stimulator:responder (S:R) cell ratio influenced both the specific cytotoxicity and the spontaneous cytotoxicity as well as the recovery of the responder cells. T lymphocytes of donor JR (HLA A23,29; B7,7; DRw5) were stimulated with JY (HLA A2,2; B7,7; DRw4,6) in primary and secondary MLC at S:R ratios of 1:50 and 1:10, respectively, since stimulations at these S:R ratios resulted in the highest specific cytotoxicity against JY, the lowest spontaneous cytotoxicity against K562 and Daudi, and in a good recovery of the responder cells. From these T-lymphocyte cultures an exponentially growing CTL line (JR-2) was obtained by weekly stimulations with irradiated JY cells at a S:R ratio of 1:1. After 2 months of culturing the growth rate of the JR-2 cells declined, but could be restored by the addition of conditioned medium, containing T-cell growth factor (TCGF). Irradiated JY cells or TCGF alone were insufficient to maintain proliferation. JR-2 cells were strongly cytotoxic for JY (50% lysis was obtained at an effector:target ratio of 1:2) but the cytotoxic activity against a classical target cell for spontaneous cytotoxicity (K562) was negligible. The cytotoxic activity of JR-2 cells against JY could be inhibited by a monoclonal antiserum W6/32, which recognizes all HLA A, B, and C specificities, and by a monoclonal antiserum directed against β2 microglobulin, whereas monoclonal anti-Ia antisera showed no inhibition. JR-2 cells lysed fresh HLA A2-homozygous lymphocytes more efficiently than HLA A2-heterozygous lymphocytes, whereas the latter were better killed than HLA A2-negative lymphocytes.

Development and characterization of allospecific long-term human cytolytic T-cell lines.

Two long-term human cytolytic T lymphocyte (CTL) lines (VE and JR), whose cytolytic activity was dependent upon both irradiated JY cells (the stimulating alloantigen) and T-cell growth factor, were established. These lines were monitored in culture for 6-8 months. Both lines were specific for HLA-A or B antigens or both and the JR line was allospecific for HLA-B7. These CTL lines killed specific target cells at an effector-to-target ratio of 0.4 (VE) or 0.08 (JR). All of the cells, which grow in suspension, rosetted with sheep erythrocytes and reacted with an antiserum specific for human T cells. The CTL line VE was used to raise rabbit antisera that immunoprecipitated two specific polypeptides (78,000 and 33,000 daltons) from labeled membranes of these CTL lines.

Monoclonal antibodies to HLA--DRw determinants.

Two monoclonal antibodies recognizing HLA-DRw (human la) antigens were produced. The DA2 antibody binds a monomorphic determinant, common to all specificities and Genox3.53 antibody binds to a cross-reacting site on the HLA-DRw1,2 and 6 specificities. Both antibodies are IgG1 and show complement dependent cytotoxicity only in the presence of rabbit anti-mouse IgG serum. Specificity of both antibodies for the HLA-DRw molecule was shown by inhibition of antibody binding by preincubation of antibody with detergent solubilized Ia from JY (HLA-DRw4,6) cells and by preincubation of target cells with F(ab')2 fragments of a rabbit anti-la serum. DA2 antibody reacted with all cells of human B cell origin tested and with peripheral blood lymphocytes of several primate species tested. Genox3.53 antibody bound only to human cells expressing HLA-DRw1,2 or 6 antigens, giving a negative reaction with all primates tested. Genox3.53 antibody detected a split in the HLA-DRw6 specificity, showing reduced binding to the Daudi cell (HLA-DRw6) in comparison with binding to several other cell lines typed as HLA-DRw6, under saturating conditions. This low reactivity with Daudi was confirmed by absorption experiments. The ratio of DA2 binding to Genox3.53 binding to homozygous and heterozygous cell lines under saturation conditions was compared. Results suggested that, on some cell lines, DA2 might be reacting with a second population of human Ia antigens in addition to the HLA-DRw antigens. When a mixture of saturating concentrations of DA2 and Genox3.53 antibodies was tested for binding to cells under saturating conditions, the number of counts bound suggested the antibodies could bind simultaneously. Direct binding experiments showed that when each antibody was iodinated, its binding was not inhibited by preincubation with the other antibody, confirming that the DA2 and Genox3.53 determinants are distinct on the Ia molecule.

Induction of secondary cytotoxic T lymphocytes by liposomes containing HLA-DR antigens.

It is evident that the gene products of the major histocompatibility complex (MHC) have a crucial role in the induction of cytotoxic T lymphocytes (CTLs) and as target antigens for CTLs1–6. Assessment of the precise molecular requirements for CTL induction and recognition, however, is difficult when intact cells are used as targets. Recently, in several model systems, it has been demonstrated that, after detergent solubilisation, cell-surface antigens can be isolated that retain the ability to stimulate a secondary CTL response. CTL stimulating activity has been isolated by detergent solubilisation from murine tumour cell plasma membranes7,8 which co-purified with H–2 antigens through lentil lectin affinity chromatography and gel filtration9,10. Highly purified HLA-A and HLA-B antigens, from the human lymphoblastoid cell line JY, have been isolated and incorporated into well characterised liposomes11. Spleen cells from mice primed to JY cells developed cytolytic activity, specific for JY targets, after co-culture with these HLA-liposomes12. In these experiments it was observed that membranes which had been solubilised with deoxycholate and then dialysed to remove the detergent (reconstituted membranes) always stimulated greater cytolytic activity than did HLA-liposomes. At the time it was suggested that this disparity might reflect the loss of other antigens involved in CTL stimulation as a consequence of HLA-A and HLA-B purification. Data are presented here which demonstrate that other antigens are indeed able to induce secondary CTLs, independently of the HLA-A and HLA-B antigens. The results suggest that this activity is present on HLA-DR-associated molecules.

Induction of secondary cytotoxic T lymphocytes by purified HLA-A and HLA-B antigens reconstituted into phospholipid vesicles.

Mouse cytotoxic T lymphocytes were induced by culturing primed spleen cells with cells, membranes, or detergent-solubilized and dialyzed membranes from the human lymphoblastoid cell line JY. Cytotoxic T cells could also be induced by coculturing primed spleen cells with phospholipid vesicles containing purified HLA-A and -B antigens derived from JY cells. The induction of killer cells by all subcellular fractions demonstrated an optimal response as a function of the amount of material added and, in the case of HLA-containing liposomes, this optimum was dependent upon the density of the HLA molecules in the liposomes. In addition, the maximal level of cytotoxicity elicited was also dependent upon this density. Finally, the cytotoxic cells demonstrated specificity as judged by a lower level of lysis on an HLA-unrelated lymphoblastoid cell line than on the appropriate target, JY. These results suggest that major histocompatibility antigens alone are sufficient for the induction of a secondary cytotoxic T lymphocyte response. It is proposed that this system may be useful in defining possible sites on the HLA molecule recognized by cytolytic T lymphocytes.