Heterogeneity of allogeneic restriction of human cytotoxic T cell clones specific for Epstein Barr virus

Clones of human cytotoxic T cells (Tc) specific for Epstein Barr virus (EBV) were isolated from peripheral blood lymphocyte (PBL) cultures stimulated repeatedly with autologous EBV-transformed lymphoblastoid cell line (LCL) cells in vitro. The method employed to clone EBV-specific Tc was a limiting dilution technique utilizing T cell growth factor (TCGF). The EBV specificity of Tc clones was determined by showing that they were significantly cytotoxic for autologous LCL cells but not for either autologous PBL or (natural killer-sensitive) K-562 cells. Eight EBV-specific Tc clones derived from a single donor exhibited distinct cytotoxic patterns against allogeneic LCL targets. Two clones were cytotoxic to LCL targets sharing both HLA-A26 and B15 antigens with effectors, and killing by two other clones was strongly restricted to autologous LCL cells. The four remaining clones showed cytotoxicities against various allogeneic LCL targets irrespective of HLA antigen expression. Eight EBV-specific Tc clones derived from a second donor also exhibited a wide spectrum of cytotoxicity to allogeneic LcL targets. We conclude that EBV-specific Tc, induced in vitro, consist of a number of clones with respect to restrictions imposed by the major histocompatibility complex. The determinants regulating these restrictions may include not only private HLA antigenic determinants that are defined by the HLA serotyping, but also undefined HLA antigenic determinants.     

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.     

Cloned lines of human cytotoxic T lymphocytes with specificity for influenza virus

The generation of human cytotoxic T cell clones with specificity for influenza virus and some of their characteristics are described. The clones were generated by limiting dilution of peripheral blood lymphocytes after two in vitro stimulations with autologous influenza A/USSR virus-infected cells and were grown in T cell growth factor. The majority of the virus-specific clones showed cross-reactivity for different influenza A virus subtypes but did not recognize influenza B virus-infected cells. The HLA specificity of two clones was further analyzed. One clone, LL33, was specific for HLA-Bw60, the other, clone WH5, for HLA-A1. Clone WH5 also seemed to recognize the serologically related HLA-A26 as restriction element for the recognition of the viral antigen. Whereas the virus-specific CTL clones had the OKT3+,4-,8+ phenotype, another clone, WH 49, exhibiting natural killer-like activity, was found to have the OKT3+,4+,8- phenotype.     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Cellular immune response to human sarcomas: cytotoxic T cell clones reactive with autologous sarcomas. I. Development, phenotype, and specificity

Human T cell clones cytotoxic for autologous sarcoma cell lines have been developed from patient JM with an osteogenic sarcoma, and from patients EG and RM with malignant fibrohistiocytoma. These clones were derived from the cocultivation of peripheral blood lymphocytes (PBL) with the respective patient's autologous irradiated established tumor cell lines (AIT). After two cycles of stimulation for 5 days in bulk culture, these "educated" lymphocytes were seeded at a density of 1 X 10(6) cells/well in 24-well plates and were cultured in the presence of highly purified natural IL 2 and AIT, the latter serving as a feeder layer. Cell numbers were reduced from the initial seeding density by one log each week until reaching a density of 10(2) cells. These cells were found to be stable in viability and cytotoxic activity, after which limiting dilution was then performed. Within 4 to 6 wk, clones were isolated with unique specificities. These clones were capable of proliferating to a total density of 10(9) cells/ml and maintained their specific cytotoxicity for more than 6 mo. Testing with a panel of target cells of various histotypes, cold-target inhibition assays, and blocking of cytotoxicity with anti-HLA monoclonal antibodies showed that the T cell clones recognize a common sarcoma-associated antigen and that the lysis is HLA restricted. Phenotypically, cytotoxic clones derived from JM were Leu-1+, Leu-2+, and Leu-3-, whereas those derived from EG exhibited either Leu-24 or Leu-3+ markers, the latter phenotype lacking cytotoxicity. RM exhibited mainly Leu-3+ clones with strong cytotoxicity. All were HNK-1- and HLA class II+, with less than 1% of cells of each clone stained by anti-TAC monoclonal antibody. The clones from each patient did not lyse autologous or allogeneic PBL, mitogen-induced T lymphoblasts, normal fibroblasts, cells isolated from benign neoplasms, carcinoma cells, Daudi B lymphoid cells, or K562 cells. With the exception of EG, all clones produced immune interferon in a range from 12 to 50 U/ml. The generation of long-term specific T cell clones can be used to further dissect the cellular immune response to sarcomas. Cytotoxic T cell clones have potential application for tumor immunotherapy.     

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.     

Differential recognition of tumor-derived and in vitro Epstein-Barr virus-transformed B-cell lines by fetal calf serum-specific T4-positive cytotoxic T-lymphocyte clones

Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3+, T4+, Dr+ phenotype and lysed autologous B blasts, the autologous LCL, and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 degrees C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens

In contrast to general findings that mouse and human cytotoxic T lymphocytes (CTL) are restricted in cytotoxic activity by major histocompatibility complex (MHC) class I antigens, we previously found that some herpes simplex virus (HSV) type I-infected cells that shared no HLA class I antigens with the HSV-1-stimulated lymphocytes were lysed. In this study, we addressed the question of the role of HLA antigens in human T cell-mediated lysis of HSV-1-infected cells by generating clones of HSV-1-directed CTL from two HSV-1-seropositive individuals. CTL clones that lysed autologous HSV-1-infected lymphoblastoid cell lines (LCL), but not natural killer-sensitive K562 cells or uninfected or influenza virus-infected LCL, were tested for cytotoxicity against a panel of allogeneic HSV-1-infected LCL. Clone KL-35 from individual KL lysed only HSV-1-infected LCL sharing the HLA class II MB1 antigen with KL. With all four CTL clones isolated from individual PM, only HSV-1-infected LCL sharing DR1 with PM were lysed. Monoclonal antibody s3/4 (directed against MB1 ), but not TS1/16 or B33 .1 (directed against a DR framework determinant), blocked lysis of autologous HSV-1-infected cells by KL-35. In contrast, B33 .1, but not s3/4, blocked lysis of autologous HSV-1-infected cells by the PM CTL clones but not by KL-35. Together, these results indicate that our five human CTL clones which are directed against HSV-1-infected cells, and which are all OKT3+, OKT4+, OKT8-, are restricted in lytic activity by HLA class II MB and DR antigens. These results suggest that the HLA D region-encoded class II antigens may be important in the recognition and destruction of virus-infected cells by human CTL.     

Differences in HLA antigen recognition by human influenza virus-immune cytotoxic T cells.

The specificity of in vitro induced human influenza-immune cytotoxic effector cells was analyzed with respect to recognition of HLA-A and -B-linked gene products. The influenza-immune cytotoxic activity observed on panels of virus-infected targets demonstrated that virus-immune effectors preferentially lyse targets with which they share HLA-A or -B specificities. Virus-immune effectors from certain donors recognized virus in conjunction with some, but not all, of their self HLA-A and -B antigens. Among donors who share a given HLA antigen (such as A2 or B7), there are differences in the ability of their virus-immune T cells to recognize the shared antigen. Virus-infected target cells from HLA-A2 or -B7 "nonresponder" donors could be lysed by virus-immune T cells obtained from other donors who shared only the HLA-A2 or -B7 antigen with these target cells. These observations suggest that the absence of cytotoxic T cell responses by some donors to influenza virus in conjunction with HLA-A2 or -B7 is not due to control by the structural genes that code for these HLA antigens, but rather may result from control by regulatory genes that act at the level of the responder and/or stimulator cell. The results are discussed in the context of Ir gene regulation of human T cell responses.     

Human myelin basic protein-specific cytolytic T lymphocyte clones are functionally restricted by HLA class II gene products

Cellular immune reactions against the autoantigen myelin basic protein (MBP) are strongly implicated in the occurrence of postinfectious and postvaccination encephalomyelitis. Clinical autoimmune encephalomyelitis in experimental animals can be transferred with cloned MBP-specific cytolytic major histocompatibility complex Class II-restricted T lymphocytes. The HLA restriction pattern of specific proliferative and cytolytic functions of two human MBP-specific cytotoxic T lymphocyte clones, derived from two different multiple sclerosis patients, was analyzed in detail. Using monoclonal antibodies against various HLA gene products and allogeneic Epstein-Barr virus-transformed B cells as antigen-presenting cells and as targets for cytolysis, it was found that MBP-specific functions of the T cell clones was restricted by HLA class II antigens, and, more specifically, by molecules encoded for by DR locus genes.     

Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients

Summary To study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4⁺ and CD8⁺ T cell subsets. To analyze this reactivity, sorter-purified CD4⁺ and CD8⁺ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4⁺ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8⁺ grew vigorously and 29 cytolytic CD8⁺ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.This work is supported by National Institutes of Health research grants CA 36 233 and EY 9031, the Lucy Adams Memorial Fund and support from the Concern Foundation     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Functional modulation of hepatitis B core antigen-specific T lymphocytes by an autoreactive T cell clone

Hepatitis B core (HBc)Ag-specific T cells present in the peripheral blood of a patient with chronic active hepatitis B were expanded by co-cultivation for 7 days with rHBcAg. After cloning at 1 cell/well in the presence of PHA and IL-2, five HBcAg-specific CD4+ cloned lines were obtained. All five lines proliferated and produced IL-2, IFN-gamma, and TNF in a dose-dependent fashion in response to HBcAg, but not to HBV envelope Ag. The cloned lines and derivative clones were HLA class II (DR1) restricted. All T cell clones were able to induce anti-HBc production by autologous B cells in response to HBcAg (helper effect). The proliferative response and the helper effect of the HBcAg-specific T cell lines and clones were augmented by co-cultivation with an autologous, autoreactive (HLA-DQ1 specific) T cell clone, even in the absence of HBcAg, and the autoreactive T cells directly stimulated anti-HBc secretion by autologous B cells, presumably due to the release of Ag-nonspecific factors. These findings define a model immunoregulatory circuit the physiologic significance of which remains to be determined.     

Stimulation of human lymphocytes with irradiated cells of the autologous Epstein-Barr virus-transformed cell line. I. Virus-specific and nonspecific components of the cytotoxic response

Peripheral blood lymphocytes were cocultivated with irradiated cells of the autologous EB virus-transformed cell line at different responder:stimulator (R:S) ratios and the cytotoxic response was assayed up to 12 days later. In cocultures set up at a R:S ratio of 4:1, the response from both EB virus antibody-positive (seropositive) and negative donors was dominated by a broad-ranging NK-like cytotoxicity which did not segregate within the E-rosette-forming subpopulation of effector cells. In contrast, cocultures set up at a R:S ratio of 40:1 and harvested after 10 to 12 days gave rise, in the case of seropositive donors only, to effector T-cell preparations which appeared to be both EB virus specific and HLA-A and B antigen restricted. Strong lysis of the autologous virus-transformed cell line and demonstrable activity against certain allogeneic HLA-A and/or B antigen-related virus-transformed lines occurred in the absence of any significant killing either of the corresponding lines from HLA-unrelated donors or of a variety of EB virus genome-negative target cells (K562, HSB2, BJAB) particularly sensitive to NK-like cytotoxicity; furthermore, lysis of the autologous cell line by these effector T cells was specifically inhibited by monoclonal antibodies binding to HLA-A, B, and C antigens on the target cell surface. This work demonstrates that an HLA-restricted EB virus-specific cytotoxic T-cell response can indeed be induced in vitro by stimulation of fresh lymphocytes with autologous EB virus-transformed cells providing cocultures are set up at the correct R:S ratio.     

Human T cell receptor-gamma delta-expressing T-cell lines recognize MHC-controlled elements on autologous EBV-LCL that are not HLA-A, -B, -C, -DR, -DQ, or -DP.

HLA-loss variants of an EBV-transformed B lymphoblastoid cell line (EBV-LCL) 721 were used to investigate whether human MHC molecules other than known class I or class II were involved in autologous T cell responses. Bulk lymphocyte cultures of purified T cells primed to an autologous variant EBV-LCL that fails to express HLA-class II and has reduced cell surface HLA-class I expression, and oligoclonal TCR-gamma delta-bearing lines derived from them, could lyse both this EBV-LCL and an independently derived, class II expressing autologous variant EBV-LCL that bears no HLA-A, -B, or -C, suggesting the presence of additional HLA-like restriction elements. Cold target inhibition of cytolysis mediated by these lines indicated that a shared or cross-reactive MHC controlled restriction element other than the known MHC determinants was retained by the EBV-LCL variants. Single-cell derived clones from these T cell lines which expressed only the TCR-gamma delta showed this same target cell specificity pattern, proving recognition of MHC-controlled determinants by autologous gamma delta T cells. Anti-gamma delta antibody could inhibit cytolysis by the gamma delta-expressing lines, suggesting that the TCR-gamma delta was involved in recognition of the EBV-LCL targets. Flow cytometric analysis with separate HLA-reactive antibodies indicated that the restriction element for these cytolytic responses is a molecule serologically cross-reactive with HLA-B and -C Ag, yet is a determinant that cannot be HLA-A, -B, -C, -DR, -DQ or -DP.     

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.     

Specific lysis of varicella zoster virus-infected B lymphoblasts by human T cells.

Epstein-Barr virus-transformed human B cells expressed cell surface varicella-zoster virus (VZV) antigens after superinfection with VZV although they did not form infectious centers in a plaque assay. The VZV-superinfected cells were lysed by autologous VZV-stimulated T-cell lines and their derivative clones. The effector cells were specific for VZV and an HLA DR antigen and were T4+. The specificity of lysis of Epstein-Barr virus-transformed, VZV-superinfected targets by prestimulated mononuclear cells in this system contrasted with the unrestricted lysis seen when the targets were VZV-infected fibroblasts.     

In vitro induction of HLA-restricted cytotoxic T lymphocytes against autologous Epstein-Barr Virus transformed B lymphoblastoid cell line

Cytotoxic T lymphocytes (CTL) against autologous EBV-transformed B lymphoblastoid cell line (LCL) were induced in vitro by culturing peripheral blood lymphocytes (PBL) of healthy donors together with mitomycin C-treated autologous LCL for 6 days. The cytotoxic cells developed only from the E-rosette-positive fraction but not from the negative fraction of PBL. These CTL killed autologous LCL but not PWM-stimulated autologous PBL. In addition, the CTL killed allogeneic LCL when at least 1 of the HLA-A antigens was identical with that of the LCL of CTL donor. However, identity of HLA-B and HLA-C antigens was not enough for a significant killing of allogeneic LCL. The specificity of the CTL was also confirmed by a cold target inhibition test. These results indicated that the CTL induced specifically recognized EBV-transformed cells with HLA restriction.     

MHC-restricted recognition of autologous melanoma by tumor-specific cytotoxic T cells. Evidence for restriction by a dominant HLA-A allele.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.     

Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones

The L3T4+, Lyt-2-, cloned BALB/c T cell lines 5.9.24 and 5.8.6 are cytotoxic for the BALB/c B cell tumor line A20/2J. The T cell cytotoxicity against A20/2J cells could be triggered either by the specific antigen ovalbumin (OVA), which is recognized by the T cell clones in association with I-Ad determinants, or by the T cell mitogens Con A and rabbit anti-mouse brain (RaMBr) antiserum. Repeated exposure of A20/2J cells to 5.9.24 and 5.8.6 T cell cytotoxicity selected variant cell lines that had developed resistance to cytotoxicity. The variant lines could be classified into four different variant phenotypes of which three were stably maintained in vitro. The type of variant obtained appeared to be related to the nature of the ligand used to trigger T cell cytotoxicity during selection. Cytotoxicity triggered by the antigen OVA generated type 1 variants that expressed abnormally low levels of I-Ad determinants at the cell surface. Type 1 variants were resistant to OVA-triggered 5.9.24 T cell cytotoxicity, but were fully susceptible to cytotoxicity triggered by Con A or RaMBr antiserum. RaMBr-triggered cytotoxicity generated two unique types of variant cell lines: type 3 variants that were deficient in cell surface Fc receptors and resistant to 5.9.24 cytotoxicity only when triggered by RaMBr antiserum, and type 4 variants that were resistant to cytotoxicity triggered by all three ligands. One type 4 variant, the IC-1 cell line, appeared to be resistant to soluble cytotoxic factors released by 5.9.24 T cells after activation by antigen. All of these variant lines retained sensitivity to cytotoxicity by classic Lyt-2+ cytotoxic T lymphocytes (CTL), a finding that indicates that L3T4a+ T cells and Lyt-2+ CTL use different molecules to attack their target cells. The variant phenotypes were inherited by clones derived from the original cell lines. Because the variants were generated without mutagenesis, they are thought to have been derived by the immunoselection of pre-existing variant cells that arose spontaneously in the parental A20/2J cell line. It is postulated that inheritable variation of A20/2J cells may represent changes that normally occur during B cell differentiation in response to T cell signals. The variant A20/2J cell lines described here provide material for the investigation of B cell surface structures that may regulate T-B cell interactions.