Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro cytotoxic immune response

The potential existence of down-regulation of cytotoxic immune response against an autologous human melanoma line was investigated as a possible explanation for cytotoxic unresponsiveness against the autologous melanoma cells. The melanoma cell line, PJ-M, was established and lymph node resident lymphocytes (LNL) were isolated from a lymph node which was partially infiltrated with the melanoma cells. Autologous peripheral blood lymphocytes (PBL) were sensitized in in vitro co-culture (IVC) against radiated PJ-M cells in the presence or absence of PJ-M-sensitized LNL and enriched suppressor (OKT8+) or inducer (OKT4+) LNL populations, and were assayed for cytotoxicity in a 4-hr 51Cr-release microcytotoxicity assay. Significant cytotoxic response against PJ-M could be generated in the PBL, but not in the LNL. The addition of sensitized, unfractionated LNL, OKT8+, or OKT4+ LNL populations abrogated cytotoxic response in the PBL against PJ-M. The suppression of cytotoxic response was induced selectively against the PJ-M targets, because IVC of PBL in the presence of the sensitized LNL did not affect the generation of polyclonal cytotoxic alloreactivities, nor did they abrogate the generation of cytotoxic response against allogeneic targets in IVC against the corresponding allogeneic targets. These results suggest the possibility that cytotoxic immune response against the autologous melanoma cells might have been suppressed by the individual's own immunoregulatory circuit.     

Autologous melanoma-induced activation of regulatory T cells that suppress cytotoxic response

The host immune response toward autologous human cancer is subject to regulation by the immunoregulatory network. We show that certain CD4+ T cell clones, derived from melanoma involved lymph node lymphocytes and from PBL stimulated by autologous melanoma cells, selectively down-regulated the induction of cytotoxic immune response of PBL against the respective autologous melanoma cells in two autologous systems. In both systems, only the generation of cytotoxic response against the autologous melanoma cells were suppressed. Cytotoxic response against EBV-infected autologous lymphoblastoid cell line in one case and cytotoxic responses against allogeneic targets in the other were not affected. In addition to suppressor activity selectively expressed against the autologous melanoma cells, the T cell clones up-regulated their Tac receptors when cocultured with the autologous melanoma cells and APC. These results support the existence of a putative tumor Ag-driven activation of regulatory T cells that affect cytotoxic immune response, in vitro, against autologous human melanoma.     

Cell-mediated amplification and down regulation of cytotoxic immune response against autologous human cancer

The cytotoxic host immune response toward autologous human cancer may be regulated by the immunoregulatory network. Here we show that helper T cells, cloned from peripheral blood lymphocytes that were sensitized in vitro against an autologous human malignant paraganglioma, proliferated against and made interleukin 2 when cocultured with the tumor-associated antigen in the presence of autologous accessory cells. Furthermore, the helper cell clones amplified cytotoxic immune response by peripheral blood lymphocytes against the paraganglioma cells in coculture with the blood lymphocytes and the paraganglioma cells. An autologous T cell line bearing suppressor phenotype, established from a lymph node that had been infiltrated with the paraganglioma tumor cells, in contrast to the helper cells, selectively suppressed the cytotoxic immune response by the blood lymphocytes against the paraganglioma cells in identical coculture. These results, therefore, demonstrate the existence of cell-mediated immunologic regulations of the cytotoxic immune response (concurrent amplification and suppression in the same host) against an autologous human tumor.     

Two distinct cytotoxic T lymphocyte subpopulations in patients with Vogt-Koyanagi-Harada disease that recognize human melanoma cells

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.     

The therapeutic effect of OK-432-combined adoptive immunotherapy against liver metastases from gastric or colorectal cancers

Twenty-four patients with liver metastases from gastric or colorectal cancer were treated with OK-432-combined adoptive immunotherapy (AIT). Lymphocytes isolated from regional lymph nodes or peripheral blood were cultured with medium containing T cell growth factor and sonicated tumor extract antigen (SE-Ag) for 9–13 days. The cultured lymphocytes were transferred mainly through the hepatic artery after the administration of OK-432, a streptococcal preparation. Sixteen of the 24 patients received a low dose of anti-cancer agents between the OK-432 injection and cell transfer. When cultured without SE-Ag, regional lymph node lymphocytes (RLNL) showed significantly (P<0.05) higher cytotoxic activity against autologous tumor cells and, on the contrary, lower cytotoxic activity against K562 than peripheral blood lymphocytes (PBL). When cultured with SE-Ag, cytotoxicity of RLNL against autologous tumor cells was nearly equivalent to that of PBL. The blastogenesis of fresh PBL to SE-Ag was significantly (P<0.05) augmented after the OK-432-combined AIT. Two patients showed complete response and 4 patients showed partial response among 19 patients who had evaluable lesions. Five patients whose liver metastases were resected were treated with OK-432-combined AIT as an adjuvant therapy. To date they are alive without recurrence in the liver.Abbreviations AIT adoptive immunotherapy - RLNL regional lymph node lymphocytes - SE-Ag sonicated tumor extract antigen     

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     

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.     

Evaluation of basic procedures for adoptive immunotherapy for gastric cancer

Peripheral blood lymphocytes (PBL) of gastric cancer patients in advanced stages showed lymphokine activated killer (LAK) activities comparable to those of healthy donors, suggesting potential applicability of LAK cells induced from PBL stimulated with recombinant interleukin-2 (rIL-2) in adoptive immunotherapy (AIT) for gastric cancer. In order to generate a large number of LAK cells from PBL, lymphocytes were cultured with both rIL-2 and phytohemagglutinin (PHA). In this culture, the numbers of cells increased to a greater extent than those in culture with rIL-2 alone but cytotoxic activity did not augment, thus suggesting that this procedure would not afford sufficient clinical effects. On the other hand, a large number of LAK cells with high anti-tumor activities were efficiently induced from spleen cells of the patients by culture of rIL-2; hence clinical usefulness of these LAK cells is anticipated. In regional lymph node lymphocytes (RLNL) cultured with rIL-2, the cytotoxic activities were lower than in those induced in PBL, and a characteristic increase of CD8 + CD11 + suppressor T cells was observed after incubation with rIL-2. Nevertheless, an increase of CD4 + 4B4⁺ helper inducer T cells was also observed in RLNL after the culture with rIL-2. Furthermore, high cytotoxic activities were induced in RLNL in some cases in which metastasis to the regional lymph nodes was not detected. When gastric cancer patients were pretreated with biological response modifiers (BRM), especially with Lentinan, LAK cells from PBL showed higher NK and LAK activities as compared with those of patients without BRM pretreatment.This work was partly supported by a grant from Hokkoku Cancer Research Foundation.     

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dentritic cell/melanoma cell hybridomas

Due to their central role in controlling immunity, dendritic cells are logical targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma cells, both of which were derived from patients with metastatic malignant melanoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interleukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activity and released Th-1-associated, but not Th-2-associated, cytokines. Furthermore they showed effective anti-melanoma activity, resulting in death of 70 +/- 9% of autologous melanoma cells. After depletion of CD4+ cells from the mixed population of primed T lymphocytes, the remaining CD8+ cells were able to kill 63+/-8% of autologous melanoma cells. Following depletion of CD8+ cells, however, the cytotoxic capacity of the remaining T lymphocytes caused death in only 32+/-6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytotoxic activity of primed T cells. These findings strongly suggest that hybridomas deliver melanoma-associated antigens via MHC class I molecules to T lymphocytes, resulting in the generation of CD8+ cytotoxic T lymphocytes with effective anti-melanoma activity in vitro. The data may serve as a basis for the use of hybridomas in the immunotherapy of malignant melanoma in vivo.     

The absence of lymphoid suppressor cells in tumor-involved lymph nodes of patients with head and neck cancer

Patients with head and neck cancer often have decreased local or regional immunocompetence. Lymphocytes obtained from tumor-involved or -uninvolved lymph nodes (LNL) of these patients showed low or undetectable levels of antitumor cytotoxicity and low proliferative responses in vitro to interleukin 2 (IL2) or mitogens in comparison to peripheral blood lymphocytes (PBL). Lymphokine-activated killer (LAK) cell activity of LNL was lower (P less than 0.05) than that of autologous PBL. Fresh LNL were neither enriched in cells with the CD8+ CD11b+ "suppressor" phenotype nor did they suppress proliferative or cytotoxic responses of autologous PBL in mixing experiments. LNL did not inhibit LAK cell generation from autologous PBL in the presence of IL2. Also, no evidence for the inhibition of autotumor-restricted responses by IL2-activated LNL was obtained. Spontaneous or in vitro-induced production of IL1 beta. TNF alpha, and IFN-tau was low or undetectable in LNL from tumor-involved and -uninvolved lymph nodes in comparison to that in normal or autologous PBL. Mitogen-induced IL2 production was normal in LNL. The depressed ability to produce certain cytokines may be in part responsible for a state of unresponsiveness present in lymph nodes obtained from patients with head and neck cancer. No evidence for the presence of lymphoid suppressor cell in LNL of these patients was obtained.     

Target level blocking of T-cell cytotoxicity for human malignant melanoma by monoclonal antibodies

Cytotoxic T lymphocytes (CTL) for autologous malignant melanoma in culture of a patient AV were induced by restimulation of PBL (peripheral blood leukocytes) with AV melanoma cells in vitro and subcultured in interleukin 2 (IL-2) conditioned media. Monoclonal antibodies detecting six antigenic systems on melanoma cell surfaces were tested for blocking activity on the effector function of subcultured cytolytic T lymphocytes for autologous melanoma cells. The monoclonal antibodies R₂₄ (γ3), specific for the G_D3 disialoganglioside on melanoma cell surfaces and I₂₄ (γM), detecting a similar antigenic determinant, blocked autologous T lymphocytotoxicity for malignant melanoma cells on the target level. The effector function of alloantigen activated cytolytic T lymphocytes generated by coculture of allogeneic PBL with Epstein-Barr virus (EBV) transformed AV B lymphocytes, was blocked by monoclonal antibody R₂₄ when tested against AV melanoma targets, but not when tested against AV B lymphocyte targets. It is concluded that blocking by mAb R₂₄ occurs in this system as a nonspecific effect, unrelated to the specific target antigen recognition by cytotoxic T lymphocytes. Steric hindrance or antibody induced membrane changes may account for the blocking effect of monoclonal antibody R₂₄.     

Major histocompatibility complex class II antigen expression on leucocyte subpopulations in the draining lymph node and tumour in the early phase of bacillus-Calmette-Guérin-induced tumour regression

Summary We investigated the cellular composition and the major histocompatibility complex (MHC) class II antigen expression in the draining lymph node and the tumour during potentiation of the immune response by intralesional bacillus Calmette-Guérin (BCG) administration in the line 10 hepatocellular carcinoma in the strain 2 guinea-pig. Five days after its injection BCG induced a ninefold increase in the number of draining lymph node cells and an increased MHC class II expression. This increased MHC class II expression was mostly due to the selective increase of B cells in the lymph nodes, and to a lesser extent to the increase of T cells expressing MHC class II antigens. Taking into account this nine-fold increase, intralesional treatment of BCG increased considerably the number of T helper/inducer (anti-CT7) and T suppressor/cytotoxic (anti-CT6) lymph node cells expressing MHC class II antigen. The percentage of tumourinfiltrating T cells expressing MHC class II antigen in the tumour was higher than the percentage of T cells in the regional draining lymph node of non-treated guinea-pigs, indicating the presence of activated T cells in the tumour. After treatment with BCG no further increase in MHC class II expression was measured in the tumour, nor was any phenotypical change of the tumour-infiltrating T cells found. In conclusion, with the use of two-colour flow cytofluorometry we have shown that the potentiation of the already existing immune response to line 10 is accompanied by a considerable increase in T helper/inducer, T suppressor/cytotoxic cells and MHC class II antigen in the regional lymph node. Whether this is essential for the potentiation of the immune response causing tumour regression and long-lasting immunity is a subject for further study.     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

Natural cytotoxic effector cell activity against Shigella flexneri-infected HeLa cells

Virus and facultative intracellular bacteria both replicate within a host cell. The recognition and killing of virus-infected cells by natural killer (NK) cells is thought to be an important host immune function. However, little is known about immune recognition of bacteria-infected cells. In this report, we show for the first time that human peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL) purified from PBL have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. This cytotoxic activity was dependent on bacterial invasion of the HeLa cells, because HeLa cells pretreated with a noninvasive isogenic variant of S. flexneri or soluble bacterial products were not killed. Pretreatment of PBL with interleukin 2 (IL 2) or interferon-alpha greatly enhanced the cytotoxic activity of PBL against Shigella-infected HeLa cells. Cytotoxic activity present in PBL or in PBL pretreated with IL 2 was shown to be associated with both Leu-11+ and Leu-11- cell populations. These results suggest that NK cell killing of bacteria-infected cells may play an important role in host defense against facultative intracellular bacterial infections.     

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)     

CD4+ Th0 cell clones,isolated from a metastatic lymph node of a melanoma patient,possess cytolytic function

In the present study T lymphocytes isolated from a metastatic lymph node (T-LNL) of a melanoma patient have been cloned. In the attempt to verify whether T-LNL may acquire in vitro functional activities in the absence of tumour-associated antigens, they were cloned utilizing allogeneic lymphocytes as feeder cells. Nineteen clones generated from T-LNL proved to be CD4+ and, among these, five were able to kill autologous and allogeneic human melanoma cells in HLA-class-II-restricted way. On the basis of their cytokine production, these CD4+ cytolytic T-LNL clones were shown to belong to the Th0 subset and three of them expersseed the V17 chain of the T cell receptor. These results suggest the presence of melanomaspecific but functionally inactive lymphocytes with T cell receptor oligoclonality in the lymph node environment. These specific T cells may acquire in vitro the capacity to kill autologous and allogeneic tumours without any induction by autologous melanoma cells.     

Natural cytotoxicity of lymphocytes from lymph nodes draining breast carcinoma and its augmentation by interferon and OK432

Summary Lymphocytes isolated from axillary lymph nodes draining breast carcinoma were tested for natural killer (NK) activity against K562 in a 4-h ⁵¹Cr-release assay, and the in vitro effects of interferon (IFN) and OK432 (a streptococcal preparation) on their cytotoxicity were examined in comparison with NK activity of autologous peripheral blood lymphocytes (PBL). The levels of NK activity were lower in lymph node lymphocytes (LNL) than in PBL of the same patients. Significant levels of LNL-mediated lysis were recorded in 14 of 42 (33%) lymph node samples and in nine of 14 (64%) patients. Purification of large granular lymphocytes (LGL) from lymph node cells by discontinuous Percoll density gradient centrifugation resulted in an induction or enhancement of cytotoxic activity, with no reactivity in LGL-depleted, small T-lymphocyte populations. Positive reactions were observed with 10 of 13 (77%) LGL samples. The low reactivity of LNL was not attributable to coexistent suppressor cells for NK function, since lymph node cells failed to suppress NK activity of normal PBL. Partially purified human IFN and OK432 augmented NK activity of patients' PBL in approximately 70% and 90% of the cases, respectively, while LNL-mediated lysis was augmented in only 7% and 36% of the lymph node samples by IFN and OK432, respectively. These results indicate that K562-reactive NK cells and/or their precursors may frequently be present at subthreshold levels in the lymph nodes draining breast carcinoma, and that the augmentation of LNL-mediated cytotoxicity by OK432 might provide a local potentiation of natural immune function at the host-tumor interface rather than IFN.     

Abnormalities of the in vitro cellular and humoral responses to tetanus and influenza antigens with concomitant numerical alterations in lymphocyte subsets in Down syndrome (trisomy 21)

Peripheral blood leukocytes (PBL) from noninstitutionalized individuals with trisomy 21, paired with closely age-matched and/or family members as controls, were analyzed for different aspects of their cellular and humoral immune responses, and were phenotypically characterized by means of various monoclonal antibodies. Both the in vitro PBL proliferative and antibody responses to a bacterial antigen (tetanus toxoid) and to viral antigens (influenza A/Bangkok and B/Singapore) were significantly decreased in trisomy 21. In addition, bacterial and viral antigen-induced in vitro interleukin 2 (IL 2) production was markedly reduced, although mitogen (PHA)-stimulated IL 2 production was not impaired. The functional abnormalities observed in trisomy 21 PBL occur concomitantly with numerical alterations in circulating lymphocyte subsets in these same individuals. Although no difference was observed between the trisomic and control groups in the percentage of total T and B lymphocytes, a decreased level of Leu-3a + 3b-positive cells (T helper/inducer cells) and an increased level of Leu-2a-positive cells (T suppressor/cytotoxic cells) that co-expressed Leu-15 (suppressor alone) were noted.     

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.