Abrogation of IFN-γ Signaling May not Worsen Sensitivity to PD-1/PD-L1 Blockade.

Programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockade is a promising therapy for various cancer types, but most patients are still resistant. Therefore, a larger number of predictive biomarkers is necessary. In this study, we assessed whether a loss-of-function mutation of the interferon (IFN)-γ receptor 1 (IFNGR1) in tumor cells can interfere with anti-PD-L1 therapy. For this purpose, we used the mouse oncogenic TC-1 cell line expressing PD-L1 and major histocompatibility complex class I (MHC-I) molecules and its TC-1/A9 clone with reversibly downregulated PD-L1 and MHC-I expression. Using the CRISPR/Cas9 system, we generated cells with deactivated IFNGR1 (TC-1/dIfngr1 and TC-1/A9/dIfngr1). In tumors, IFNGR1 deactivation did not lead to PD-L1 or MHC-I reduction on tumor cells. From potential inducers, mainly IFN-α and IFN-ß enhanced PD-L1 and MHC-I expression on TC-1/dIfngr1 and TC-1/A9/dIfngr1 cells in vitro. Neutralization of the IFN-α/IFN-ß receptor confirmed the effect of these cytokines in vivo. Combined immunotherapy with PD-L1 blockade and DNA vaccination showed that IFNGR1 deactivation did not reduce tumor sensitivity to anti-PD-L1. Thus, the impairment of IFN-γ signaling may not be sufficient for PD-L1 and MHC-I reduction on tumor cells and resistance to PD-L1 blockade, and thus should not be used as a single predictive marker for anti-PD-1/PD-L1 cancer therapy.

Development of T cell lines sensitive to antigen stimulation.

Immortalized T cells such as T cell hybridomas, transfectomas, and transductants are useful tools to study tri-molecular complexes consisting of peptide, MHC, and T cell receptor (TCR) molecules. These cells have been utilized for antigen discovery studies for decades due to simplicity and rapidness of growing cells. However, responsiveness to antigen stimulation is typically less sensitive compared to primary T cells, resulting in occasional false negative outcomes especially for TCRs having low affinity to a peptide-MHC complex (pMHC). To overcome this obstacle, we genetically engineered T cell hybridomas to express additional CD3 molecules as well as CD4 with two amino acid substitutions that increase affinity to MHC class II molecules. The manipulated T cell hybridomas that were further transduced with retroviral vectors encoding TCRs of interest responded to cognate antigens more robustly than non-manipulated cells without evoking non-antigen specific reactivity. Of importance, the manipulation with CD3 and mutated human CD4 expression was effective in increasing responsiveness of T cell hybridomas to a wide variety of TCR, peptide, and MHC combinations across class II genetic loci (i.e. HLA-DR, HLA-DQ, HLA-DP, and murine H2-IA) and species (i.e. both humans and mice), and thus will be useful to identify antigen specificity of T cells.

Interactions of Streptococcus iniae with phagocytic cell line.

Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1ß, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection.

Generation and characterization of immortalized rat retinal microglial cell lines.

Retinal microglia play an important role as resident immunocompetent and phagocytic cells in the event of injury and disease. Retinal microglia and microglia precursor transplantation show a rescue effect in ischemic retina and retinal degeneration. However, studies of retinal microglia have been hampered by the difficulty of obtaining sufficient numbers of microglia. One way to circumvent this difficulty is to establish permanent retinal microglia cell lines. In the present study, we report the generation of immortalized retinal microglia, T-MG cells, from postnatal day 3 rat retinal tissue using a lentiviral vector encoding SV40 large T antigen. The T-MG cells exhibited cell-type-specific antigens for monocyte/macrophage lineage cells, including CD11b (OX42), ED1 (OX6), and Iba1, and actively phagocytosed latex beads. In addition to primary retinal microglia, T-MG cells also have the ability to recruit into chemokines. Treatment of T-MG cells with lipopolysaccharide (LPS) led to increased levels of tumor necrosis factor-α, interleukin-1ß, and inducible nitric oxide synthase. Genome-wide microarray analysis showed a less than 1% difference in the genes between the T-MG cells and the control primary retinal microglia. The T-MG cells exhibited properties similar to those of the primary retinal microglia and should have considerable utility as an in vitro model for the study of retinal microglia in health and as a curative therapy and an in vivo model for the study of retinal microglia in disease.

Inflammatory response and barrier properties of a new alveolar type 1-like cell line (TT1).

PURPOSE: To evaluate the inflammatory response and barrier formation of a new alveolar type 1-like (transformed type I; TT1) cell line to establish its suitability for toxicity and drug transport studies. METHODS: TT1 and A549 cells were challenged with lipopolysaccharide (LPS). Secretion of inflammatory mediators was quantified by ELISA. The barrier properties of TT1 cells were evaluated by transepithelial electrical resistance (TEER), fluorescein sodium (flu-Na) apparent permeability (P(app)) and staining of zona occludens-1 (ZO-1). RESULTS: LPS stimulated similar levels of secretion of IL-6 and IL-8 in TT1 and A549 cells. TNF-alpha was not produced by either cell line. In contrast to A549 cells, TT1 cells did not secrete SLPI or elafin. TT1 cells produced maximal TEER of approximately 55 ohms cm(2) and flu-Na P(app) of approximately 6.0 x 10(-6) cm/s. ZO-1 staining was weak and discontinuous. Attempts to optimise culture conditions did not increase the barrier properties of the TT1 cell layers. CONCLUSIONS: The TT1 cell line models the alveolar inflammatory response to LPS challenge and provides a valuable complement to cell lines currently used in toxicity assays. However, under the experimental conditions used the TT1 cell line did not form the highly restrictive tight junctions which exist in vivo.

Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts.

Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed.

[Reorganization of actin cytoskeleton in 3T3-SV40 cells and their sensitivity to lysis by natural killer cells].

The present work was aimed to examine whether the actin reorganization of 3T3-SV40 cells influences their sensitivity to natural killer (NK) cells activity. The effects of N-acetylcystein (NAC) and latrunculin B, actin depolimerizator, on both cellular parameters were studied. Experiments with NAC demonstrated that 3T3-SV40 sensitivity to NK cells activity remained unchanged under the disordered microfilaments but decreased upon the appearance of structured stress-fibres. The data on latrunculin B action resulted in the opposite conclusion: the more microfilaments disorganization in the presence of latrunculin B the lesser 3T3-SV40 sensitivity to lysis by NK cells. These facts suggest that relations between microfilament integrity in 3T3-SV40 cells and their sensitivity to NK cells are rather independent. The latter confirms our previous conclusion (Gamaley et al., 2006). Decrease in 3T3-SV40 sensitivity to NK cells activity accompanied by actin reorganization resulted from both latrunculin B and NAC action suggests changes in cellular surface, which ultimately lead to inactivation (or loss) of the molecules being activating signals to NK cells.

Assessment of CD4+ T cells specific for the tumor antigen SSX-1 in cancer-free individuals.

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.

Identification of new NY-ESO-1 epitopes recognized by CD4+ T cells and presented by HLA-DQ B1 03011.

NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in patients with NY-ESO-1-expressing malignancies. Since CD4+ T cells play a critical role in generating and maintaining antigen-specific cellular and humoral immune responses, we searched for new NY-ESO-1 epitopes presented by MHC class II molecules. CD4+ T cells of patients with NY-ESO-1-expressing cancer were presensitized with 18-mer overlapping synthetic peptides spanning the entire sequence of NY-ESO-1. Two partly overlapping NY-ESO-1 epitopes p49-66 and p55-72 were identified as targets for NY-ESO-1-specific CD4+ T cells. Peptide-specific CD4+ T-cell clones were generated by repetitive stimulation with NY-ESO-1 p49-66 and p55-72. Further experiments confirmed distinct specificities for the CD4+ T-cell clones indicating that at least 2 different CD4+ T-cell epitopes are located in the region p49-72 of the NY-ESO-1 sequence. Using a set of partially histocompatible EBV-B cell lines and MHC class II-specific antibodies, we found that both CD4+ T-cell epitopes were presented in the context of HLA-DQ B1 03011(DQ7). Natural processing and presentation of these epitopes was demonstrated by recognition of an HLA-DQ B1 03011- and NY-ESO-1-expressing lymphoma cell line and by recognition of dendritic cells (DC) exogenously loaded with NY-ESO-1 protein or infected with recombinant NY-ESO-1 adenoviral constructs. The specific production of IFN-gamma and TNF-alpha suggests that the NY-ESO-1-specific CD4+ T-cell clones belong to the Th1 subtype. The characterization of the new HLA-DQ B1 03011-restricted NY-ESO-1 peptides broadens the repertoire of epitopes that can be used to monitor NY-ESO-1-specific spontaneous and vaccine-induced T-cell responses in cancer patients.

[Therapeutic monoclonal antibodies: a little history, a lot of engineering, and... some clinical successes]. / Anticorps monoclonaux à usage thérapeutique: un peu d'histoire, beaucoup d'ingénierie, et... quelques succès cliniques.

Thirty years after their discovery by Milstein and Köhler, monoclonal antibodies have now come of age as therapeutics. Nineteen monoclonal antibodies are on the market and/or have got authorization to be used for the treatment of severe diseases. Many technical efforts have been devoted over the last two decades to the generation of second generation mAbs with better affinities, decreased immunogenicity and optimized effector functions. The development of molecular engineering techniques applied to antibody molecules has also made it possible to design bi-specific antibodies and fusion molecules exhibiting different modules with bi-functional activities. The use of proteomics and genomics combined with phage display allows now the rapid selection of antibodies directed against new targets at a high rate. Many efforts are currently focused on the selection of high-responder patients, the optimization of antibody delivery, schemes of infusion, antibody pharmaco-kinetics and bio-distribution, as well as on a better control of the severe side-effects generated by some antibody treatments.

Mouse serum factor(s) down-modulate the CD4 and CXCR4 molecules on human T cells conferring resistance to HIV infection in NOG mice.

Human cells have developed innate immunity, exploiting several means to block virus infection, and viruses have evolved diverse strategies to resist these. We show here that the human immunodeficiency virus 1 (HIV-1) could neither progressively infect engrafted human leukemic T cells nor repress their growth in NOG mice. However, ED-40515(-) cells infected with HIV-1 before inoculation were found to significantly delay the onset of tumor growth and increased the survival period of NOG mice. ED-40515(-) tumor cells showed resistance to HIV-1 which was apparently correlated with the down-regulation of CD4 and CXCR4 molecules in NOG mice. Serum from three different mouse strains, including NOG, retained a suppressive effect on the CD4 molecule of ED-40515(-) cells in vitro. ED-40515(-) cells obtained from mice re-expressed CD4 and CXCR4 molecules upon in vitro culture and were again successfully infected with HIV-1. These findings indicate that HIV-1 may initially successfully delay or regress tumor growth in NOG mice, but eventually fails to do so because of the evolution of HIV-resistant cells due to a rapid down-modulation of CD4 and CXCR4. Our data also demonstrated that some unknown soluble factor(s) present in mouse serum was responsible for conferring resistance to HIV infection to human T cells.

Establishment of HTLV-I-infected cell lines from peripheral blood mononuclear cells of Brazilian patients.

To investigate epidemiological and pathogenetic features of HTLV-I infection, a cohort of carriers has been followed at the USP Teaching Hospital since 1991. This study describes the establishment of cell lines from peripheral blood mononuclear cells (PBMC) of infected subjects. Ex vivo PBMC were cultured with those from a seronegative donor and morphologic evidence of cell transformation was obtained after 90 days with detection of multinucleated cells exhibiting cerebriform nuclei. Integration of HTLV-I proviral DNA and expression of viral antigens was demonstrated in culture by PCR and immunofluorescence. Cell lines were maintained for 240 days, gradually weaned from exogenous IL-2. Immunophenotyping of cell lines on flow cytometry yielded evidence of cell activation. Establishment of HTLV-I-infected cell lines from ex vivo PBMC is feasible and may be useful for studies on lymphocyte phenotypic changes and on mechanisms of HTLV-induced cell proliferation. Moreover they may be used with diagnostic purposes in immunofluorescence tests.

Successful control of Epstein-Barr virus (EBV)-infected cells by allogeneic nonmyeloablative stem cell transplantation in a patient with the lethal form of chronic active EBV infection.

Chronic active Epstein-Barr virus infection (CAEBV) is a heterogeneous EBV-related disorder, ranging from mild/moderate forms to rapidly lethal disorders. The lethal form of CAEBV is characterized by multiple organ failure, hemophagocytic syndrome, and development of lymphomas. Allogeneic stem cell transplantation is considered as the only potentially curative treatment for the lethal form of CAEBV, but it is not always desirable because of the high incidence of regimen-related toxicities. A 17-year-old female with CAEBV, who was refractory to conventional therapies and considered to be unable to receive a myeloablative regimen because of multiple organ dysfunction, underwent allogeneic nonmyeloablative stem cell transplantation (allo-NST) before developing a hematological malignancy. She has been well without any signs of CAEBV for 27 months after allo-NST, and we confirmed that specific cytotoxic T lymphocyte activity against EBV was reconstituted. This outcome suggests that allo-NST can control CAEBV by reconstituting the host immunity against EBV.

T-cell responses to multiple antigens presented by RNA-transfected APCs: a possible immunomonitoring tool.

The increasingly deeper understanding of how the immune system recognizes and destroys tumors promises to enable the development of new approaches for gene therapy and immunotherapy. However, a treatment that induces safe and potentially beneficial antitumor responses is expected to require stepwise refinements. As part of this challenge, assays are needed to measure specific antitumor immune responses in patients. This becomes problematic because most tumors express unknown tumor antigens and it is often difficult to obtain sufficient amounts of viable tumor material for in vitro assays. Recently it was demonstrated that RNA derived from tumor cells stimulated T cells in an antigen-specific manner. These studies have formed the basis for the development of dendritic cell vaccines that express tumor antigens following translation of tumor RNA. Therefore, it occurred to us that antigen-presenting cells transfected with total tumor RNA might also be valuable in monitoring the antitumor responses induced in patients who participate in clinical trials. To test this hypothesis, we developed a model in which Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines were used as a source of RNA. Since this RNA encodes for known EBV antigens, it was possible to determine whether the expected responses were observed. Our results show for the first time that T cells primed to APC transfected with RNA isolated from EBV-infected lymphocytes exhibited a fine specificity that enabled them to recognize individual EBV antigens.

Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines.

Adoptive transfer of CMV-specific T cells offers the potential for reconstitution of viral immunity after allogeneic transplantation. However, the logistics of producing virus-specific T-cell clones has limited the application of cellular therapies. We treated 16 patients for CMV infection with polyclonal CMV-specific T-cell lines generated by short-term culture. Massive in-vivo expansions of CMV-specific cytotoxic T lymphocytes were observed, resulting in reconstitution of viral immunity. In eight cases antiviral drugs were not required, and subsequent episodes of reactivation occurred in only two patients. Our findings indicate that application of CMV-specific cell lines is both feasible and effective in a clinical environment.

Study of Herpesvirus saimiri immortalization of gammadelta T cells derived from peripheral blood and CSF of multiple sclerosis patients.

Human gammadelta T cells are an integral part of the innate immune system and have been difficult to study owing primarily to their relatively low abundance and their fastidious culture properties associated with short in vitro lifespan. Their increased presence within multiple sclerosis (MS) white matter plaques compared to peripheral blood (PB) suggests a specific interaction with central nervous system (CNS) tissues. This fact, together with their innate ability to lyse human oligodendrocytes in culture implicate them possibly in the pathogenesis of MS. To further investigate their potential role in MS, we studied whether gammadelta T cells could be effectively immortalized using Herpesvirus saimiri (HVS), so that they could be studied in longer-term cultures. Effective culture conditions were established resulting in efficient HVS growth transformation of multiple PB and CSF gammadelta T cell lines and clones that could exist in IL-2-dependent culture for periods in excess of 2 years. Phenotypic and functional comparison studies with parental nontransformed gammadelta T cells were performed to characterize the changes that possibly induced by viral transformation. Using panels of transformed gammadelta T cell clones representing discrete gammadelta TcR subtypes, there was no apparent correlation between intracytoplasmic cytokine expression or tumor cell cytotoxicity with a specific TcR. All transformed gammadelta T cells analyzed, regardless of their compartment of origin, strongly expressed intracytoplasmic IFN-gamma and TNF-alpha, but little IL-2 or anti-inflammatory IL-4 or IL-10. These results indicate that HVS transformation of gammadelta T cells can be used to generate lines and clones from both the CSF and PB compartments for further study and elucidation of their potential role in MS pathogenesis.

CD4+ T cell-induced differentiation of EBV-transformed lymphoblastoid cells is associated with diminished recognition by EBV-specific CD8+ cytotoxic T cells.

EBV transformation of human B cells in vitro results in establishment of immortalized cell lines (lymphoblastoid cell lines (LCL)) that express viral transformation-associated latent genes and exhibit a fixed, lymphoblastoid phenotype. In this report, we show that CD4(+) T cells can modify the differentiation state of EBV-transformed LCL. Coculture of LCL with EBV-specific CD4(+) T cells resulted in an altered phenotype, characterized by elevated CD38 expression and decreased proliferation rate. Relative to control LCL, the cocultured LCL were markedly less susceptible to lysis by EBV-specific CD8(+) CTL. In contrast, CD4(+) T cell-induced differentiation of LCL did not diminish sensitivity of LCL to lysis by CD8(+) CTL specific for an exogenously loaded peptide Ag or lysis by alloreactive CD8(+) CTL, suggesting that differentiation is not associated with intrinsic resistance to CD8(+) T cell cytotoxicity and that evasion of lysis is confined to EBV-specific CTL responses. CD4(+) T cell-induced differentiation of LCL and concomitant resistance of LCL to lysis by EBV-specific CD8(+) CTL were associated with reduced expression of viral latent genes. Finally, transwell cocultures, in which direct LCL-CD4(+) T cell contact was prevented, indicated a major role for CD4(+) T cell cytokines in the differentiation of LCL.

Immunisation with modified HPV16 E7 genes against mouse oncogenic TC-1 cell sublines with downregulated expression of MHC class I molecules.

Human papillomavirus type 16 (HPV16)-transformed mouse TC-1 cells are extensively used in the evaluation of efficacy of experimental vaccines against tumours induced by HPVs. As these cells strongly express MHC class I molecules and downregulation of MHC class I surface expression is one of the important mechanisms that enable tumour escape from the host immune system, we undertook to derive TC-1 clones with reduced expression of MHC class I antigens. TC-1 cells were inoculated into mice preimmunised with an E7 gene-based DNA vaccine and from tumours developing in a portion of the animals, cell clones with downregulated MHC class I surface expression were isolated. Treatment with IFN-gamma resulted in an upregulation of MHC class I molecules in these cells, but after IFN-gamma removal, their expression gradually dropped again. When the expression of some components of the antigen-processing machinery (APM; LMP-2, TAP-1, and TAP-2) was tested, a reduced TAP-1 production was detected in cell lines with downregulated MHC class I expression. An enhanced immunoresistance of TC-1-derived clones with reduced MHC class I expression was observed in animals immunised with plasmids carrying modified E7 genes. Apart from the previously described fusion gene Sig/E7/LAMP-1, a new construct, Sig/E7GGG/LAMP-1, with a mutated Rb-binding site, was also used for immunisation. No significant change of immunogenicity was recorded for Sig/E7GGG/LAMP-1. Cell lines with downregulated MHC class I expression derived from TC-1 cells may represent a useful model for testing therapeutic anti-HPV vaccines in settings more relevant to clinical requirements.

Stable transgenic expression of IL-2 and HSV1-tk by single and fusion tumor cell lines bearing EWS/FLI-1 chimeric genes.

In augmenting systemic anti-tumor immune response, the authors evaluated the genetic modification of Ewing family tumor (EFT) cell lines for use as allogeneic vaccines. EFT cell lines A673 and RD-ES were transfected with cDNAs for human interleukin (IL)-2 and/or HSV1 thymidine kinase (HSV1-tk), respectively. Clones with high and stable secretion of IL-2 alone or with coexpression of functional HSV1-tk were obtained and their features were analyzed. IL-2 expressing clones derived from the A673 cell line demonstrated decreased expression of HLA class I molecules compared with the parental cell line and corresponding clones derived from RD-ES. However, IFN-gamma could upregulate the expression of HLA class I antigens by IL-2 transfected A673 cells. Ganciclovir induced apoptosis in double-transfected cell clones. IL-2/HSV1-tk cells continued to produce and release IL-2 after initial ganciclovir treatment. After gamma-irradiation, transfected clones released bioactive IL-2 in a quantity sufficient to activate T and natural killer cells in culture. A polyvalent allogeneic vaccine was also obtained using fusion of two different transgenic cell lines. The resulting hybrids inherited antigenic and transgenic characteristics of both parental cell lines. It is presumed that the cell lines generated here could be used as allogeneic vaccines for treatment of patients with EFTs.