Mixed Lymphocyte Tumor Cell Culture Research Articles

Characterization of Renal-Cell Carcinoma (RCC)-Reactive Cytotoxic T-Lymphocyte Responses Generated from Peripheral Blood of HLA-Matched Sibling and Unrelated Healthy Individuals in Vitro.

Although current allo-transplantation therapy can induce considerable graft-versus-tumor (GvT) effects in RCC patients, it is also accompanied by severe, even life-threatening side effects, mainly due to graft-versus-host disease (GvHD). Efforts aiming to improve the specificity and efficiency of allogeneic cell therapy in this disease (e.g. specific donor lymphocyte infusion or vaccination) will certainly benefit from the identification of potential anti-tumor effector mechanisms and their corresponding target structures. We recently demonstrated that RCC-reactive cytotoxic T-lymphocyte (CTL) clones can be isolated from peripheral blood of healthy donors matched with the RCC stimulators for HLA-class I. These CTL were found to recognize a broad panel of RCC antigens with restricted or ubiquitous tissue expression (Doerrschuck A, et al., Blood July 1, 2004, Epub).We now extended our analyses on peripheral blood mononuclear cells (PBMC) of further HLA-matched healthy sibling (1) and unrelated individuals (4) and compared these results with available autologous patient PBMC. While mixed lymphocyte/tumor-cell culture (MLTC) responders derived from allogeneic donors showed a robust antigen-dependent proliferation over several weeks, a weak if any proliferative response was seen with autologous MLTC populations. By analysing the fine specificity of MLTC-derived clonal CTL the majority of allogeneic effectors recognized exclusively their RCC stimulators, but not corresponding lymphoblastoid-cell lines or natural killer target K562. These CTL were restricted by various HLA-A, -B or -C molecules. We further isolated CTL clones that exhibit an extraordinary strong recognition of RCC and various epithelial tumor-cell lines. Antibody blocking experiments provided clear evidence that these CTL are restricted by a not yet defined HLA-Ib molecule and, simultaneously, by a NKG2D-dependent mechanism. Other rapidly proliferating CD3+ CD8+ CTL clones were obtained that showed a non-HLA-restricted reactivity against RCC and a minor but consistent reactivity against targets with low or absent HLA-class I expression (e.g. K562).In conclusion, our results demonstrate that a heterogeneous panel of RCC-reactive HLA-Ia/Ib-restricted CTL can be isolated from PBMC of HLA-class I-matched healthy individuals. Alternatively, CTL recognizing RCC in a non-HLA restricted manner can be obtained. Our observations might reflect the superior ability to activate and expand RCC-directed T cells from PBMC of allogeneic healthy donors compared to the autologous setting. At this point, we cannot conclude whether these various CTL populations contribute to effective anti-RCC immune responses occurring in vivo. Answering this question will certainly require to identify further CTL-defined target structures at the molecular level. This would allow us to analyse the expression of candidate antigens and the frequency of specific CTL in RCC patients after allogeneic blood stem-cell transplantation, and to correlate these findings with clinical GvT and GvH events.

CD8+ cytotoxic T lymphocytes isolated from allogeneic healthy donors recognize HLA class Ia/Ib–associated renal carcinoma antigens with ubiquitous or restricted tissue expression

AbstractAllogeneic hematopoietic stem cell transplantation can induce considerable tumor remissions in metastatic renal-cell carcinoma (RCC) patients. The precise effector mechanisms mediating these graft-versus-tumor reactions are unknown. We studied RCC-directed CD8+ T-cell responses in blood lymphocytes of healthy individuals matched with established RCC cell lines for HLA-class I. In 21 of 22 allogeneic mixed lymphocyte/tumor-cell cultures (MLTCs), RCC-reactive cytotoxic T-lymphocytes (CTLs) were readily obtained. From MLTCs, 121 CD8+ CTL clones with memory phenotype were isolated. Their anti–RCC reactivity was restricted by multiple classical HLA-Ia molecules, in particular by HLA-A2, -A3, -B7, -B44, -Cw7, and by a nonclassical HLA-Ib determinant. Extensive cross-reactivity analyses on a broad target panel identified CTLs that recognize antigens with expression restricted to renal tissue or to renal and colon tumors. Other CTLs were directed against antigens with broader tissue distribution being expressed in various epithelial and nonepithelial tumors or, additionally, in hematopoietic cells. With microcapillary liquid chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/TOF mass spectrometry, we identified the HLA-A*0301-associated nonpolymorphic peptide KLPNSVLGR encoded by the ubiquitously expressed Eps15 homology domain–containing 2 gene as a CTL target. Defining human RCC antigens recognized by alloreactive CTLs may allow to improve the specificity and efficiency of allogeneic cell therapy (eg, specific donor-lymphocyte infusions or vaccination) in metastatic RCC patients.

Growth and functional reactivity of lymphocytes obtained from three anatomic compartments in patients with non-small-cell lung cancer (NSCLC).

Non-small-cell lung cancer (NSCLC) claims the lives of both men and women worldwide. In this study, we describe the ability to expand lymphocytes from solid tumor biopsies, pleural fluid, and peripheral blood of patients with NSCLC. While lymphocytes readily expanded from both solid biopsies and pleural fluids, the incidence of obtaining tumor-specific lymphocytes was low. Specific cytolytic and/or cytokine-releasing activity was observed in tumor-infiltrating lymphocytes (TIL) from 3/15 solid tumor biopsies (20%) and 2 of 4 pleural fluid samples (50%). Two of the CTL derived from solid tumor were cytolytic, one being HLA-A2 restricted while the other was either restricted at HLA-A30 or - B18. One of the pleural fluid cytotoxic T lymphocyte (CTL) was also HLA-A2 restricted. Peripheral blood from NSCLC patients was studied using mixed lymphocyte tumor cell cultures (MLTC) as reported previously. Peripheral blood lymphocytes were cultured with allogeneic NSCLC-TC line 29.7, which expressed by gene transfer the lymphocyte costimulatory molecule CD80. Of 9 HLA-A2 patient samples tested, 8 gave rise to lymphocytes, which lysed NSCLC tumor cells expressing HLA-A2 in an major histocompatibility complex (MHC)-restricted fashion. While specific CTL can be generated from all anatomic sites, for clinical trials peripheral blood appears to be the site of choice for obtaining specific precursors.

Apoptotic cell death in conjunction with CD80 costimulation confers uveal melanoma cells with the ability to induce immune responses.

Uveal melanoma is a rare malignancy with a poor prognosis despite current therapeutic intervention. The current investigation focuses on the immunogenicity of uveal melanoma cells genetically modified with recombinant adenovirus encoding CD80 (AdCD80) in contrast to their parental counterpart. We demonstrate that costimulation provided by uveal melanoma cells improved immune responses in vitro as determined by mixed lymphocyte tumour cell cultures and cytotoxic T-cell assays using lymphocytes from healthy donors and uveal melanoma patients. Flow cytometry revealed T-cell stimulation by activated CD4+ and CD8+ T cells. Additionally, autologous lymphocytes proliferated in response to CD80-expressing primary uveal melanomas, indicating that this patient group is suitable for immunotherapy. Moreover, this study utilized AdCD80 modified and parental apoptotic tumour cells, loaded onto immature dendritic cells, as a source of tumour antigen. The ability of live or apoptotic tumour cells to stimulate lymphocyte proliferation and activation was determined. Apoptotic uveal melanoma cells expressing CD80 were efficient at inducing an immune response and served as a potent immunogen. The use of apoptotic uveal melanoma cells in combination with expression of costimulatory molecules could prove a novel adjuvant therapy for the treatment of this disease.

Human Urinary Bladder Transitional Cell Carcinomas Acquire the Functional Fas Ligand during Tumor Progression

The interaction between FasL on tumor cells and Fas on lymphocytes may represent a tumor immune escape mechanism. We explored FasL expression and function in human urinary bladder transitional cell carcinomas (TCCs). FasL expression was observed in situ in 45% of TCCs (n = 45) and was absent in normal urothelium (n = 20). A correlation existed between FasL expression and high tumor grade (0% in G1, 14% in G2, and 75% in G3; P < 0.0001) and stage (13% in superficial Ta-T1 versus 81% in invasive T2-T4; P < 0.0001). FasL function was shown by the ability of two FasL-positive primary culture TCC cell lines (established from two FasL-positive invasive TCCs) to induce Fas-mediated killing not only of conventional Fas-sensitive targets (such as Jurkat cells or phytohemagglutinin-lymphoblasts), but also of autologous T lymphocytes generated in a mixed lymphocyte tumor-cell culture. In addition, an association between FasL expression by TCC cells and activated caspase-8, -9, and -3 expression by interferon-gamma-producing CD8-positive tumor-infiltrating lymphocytes was observed in situ. Our results show a functional expression of TCC-expressed FasL that correlates with tumor progression. These results suggest that TCC-expressed FasL may induce apoptosis of anti-tumor T lymphocytes in vivo, providing new insights on the mechanisms involved in bladder TCC progression.

C-kit+ FcR+ myelocytes are increased in cancer and prevent the proliferation of fully cytolytic T cells in the presence of immune serum.

Immunogenic cancers induce both IgG antibodies and CD8(+) cytotoxic T lymphocytes (CTL). Rejection of almost all immunogenic tumors depends ultimately on CTL. When tumors grow progressively, IgG continues to be produced but CTL may no longer be demonstrable. Using syngeneic mixed lymphocyte tumor cell cultures, we found that proliferation of fully activated proliferating CTL is prevented by a small subpopulation of immature myeloid c-kit(+) FcR(+) cells, for convenience referred to as "barrier cells". Both, FcR on barrier cells and IgG linked to TGF-beta (IgG-TGF-beta) present in immune serum, are obligatory for barrier cells to prevent proliferation of CTL, suggesting that IgG-TGF-beta binds FcR to activate suppression. Growing tumors increase barrier cells in the spleen. Interfering with the cells or molecules essential for barrier cells to prevent proliferation of CTL may enhance tumor and other CD8(+) CTL-mediated immunity.

Effective anti-tumor adoptive immunotherapy: utilization of exogenous IL-2-independent cytotoxic T lymphocyte clones.

To attain one of the final goals for cancer immunotherapy, cytotoxic T lymphocyte (CTL) clones were selected on the basis of exogenous IL-2 independence after limiting dilution culture from mixed lymphocyte tumor cell culture cells of FBL-3 tumor-immune spleens. About 10% of the clones could be propagated up to >5 times by weekly passages in the presence of splenic feeder and stimulating tumor cells. Two of the representative FBL-3-specific CTL clones that were able to undergo the fifth passage were expanded in large numbers for adoptive transfer by two rounds of a weekly passage with medium containing IL-2. FBL-3-specific CTL clones thus obtained showed a strong ability to eliminate the established tumors when transferred into tumor-bearing nude mice. In addition, the cells were recovered from the mouse spleen even 8 months after the transfer. The most striking differences between the CTL clones used in this experiment and those maintained conventionally in the presence of IL-2 were the abilities to produce IL-2 by themselves and the high expression level of the integrin molecule, VLA-4, that disappeared when cultured completely in the continuous presence of IL-2 in vitro during 12 weeks. In addition, concomitant with the disappearance of exogenous IL-2 independence and VLA-4 expression, the CTL clones lost their capacity to eradicate the tumor in vivo. Thus, the higher capacity of CTL clones to produce IL-2 on their own seemed to be correlated with the in vivo efficacy for tumor eradication and the long-term maintenance of their physiological profiles typical of memory T cells.

Generation of chronic myelogenous leukemia-specific T cells in cytokine-modified autologous mixed lymphocyte/tumor cell cultures.

Chronic myelogenous leukemia (CML) may be amenable to cell-based adoptive immunotherapy, as suggested by the graft-versus-leukemia effect of bone marrow transplantation and the therapeutic benefit of donor leukocyte infusions. Specific adoptive immunotherapy without bone marrow transplantation might be more effective and less cost-intensive. Professional antigen-presenting cells, the dendritic cells, from patients with CML are derived from the malignant clone and may stimulate antileukemia T-cell responses. Autologous T cells may also be able to recognize tumor antigens on CML cells directly. Here, the authors show that CD4 and CD8 T-cell responses to autologous CML cells can be generated in vitro rapidly and effectively by performing modified autologous mixed lymphocyte/tumor cell cultures (MLTC) in serum-free medium in the presence of cytokines known to support dendritic cell differentiation. MLTC-sensitized T cells secreted large amounts of the type 1 cytokine interferon-gamma, as well as interleukin (IL)-2. However, they also secreted a variety of other cytokines, including the type 2-subtype cytokine IL-13 but not the classic type 2 cytokines IL-4, IL-5, and IL-10. Monoclonal populations of CML-specific CD4 cells could be derived from these lines in limited numbers but showed markedly enhanced reactivity. This suggests that CML-specific T cells are relatively rare in these autologous MTLC-derived sensitized populations, but that their isolation and propagation would yield much more potent antitumor effector cells for use in adoptive immunotherapy without the need for bone marrow transplantation.

Expression of B7.1 (CD80) in a renal cell carcinoma line allows expansion of tumor-associated cytotoxic T lymphocytes in the presence of an alloresponse.

We have selected a well-characterized human renal cell carcinoma (RCC) line as the basis for development of a genetically engineered tumor cell vaccine to be applied in an allogeneic setting. This cell line was genetically modified by retroviral transduction to express B7.1 costimulatory molecules. The unmodified tumor cells and B7.1-expressing tumor cells were compared for their ability to induce tumor-associated responses in allogeneic peripheral blood mononuclear cells (PBMC) of two normal control donors having single MHC class I allele matches with the tumor cells. PBMC primed using B7.1-modified tumor cells showed a preponderance of CD3+CD8+ cytotoxic T lymphocytes (CTL) that proliferated over extended periods of time in mixed lymphocyte tumor cell (MLTC) cultures. Strong cytolytic activity developed in the primed populations and included allospecific CTL with specificity for mismatched HLA-A, -B and -C molecules. Nevertheless, it was possible to isolate CTL clones that were able to lyse tumor cells but not lymphoblastoid cells that expressed all the corresponding allospecificities. Thus, induction of complex allospecific responses did not hinder the development of tumor-associated CTL in vitro. These results support the use of this genetically modified allogeneic tumor cell line for vaccination of partial-MHC matched RCC patients.

Autologous dendritic cells or cells expressing both B7-1 and MUC1 can rescue tumor-specific cytotoxic T lymphocytes from MUC1-mediated apoptotic cell death

We attempted to induce MUC1-specific cytotoxic T lymphocytes (CTLs) by mixed-lymphocyte tumor cell culture (MLTC) using two allogeneic MUC1-positive cancer cell lines, T-47D and MCF7. The induced CTLs exhibited MUC1-specific cytotoxicity 16 days after the initial stimulation. However, these CTLs underwent apoptotic death within 16 days. To examine whether the B7-1 molecule is required for the expansion of the responder cells, a B7-1(+)/MUC1(-) cell line was transfected with MUC1 cDNA, and the resulting transfectant was employed as a stimulator in an autologous MLTC. The CTLs exhibited MUC1 specificity but also continued to propagate. In parallel, autologous dendritic cells (DCs) were added to an MLTC containing peripheral blood lymphocytes (PBLs) and the allogeneic MUC1-positive stimulators. The CTLs demonstrated MUC1 specificity and their number increased. This suggests that the B7-1 molecule is required for rescuing CTLs from MUC1-mediated apoptotic death, but not for the induction of MUC1-specific responsiveness. This strategy to obtain the CTLs efficiently may be useful for adoptive immunotherapy against cancer.

B7.1 costimulation increases T-cell proliferation and cytotoxicity via selective expansion of specific variable alpha and beta genes of the T-cell receptor

Background: Optimal T-cell activation requires not only ligation of the T-cell receptor (TcR) but also delivery of costimulatory signals by various accessory molecules. The interaction of the costimulatory molecule B7.1 (CD80) with its receptor CD28 provides a strong positive signal to T cells. Methods: The B7.1 gene was transduced into cultured human ovarian, breast, and pancreatic tumor cells by using a retroviral vector. Autologous as well as allogeneic naive T-cells were stimulated with either wild-type or B7.1-transduced tumor cells in a mixed lymphocyte tumor cell culture (MLTC). In addition to cytolytic activity, T-cell proliferation, T-cell subset composition, and the frequencies of TcR variable (V) alpha and beta genes were compared in T cells from both types of MLTC. Results: Introduction of the B7.1 gene into tumor cells was successful in all tumors to a varying degree. Those tumors expressing high levels of B7.1 induced significantly higher levels of T-cell proliferation than wild-type tumor cells. T-cell subset composition did not markedly differ between T cells stimulated with wild-type tumor cells or B7.1-expressing tumor cells. However, T cells stimulated with B7.1-expressing tumor cells showed a significantly increased cytolytic potential. The increased cytotoxic T lymphocyte activity was associated with a higher frequency of specific TcR Vα and Vβ genes. In addition, B7.1 costimulation promoted oligoclonality among the responding T cells. Conclusions: These data suggest that costimulation through B7.1 promotes T-cell proliferation and cytotoxic activity through clonal expansions of T cells bearing antigen-specific TcR Vα and Vβ genes and through promotion of oligoclonality. The data also suggest that promoting B7.1-mediated costimulation is an important aspect of immune therapies. (Surgery 2000;127:342-50.)

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.

Tumor stimulator cell modification by infection with Newcastle Disease Virus: analysis of effects and mechanism in MLTC-CML cultures.

Effects of tumor stimulator cell modification by infection with Newcastle Disease Virus (NDV) are described as analysed in vitro in mixed lymphocyte tumor cell cultures (MLTC). Direct antitumor effects were seen with human melanoma or colon-carcinoma cells in a dose- and time-dependent manner when using live but not UV inactivated virus. When T cell stimulation was measured by [3H]-thymidine uptake, NDV infected tumor stimulator cells did not show an augmentation but rather an inhibitory effect in comparison to non-infected stimulator cells. Virus infected tumor stimulator cells were, however, capable of augmenting the induction of tumor specific cytotoxic T cells in MLTC-CML assays when using murine ESb lymphoma immune cells and syngeneic NDV modified ESb cells as stimulators. A CML stimulatory effect was also shown for NDV modified third party cells and thereof derived conditioned medium. These effects are most likely explained by interferon- which is induced in tumor cells by NDV infection and by interferon-á which is induced in responder cells when stimulated with NDV infected stimulator cells.

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.

CD80 expression in an HLA-A2-positive human non-small cell lung cancer cell line enhances tumor-specific cytotoxicity of HLA-A2-positive T cells derived from a normal donor and a patient with non-small cell lung cancer.

To generate non-small cell lung cancer (NSCLC)-reactive lymphocytes, we transfected an HLA-A2-expressing human NSCLC line (1355) with the cDNA encoding the lymphocyte co-stimulatory molecule CD80. Following selection in G418, 1355.7 demonstrated stable cell-surface expression of CD80. Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established in 600 IU/ml IL-2 using HLA-A2+ normal donor peripheral blood mononuclear cells (PBMCs) stimulated with 1355-P (parental), 1355.7 or IL-2 alone. In 7 of 9 MLTCs, those stimulated with 1355.7 demonstrated enhanced growth after 30 to 45 days of culture. The predominant lymphocyte to grow in all MLTCs was a CD3+alphabeta+CD4+ T cell. In one case, lymphocytes stimulated with 1355.7 (MLTC 2389.7) exhibited preferential lysis of 1355. MLTC 2389.7 was cloned by limiting dilution, and 2 resultant cloids were shown to be NSCLC-reactive and dependent on both MHC class 1 and CD3 in their recognition of tumor cells. Additionally, allogeneic MLTCs were established using three HLA-A2+ NSCLC patients' PBMC. The predominant lymphocyte to grow in these MLTCs was a CD3+ alphabeta+CD8+ T cell. In cytotoxicity studies, MLTC-UKY25.7 demonstrated preferential lysis of 1355-P, 1355.7 and an HLA-A2+ NSCLC cell line, 1650. Lymphocytes from this MLTC did not lyse K562, Daudi or an HLA-A2- NSCLC cell line, 647. Our data suggest that CD80-expressing NSCLC tumor cells may enhance the generation of specific CTLs in vitro. These CTLs could be important reagents for use in cellular immunotherapy and/or in isolating tumor antigens for potential tumor vaccine development.

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.

Adenoviral delivery of B7-1 (CD80) increases the immunogenicity of human ovarian and cervical carcinoma cells.

The majority of tumour cells do not express immune costimulatory molecules and this may account for their inability to stimulate directly an antitumour T cell response. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding the human B7-1 costimulatory molecule. We explored the use of this vector for gene transfer to a number of human ovarian and cervical tumour cell lines, and to primary ovarian tumour material. Rapid and efficient gene transfer and expression was obtained in the majority of cases using a multiplicity of infection of 30 plaque forming units per cell. B7-1 expression was detectable at the cell surface within 12 h and was still detectable 10 days after infection. The immunogenicity of gene-modified tumour cells was tested in an allogeneic mixed lymphocyte tumour cell culture. Tumour cells expressing B7-1 were found to induce significantly higher levels of T cell proliferation than tumour cells modified with a control adenovirus carrying the beta-galactosidase gene. B7-1-induced T cell proliferation could be blocked by the addition of anti-B7-1 antibodies at the initiation of cocultures. These results support the rationale for use of adenovirally delivered B7-1 for genetic immunotherapy of ovarian and cervical cancer.

Melanomas that develop within the eye inhibit lymphocyte proliferation.

Experiments were performed to compare the ability of ocular and skin melanoma cells to stimulate T cells. Primary melanoma cell lines were obtained from a series of patients with either eye or skin melanoma. The ability of tumor cells to stimulate T cells in the absence of exogenous growth factors was assessed in mixed-lymphocyte tumor cell cultures in which allogeneic lymphocytes were stimulated with irradiated ocular or skin melanoma cells. Expression of HLA class I and class II on tumor cells, in the presence or absence of IFN-gamma, was determined by flow cytometry. The ability of tumor cells to inhibit T-cell proliferation was determined by adding various concentrations of irradiated tumor cells to standard mixed-lymphocyte cultures. Our results indicate that primary skin melanoma cells induce vigorous proliferation of allo-antigen-specific T cells. By contrast, ocular melanoma cells failed to induce significant T-cell proliferation. The failure of ocular melanoma cells to stimulate lymphocyte proliferation was not due to low levels of either class I or class II on tumor cells since tumor cells treated with IFN-gamma expressed high levels of class I and class II but still failed to induce lymphocyte proliferation. Ocular melanoma cells inhibited lymphocyte proliferation, as shown by experiments in which a small number of tumor cells prevented proliferation of T cells in mixed-lymphocyte cultures. Inhibition of lymphocyte proliferation required cell-to-cell contact, and supernatants from tumor cell cultures did not prevent lymphocyte proliferation. Moreover, the ability of ocular melanoma cells to inhibit T-cell proliferation was lost when tumor cells migrated from the eye and formed hepatic metastases. We conclude that there is a fundamental difference in the immunogenicity of ocular and skin melanoma cells. Ocular melanomas, but not primary skin melanomas, are poorly immunogenic tumors that inhibit T-cell proliferation. Our results imply that the immunogenicity of melanoma cells is altered when they develop within the unique ocular micro-environment.

Dominant TCRB-V-J chain usage and clonal expansion of sarcoma-reactive CD4+ HLA-DR-restricted T cells suggest a limited set of immunodominant sarcoma antigens.

Tumor-reactive CD4+ T cells have been isolated from tumor patients, and their specifity but not T-cell receptor (TCR) repertoire has been analyzed. Since we have described CD4+ sarcoma-reactive T-cell clones, we now sought to determine whether the TCR repertoire of these clones provides information on the spectrum of recognized sarcoma antigens. We analyzed the TCR beta (TCRB) chain repertoire of 19 CD4+ HLA-DR-restricted T-cell clones reactive with the autologous sarcoma cell line MZ-MES-1, with HLA-DR-matched tumor cell lines of different tissue origins and B-cell blasts. We identified 7 different clonotypes, which used a limited set of TCRBV and TCRBJ segments. Although the CDR3 of the different clonotypes was diverse, repeated restimulation with sarcoma cells led to a monoclonal expansion of T cells with particular TCR clonotypes in 5/6 mixed lymphocyte tumor cell cultures (MLTC). One clonotype was found in 2 independent MLTC experiments. Sarcoma-reactive T cells were demonstrated in patient tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) by clonotypic PCR. Our results indicate a limited number of immunodominant antigens expressed by the sarcoma cells. The junctional diversity of the TCR clonotypes shows that these antigens did not lead to extensive negative thymic selection as classical autoantigens would have. Therefore, the recognized antigens might represent cryptic autoantigens related to cellular transformation or proliferation.

Dominant TCRB‐V‐J chain usage and clonal expansion of sarcoma‐reactive CD4 HLA‐DR‐restricted T cells suggest a limited set of immunodominant sarcoma antigens

Tumor-reactive CD4+ T cells have been isolated from tumor patients, and their specifity but not T-cell receptor (TCR) repertoire has been analyzed. Since we have described CD4+ sacoma-reactive T-cell clones, we now sought to determine whether the TCR repertoire of these clones provides information on the spectrum of recognized sarcoma antigens. We analyzed the TCR beta (TCRB) chain repertoire of 19 CD4+ HLA-DR-restricted T-cell clones reactive with the autologous sarcoma cell line MZ-MES-1, with HLA-DR-matched tumor cell lines of different tissue origins and B-cell blasts. We identified 7 different clonotypes, which used a limited set of TCRBV and TCRBJ segments. Although the CDR3 of the different clonotypes was diverse, repeated restimulation with sarcoma cells led to a monoclonal expansion of T cells with particular TCR clonotypes in 5/6 mixed lymphocyte tumor cell cultures (MLTC). One clonotype was found in 2 independent MLTC experiments. Sarcoma-reactive T cells were demonstrated in patient tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) by clonotypic PCR. Our results indicate a limited number of immunodominant antigens expressed by the sarcoma cells. The junctional diversity of the TCR clonotypes shows that these antigens did not lead to extensive negative thymic selection as classical autoantigens would have. Therefore, the recognized antigens might represent cryptic autoantigens related to cellular transformation or proliferation. Int. J. Cancer 72:403–407, 1997. © 1997 Wiley-Liss, Inc.