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)     

In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma

Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.     

Auto-tumor lysis by blood lymphocytes in vitro

Summary Selectivity of the lysis of the tumor cells by autologous blood lymphocytes and its various subsets was investigated by means of the cold target competition assay. The effectors were autologous lymphocytes passed through a nylon-wool column (unfractionated: U) and their low-and high-density subsets, either without or after activation. The lymphocytes were activated (a) in autologous mixed lymphocyte tumor cell culture in autologous (MLTC), (b) in mixed lymphocyte culture (MLC), without and with interleukin-2, for 6 days, or (c) by phytohaemagglutinin for 3 days. Autologous-lymphocyte-mediated cytotoxicity (auto-tumor lysis: ALC) by the unfractionated, unmanipulated blood lymphocyte (U) population, its high-density fraction and those induced for auto-tumor lysis in the MLTC is regularly weak and affects only the autologous tumor cells. Their ALC function was inhibited only by the target identical unlabelled cells while the effect of separated low-density lymphocytes was inhibited also by allogeneic tumor cells.The cold-target competition assay indicated that several subsets with different specificities exist simultaneously in the effector populations activated in MLC, because the various targets did not cross-compete or did so only partially. Whenever interleukin-2 was added, at the start of the mixed cultures (MLTC or MLC), the lytic effects were no longer selective. Phytohaemagglutinin-activated effectors lysed several targets. These targets were inhibitory in a criss-cross fashion. Generally, populations showing auto-tumor selectivity had weak lytic effects, while the strongly activated effectors, with strong cytotoxic function, were not selective.     

Lysis of fresh human tumour cells by autologous tumour-associated lymphocytes: Two distinct types of autologous tumour killer cells induced by co-culture with autologous tumour

Summary The specific and natural killer (NK)-restricted nature of auto-tumour cytotoxicity of tumour-associated lymphocytes was studied in cancer patients with malignant pleural effusions. Large granular lymphocytes (LGL) and small T lymphocytes were isolated from carcinomatous pleural effusions by centrifugation on discontinuous Percoll gradients. Tumour cells freshly isolated from pleural effusions were classified according to their susceptibility to lysis by Percoll-purified LGL from the blood of normal donors in a 4-h ⁵¹Cr release assay. Of 12 NK-sensitive tumour samples, 11 were killed by autologous fresh effusion LGL, whereas only 2 were lysed by autologous T cells. Neither LGL nor T cells were cytotoxic to NK-resistant autologous tumour cells. T cells and LGL were each cultured in vitro with autologous tumour cells for 6 days. Effusion LGL maintained their auto-tumour killing activity in 10 of 12 autologous mixed lymphocyte-tumour cultures (MLTC) with NK-sensitive tumour, while LGL lost the activity when cultured alone. Removal of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL enriched effector cells. Autologous MLTC-derived LGL could also kill NK-sensitive allogeneic effusion tumour cells and K562 cells, as did fresh LGL. In autologous MLTC LGL failed to acquire lytic function to NK-resistant autologous tumour cells. In contrast, in vitro activation of effusion T cells with autologous tumour cells induced auto-tumour killer cells in 9 of 12 NK-sensitive tumour samples and 3 of 6 NK-resistant tumour cases. However, cultured T cells were incapable of killing allogeneic tumour cells and K562 cells. In the autologous MLTC effusion T cells proliferated vigorously in response to autologous tumour cells, whereas LGL showed no proliferation. The enrichment of blasts from cultured T cells on discontinuous Percoll gradients resulted in an enhancement of auto-tumour cytotoxicity, with no reactions recorded in blast-depleted, small, resting T cells. These results indicate that two distinct types of auto-tumour-recognising lymphocytes, LGL and T cells, are present in carcinomatous pleural effusions of cancer patients and that each effector type recognises different membrane moieties of autologous effusion tumour cells.     

Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells

Summary The kinetics of interleukin-2 receptor (IL-2R) expression and the [³H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R⁺ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R⁺ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.     

Induction of preferential cytotoxicity against allogeneic mouse lymphoma cells: in vitro and in vivo studies

The aim of this study was to activate, in mixed leukocyte/tumor cell cultures (MLTC), cytotoxic lymphocytes exhibiting preferential activity in vitro and in vivo towards allogeneic mouse lymphoma cells. Whereas the lymphoma target cells were readily lysed by the MLTC-derived lymphocytes, the cytotoxicity against the corresponding allogeneic concanavalin-A(ConA)-induced lymphoblasts was more than tenfold lower. Both activities were mediated by CD3⁺, TCR⁺, CD8⁺, CD4⁻ cytotoxic T cells (CTL). ConA-induced lymphoblasts were readily lysed by anti-Thy1.2 antibodies and complement, by CTL derived from mixed leukocyte cultures (MLC) and by the MLTC-derived CTL in the presence of ConA, indicating that the lymphoblasts are not merely less lysable than the lymphoma cells but that the latter are specifically recognized by the CTL. Lymphoblasts poorly competed with ⁵¹Cr-labeled lymphoma cells in a “cold”-target competition assay, suggesting that the MLTC-derived CTL largely recognize epitopes expressed only by the lymphoma cells. Furthermore, analysis of the cytotoxic activity of more than 500 MLTC-derived CTL oligoclones and over 30 clones revealed that one-third of them were cytotoxic only against the allogeneic lymphoma cells, one-third were reactive against both the lymphoma and the allogeneic lymphoblast target cells and the remainder were not cytotoxic at all. Upon injection into sublethally irradiated, lymphoma-bearing allogeneic mice, the MLTC-derived CTL cured 56% of the recipients and caused graft versus host disease (GVHD) is only 22%, whereas CTL activated in MLC against allogeneic splenocytes were therapeutically ineffective and caused lethal GVHD in 89% of the recipients. Although the therapeutic efficacy of the in vitro-generated antitumor CTL was demonstrated against experimental lymphoma lines, this strategy might prove effective in tumor immunotherapy in conjunction with other modalities. Received: 24 December 1998 / Accepted: 5 April 1999     

Human T-cell cultures with selective autotumor reactivity

Summary T-cell cultures derived from the blood of 14 patients with solid tumors were propagated with T-cell growth factor (TCGF). The cultures were initiated from lymphocytes exposed to autologous tumor-biopsy cells. TCGF was added either immediately or 3–10 days later. In the former culture type the cell yield on day 7 was considerably higher. The cytotoxic potential of the cultured cells was assayed on two occasions, between days 7 and 10 and between weeks 5 and 8. Cells of all but two cultures had the potential to lyse autologous tumor-biopsy cells.On the population level, cytotoxicity was specific for autologous tumor in those cultures that were driven to growth with TCGF after the 3rd day. These lymphocytes did not lyse allogeneic tumor-biopsy cells. In contrast, all five cultures initiated in the presence of TCGF exhibited a broader cytotoxic potential, i.e., in addition to the stimulator autologous-tumor cells, they also lysed other targets. Another difference between the two culture types was their behavior toward K562. Tested on the 7th day they all lysed K562; however, this function declined in strength or disappeared later in the cultures exposed to TCGF after the 3rd day.Reexposure of the lymphocytes to autologous tumor-biopsy cells after 2 weeks of culture period, but not on the 7th day, induced DNA synthesis. This secondary response was specific inasmuch as allogeneic tumor cells had little or no effect.One of the autotumor restimulated cultures was tested for cytotoxic potential. It increased against the autologous but not against other tumors or K562 cells.     

Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro

 Using a modification of the autologous mixed lymphocyte/tumour cell culture (MLTC), it is demonstrated here that lymphocytes from chronic-phase myelogenous leukaemia (CML) patients (n = 58), but not from their HLA-identical siblings, proliferated upon coculture with autologous tumour cells. However, in most cases, the level of proliferation measured was low (stimulation index <3, n = 37). This was most likely related to the amount of interleukin-10 (IL-10) released into the culture medium by the CML cells, because addition of neutralizing anti-IL-10 serum to MLTC markedly enhanced proliferative responses. In addition, supplementation of media with IL-1α further enhanced proliferative responses and a combination of anti-IL-10 serum and IL-1α was more effective than either agent alone. Only HLA-DR-matched CML cells, but not HLA-DR-mismatched CML cells or matched or mismatched PBMC restimulated proliferation of IL-2-dependent T cell lines derived from MLTC supplemented with IL-1α and anti-IL-10 serum. The responding cells under these conditions were predominantly CD4⁺ and secreted IL-2, and interferon γ; some secreted IL-4, but none secreted IL-10. These data therefore suggest the existence of an HLA-DR-restricted DTH/Th1-type of tumour-specific immunity in CML patients, which may be down-regulated in vitro by excessive secretion of IL-10 together with depressed secretion of IL-1. Received: 9 November 1995 / Accepted: 8 February 1996     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Non-fastidious,melanoma-specific CD8+ cytotoxic T lymphocytes from choroidal melanoma patients

To characterize the anti-melanoma reactivity of CD8⁺ cytotoxic T lymphocytes (CTL) from choroidal melanoma patients, CTL clones were isolated from the peripheral blood of three patients after mixed lymphocyte/tumor cell culture (MLTC). Clones were derived from lymphocytes stimulated by allogeneic (OCM-1, A24, A28) or autologous (OCM-3, Al, A30) melanoma cells. Their reactivity against a panel of HLA-typed melanoma and nonmelanoma cells was assessed, to determine whether a single CTL clone could recognize and lyse a variety of allogeneic melanoma cell lines. While proportionately more clones derived from autologous MLTC were melanoma-specific than allogeneic MLTC (42% versus 14%), melanoma-specific CTL were recovered from both. Notably, a novel melanoma specificity was identified. These CTL clones were termed non-fastidious because they were capable of lysing melanoma cells with which they had no HLA class I alleles in common. Nonetheless, lysis was mediated by the HLA class I molecule. Since lysis was specific for melanoma cells, these CTL appeared to recognize a shared melanoma peptide(s). Because of their prevalence, we propose that non-fastidious CTL are integral to human anti-melanoma T cell immunity. This reinforces clinical findings that allogeneic melanomas can substitute for autologous tumors in active specific immunotherapy. By circumventing the need for autologous melanoma, it is possible to treat patients after removal of the primary choroidal melanoma in an attempt to prevent metastasis.Supported by USPHS grants EY-09031 and EY-09427, and the Lucy Adams Choroidal Melanoma Research Fund to J. K.-M.     

Clonal analysis of human tumor infiltrating lymphocytes reactive with autologous tumor cells: different target cell specificities of NK-like and cytotoxic T-cell clones

Lymphocytes, derived from surgically resected lung carcinoid tissue, were stimulated in mixed culture with irradiated autologous tumor cells (MLTC). The autologous MLTC-stimulated lymphocytes were found to have killing activity against both autologous tumor cells and NK-sensitive target cells. The lymphoblasts generated during MLTC were isolated and cloned under limiting dilution conditions in the presence of interleukin 2. The cloned cell lines were analyzed for cell phenotype and tested for cytotoxic activity. Three cloned cell lines, out of 19 tested, were found to be cytotoxic either against NK-sensitive target cells (natural killers) or the autologous tumor cells. Two clones, having OKT8 phenotype, caused no lysis of the autologous tumor cells, though both exerted NK-like activity against K562 cells. Only one clone with OKT4 phenotype showed specific cytotoxic activity against the autologous tumor, but no NK-like activity against a panel of tumor target cells. These results suggest the coexistence of two types of antitumor cytotoxic lymphocytes at the tumor site: precursors of NK-like cells and specific cytotoxic T cells. Target cell specificity provided a means of distinguishing between the two types.     

Conditions for the generation of autoreactive human T cell clones: requirement for lymphokines other than interleukin 2 alone

In an attempt to determine whether factors other than interleukin (IL) 2 alone were necessary for the generation of autoreactive suppressive T cell clones, lymphocytes from HLA-Dw-matched allogeneic mixed leukocyte cultures (MLC) were propagated and cloned in purified IL 2, partially purified IL 2, conditioned medium (CM) from stimulated peripheral blood mononuclear cells (PBMC), or with purified IL 2 plus IL 3, IL 4, or interferon-gamma (IFN-gamma). Cloning efficiencies were very low in all cases (less than 10%) and of 48 clones tested only 6 were capable of autocrine proliferation after stimulation with autologous PBMC. Four of these clones were derived from populations expanded and cloned in CM, one from cultures with partially purified IL 2, and one with purified IL 2. All were CD4+ alpha/beta-T cell receptor. Their stimulation was blocked by anti-DR and broadly reactive MHC class II-specific monoclonal antibodies (mAb) but not by anti-DQ or anti-DP mAb. One clone was blocked exclusively by broad mAb but not by anti-DR, -DQ, or -DP mAb, and this was the only clone to suppress lymphocyte proliferation in allogeneic MLC, a property previously described for autoreactive clones derived under similar conditions detecting potentially novel lymphocyte activating determinants designated "DY." These results therefore suggest that DY-specific autoreactive suppressive clones are produced under these conditions only at a low frequency and that an unidentified factor other than IL 2, IL 3, IL 4, or IFN-gamma is involved in their generation.     

Clonal analysis of cytotoxic T lymphocytes (CTL) against autologous melanoma

Summary This study investigates the nature and specificity of cytotoxic T lymphocytes (CTL) in patients with melanoma which are able to kill autologous melanoma cells. Interleukin 2 (IL2)-dependent T cell clones from two melanoma patients and a normal subject were generated in mixed lymphocyte cultures (MLC) or mixed lymphocyte tumor cell cultures (MLTC) and propagated for prolonged periods in tissue culture. Analysis of their phenotype by a wide range of monoclonal antibodies (M.Abs) revealed two main phenotypes which depended on whether they expressed Fc receptors detected by Leu 11 M.Abs or not. Leu 11^– T cells (referred to as Type 1) were inhibited by M.Abs to T3, T8, and a common HLA, ABC antigen. Conversely Leu 11⁺ T cells (referred to as Type 2) were inhibited by M.Ab to Leu 11 but not by M.Ab to T3, T8 and the HLA, ABC antigen. Subtypes among Type 1 cells were recognized which depended on their specificity. The most restricted were CTL [Type 1(a)] clones generated only in MLTC which recognized the autologous melanoma cell plus 1 of 11 other melanoma target cells. Type 1(b) CTL clones recognized a larger proportion (approximately 50%) of the melanoma cells. A third category [Type 1(c)] recognized antigens on melanoma cells shared with that on the EBV-transformed B cells used as stimulators in the MLC. Type 2 CTL clones had broad specificity to melanoma and nonmelanoma cells, characteristic of that described for lymphokine activated killer (LAK) cells. The latter were MHC unrestricted but further studies are required to clarify whether the Type 1 CTL clones are MHC restricted or not. The CTL activity of all clones was inhibited by M.Ab to the sheep red blood cell receptor and to the T10 antigens. It is suggested that recognition of these different types of CTL clones may assist future studies on the immune response against melanoma and the nature of antigens recognized by CTL.     

Conditions for antitumor cytotoxic T-lymphocyte formation and inhibition of their activity by T-suppressors in mixed culture of human lymphocytes and tumor cells

Conditions for antitumour autolytic T lymphocytes (CTL) induction during human mixed lymphocyte tumour cell culturing (MLTC), as well as the patterns of CTL activation abolition in the experimental system by suppressor cells were investigated. The responders used in MLTC were peripheral blood lymphocytes of colorectal cancer patients fractionated by means of multilayer Percoll gradients centrifugation (the density of layers being 1.077, 1.067, 1.056 g/ml). The cells of the second fraction collected in the density interphase of 1.077-1.067 g/ml (more than 90% of population belonging to T lymphocytes), when used as responders in MLTC, developed an autolytic activity against autologous and allogeneic tumour target cells. The cell of the first fraction (collected in the interphase of 1.067-1.056 g/ml) added to the second fraction cells at the beginning of MLTC, prevented the following CTL induction. The first fraction contained T-suppressor cells capable of strongly interfering with antitumour CTL activity.     

Human anti-lymphoma responses generated in vitro and in vivo following sensitization with allogeneic leukocytes

Peripheral blood lymphocytes (PBL) from a patient with poorly differentiated lymphocyte lymphoma (PDLL), after stimulation for 7 days with X-irradiated allogeneic lymphocytes pooled from three or ten donors (poolx), were cytotoxic for autologous lymphoma cells. Some clones lytic for autologous lymphoma cells, that were derived from this patient's pool-stimulated cells, resembled cytotoxic T lymphocytes (CTL), while other clones resembled natural killer (NK)-like cells in that they also lysed NK-sensitive HLA-negative K562 cells. In a second patient with more advanced PDLL, PDL cultured with T-cell growth factor (which is produced following stimulation with mitogens or alloantigens) lysed autologous lymphoma cells. On the basis of these in vitro findings, we asked whether IV transfusions with X-irradiated allogeneic leukocytes would result in anti-lymphoma responses in vivo. Ten days after transfusions with X-irradiated leukocytes from four unrelated donors, the first patient's two previously palpable nodes were no longer palpable and he remained in complete clinical remission for 6 months. The second patient had a temporary partial remission with dramatic reduction in size of multiple cervical and axillary nodes within 2 weeks after receiving the leukocyte transfusions.     

Establishment and functional characterization of human herpesvirus 6-specific CD4+ human T-cell clones.

In order to clarify the protective immune responses against a newly identified herpesvirus, human herpesvirus 6 (HHV-6), we established HHV-6-specific human T-cell clones and examined their functional properties. Five CD3+CD4+CD8- T-cell clones, which proliferated in response to stimulation with two different strains of HHV-6 in the presence of autologous antigen-presenting cells but not with herpes simplex virus type 1 or human cytomegalovirus, were established from peripheral blood lymphocytes of a healthy individual. The proliferative response of all T-cell clones to HHV-6 antigen was inhibited by addition of anti-HLA-DR monoclonal antibody, indicating that these clones were human leukocyte antigen (HLA) class II DR restricted. Of the five clones, two lysed HHV-6-infected autologous lymphoblasts, but not HHV-6-infected allogeneic cells or natural killer-sensitive K562 cells (group 1); one showed cytotoxicity against HHV-6-infected autologous lymphoblasts as well as HHV-6-infected allogeneic cells and K562 cells (group 2); and the remaining two showed no cytotoxic activity (group 3). The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR monoclonal antibody to the culture, whereas this monoclonal antibody had no effect on the cytotoxicity of group 2 and did not induce the cytotoxicity of group 3. Perforin, which is one of the mediators of cytotoxicity, was abundantly expressed in group 1 and 2 clones. Moreover, all groups of clones produced gamma interferon after culture with antigen-presenting cells followed by HHV-6 antigen stimulation. These results suggest that HHV-6-specific CD4+ T cells have heterogeneous functions.     

In vitro induction of immunological tolerance

IL-2 was previously shown to induce cytotoxic effectors with a broad spectrum of target specificities in thymus and spleen cell cultures. This study was designed to show whether T cells activated by H-2 allogeneic cells in MLC or by syngeneic tumor cells in MLTC are also potential targets for these cytotoxic effectors. We found that thymocytes activated in vitro for 5 days by rIL-2 were capable of killing tumor cells as well as activated T cells. Thymocytes activated by IL-2 were accordingly utilized as a means of effecting clonal deletion of T cells activated by H-2 allogeneic target cells in MLC. To establish whether the unresponsiveness is specific. IL-2-activated thymocytes were added as third party cells to MLC and MLTC. The results showed that both T cells, proliferating in response to H-2 allogeneic cells, and CTL, reactive against syngeneic tumors or H-2 allogeneic cells, are eliminated from the T cell pool. Only alloreactive T cells are specifically eliminated in MLC by IL-2-activated thymocytes, as the remaining T cells are capable of proliferating and generating CTL in response to antigenically unrelated third party allogeneic cells. The possibility that unresponsiveness might be due to soluble factors was ruled out by studies performed with a diffusable "chamber insert" culture system. The results provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance.     

Lymphocyte transformation induced by autologous cells.

Human peripheral blood T lymphocytes are stimulated to proliferate when cultured with autologous B-lymphoblastoid cell lines, autologous mitogen-induced lymphoblasts, or autologous non-T blood lymphocytes. This reaction, the autologous mixed lymphocyte reaction, has attributes of an immune response possessing both memory and specificity. The capacity to stimulate autologous T lymphocyte proliferation depends on the lineage of the lymphoid cell and not on its establishment in continuous culture or carriage of the EB viral genome. The determinant on non-T lymphocytes which stimulates the autologous mixed lymphocyte reaction appears to be an Ia determinant. Thus, allogeneic graft rejection and the allogenic mixed lymphocyte reaction are very likely extensions of an immune response expressed within the host.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes