Autologous Mixed Lymphocyte Tumor Cell Research Articles

HLA class I loss in metachronous metastases prevents continuous T cell recognition of mutated neoantigens in a human melanoma model.

T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient's HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1G161S, INSIG1S238F, MMS22LS437F and PRDM10S1050F, were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy.

Abstract PR11: Neo-antigen landscape dynamics during human melanoma-T cell interactions

Abstract Recognition of the neo-antigens that are formed as a consequence of DNA damage is likely to be a major driving force behind the clinical activity of T cell checkpoint blockade and adoptive T cell therapy for treatment of non-virally induced solid cancers. Consequently, strategies to selectively enhance T cell reactivity against genetically defined neo-antigens are currently under development. In mouse models, T cell pressure has been shown to sculpt the antigenicity of tumors through selection of tumor cell variants that do not express the relevant neo-antigens. However, it is unclear whether the T cell-recognized neo-antigen repertoire is constant over time in human cancers. To address this issue, we analyzed the stability of neo-antigen specific T cell responses and the antigens they recognize in stage IV melanoma patients treated by adoptive T cell transfer. Fresh tumor tissue resected before and after treatment was used to establish autologous melanoma cell lines, to culture tumor-infiltrating lymphocytes (TIL) and to generate T-cell infusion products by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Material from 2 patients with objective clinical responses after infusion of autologous tumor-specific CD4+ and CD8+ T cells was analyzed by whole exome and RNA sequencing. Subsequently, 31-mer peptides covering all the non-synonymous somatic mutations were used to test the reactivity of both the T-cell infusion products and TIL isolated from subsequent tumor lesions by ELISA and flow cytometry. The expression of genes encoding T cell-recognized neo-antigens was then analyzed within the tumor cell lines and within the corresponding paraffin-embedded tumor tissue, by real-time PCR analysis, a competitive allele-specific PCR assay, and by analysis of heterozygosity and next generation amplicon sequencing. Analysis of the infusion products showed that 50-80% of T cells reacted to autologous tumor cells, with only a very low percentage of these cells recognizing known shared tumor-associated epitopes. Instead, tumor reactivity was predominantly mediated by CD4+ and CD8+ T cells recognizing clonally expressed neo-antigens formed by private mutations. CD8+ T cells within the infusion products and within TIL populations generated from subsequent tumor lesions of these two patients recognized a total of 6 different neo-antigens, and analysis of these neo-antigens in both the index lesion and subsequent lesions revealed profound alterations in the T cell-recognized neo-antigen landscape. In particular, in 2 cases, the mutant allele was lost from a subsequent tumor, in one case, expression of the mutant gene was substantially reduced in a recurring lesion, and in another case expression of the mutant gene was increased approx. 40-fold in a subsequent tumor. Notably, in 4 out of 4 cases these changes in neo-antigen expression were paralleled by loss or gain of the respective neo-antigen specific T cell response. Collectively, our data demonstrate that under conditions in which a high frequency of tumor-specific T cells is present, tumor cell variants with reduced or lost neo-antigen expression can emerge, similar to what has been observed in mice. This, and the observation of concurrent acquisition of novel T cell reactivity, reveal the dynamics of T cell - neo-antigen interaction in human cancer, and indicate that immunotherapies that maximize the capacity to respond to an altering neo-antigen landscape may offer the highest chance to achieve long-term tumor control. Citation Format: Els M.E. Verdegaal, Noel de Miranda, Marten Visser, Tom Harryvan, Marit van Buuren, Rikke Andersen, Sine Hadrup, Caroline van der Minne, Remko Schotte, Hergen Spits, John Haanen, Ellen Kapiteijn, Ton Schumacher, Sjoerd H. Van Der Burg. Neo-antigen landscape dynamics during human melanoma-T cell interactions [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR11.

Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha

A phase I/II study was conducted to test the feasibility and safety of the adoptive transfer of tumor-reactive T cells and daily injections of interferon-alpha (IFNα) in metastatic melanoma patients with progressive disease. Autologous melanoma cell lines were established to generate tumor-specific T cells by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Ten patients were treated with on average 259 (range 38–474) million T cells per infusion to a maximum of six infusions, and clinical response was evaluated according to the response evaluation criteria in solid tumors (RECIST). Five patients showed clinical benefit from this treatment, including one complete regression, one partial response, and three patients with stable disease. No treatment-related serious adverse events were observed, except for the appearance of necrotic-like fingertips in one patient. An IFNα-related transient leucopenia was detected in 6 patients, including all responders. One responding patient displayed vitiligo. The infused T-cell batches consisted of tumor-reactive polyclonal CD8+ and/or CD4+ T cells. Clinical reactivity correlated with the functional properties of the infused tumor-specific T cells, including their in vitro expansion rate and the secretion of mainly Th1 cytokines as opposed to Th2 cytokines. Our study shows that relatively low doses of T cells and low-dose IFNα can lead to successful treatment of metastatic melanoma and reveals a number of parameters potentially associated with this success.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-011-1004-8) contains supplementary material, which is available to authorized users.

The response of autologous T cells to a human melanoma is dominated by mutated neoantigens

Our understanding of pathways leading to antitumor immunity may depend on an undistorted knowledge of the primary antigenic targets of patients' autologous T cell responses. In the melanoma model derived from patient DT, we applied cryopreserved short-term autologous mixed lymphocyte-tumor cell cultures (MLTCs) in combination with an IFN-gamma enzyme-linked immunospot (ELISPOT) assay to cDNA expression screening. We identified three previously unknown peptides processed from melanosomal proteins tyrosinase (presented by HLA-A(*)2601 and -B(*)3801) and gp100 (presented by HLA-B(*)07021) and five neoantigens generated by somatic point mutations in the patient's melanoma. The mutations were found in the genes SIRT2, GPNMB, SNRP116, SNRPD1, and RBAF600. Peptides containing the mutated residues were presented by HLA-A(*)03011, -B(*)07021, and -B(*)3801. Mutation-induced functional impairment was so far demonstrated for SIRT2. Within MLTC responder populations that were independently expanded from the patient's peripheral blood lymphocytes of different years, T cells against mutated epitopes clearly predominated. These results document a high degree of individuality for the cellular antitumor response and support the need for individualizing the monitoring and therapeutic approaches to the primary targets of the autologous T cell response, which may finally lead to a more effective cancer immunotherapy.

Cytolytic T Lymphocytes Recognize an Antigen Encoded by MAGE-A10 on a Human Melanoma

Abstract From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254–262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.

Derivation of Tumor-specific Cytolytic T-Cell Clones from Two Lung Cancer Patients with Long Survival

We derived lung carcinoma cell lines from tumor material resected from a patient with small-cell lung cancer (SCLC) and from a patient with non-small-cell lung cancer (NSCLC). The patient with NSCLC was vaccinated with irradiated autologous tumor cells. The two patients enjoyed an exceptionally favorable clinical evolution and are currently without signs of cancer 10 and 8 yr after their diagnoses, respectively. Autologous mixed lymphocyte-tumor cell cultures (MLTC) were produced with blood lymphocytes stimulated with irradiated autologous tumor cells. The first patient's SCLC cells, which carried a small amount of human leukocyte antigen (HLA) class I molecules, were incubated with interferon-gamma (IFN-gamma) before being used as stimulator cells. A cytolytic T-lymphocyte (CTL) clone was derived that specifically lysed the IFN-gamma-treated SCLC cells but did not lyse untreated tumor cells or autologous lymphoblasts. Clones of autologous tumor-specific CTL, directed against the NSCLC cells of the other patient, were also obtained. These tumor cells carried a higher level of HLA class I molecules and were lysed by the CTL without incubation with IFN-gamma. Altogether, these results indicate that SCLC and NSCLC cancer cells can be recognized by autologous CTL, and might therefore be susceptible to specific immunotherapy.

Introduction of adhesive and costimulatory immune functions into tumor cells by infection with Newcastle Disease Virus.

We demonstrate in this study that infection of tumor cells by Newcastle Disease Virus (NDV) leads to changes in tumor cell surface adhesiveness and tumor immune costimulatory function. While adsorbtion of virions to the cell surface occurs after short-term (10 min) incubation and leads to cells expressing viral antigens at low antigen density (LAD), viral replication in the cytoplasm occurs within 5-24 h leading to tumor cells expressing viral antigens at high antigen density (HAD) as shown by quantitative FACS flow cytometry. Virus infected tumor cells showed an increased adhesiveness for erythrocytes and lymphocytes. When IL-2 preactivated human lymphocytes with cytotoxic potential were coincubated with 51Cr-labeled NDV-infected or non-infected human colon carcinoma cells increased lysis of the virus infected targets was observed. The virus mediated cell adhesion could be inhibited by monoclonal antibody (mAb) against the hemagglutinin-neuraminidase (HN) molecule but not by antibody against the fusion protein. HN cDNA transfectants also mediated increased lymphocyte adhesion in comparison to wild-type or neo-vector transfected control cells. Further experiments demonstrated that not only the adhesion domain of HN but also the neuraminidase plays a role in cell-cell interactions. A comparison of an NDV neuraminidase mutant of the strain Australian Victoria (AV-L1) with the parental AV strain revealed pronounced differences in their capacity to mediate lymphocyte binding and costimulatory activity. The mutant with highly decreased neuraminidase activity was very similar to NDV Ulster in adhesive and costimulatory activity while the parental line with high neuraminidase activity was negative for both functions. Costimulatory effects of NDV Ulster and AV-L1 were revealed when virions and suboptimal concentrations of anti-CD3 mAbs were coated to microtiter plates for induction of murine CD4 T cell proliferation. In human autologous mixed lymphocyte-tumor cell cultures up-regulation of T cell activation markers CD69 and CD25 was seen with NDV modified but not with non-modified tumor cells.

Relative roles of natural killer- and T cell-mediated anti-leukemia effects in chronic myelogenous leukemia patients treated with interferon-alpha.

Potential anti-leukemia effects mediated by T cells or by natural killer (NK) cells were investigated in chronic myelogenous leukemia (CML) patients treated with interferon-alpha. Therapy-associated modulation of T cell and NK reactivity was monitored for one year from initiation in autologous mixed lymphocyte-tumor cell reactions and cytotoxicity directed against autologous CML cells, respectively. During the course of IFN-therapy, NK activity against autologous CML cells increased steadily, whereas T cell reactivity fluctuated randomly. Despite the high level of T cell reactivity to autologous tumor cells in short-term (6 days) culture, 1) they failed to respond to synthetic peptides corresponding to the bcr/abl fusion sequence of the patient, and 2) only one proliferative T cell clone (TCC) was isolated which specifically recognized HLA-DR-matched CML cells. This TCC appeared not to recognize synthetic peptides corresponding to the bcr/abl fusion sequence of the patient; the antigen to which it responds remains unknown. To assess potential immunogenicity of bcr/abl peptides, it was attempted to sensitize T cells from normal donors in vitro. Of 109 cell lines obtained from seven different donors, eleven showed peptide-dependent proliferation. Therefore, although these results show that it is possible to isolate apparently CML-specific T cells from patients, as well as to prime T cells against tumor-specific peptide in vitro, the frequency of such T cell-mediated reactivity appears low and its relevance to anti-leukemic effects questionable. On the other hand, the strong time-dependent enhancement of natural killing of autologous CML blasts during IFN-alpha treatment, a phenomenon not observed for T cell reactivity, suggests that natural immunity may be more important in controlling disease.

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.

Genes Coding for Tumor Antigens Recognized by Human Cytolytic T Lymphocytes

In order to define the antigens recognized by cytolytic T lymphocytes (CTLs) on autologous tumors, we derived tumor-specific CTL clones from autologous mixed lymphocyte tumor cell cultures. The gene coding for a tumor rejection antigen expressed on a melanoma was isolated by transfecting genomic DNA of the tumor into an antigen-loss variant of the melanoma. Transfectants were identified on the basis of their ability to stimulate tumor necrosis factor release by the CTL clone. The gene that transferred the expression of the antigen was named MAGE-1. It is a new gene, silent in normal tissues with the exception of testis, but expressed in several types of tumors. The antigen recognized by the CTL clone is a nonapeptide derived from the protein encoded by gene MAGE-1, and presented by the HLA class I molecule HLA-A1. Using two other antimelanoma CTL clones, we identified the tyrosinase gene as coding for an antigen presented by HLA-A2 on this type of tumors. The identification of these tumor rejection antigens open new possibilities for the specific immunotherapy of cancer.

MHC class-I-restricted auto-tumor-specific CD4+CD8- T-cell clones established from autologous mixed lymphocyte-tumor-cell culture (MLTC).

Autologous mixed lymphocyte-tumor cell cultures (MLTC) were initiated with cytokine (IFN gamma and TNF alpha)-treated ex-vivo tumor cells of lung, ovarian, breast and stomach carcinomas. The cytokine-treated tumors expressed class-I but not class-II molecules. Although the proportion of CD8+ lymphocytes increased in the bulk culture of MLTCs, in 5/7 experiments the majority of the established T-cell clones were CD4+. Among the CD8+ clones a high proportion (77%) was cytotoxic, while the proliferative response was more frequent among the CD4+ clones (70%). In 4/26 cytotoxic T-lymphocyte (CTL) clones (3/17 CD4+ and 1/9 CD8+), derived from a patient with class I+ class II- stomach carcinoma, lysis was restricted to the autologous tumor cells. These auto-tumor-specific clones did not lyse the autologous ConA blasts, the 5 allogeneic ex-vivo tumors, the NK-sensitive K562 or the relatively sensitive Daudi cells. The cytotoxicity of these clones was inhibited by pre-incubation of the tumor cells with W6/32 (alpha-class I) MAb, or by preincubation of the lymphocytes with OKT3 (alpha-CD3) MAb. The alpha-CD4 (OKT4) MAb had only a marginal effect on the CD4+ clones, while the lytic function of the CD8+ clone was inhibited by the alpha-CD8 (OKT8) MAb. The 3 CD4+ CTL clones also responded with proliferation to the autologous tumor cells. This proliferative response was inhibited by the presence of W6/32 MAb. Our results indicate that the auto-tumor lysis exerted by CD4+ CTL clones was restricted by the class-I antigens, and that the CD4 molecules of the clones were not essential for the lytic interaction.

Clonal expansion of T cells following in vitro stimulation with autologous melanoma cells and interleukin-2 studied by molecular analysis of a T cell receptor

Twelve autologous mixed peripheral blood (PBL) tumor cell interactions (MLTC) followed by in vitro expansion of the stimulated T cells in recombinant interleukin-2 (IL-2) were analyzed for the potential emergence of oligoclonal or monoclonal T cell populations in the PBL by Southern blot analysis of the T cell receptor (TCR) beta gene. The emergence of oligoclonal or monoclonal TCR beta gene rearranged populations was seen in 5 of the 12 cases. In 2 of these 5 cases only one dominantly rearranged band was observed. The emergence of oligoclonal or monoclonal T cell populations following stimulation with autologous melanoma cells was associated with predominant CD4 phenotype of the stimulated PBL exhibiting a varied degree of cytotoxicity toward the respective autologous melanoma cells. The evidence of emergence of monoclonal or oligoclonal T cell populations following stimulation with autologous tumor cells strongly supports the existence of T cell-mediated responses against autologous melanomas. Furthermore, cellular and molecular analyses of T cell responses in autologous mixed lymphocyte tumor cell interactions will provide valuable information on the nature of the T cell responses and on the pattern of gene segment usages by the T cells in response to the autologous tumor cells.

Auto-tumor lysis by blood lymphocytes in vitro. Strongly activated lymphocytes lack selectivity.

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.

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.

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 gamma) and tumor necrosis factor (TNF alpha) 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.

Arginine, protein malnutrition, and cancer

The amino acid arginine has anabolic and immunostimulatory properties. This study evaluated the potency of arginine in limiting the severe nutritional and immunological insults of protein calorie malnutrition and increasing tumor load. In protein-depleted A/J mice ( n = 340) bearing either an immunogenic (C1300) or poorly immunogenic (TBJ) neuroblastoma, arginine supplementation [1%] significantly augmented T lymphocyte responses (mitogenesis, interleukin-2 production) compared with both a glycine-supplemented and nonsupplemented group. Arginine supplementation significantly retarded the growth of C1300 and prolonged median host survival. These results correlated with augmented autologous mixed lymphocyte tumor cell responses and enhanced specific cytotoxicity. This anti-tumor effect was not demonstrated in mice bearing TBJ where both arginine and glycine stimulated tumor growth compared with nonsupplemented mice. There was no significant difference between arginine and glycine in preservation of carcass weight. These studies suggest that the immunostimulatory effects of arginine are not due to supplemental nitrogen and that an associated antitumor effect is dependent on tumor antigenicity.

Suppressor cells in the effector phase of autologous cytotoxic reactions in cancer patients.

Cytotoxicity was induced in lymphocytes (CL) from 10 out of 15 patients by autologous mixed lymphocyte tumor cell culture and further cultivation with recombinant interleukin-2. In cells from 3 of the 10 patients, cytotoxicity was suppressed by more than 50% when autologous peripheral blood mononuclear cells (PBMC) from the patients with large tumors were added to the autologous killing system. The cells responsible for suppressing the cytotoxicity in the effector phase were adherent or nonadherent to plastic depending on the patient examined. The T cell fraction from 1 patient significantly suppressed the cytotoxic activity, and this suppression was seen only in the autologous system. On the other hand, plastic adherent cells but not T cells from PBMC of 2 subjects suppressed the cytotoxic activity of CL. The reason why the main cell population suppressing the CL activity differed among the patients is unclear. However, the findings that the suppression was mostly abrogated following resection of the tumor mass suggested that suppressor cells, either of macrophage lineage or T cells, are induced in patients with a large tumor mass. This speculation is supported by the finding that the PBMC from a patient with tumor recurrence regained the suppressive activity.

Lysis of autologous tumor cells by blood lymphocytes activated in autologous mixed lymphocyte tumor cell culture--no correlation with the postsurgical clinical course.

Mixed lymphocyte tumor cell cultures (MLTC) were initiated with cells collected at the time of surgery from 62 patients with the following diagnoses: 12 squamous cell carcinoma, 14 adenocarcinoma of the lung, 17 osteosarcomas, and 16 soft tissue sarcomas. The lytic effect generated against autologous tumor cells was tested on the 7th day. These patients had been part of previous evaluation, which revealed that the lysis of autologous tumor cells by freshly collected lymphocytes correlated with the postsurgical clinical course. Patients with long survival exhibited autotumor lysis without previous activation. Blood lymphocytes of about half of the primarily nonreactive patients were stimulated for lytic function in MLTC. However, this parameter showed no correlation with the clinical course.

Differences of Sensitivity to Autologous Cytotoxic Lymphocytes Between Primary Tumor and its Cervical Lymph Node Metastases

Antigenic differences between primary tumors and their cervical lymph node metastases of 12 patients with head and neck cancers were examined by measuring their sensitivity to cytotoxic lymphocytes (CL). Cytotoxicity was induced by autologous mixed lymphocyte (CL). Cytotoxicity was induced by autologous mixed lymphocyte tumor cell culture (MLTC), and further cultivation with recombinant interleukin-2 (rIL-2). The effector cells which were used in this study consisted of OKT3+8+ and OKT3+4+ subpopulations. Their cytotoxic nature was different from lymphokine activated killer cell (LAK cell) activity. Cytotoxicity of CLs stimulated by autologous primary tumor cells (CLP) was observed in 7 out of 12 patients (58.3%). In contrast, cytotoxicity of CLs stimulated by metastatic tumor cells (CLM) was observed in 4 out of 12 patients (33.3%). In the cases in which both CLP and CLM were successfully induced, cross-reactivity tests and cold target inhibition tests were performed. These results suggested that a reduction in immunogenicity had occurred at the metastatic site, and sensitivity against autologous CL was different between primary and metastatic tumor cells.

Generation of Human Cytotoxic T Lymphocytes Against Autologous Solid Tumors by Autologous Mixed Lymphocyte Tumor Cell Culture with T Cell Growth Factor

Autologous mixed lymphocyte tumor cell cultures (MLTC) generated cytotoxicity against autologous tumors in 5 out of 15 (33%) patients with head and neck cancer. Further cultivation of MLTC-activated lymphocytes with T cell growth factor (TCGF) resulted not only in the propagation of cytotoxic T lymphocytes (CTL) and an increase of cytotoxicity against autologous tumors, but also in the induction of killer cells against allogeneic tumor cells. The results of crisscross tests using fresh tumor cells from 17 donors and lymphocytes from 10 donors indicate that cultivation of MLTC-activated lymphocytes with TCGF generated killer cells cytotoxic for allogeneic tumors in most cases, but not for autologous or allogeneic phytohemagglutinin-induced lymphoblasts. Further, the results of a cold target inhibition test undertaken in an autologous tumor killing system suggest that at least 2 different subsets, specifically cytotoxic for autologous tumors and cytotoxic for both autologous and allogeneic tumors, were developed in the CTL induced by autologous MLTC followed by cultivation with TCGF. The phenotypes of the effector cells were shown to be OKT3+, Leu2a+, OKT8+, OKM1-, and Leu7- by blocking assay using complement and monoclonal antibody against the T cell surface marker.

Generation of human cytotoxic T lymphocytes against fresh autologous and allogeneic solid tumors by mixed lymphocyte tumor cell culture with T cell growth factor.

Autologous mixed lymphocyte tumor cell culture (MLTC) induced cytotoxic lymphocytes against autologous tumor cells in 4 out of 14 donors (29%). Further cultivation of MLTC-activated lymphocytes with T cell growth factor (TCGF) resulted not only in the propagation of cytotoxic T lymphocytes (CTL) and an increase of cytotoxicity against autologous tumors, but also in the induction of killer cells against allogeneic tumor cells. Briefly, the results of crisscross tests using fresh tumor cells from 17 donors and lymphocytes from 10 donors indicate that cultivation of MLTC-activated lymphocytes with TCGF generated killer cells against allogeneic tumor cells in most cases, but not against autologous or allogeneic phytohemagglutinin-induced lymphoblasts. These effector cells were mainly OKT3+, OKT8+, OKM1- and Leu7-. Further, the results of cold target inhibition tests undertaken in an autologous tumor killing system suggest that at least 2 different subsets, specifically cytotoxic for autologous tumors and cytotoxic for both autologous and allogeneic tumors, were developed in the CTL induced by autologous MLTC followed by cultivation with TCGF.