Bone Marrow-derived CD8+ T Cells From Pediatric Leukemia Patients Express PD1 and Expand Ex Vivo Following Induction Chemotherapy.

Adoptive cell therapy (ACT) of chimeric antigen receptor T cells has demonstrated remarkable success for the treatment of pediatric B-cell leukemia. For patients who are not candidates for chimeric antigen receptor T-cell therapy, ACT using tumor antigen-experienced polyclonal T cells may be a treatment option. Since leukemic blasts reside in the bone marrow and bone marrow is a preferred site for homeostatic proliferation of cytotoxic memory CD8 T cells, we hypothesized that bone marrow would be a source of activated T cells. The aim of this study was to determine the feasibility of using bone marrow-derived T cells following postinduction chemotherapy for use in adoptive cell transfer. Matched patient samples of bone marrow and peripheral blood-derived T cells expanded ex vivo and displayed similar apoptotic profiles. Before activation and expansion, there was a significant increase in the percentage of bone marrow-derived CD8 T cells expressing activation markers PD1, CD45RO, and CD69 as compared with peripheral blood CD8 T cells. Considering, melanoma-reactive CD8 T cells reside in the subset of PD1CD8 T cells, the bone marrow may be an enriched source leukemic-specific T cells that can be used for ACT.

Programmed death receptor-1/programmed death receptor ligand-1 blockade after transient lymphodepletion to treat myeloma.

Early phase clinical trials targeting the programmed death receptor-1/ligand-1 (PD-1/PD-L1) pathway to overcome tumor-mediated immunosuppression have reported promising results for a variety of cancers. This pathway appears to play an important role in the failure of immune reactivity to malignant plasma cells in multiple myeloma patients, as the tumor cells express relatively high levels of PD-L1, and T cells show increased PD-1 expression. In the current study, we demonstrate that PD-1/PD-L1 blockade with a PD-L1-specific Ab elicits rejection of a murine myeloma when combined with lymphodepleting irradiation. This particular combined approach by itself has not previously been shown to be efficacious in other tumor models. The antitumor effect of lymphodepletion/anti-PD-L1 therapy was most robust when tumor Ag-experienced T cells were present either through cell transfer or survival after nonmyeloablative irradiation. In vivo depletion of CD4 or CD8 T cells completely eliminated antitumor efficacy of the lymphodepletion/anti-PD-L1 therapy, indicating that both T cell subsets are necessary for tumor rejection. Elimination of myeloma by T cells occurs relatively quickly as tumor cells in the bone marrow were nearly nondetectable by 5 d after the first anti-PD-L1 treatment, suggesting that antimyeloma reactivity is primarily mediated by preactivated T cells, rather than newly generated myeloma-reactive T cells. Anti-PD-L1 plus lymphodepletion failed to improve survival in two solid tumor models, but demonstrated significant efficacy in two hematologic malignancy models. In summary, our results support the clinical testing of lymphodepletion and PD-1/PD-L1 blockade as a novel approach for improving the survival of patients with multiple myeloma.

Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

Recurrent exposures to a pathogenic antigen remodel the CD8+ T cell compartment and generate a functional memory repertoire that is polyclonal and complex. At the clonotype level, the response to the conserved influenza antigen, M158-66 has been well characterized in healthy individuals, but not in patients receiving immunosuppressive therapy or with aberrant immunity, such as those with juvenile idiopathic arthritis (JIA). Here we show that patients with JIA have a reduced number of M158-66 specific RS/RA clonotypes, indicating decreased clonal richness and, as a result, have lower repertoire diversity. By using a rank-frequency approach to analyze the distribution of the repertoire, we found several characteristics of the JIA T cell repertoire to be akin to repertoires seen in healthy adults, including an amplified RS/RA-specific antigen response, representing greater clonal unevenness. Unlike mature repertoires, however, there is more fluctuation in clonotype distribution, less clonotype stability, and more variable IFNy response of the M158-66 specific RS/RA clonotypes in JIA. This indicates that functional clonal expansion is altered in patients with JIA on immunosuppressive therapies. We propose that the response to the influenza M158-66 epitope described here is a general phenomenon for JIA patients receiving immunosuppressive therapy, and that the changes in clonal richness and unevenness indicate a retarded and uneven generation of a mature immune response.

Corrigendum: Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

[This corrects the article DOI: 10.3389/fimmu.2024.1306490.].

Depletion of CD25⁺ T cells from hematopoietic stem cell grafts increases posttransplantation vaccine-induced immunity to neuroblastoma.

A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4⁺ T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4⁺ T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25⁺ Treg cells was similar to that achieved by in vivo depletion of all CD4⁺ T cells. Depletion of CD25⁺ Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4⁺ T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8⁺ T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.

Depletion of CD4 T cells enhances immunotherapy for neuroblastoma after syngeneic HSCT but compromises development of antitumor immune memory.

High-risk neuroblastoma remains a clinically challenging disease. Here, we report that a multifaceted immunotherapeutic approach including syngeneic hematopoietic stem cell transplantation (HSCT), adoptive transfer of sensitized T cells (from syngeneic donors vaccinated to tumor antigens), and early posttransplantation tumor vaccination can effectively treat mice with established neuroblastoma. Vaccination was an important component of this immunotherapy, as it resulted in enhanced and prolonged tumor-specific CD8 T-cell activity and improved antitumor efficacy. Surprisingly, CD4 cell depletion of mice given sensitized T cells resulted in better tumor-free survival, which was associated with an early increased expansion of CD8 T cells with an effector phenotype, increased numbers of tumor-reactive CD8 T cells, and increased tumor infiltration by CD8 T cells. However, in the absence of CD4 T cells, development of long-term tumor immunity (memory) was severely compromised as reflected by diminished CD8 T-cell recall responses and an inability to resist tumor rechallenge in vivo. Based on these results, a major challenge with this immunotherapeutic approach is how to obtain the ideal initial antitumor response but still preserve antitumor immune memory. These data suggest that identification and selective depletion of immune inhibitory CD4 T cells may be a strategy to enhance early antitumor immunity and induce a long-lasting tumor response after HSCT.

A Serum-Induced Transcriptome and Serum Cytokine Signature Obtained at Diagnosis Correlates with the Development of Early Pancreatic Ductal Adenocarcinoma Metastasis.

BACKGROUND: Despite the accessibility of blood, identification of systemic biomarkers associated with cancer progression has been especially challenging. The aim of this study was to determine a difference in baseline serum immune signatures in patients that experienced early pancreatic ductal adenocarcinoma (PDAC) metastasis compared with patients that did not. We hypothesized that immune mediators would differ in the baseline serum of these patient cohorts. To test this hypothesis, novel approaches of systemic immune analysis were performed. METHODS: A serum-induced transcriptional assay was used to identify transcriptome signatures. To enable an understanding of the transcriptome data in a global sense, a transcriptome index was calculated for each patient taking into consideration the relationship of up- and downregulated transcripts. For each patient, serum cytokine concentrations were also analyzed globally as a cytokine index (CI). RESULTS: A transcriptome signature of innate type I IFN inflammation was identified in patients that experienced early metastatic progression. Patients without early metastatic progression had a baseline transcriptome signature of TGFß/IL10-regulated acute inflammation. The transcriptome index was greater in patients with early metastasis. There was a significant difference in the CI in patients with and without early metastatic progression. CONCLUSIONS: The association of serum-induced transcriptional signatures with PDAC metastasis is a novel finding. Global assessment of serum cytokine concentrations as a CI is a novel approach to assess systemic cancer immunity. IMPACT: These systemic indices can be assessed in combination with tumor markers to further define subsets of PDAC that will provide insight into effective treatment, progression, and outcome.

Frontline Science: PECAM-1 (CD31) expression in naïve and memory, but not acutely activated, CD8+ T cells.

Inhibitory cell surface proteins on T cells are often dynamically regulated, which contributes to their physiologic function. PECAM-1 (CD31) is an inhibitory receptor that facilitates TGF-ß-mediated suppression of T cell activity. It is well established in CD4+ T cells that PECAM-1 is expressed in naïve recent thymic emigrants, but is down-regulated after acute T cell activation and absent from memory cells. The extent to which PECAM-1 expression is similarly regulated in CD8+ T cells is much less well characterized. We evaluated T cells recovered from mice after infection with a model intracellular pathogen and determined that, in CD8+ T cells, PECAM-1 expression was strongly down-regulated during acute infection but re-expressed to intermediate levels in memory cells. Down-regulation of PECAM-1 expression in CD8+ T cells was transcriptionally regulated and affected by the strength and nature of TCR signaling. PECAM-1 was also detected on the surface of human activated/memory CD8+ , but not CD4+ T cells. These data demonstrate that PECAM-1 expression is dynamically regulated, albeit differently, in both CD4+ and CD8+ T cells. Furthermore, unlike memory CD4+ T cells, memory CD8+ T cells retain PECAM-1 expression and have the potential to be modulated by this inhibitory receptor.

T Cells Deficient in Diacylglycerol Kinase ζ Are Resistant to PD-1 Inhibition and Help Create Persistent Host Immunity to Leukemia.

Efforts to improve the efficacy of adoptive T-cell therapies and immune checkpoint therapies in myelogenous leukemia are desired. In this study, we evaluated the antileukemia activity of adoptively transferred polyclonal cancer antigen-reactive T cells deficient in the regulator diacylglycerol kinase zeta (DGKζ) with or without PD-1/PD-L1 blockade. In the C1498 mouse model of myeloid leukemia, we showed that leukemia was eradicated more effectively in DGKζ-deficient (DGKζ-/-) mice than wild-type mice. T cells transferred from DGKζ-deficient mice to wild-type tumor-bearing recipients conferred this benefit. Leukemia clearance was similar to mice treated with anti-PD-L1. Strikingly, we found that the activity of adoptively transferred DGKζ-/- T cells relied partly on induction of sustainable host T-cell immunity. Transferring DGKζ-deficient T cells increased the levels of IFNγ and other cytokines in recipient mice, especially with coadministration of anti-PD-L1. Overall, our results offered evidence that targeting DGKζ may leverage the efficacy of adoptive T-cell and immune checkpoint therapies in leukemia treatment. Furthermore, they suggest that DGKζ targeting might decrease risks of antigen escape or resistance to immune checkpoint blockade. Cancer Res; 77(20); 5676-86. ©2017 AACR.

Adoptive cell therapy using PD-1+ myeloma-reactive T cells eliminates established myeloma in mice.

BACKGROUND: Adoptive cellular therapy (ACT) with cancer antigen-reactive T cells following lymphodepletive pre-conditioning has emerged as a potentially curative therapy for patients with advanced cancers. However, identification and enrichment of appropriate T cell subsets for cancer eradication remains a major challenge for hematologic cancers. METHODS: PD-1+ and PD-1- T cell subsets from myeloma-bearing mice were sorted and analyzed for myeloma reactivity in vitro. In addition, the T cells were activated and expanded in culture and given to syngeneic myeloma-bearing mice as ACT. RESULTS: Myeloma-reactive T cells were enriched in the PD-1+ cell subset. Similar results were also observed in a mouse AML model. PD-1+ T cells from myeloma-bearing mice were found to be functional, they could be activated and expanded ex vivo, and they maintained their anti-myeloma reactivity after expansion. Adoptive transfer of ex vivo-expanded PD-1+ T cells together with a PD-L1 blocking antibody eliminated established myeloma in Rag-deficient mice. Both CD8 and CD4 T cell subsets were important for eradicating myeloma. Adoptively transferred PD-1+ T cells persisted in recipient mice and were able to mount an adaptive memory immune response. CONCLUSIONS: These results demonstrate that PD-1 is a biomarker for functional myeloma-specific T cells, and that activated and expanded PD-1+ T cells can be effective as ACT for myeloma. Furthermore, this strategy could be useful for treating other hematologic cancers.

A novel family of mobile genetic elements is limited to the germline genome in Tetrahymena thermophila.

In the ciliated protozoan Tetrahymena thermophila, extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germline micronucleus. This study describes the isolation and complete characterization of Tlr elements, a family of approximately 30 micronuclear DNA sequences that are efficiently eliminated from the developing macronucleus. The data indicate that Tlr elements are comprised of an approximately 22 kb internal region flanked by complex and variable termini. The Tlr internal region is highly conserved among family members and contains 15 open reading frames, some of which resemble genes encoded by transposons and viruses. The Tlr termini appear to be long inverted repeats consisting of (i) a variable region containing multiple direct repeats which differ in number and sequence from element to element and (ii) a conserved terminal 47 bp sequence. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the Tetrahymena germline genome. Possible mechanisms of developmentally programmed Tlr elimination are discussed.

E-cadherin re-expression shows in vivo evidence for mesenchymal to epithelial transition in clonal metastatic breast tumor cells.

Substantial experimental evidence has shown that dedifferentiation from an epithelial state to a mesenchymal-like state (EMT) drives tumor cell metastasis. This transition facilitates tumor cells to acquire motility and invasive features. Intriguingly, tumor cells at the metastatic site are primarily epithelial, and it is believed that they differentiate back to an epithelial state by a process called mesenchymal to epithelial transition (MET). However, there is little in vivo evidence to support the MET process. To investigate EMT and MET in vivo, we generated two epithelial (E) and two mesenchymal (M) primary clonal cell lines from a spontaneous mouse mammary tumor (Tg MMTV/neu). These cells were labeled with reporters (GFP and luciferase), and tracked in vivo during primary tumor growth and subsequent secondary metastasis. Once E cells were implanted into the mammary fat pad, E-cadherin expression progressively decreased and continued to decrease as the primary tumor enlarged over time. A greater percentage of E tumor cells expressed E-cadherin at the secondary metastatic site as compared to the corresponding primary tumor site. Collectively, these data provide direct in vivo evidence that epithelial tumor cells have metastatic potential, undergo EMT at the primary tumor site, and MET at the metastatic site.

Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines.

BACKGROUND: The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. We sought to develop non-viral transfection methods that could transform patient tumor cells into cancer vaccines, paving the way for rapid production of autologous cell-based vaccines. METHODS: As the extended culture and expansion of most patient tumor cells is not possible, we sought to first evaluate a new technology that combines electroporation and chemical transfection in order to determine if plasmid-based gene vectors could be instantaneously delivered to the nucleus, and to determine if gene expression was possible in a cell-cycle independent manner. We tested cultured cell lines, a primary murine tumor, and primary human leukemia cells from diagnostic work-up for transgene expression, using both RFP and CD137L expression vectors. RESULTS: Combined electroporation-transfection directly delivered plasmid DNA to the nucleus of transfected cells, as demonstrated by confocal microscopy and real-time PCR analysis of isolated nuclei. Expression of protein from plasmid vectors could be detected as early as two hours post transfection. However, the kinetics of gene expression from plasmid-based vectors in tumor cell lines indicated that optimal gene expression was still dependent on cell division. We then tested to see if pediatric acute lymphocytic leukemia (ALL) would also display the rapid gene expression kinetics of tumor cells lines, determining gene expression 24 hours after transfection. Six of 12 specimens showed greater than 17% transgene expression, and all samples showed at least some transgene expression. CONCLUSION: Given that transgene expression could be detected in a majority of primary tumor samples analyzed within hours, direct electroporation-based transfection of primary leukemia holds the potential to generate patient-specific cancer vaccines. Plasmid-based gene therapy represents a simple means to generate cell-based cancer vaccines and does not require the extensive infrastructure of a virus-based vector system.

Immune modulating effects of cyclophosphamide and treatment with tumor lysate/CpG synergize to eliminate murine neuroblastoma.

BACKGROUND: Neuroblastoma is a pediatric cancer of neural crest origin. Despite aggressive treatment, mortality remains at 40 % for patients with high-risk disseminated disease, underscoring the need to test new combinations of therapies. In murine tumor models, our laboratory previously showed that T cell-mediated anti-tumor immune responses improve in the context of lymphopenia. The goal of this study was to incorporate lymphodepletion into an effective immune therapy that can be easily translated into neuroblastoma standard of care. Based on the lymphodepleting effects of cyclophosphamide, we hypothesized that cyclophosphamide would synergize with the TLR9 agonist, CpG oligodeoxynucleotide (ODN), to produce a T cell-mediated anti-neuroblastoma effect. METHODS: To test this hypothesis, we used the AgN2a aggressive murine model of neuroblastoma. Mice bearing subcutaneous tumors were treated with cyclophosphamide followed by treatment with tumor cell lysate mixed with CpG ODN injected at the tumor site. RESULTS: Subcutaneous neuroblastoma regressed only in mice that were treated with 100 mg/kg cyclophosphamide prior to receiving treatments of tumor lysate mixed with CpG ODN. The anti-neuroblastoma response was T cell-mediated. Synergy between cyclophosphamide and the tumor lysate/CpG ODN treatment influenced the production of anti-tumor CD8 T cell effectors, and dendritic cell homeostasis. For clinical consideration, an allogeneic tumor lysate was used effectively with this protocol to eliminate AgN2a tumor in vivo. CONCLUSION: Synergistic immune modulating effects of cyclophosphamide and a treatment containing tumor cell lysate and CpG ODN provide T cell-mediated anti-tumor activity against murine neuroblastoma.

Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma.

BACKGROUND: Multiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity. METHODS: To test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4. RESULTS: Temporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies. CONCLUSIONS: These data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies.

Examining T cells at vaccine sites of tumor-bearing hosts provides insights to dysfunctional T-cell immunity.

When tumor vaccines are administered as cancer immunotherapy, cellular interactions at the vaccine site are crucial to the generation of antitumor immunity. Examining interactions at the vaccine site could provide important insights to the success or failure of vaccination. Our laboratory previously showed that while administration of a cell-based vaccine to tumor-free mice leads to productive antineuroblastoma immunity, vaccination of tumor-bearing mice does not. The goal of this study was to examine immune effectors at the vaccine site to identify mechanisms responsible for the generation of ineffective antitumor immunity in tumor-bearing mice. The results of this study show that vaccine sites of tumor-bearing mice contained significantly fewer T cells than vaccine sites of tumor-free mice. Similar migration and proliferation of T cells was observed in the vaccine sites of tumor-bearing and tumor-free mice, but T cells in the sites of tumor-bearing mice were more apoptotic. T cells at the vaccine sites of both tumor-free and tumor-bearing mice had an effector-memory phenotype and expressed activation markers. Despite the activated phenotype, T cells from tumor-bearing mice elicited defective antitumor immune responses. Although T cells from vaccine sites of tumor-bearing mice were capable of producing inflammatory cytokines, the T cells from tumor-bearing mice produced lower levels of cytokines compared with T cells from the tumor-free mice. Remarkably, this defect seems to be systemic, affecting distal T cells in tumor-bearing mice. This study demonstrates that the defective vaccine-induced immune response to neuroblastoma in tumor-bearing hosts originates as a result of tumor burden, resulting in poor antitumor immunity.

Separation and Characterization of Epithelial and Mesenchymal-like Murine Mammary Tumor Cells Reveals Epithelial Cell Differentiation Plasticity and Enhanced Tumorigenicity of Epithelial-enriched Tumor Cells.

Tumors are composed of heterogeneous populations of cells including tumor-initiating cells (TICs) and metastatic precursors. While the origin of these cells is unknown, there is evidence that tumor cells can transdifferentiate from an epithelial to a mesenchymal phenotype, a property referred to as epithelial-to-mesenchymal transition (EMT). This cellular plasticity may explain the heterogeneous nature of tumors and differences in the tumorigenic and invasive properties of cells. Understanding the origin of these cells and the contribution of external factors that influence the acquisition of cellular properties is critical for the development of therapeutics to eradicate cancer. In this study, we show that primary murine tumor cells harvested from FVB/N Tg (MMTV/Neu) spontaneous mammary tumors possess differentiation plasticity and can be enriched to be epithelial or mesenchymal-like using selected culture media conditions, and we show evidence of EMT in a clonal population of primary epithelial tumor cells when cultured in fibroblast growth factor-1 (FGF-1) or transforming growth factor-ß (TGF-ß). We also determined that in contrast to the identification of mesenchymal-like tumor cells as TICs in orthotopic xenograph models of tumorigenicity, epithelial-enriched murine mammary tumor cells were more tumorigenic as compared to mesenchymal-enriched cells when transplanted back subcutaneously into syngeneic immune competent mice. Together, these data suggest that EMT plasticity can be induced in primary murine mammary tumor cells, and that tumorigenicity of epithelial or mesenchymal-like cells may be influenced by factors such as the site of tumor inoculation or the immune state of the host (xenogenic immune compromised versus syngeneic immune competent).

Neuroblastoma cells transiently transfected to simultaneously express the co-stimulatory molecules CD54, CD80, CD86, and CD137L generate antitumor immunity in mice.

The goal of this study was to show that nonviral gene transfection technology can be used to genetically modify neuroblastoma cells with immune stimulatory molecules, and that the modified cells can generate an antitumor immune response. The authors found that an electroporation-based gene transfection method, nucleofection, could be used to modify mouse AGN2a (an aggressive variant of Neuro-2a) neuroblastoma cells to simultaneously express as many as four different immune stimulatory molecules encoded by separate plasmid vectors. Within 18 hours after nucleofection, greater than 60% of the cells typically expressed the transfected gene products, and the percentages of cells expressing the products often exceeded 96%. High levels of plasmid in cell nuclei immediately after nucleofection documented instantaneous availability of gene vectors to the transcriptional machinery. AGN2a cells nucleofected to express the co-stimulatory molecules CD80 and CD86 expressed higher levels of these molecules than cells that had been permanently transfected with these same plasmid vectors, and the nucleofected cells were as effective as the permanently transfected cells at inducing an antitumor response in vivo in a tumor prevention model. AGN2a cells nucleofected with four separate plasmid vectors encoding CD54, CD80, CD86, and CD137L induced a T-cell immune response in vitro and served as a potent tumor vaccine in the tumor prevention model. These data show that transient transfection using a nonviral based method, nucleofection, can be used to rapidly generate novel cell-based tumor vaccines.