Expression of the major histocompatibility antigens HLA-A2 and HLA-B7 by DNA-mediated gene transfer

Genes coding for the heavy chain of the class I antigens HLA-A2 or HLA-B7 of the human major histocompatibility complex have been introduced into mouse LtK- cells by cotransfection with the herpes simplex virus thymidine kinase gene. HAT-resistant colonies were isolated expressing either HLA-A2 or HLA-B7 as monitored by indirect immunofluorescence. Immunoprecipitation analysis of both antigens by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing (IEF) showed that they were identical to the HLA-A2 and HLA-B7 expressed in the human lymphoblastoid cell line JY (homozygous HLA-A2, HLA-B7). However, human cytotoxic T lymphocytes (CTL) generated against JY and CTL clones specific for HLA-A2 or HLA-B7 were unable to recognize the transfectants as targets. These results indicate that the human HLA-A2 (or B7) complexed with the murine beta 2-microglobulin could be an inappropriate target structure for the CTL. However, because the transfectants are not killed by human CTL even in the presence of lectins, it is suggested that other molecules that are not able to overcome the human-mouse species barrier may be involved in the killing mechanism.     

In vitro-isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.Abbreviations used in this paper: CTL cytotoxic - T lymphocytes - BSA bovine serum albumin - PHA phytohemagglutinin - Con A concanavalin A.     

Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones

Human cytolytic T lymphocytes (CTL) clones and HLA-A2- and HLA-B7-transfected human, monkey, and mouse cell lines were used to investigate the basis for species-restricted antigen recognition. Most allospecific CTL clones obtained after stimulation with the human JY cell line (source of HLA-A2 and HLA-B7 genomic clones) recognized HLA antigens expressed in human and monkey cell lines but did not recognize HLA expressed in murine cells. By initially stimulating the responder cells with HLA-transfected mouse cells, two CTL clones were obtained that recognized HLA expressed in murine cells. Functional inhibition of these CTL clones with anti-class I monoclonal antibodies (MAb) indicated that clones reactive with HLA+ murine cells were of higher avidity than clones that did not recognize HLA+ murine target cells. MAb inhibition of accessory molecule interactions demonstrated that the LFA-1 and T8 surface molecules were involved in CTL-target cell interactions in all three species. In contrast, the LFA-2/CD2 molecule, previously shown to participate in a distinct activation pathway, was involved in the cytolysis of transfected human and monkey target cells, but not in the lysis of HLA+ murine cells. Thus transfection of HLA genes into different recipient species cell lines provides us with the ability to additionally delineate the functional requirements for allospecific CTL recognition and lysis.     

Multiple epitopes on human and murine cells expressing HLA-B7 as defined by specific murine cytotoxic T cell clones

Eleven cytotoxic T lymphocyte (CTL) clones were derived from C57BL/6 spleen cells immunized with HLA-B7 expressing human lymphoblastoid cell lines. Reactivity against HLA-B7 was initially established because the clones lysed 2 target cells that shared only HLA-B7 with the immunizing cell line and they did not lyse five other cell lines that were HLA-B7 negative but expressed other class I or class II antigens found on the immunizing cell. Six of the clones were subsequently shown to lyse all tested HLA-B7-positive B and T lymphoid cell lines, peripheral blood lymphocytes, and a murine L cell that expressed HLA-B7 as a consequence of DNA-mediated gene transfer. On the basis of the inability of the clones to lyse a panel of HLA-B7-negative cell lines, up to 18 other class I antigens could be eliminated as being cross-reactively recognized. However, two of the clones recognized a single HLA-B7-negative cell line. It is suggested that in these cases the clones were cross-reactively recognizing the HLA-B27 or HLA-B40 antigens that were present on these target cells. The remaining five CTL clones failed to lyse one out of seven tested HLA-B7-positive lymphoid lines (either RPMI-1788 or DR1B) and failed to lyse peripheral blood lymphocytes from one out of three tested HLA-B7-positive individuals. These five clones also did not recognize the HLA-B7-positive murine L cell. However, based on analysis with a large target cell panel, the reactivity pattern of these five clones could only be correlated with recognition of HLA-B7. This conclusion is further supported by antibody-blocking studies to be reported elsewhere. As before, lysis of single HLA-B7-negative target cells by two of the clones could be ascribed to recognition of HLA-B27 or HLA-B40. The results show that murine clones raised against HLA-B7 exhibit a high degree of specificity for determinants that are unique or largely confined to the HLA-B7 alloantigen. In addition, these clones define different antigenic determinants on the molecule. Thus, such clones appear to be excellent candidates for use as human tissue typing reagent. The results further show that there is a strong correlation between recognition of particular HLA-B7-positive human cell lines and recognition of the HLA-B7 expressing murine L cell. Possible reasons for such a correlation and their relationship to the general phenomenon of CTL recognition are discussed.     

Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of HLA-B7 X human beta 2m double transgenic mice

The cytolytic responses of either normal (non transgenic), HLA-B7 (single transgenic) or HLA-B7 x human beta 2 microglobulin (double transgenic) DBA/2 mice induced by transfected HLA-Cw3 P815 (H-2d) mouse mastocytoma cells were compared, to evaluate whether the expression of an HLA class I molecule in responder mice would favor the emergence of HLA-specific, H-2-unrestricted CTL. Only 8 of 300 HLA-Cw3-specific CTL clones tested could selectively lyse HLA-Cw3-transfected cells in an H-2-unrestricted manner, all having been isolated after hyperimmunization of double transgenic mice. These clones also lysed HLA-Cw3+ human cells. Unexpectedly, the lysis of the human but not that of the murine HLA-Cw3 cells was inhibited by Ly-2,3-specific mAb. Despite significant expression of HLA-B7 class I molecules on transgenic lymphoid cells, including thymic cells, limiting dilution analysis and comparative study of TCR-alpha and -beta gene rearrangements of the eight isolated clones (which suggested that they all derived from the same CTL precursor) indicated that the frequency of HLA-Cw3-specific H-2 unrestricted cytotoxic T lymphocytes remained low (even in HLA-B7 x human beta 2-microglobulin double transgenic mice). This suggests that coexpression of HLA class I H and L chain in transgenic mice is not the only requirement for significant positive selection of HLA class I-restricted cytotoxic mouse T lymphocytes.     

Study of HLA class I restriction and the directed antigens of cytotoxic T lymphocytes at the tumor sites of ovarian cancer

The molecular basis of T-cell-mediated recognition of ovarian cancer cells remains to be fully addressed. In this study we investigated HLA class I restriction and directed antigens of cytotoxic T lymphocytes (CTL) at the sites of ovarian cancer. Three HLA-class-I-restricted CTL lines were established from the tumor sites of ovarian cancer by culturing tumor-infiltrating lymphocytes or tumor-associated ascitic lymphocytes with interleukin-2: (1) HLA-A2402-restricted and ovarian-adenocarcinoma-specific CTL, (2) HLA-A2-restricted CTL recognizing histologically different cancers, and (3) HLA-B52-restricted and ovarian-cancer-specific CTL. HLA-A0201, HLA-A0206 and HLA-A0207 tumor cells were lysed by the HLA-A2-restricted CTL. HLA-B52 restriction of the third CTL line was confirmed by the transfection of HLA-B5201 cDNA into the tumor cells. The HLA-A2-restricted CTL recognized the SART-1, but not the MAGE-1 or MAGE-3 antigen. These results may facilitate a better understanding of the molecular basis of tumor-specific immunity at the tumor site of ovarian cancer. Received: 30 December 1998 / Accepted: 2 March 1999     

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.     

CTL adhesion and antigen recognition are discrete steps in the human CTL-target cell interaction

Th initial step in cytolytic T lymphocyte (CTL)-mediated cytolysis involves target cell adhesion and antigen recognition. To investigate these initial events in the CTL-target interaction, we used HLA-A2- and HLA-B7-specific human CTL clones and HLA-typed B lymphoblastoid target cells. By using two different adhesion assays, we demonstrated antigen nonspecific CTL-target cell adhesion. To more precisely define the contribution of the antigen-specific receptor to CTL-target cell adhesion, we used the HLA-A2, HLA-B7, and mock transfected RD target cells. Consistent with the results when using B lymphoblastoid target cells, the CTL clones demonstrated equivalent adhesions to the RD target cells whether or not they expressed HLA-A2 or HLA-B7. These results suggested that CTL-target cell adhesion occurred independent of the T cell receptor. By using the calcium-sensitive dye Indo-1 and flow cytometry, we assessed CTL-target cell adhesion and CTL activation. Simultaneous measurement of adhesion and intracellular free calcium demonstrated that CTL-target cell adhesion alone did not activate CTL clones. Both CTL-target cell adhesion and the presence of the appropriate HLA target molecule were necessary for the efficient activation of human CTL. MAb inhibition studies indicated that antigen nonspecific adhesion is largely regulated by the LFA-1, CD2 (LFA-2/T11), and LFA-3 cell surface molecules. These antigen nonspecific cell-cell interaction molecules appear to play an important role in facilitating antigen recognition and subsequent target cell lysis.     

The ability of cytotoxic T cells to recognize HLA-A2.1 or HLA-B7 antigens expressed on murine cells correlates with their epitope specificity

Two groups of human and murine cytotoxic T lymphocyte (CTL) clones specific for human leukocyte antigen (HLA)-A2 or -B7 can be distinguished based on their ability to kill murine transfectants expressing these molecules. The clones which do not recognize murine transfectants exhibited greatly reduced conjugate formation with these cells, indicating that the inability to lyse these cells occurs in recognition and binding. No systematic differences in inhibitory titer between the two types of CTL clones were seen with anti-CD8 (Lyt-2), anti-LFA-1, or monoclonal antibodies against HLA class I molecules. However, blocking with anti-HLA class I monoclonal antibodies suggested that different CTL clones recognized spatially separate epitopes on HLA-A2 and -B7. In addition, a correlation between the inability to recognize murine transfectants and fine specificity was seen. Eight of nine clones which did not lyse murine transfectants also failed to recognize human cells expressing HLA-A2.2 or -A2.3. In contrast only 5 of 12 clones which lysed transfectants failed to recognize the variant molecules. Analogous data were obtained with human CTL clones raised against HLA-A2.1. These findings suggest that CTL clones that do not lyse murine cells expressing appropriate antigens recognize epitopes that have been altered or lost as a consequence of expression on the murine cell surface. It is suggested that the loss of HLA-associated epitopes on the murine cell surface may be due to differences between mouse and human cells in the processing or presentation of class I-associated peptides.     

Serological expression after sequential double transfection with purified HLA-11 gene of mouse fibroblasts carrying human beta-2 microglobulin

A genomic cosmid library constructed from DNA from a genotyped individual (JF = HLA-A11, Cw–, B38/A26, Cw7, B51) was screened for clones containing class I histocompatibility genes. Among these clones, one was found to carry a 4.8 kb Hind III fragment which is highly correlated with HLA-A11. This clone was used to transfect LMTK⁺ cultured mouse fibroblast transformants expressing human beta-2 microglobulin. The human beta-2 microglobulin heavy chain-associated determinant was positively detected by the M18 monoclonal antibody. HLA-A11 expression on these doubly transformed cells was specifically demonstrated by complement-dependent cytotoxicity with HLA-A11 + A3-specific but not with HLA-A3-specific monoclonal antibodies. Absorption studies with human alloantisera confirmed the presence on these cells of HLA-A11 determinants and of cross-reacting determinants which absorbed anti-HLA-A1 and –A3 alloantisera. The JF5-J27 transfected cell expressed both heavy and light chains of human class I histocompatibility genes.Abbreviations used in this paper ₂m beta-2 microglobulin - CTL cytolytic T lymphocytes - FCS fetal calf serum - HAT hypoxanthine-azaguanine-thymidine - kb kilobase pair - MHC major histocompatibility complex - MoAb monoclonal antibodies - PBL peripheral blood lymphocytes - PEG polyethylene glycol - r correlation coefficient This study is dedicated to the memory of Jean-Jacques Metzger.     

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.     

Cytotoxic activity and lymphokine production of T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ cytotoxic T lymphocyte (CTL) clones recognizing HLA-A2 and HLA-A2 mutants. Recognition of TCR-gamma delta+ CTL clones is affected by mutations at positions 152 and 156

TCR-gamma delta+ CTL clones were generated from CD4-CD8- T cells that were stimulated twice with the cell line JY. Either IL-2 or IL-4 was used as growth factor. A number of TCR-gamma delta+ clones were found to lyse the stimulator cell line JY. Two of these clones secreted N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase activity after stimulation with JY cells. The cytotoxic activity of these two clones was blocked by a mAb specific for HLA-A2. Moreover, these two TCR-gamma delta+ clones selectively lysed human fibroblast line M1 and murine P815 cells transfected with DNA fragments encoding HLA-A2 but not those transfected with HLA-B7 encoding DNA, indicating that these clones recognize HLA-A2. Analysis of the recognition of HLA-A2 by using target cells transfected with mutated HLA-A2 encoding genes revealed that the nature of the amino acid at position 152 of the molecule is critical for recognition of the TCR-alpha beta+ as well as the TCR-gamma delta+ CTL clones since replacement of Val for Ala at that position resulted in abrogation of recognition of one TCR-gamma delta+ and one TCR-alpha beta+ clone and substitution of Val for Glu affected recognition of all clones. Substitution of Leu for Trp at position 156 abrogated recognition by one TCR-gamma delta+ and one TCR-alpha beta+ T cell clone, but recognition by the other clones was not changed. All clones were able to secrete IL-2, IFN-gamma, and GM-CSF but not IL-4 after activation.     

T3 monoclonal antibody activation of nonspecific cytolysis: a mechanism of CTL inhibition

The T3 antigen is expressed on all cytotoxic T lymphocytes (CTL). Monoclonal antibodies (MAb) to the T3 antigen previously have been shown to inhibit CTL-mediated killing of cells expressing the relevant target antigens. The mechanism of T3 MAb inhibition, however, remains undefined. In this report, we describe a novel effect of the T3 MAb: the stimulation of allospecific CTL clones to kill target cells that do not express the relevant HLA antigens. The stimulation of nonspecific killing was seen only with MAb to the T3 antigen; MAb to other function-associated antigens (e.g., LFA-1, LFA-2, LFA-3, T4, T8, HLA-A,B,C, and DR) had no effect. T3 MAb stimulated nonspecific killing by CTL clones expressing both the T4+ and T8+ phenotype and by CTL clones specific for both class I and class II HLA alloantigens. Target cell susceptibility to T3 MAb stimulated killing was variable. CTL clones lysed some target cell lines very efficiently (e.g., K562, Daudi, and M124.1) but lysed other cell lines much less efficiently (e.g., 23.1, Mann, and L cells). In CTL-mediated cytotoxicity assays with target cells expressing the relevant HLA antigens, T3 MAb demonstrated the expected inhibition of cytolysis. Thus, the ability of T3 MAb to stimulate and inhibit CTL-mediated cytolysis suggests that both effects may be the result of a common mechanism of activation.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. II. Bifunctional clones with cytotoxic and virus-induced proliferative activities exhibit herpes simplex virus type 1 and 2 specific or type common reactivities

Human cytotoxic T lymphocyte (CTL) clones directed against herpes simplex virus (HSV)-infected cells were generated after stimulation of peripheral blood lymphocytes (PBL) with HSV type 1 (HSV-1) and HSV type 2 (HSV-2). These CTL clones were studied with regard to HSV type specificity and with regard to whether they also express helper cell activity. Some clones, generated after stimulation with HSV-1, were cytotoxic for autologous cells infected with either HSV-1 or HSV-2 ("HSV type common clones"), whereas other clones lysed HSV-1-infected cells only ("type-specific clones"). Similarly, after HSV-2 stimulation, both HSV-2 specific and HSV type common clones were obtained, indicating the heterogeneity of human cytotoxic T cells to HSV. All CTL clones tested were found to be bifunctional in that they also proliferated in response to stimulation with HSV. The HSV type specificity of the proliferative response was identical to that of the cytotoxic activity of the clones. An HSV type common clone, when stimulated with either HSV-1 or HSV-2, and an HSV-1 specific clone, when stimulated with HSV-1 but not with HSV-2, produced a factor, presumably interleukin 2 (IL 2), which induced proliferation of CTLL, an IL 2-dependent T cell line, providing evidence that our HSV-directed CTL clones also express helper cell activity. CTL clones that we previously reported were restricted in cytotoxic activity by HLA class II DR-1 or MB-1 antigens were found, in this study, to be restricted in proliferative response to HSV by these same HLA antigens. These results suggest that our bifunctional T cell clones directed against HSV may recognize the same viral antigenic determinants and the same HLA antigens for both cytotoxic and virus-induced proliferative activities. This is the first demonstration of human HSV type specific and HSV type common T cell clones and HSV specific T cell clones with both cytotoxic and helper cell activities.     

Preferential HLA usage in the influenza virus-specific CTL response

To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8(+) T cell population, the proportion of IFN-gamma- and TNF-alpha-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8(+) T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied.     

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.     

Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

A proteolytically modified form of beta 2-microglobulin (beta 2-m) present in the serum of patients suffering from autoimmune, immunodeficient diseases and cancer has been reported in the literature. In the present study we show that human beta 2-m as well as the proteolytically modified human form (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native human beta 2-m; and the modifying activity of murine MLC responder cells was blocked in an intermediary step by an alloantibody, which reacts specifically with murine major histocompatibility complex, class I-associated beta 2-m. These findings suggest that the modification process is preceded by an association of human beta 2-m with the cell surface of the responder cells. Our data indicate that the modification of beta 2-m might reflect early events in allospecific responder cell activation.     

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)     

H2-restricted recognition of cloned HLA class I gene products expressed in mouse cells

Long-term syngeneic mouse cytolytic T lymphocyte (CTL) clones were obtained from DBA/2 (H2d) mice immunized with P815 (H2d) cells transfected with cloned human class I histocompatibility genes, HLA-CW3 or HLA-A24. Three distinct patterns of specificity were defined on P815 HLA transfectant target cells. One clone lysed HLA-CW3 but not -A24 transfectants, and a second lysed HLA-A24 but not -CW3 transfectant target cells. The third clone lysed P815 targets transfected with either HLA gene. None of the CTL clones lysed L cells (H2k) transfected with the same HLA genes or human targets that expressed these HLA specificities. Several lines of evidence indicated that recognition of HLA transfectants by these CTL clones was H2 restricted. First, lysis of P815 HLA transfectants could be inhibited by anti-H2Kd monoclonal antibody. In addition, the anti-P815-HLA CTL clones could lyse a (human X mouse) hybrid target that expressed both HLA class I and H2Kd antigens, but not a clonal derivative that no longer expressed H2Kd. The most direct evidence for H2-restricted recognition of P815-HLA transfectants by the syngeneic CTL clones was obtained by double transfection of mouse L cells (H2k) with both HLA and H2 class I genes. L cells transfected with HLA and H2Kd genes were susceptible to lysis by the same CTL clones that lysed the corresponding P815-HLA transfectant targets. Thus under certain conditions, CTL recognition of xenogeneic class I histocompatibility gene products can be restricted by other class I gene products.     

Simultaneous transduction of B7-1 and IL-2 genes into human melanoma cells to be used as vaccine: enhancement of stimulatory activity for autologous and allogeneic lymphocytes

 In order to construct an immunogenic cellular vaccine, we transduced three HLA-A*0201 human melanoma lines, selected for expression of classes I and II HLA, adhesion molecules and the T cell-defined melanoma antigens Melan/MART-1, gp100 and tyrosinase, with both interleukin-2 (IL-2) and B7-1 genes by the use of a polycistronic retroviral vector. The lines were selected to share only the HLA-A*0201 allele to avoid generation of strong alloreactivity in case of their multiple in vivo use in HLA-A*0201 + patients. Phenotypic and functional analysis of B7-1-IL2 transduced melanoma lines in comparison with B7-1 transduced and/or parental untransduced counterparts were then carried out. Tumor cells expressing either B7-1 or both genes did not change their original antigenic profile. From a functional point of view, expression of both genes in melanoma lines: (1) improved the response of anti-melanoma cytotoxic T lymphocytes (CTL) over singly transduced or untransduced melanoma cells when subthreshold levels of MHC-peptide complexes were expressed by melanoma cells; (2) conferred a distinct advantage in the ability to stimulate cytotoxicity and interferon-γ release by autologous and/or HLA-A*0201-compatible allogeneic lymphocytes; (3) allowed the generation of a high number of specific CTL by in vitro stimulation of lymphocytes of HLA-A*0201-melanoma patients. Thus, B7-IL2 gene-transduced melanoma lines appear to display a high immunogenicity and could be used as vaccine in melanoma patients. Received: 17 August 2000 / Accepted: 1 February 2001