The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-Ingative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%–42.3% and 32.7%–53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8⁺ or CD4⁺ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.     

DR antigen expression on ovarian carcinoma cells does not correlate with their capacity to elicit an autologous proliferative response

Summary Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8=50%) and negative MLTC (7/13=53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens.     

Generation of natural killer-like activity in mixed lymphocyte-tumor cell cultures. II. Correlation between expression of HLA-DR antigens on the activated T cells and their cytotoxic capability

In this study, we investigated the role of DR antigens in human mixed lymphocyte reactions (MLR) at the responder cell level. Upon stimulation by allogeneic lymphocytes or leukemic cell lines, a large proportion of T cells underwent blastogenesis and began expressing DR antigens. Analysis by a fluorescence-activated cell sorter revealed that both subpopulations of large activated T cell blasts and of small T lymphocytes became DR+ by synthesis and/or uptake. Depletion of DR+ responder cells from 6-day-old MLRs by treatment with anti-DR monoclonal antibodies (mAbs) plus complement (C) reduced but did not completely abrogate the natural killer (NK)-like activity of the responder lymphocytes, suggesting that the MLR-induced cytotoxic cells include both DR+ and Dr- populations. The expression of NK-like activity by the responder cells was also greatly reduced upon addition of anti-DR mAbs (without C) at the start of the mixed cultures. This effect was observed regardless of the presence of DR antigens on the stimulator cells, indicating that the anti-DR mAbs can interact with the antigens present on both the stimulator and responder populations. These data show that during an MLR, the continued presence of DR antigens on the responding population is essential for the expression and maintenance of the proliferative and cytotoxic capabilities of these cells.     

Role of HLA-DR bearing Langerhans and epidermal indeterminate cells in the in vitro generation of alloreactive cytotoxic T cells in man

Human epidermal cells (EC) act as stimulator cells in the mixed-skin cell lymphocyte culture reaction (MSLR). To analyze the role of human epidermal Langerhans cells (LC) and indeterminate cells (IC), which are the only cells expressing the DR-Ia-like antigens in normal epidermis, in the generation of alloreactive cytotoxic T lymphocytes (CTL) in cell-mediated cytolysis, 18-hr 51Cr-release assays against PBL targets (targets autologous to stimulator EC) were conducted after allogeneic human MSLR. MSLR and CTL assays were conducted with, as stimulator cells, suspensions of normal human EC as controls, and EC after: (1) preincubation with anti-HLA-DR or OKT6 (specific for LC in EC suspension) monoclonal antibodies; (2) panning, a monolayer technique used to deplete EC suspensions in OKT6 or DR-positive cells. The generation of alloreactive CTL was found to occur only after allogeneic MSLR and when targets and stimulator cells were from the same donor; it was reduced by EC incubation: cytotoxic activity 26.66 +/- 3.84 (controls); 8.8 +/- 3.6 and 7.7 +/- 3.7 (EC incubated with OKT6 or anti-DR, respectively); it was reduced or abolished when the EC used in MSLR were depleted in OKT6 or DR-positive cells by panning. These findings demonstrate that human LC and IC are necessary for an optimal in vitro sensitization in MSLR and the subsequent in vitro generation of alloreactive CTL in man.     

Auto-tumor recognition following in vitro induction of MHC antigen expression on solid human tumors: stimulation of lymphocytes and generation of cytotoxicity against the original MHC-antigen-negative tumor cells

Summary Expression of major histocompatibility complex (MHC) class I antigens was induced in eight out of nine freshly prepared tumor cell suspensions by exposure to interferon (IFN) and tumor necrosis factor (TNF) in vitro. The untreated, class-I-antigen-negative, and the treated, antigen-positive, cells of three tumors (one breast carcinoma, one plasmocytoma and one ovarian carcinoma) were compared for the capacity to stimulate autologous and allogeneic blood lymphocytes, to generate auto-tumor cytotoxicity and for sensitivity to the lytic effect induced in autologous mixed lymphocyte tumor cell culture (MLTC). The MHC class I-negative cells did not stimulate, while the cells induced for expression of antigens did. On the other hand, when the autologous cytotoxic cells were generated in the MLTC by the class I antigen-positive tumor cells the class I-negative tumor cells were also damaged. Lysis of the class-I-positive tumor cells was abrogated by the W6/32 monoclonal antibody directed against the monomorphic part of the class I molecules.     

HLA-DR antigens induce proliferation and cytotoxicity of T cells against haptenated (TNP and FITC) self structures

Antisera directed against the heavy, the light, or reactive against the complex of both chains of HLA-DR antigens strongly inhibited proliferation of T cells induced by TNP- or FITC-labeled autologous cells when added at initiation of the cultures, but not 72 h later. T cells from cultures treated with the anti-DR sera were unresponsive to interleukin-2 (IL-2). Nonetheless, the anti-DR sera did not inhibit proliferation of T cells that had already acquired sensitivity to IL-2. The DR antibodies abrogated the synthesis of IL-2 induced by both TNP- and FITC-conjugated autologous cells. Treatment of TNP- and FITC-labeled autologous cell cultures with the four different types of anti-DR sera significantly inhibited the induction of cytotoxic T cells. However, DR antibodies added at the effector phase of cytotoxicity assays did not inhibit the cytotoxic activity. Effector T cells from cultures treated with the anti-DR sera were unresponsive to IL-2 and addition of IL-2 to these cultures did not restore the cytotoxic activity. In contrast, effector T cells from cultures performed in the absence of the anti-DR sera proliferated to IL-2 stimulation and addition of IL-2 to these cultures significantly increased the generation of killer cells specific for hapten-labeled self structures. From these results we concluded the following: (1) Both the heavy and the light chains of DR antigens participate actively in the activation of T cells by rendering resting T cells sensitive to IL-2 and by inducing production of the growth factor in TNP- and FITC-conjugated autologous cell cultures. (2) The heavy and light chains of the DR antigens play an essential role in the induction of cytotoxic T cells specific for hapten-labeled self structures, most likely by enabling cytotoxic T cells to respond to IL-2 and by inducing the IL-2 producer T cells to synthesize the growth factor.     

Depletion of human NK cells with monoclonal antibodies allows the generation of cytotoxic T lymphocytes without NK-like cells in mixed cultures

In vitro stimulation of human mononuclear cells with x-irradiated autologous lymphoblastoid cell line (LCL) or allogeneic normal cells in mixed leukocyte cultures (MLC) was previously shown to result in the generation of OKT3+ OKT8+ cytotoxic T lymphocytes (CTL) lytic for allogeneic and autologous LCLs and also of natural killer- (NK) like cells that are OKT3- and primarily OKT8- and are lytic for HLA- NK-sensitive K562 cells. The origin of the NK-like cells was not previously known because, although the majority of fresh human NK cells react with monoclonal antibodies OKM1 and B73.1, lymphocytes bearing these markers are not detected several days after the onset of MLC, when NK-like cells are present. In this study, experiments were undertaken to determine whether NK-like cells generated after stimulation with x-irradiated pooled allogeneic normal cells (poolx) or with autologous LCL are derived from cells expressing antigens reactive with monoclonal antibodies OKM1 or B73.1, which react with fresh NK cells. Mononuclear cells, depleted of monocytes, were stained with OKM1 or B73.1 and fluorescein-labeled goat anti-mouse IgG. Lymphocytes depleted of OKM1+ or B73.1+ cells, by fluorescence-activated cell sorting, and lymphocytes that were stained but not sorted were stimulated for 7 days with either poolx or autologous LCL. The generation of NK-like activity was decreased at least 90% after depletion of cells reactive with OKM1 or B73.1, whereas the generation of CTL against autologous and allogeneic LCL was minimally affected. These findings show that NK-like cells generated in MLC are derived from cells that express the phenotype of fresh NK cells (OKM1+ or B73.1+) and that CTL can be generated in cultures in which relatively little NK-like activity is concomitantly detected, by depleting NK cells with monoclonal antibodies before stimulation.     

Generation of cytotoxic effector cells against human melanoma

Metastatic or tumor-draining lymph nodes from six of nine melanoma patients undergoing lymph node dissection for metastatic melanoma generated cytotoxic T cells against autologous melanoma when these lymph node cells were treated by in vitro sensitization and recombinant interleukin-2 (IL-2). During the initial lymphocyte culture (2–6 weeks), cross-reactivity with autologous tumor cells, K562 and Daudi cells was usually noted. Cold-target inhibition assay with K562 and Daudi showed K562/Daudi-associated antigens on melanoma cells. During the later phase of lymphocyte culture with repeated in vitro sensitization (over 6–10 weeks), cytotoxicity was noted against autologous and allogeneic melanoma cells but not against K562, Daudi cells or autologous fibroblasts. Repeated in vitro sensitization resulted in the selection of specific cytotoxic lymphocytes against melanoma. Cold-target inhibition assay with autologous and allogeneic melanoma cells revealed shared and individual antigens. Using blocking monoclonal antibodies, MHC-restricted killing was noted in the autologous system. Further, both the autologous and allogeneic systems could be mediated through adhesion molecules such as ICAM-1 and LFA-3 on melanoma cells and LFA-1 on T cells. This study suggests that a constellation of cytotoxic effector cells and melanoma-associated antigens may be pivotal in tumor killing. Thus, future adoptive immunotherapy should modulate and enhance this complex interaction.This work was supported by an Elsa, U. Pardee Foundation grant, the Arizona Chronic Disease Research Commission grant and partly by grant CA23074 from the National Institutes of Health, Bethesda, MD, 20892     

Activated T lymphocytes within human solid tumors

Summary The majority of lymphocytes separated from tumor cell suspensions were T cells. Conjugates of T lymphocytes and tumor cells were often seen. Variable numbers of T cells exhibited signs of activation such as the ability to form stable E rosettes and attachment to normal and malignant cells (a phenomenon designated natural attachment: NA). A proportion of T cells activated in vitro by allogeneic stimulation regularly exhibit these properties. The T cell-tumor conjugates in the suspensions may represent the NA phenomenon, but they could also be the product of T cells that adhere on the basis of specific recognition of cell surface antigens.Abbreviations BBS balanced salt solution - E rosettes rosettes formed with sheep erythrocytes - EA rosettes rosettes formed with ox erythrocytes coated with anti-ox IgG - FCS fetal calf serum - MLC mixed lymphocyte cultures - NA natural attachment - PBL peripheral blood lymphocytes - SRBC sheep erythrocytes - T lymphocytes thymusderived lymphocytes     

Macrophage antigens associated with adhesion: identification by a monoclonal antibody specific for Lewis lung carcinoma cells

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11-12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.     

Propagation of cytotoxic effectors from chronic myeloid leukemia patients and cloning of cytotoxic T cells

Summary Cytotoxic cells (CTCs) generated from peripheral blood lymphocytes of 5 chronic myeloid leukemia (CML) patients in remission on stimulation with autologous leukemic cells and allogeneic lymphocytes (3-cell assay), were propagated in vitro in interleukin-2 (IL-2)-containing medium and periodic stimulation with autologous leukemic cells, for a period of 4 to 6 months. During this period, the cells were assessed for phenotype and for cytotoxic responses in a 4-h ⁵¹Cr release microcytotoxicity assay. The CTCs continued to show specific lysis of autologous leukemic cells and bone marrow (BM) cells. However, the nonspecific lysis of natural killer (NK) targets and the proportion of cells showing NK phenotype (HNK-1 antigen) increased progressively on cultivation in IL-2-containing medium. Therefore cells showing CD8 phenotype and specific cytotoxic function were segregated by cloning CTCs under the condition of limiting dilution in the presence of allogeneic feeder cells and IL-2-containing medium. Three cytotoxic T cell (CTL) clones expressing CD3+, CD8+, and HLA DR+ phenotypes were obtained from CTCs of 2 CML patients. These clonoid populations, maintained in IL-2-containing medium and periodic antigenic stimulation with autologous leukemic cells, showed specific lysis of autologous leukemic cells and BM cells even at lower (10:1) effector:target ratios. They did not kill K562 (erythroblastoid leukemic NK target cell line) cells and autologous phytohemagglutinin-induced blasts. These clones apparently functioned in an MHC-restricted manner as they did not lyse allogeneic CML cells which would also express a similar set of maturation antigens if sensitization was, as it appeared, against these antigens. Finally, interaction of autologous BM cells with CTL clones reduced the colony forming potential of BM cells only to the extent of 18%–30%. The results therefore indicate that such CTL clones can possibly be used in adoptive immunotherapy as they showed minimal BM toxicity.     

Lysis of fresh human solid tumor cells by autologous lymphocytes activated in vitro by allosensitization

Summary We have previously demonstrated that cancer patients' peripheral blood lymphocytes (PBL) allosensitized against single or pool normal donor PBL are capable of lysing fresh autologous tumor cells in a 4-h ⁵¹Cr-release assay. In this report, we present further investigations into this phenomenon. These alloactivated killer cells (A-AK cells) lysed autologous and allogeneic tumors and allogeneic but not autologous PBL. Furthermore, cold target inhibition studies demonstrated that autologous and allogeneic tumors were lysed by the same effector cells. Multiple metastases from the same patient were equivalently lysed by these A-AK cells. The presence of adherent cells and proliferation of the precursors were necessary to generate A-AK cells, although the effector cell itself was radioresistant and nonadherent. The effects of allosensitization were enhanced by the addition of lectin-free interleukin-2 preparations to the in vitro culture by partial depletion of adherent cells prior to sensitization. The A-AK effector cell was OKT3+, OKT8+, OKT4–, OKM1– and could be generated by just 3 days of allosensitization. The precursors for A-AK cells could be separated from NK cells on percoll gradients and lysis could be generated from thoracic duct lymphocytes, a population devoid of NK cells. The phenotype of the majority of the precursor cells was OKT3+, OKT4–. These allocatived PBL could be expanded in crude or lectin-free interleukin-2 without loss of cytotoxicity for fresh autologous tumor cells. Activated T cells represent a population of non-NK cells with broad lytic specificity for fresh tumor cells. Such cells may be of value in the adoptive immunotherapy of human solid tumors and may play a role in immune surveillance.     

Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas

Summary T lymphocytes were isolated from tumor biopsies in 13 patients with breast carcinomas. Immunohistology with monoclonal antibodies confirmed the presence of mononuclear cell infiltrates composed primarily of T lymphocytes in all tumors studied. While the proportion of T lymphocytes expressing the T4 or the T8 surface marker varied from tumor to tumor as determined by morphometric analysis, T8+ cells were more numerous than T4+ cells in 8/12 breast tumors studied. Relatively few T cells (<10% in 11/12 tumors) were in an activated state as judged by the surface expression of HLA-DR antigens or the receptor for interleukin-2 (IL-2). In 1 case 20% of the infiltrating mononuclear cells were expressing the IL-2 receptor. The tumor infiltrating lymphocytes (TIL) recovered from 10 tumors were cloned in a microculture system that permits proliferation of nearly 100% of normal peripheral blood T lymphocytes (PBL-T). In contrast to normal and autologous PBL-T, frequencies of proliferating T lymphocyte precursors (PTL-P) were depressed (<0.01) in 7/10 TIL preparations indicating a decreased responsiveness of TIL to phytohemagglutinin at the single-cell level. The frequency of PTL-P was noticeably higher in 2 cases (0.03 and 0.09) and close to normal in 1 case (0.39).A total of 170 clones were expanded in vitro and analyzed for different functional capabilities. Most of these clones expressed the T4+/T8-phenotype (73%) and strikingly 53% of these T4+/T8– clones were cytolytic in a lectin-dependent assay, a functional subset which is uncommon among normal PBL-T. Some clones (10%) lysed allogeneic breast tumor cells (MCF7). Only 15% of the clones displayed natural killer activity. Among the cytolytic clones, 17 of 31 tested were also IL-2 producers irrespective of the T4 or T8 phenotype. Our results show that human mammary carcinomas contain many infiltrating T cells with cytolytic potential. Interestingly, among the proliferating cytolytic T cell clones (56% of the microcultures), many expressed the T4+/T8– phenotype. These findings may indicate that the in situ cytolytic reaction (against unknown antigens) is associated preferentially with class II antigens.Fogarty International Fellow of NIH, 1984–1985; on leave from Dept of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pa, USA     

Induction of CD4+ cytotoxic T cells by sensitization with allogeneic melanomas bearing shared or cross-reactive HLA-A.

Human melanoma is an immunogenic neoplasm whereby enhancement of specific cell-mediated immunity can alter tumor progression. HLA-A2-restricted CTL have been demonstrated to kill allogeneic HLA-A2-matched melanoma. We investigated the ability of allogeneic melanoma cells sharing HLA-A antigens to sensitize melanoma patients' lymphocytes to induce HLA-A-restricted CTL to autologous melanoma. PBL from melanoma patients were cocultured with autologous melanoma cells in defined "cocktail medium" to generate melanoma-specific HLA-A-restricted CTL lines. CTL generated by sensitization with allogeneic melanoma bearing shared HLA-A2, A11, A24, or "cross-reactive" HLA-A antigens could kill almost as many autologous melanoma cells as CTL sensitized with autologous melanoma. There are HLA-A antigens that are immunogenically cross-reactive because they share determinant epitopes. CTL were not activated NK or LAK cells. The HLA restriction and melanoma cell specificity of the CTL were demonstrated by cold target inhibition with autologous and allogeneic melanoma and B lymphoblasts. Anti-CD3 and anti-HLA AB inhibited CTL killing of melanoma. The CTL were predominantly CD3+CD4+ TCR alpha/beta+. These studies demonstrate that melanomas being shared or cross-reactive HLA-A can be used for in vitro generation of HLA-restricted CTL that recognize melanoma-associated antigens. The findings have very important implications in human tumor immunotherapy.     

Membrane structures involved in auto-tumor recognition

Tumor patients' blood lymphocytes have the capacity to recognize autologous tumor cells in vitro. A consequence of this recognition is the proliferation of small-size, high-density, resting T cells. Both helper (CD4+) and cytotoxic/suppressor (CD8+) T lymphocytes proliferate in the mixed lymphocyte-tumor cell cultures. In contrast to the autologous mixed lymphocyte cultures, both the auto-erythrocyte rosetting and non-rosetting (AE+ and AE-) T cells participate in the auto-tumor response. In contrast to stimulation by virus-infected or hapten-modified cells, DR antigen expression is not essential for stimulation by autologous tumor cells. In a proportion of cancer patients, blood lymphocytes have the capacity to lyse the patients' own tumor cells in vitro. There are two populations of lymphocytes with auto-tumor cytotoxic function. The first is characterized by low buoyant density and by non-adaptive cytotoxicity. In contrast to the recognition of hapten-modified or virus-infected target cells by the CTL, recognition of autologous tumor cells by the cytotoxic LD cells occurs even when the MHC class I antigens are blocked by mAb. The CD3 complex is also not involved in LD-mediated lysis. The other population with auto-tumor cytotoxic function comprises high-density, resting T cells. Recognition of autologous tumor cells by cytotoxic HD lymphocytes shares the characteristics of CTLs, i.e., their function is abrogated by pretreatment of the effectors with mAbs directed to the T3 receptor complex and by preincubation of the targets with mAb to the MHC class I antigens. Cytotoxicity of HD cells is restricted to the autologous tumor cells. This selectivity and the characteristics shared with CTL suggest that the auto-tumor reactivity of HD lymphocytes reflects an immune response against the autologous tumor.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors: effects of tumor cells and their supernatants on proliferative responses of lymphocytes

Tumor-infiltrating lymphocytes (TIL) were obtained from 22 humans with solid tumors. In three cases only, one colon and two lung carcinomas, TIL which contained from 3 to 10% of T cells expressing the interleukin 2 receptor (IL 2R) were obtained, and these proliferated in the presence of exogenous IL 2. In most TIL preparations, however, the T lymphocytes did not express the IL 2R and failed to proliferate in response to IL 2. In contrast, TIL were able to proliferate in response to irradiated allogeneic spleen cells in mixed lymphocyte culture. Proliferative responses of autologous PBL were not inhibited by the addition of TIL. In most tumors, the TIL showed no response or had significantly lower (p less than 0.01) responses to PHA, Con A, and the phorbol ester TPA than did autologous peripheral blood lymphocytes (PBL). A limiting-dilution microculture system which allows clonal growth of every T cell was used to demonstrate decreased responses of the TIL to PHA at a single-cell level. In contrast to normal PBL-T with proliferating frequencies from 0.46 to 1.0, those for T cells in three TIL preparations were zero, 0.005, and 0.01. Normal PBL exposed in vitro to tumor cells or their supernatants lost the ability to respond to mitogens and to clone normally (e.g., proliferating frequency of 0.147 vs 0.863 in control). The TIL isolated from solid tumors resemble normal PBL exposed in vitro to tumor cells or their supernatants in terms of decreased responses to mitogens and poor clonogenicity in the PHA-dependent microculture system. It is possible that tumor cells may inhibit certain functions of the TIL in human solid tumors.     

Identification of specific cytolytic immune responses against autologous tumor in humans bearing malignant melanoma

Tumor-infiltrating lymphocytes from six patients with metastatic malignant melanoma were expanded by culture in recombinant interleukin 2. Three of the preparations were highly cytotoxic against autologous fresh melanoma tumor cells, but not against autologous fresh normal cells or allogeneic fresh tumor targets. The other three were highly cytotoxic against autologous fresh melanoma tumor cells and also had a limited capacity to kill allogeneic fresh tumor targets. The tumor-associated specific killer cells could be expanded from threefold to 95,652-fold with maintenance of specific antitumor lysis. The expanded tumor-infiltrating cells were Leu-4+ T cells, and in five of six patients the majority were Leu-3+. These studies demonstrate that the melanoma-bearing patient raises an immune response against autologous tumor and presents a method for the generation of human lymphocytes with antitumor reactivity that may be useful in the adoptive immunotherapy of tumors.     

Abnormalities of T cells isolated from mediastinal lymph nodes and peripheral blood of patients with lung carcinoma: deficit in PHA-induced expression of HLA class II antigens and in autologous mixed lymphocyte reactions

Summary A reduced percentage of T cells isolated from mediastinal lymph nodes and peripheral blood of patients with lung carcinoma acquired HLA Class II antigens following in vitro stimulation with PHA. Furthermore T cells were functionally abnormal in autologous and allogeneic mixed lymphocyte reactions (MLR). The immunoregulatory properties of HLA Class II antigens and autologous MLRs suggest that these abnormalities may affect the interaction of the host's immune system with tumor cells.     

Effects of sodium periodate modification of lymphocytes on the sensitization and lytic phases of T cell-mediated lympholysis.

Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.