Local costimulation reinvigorates tumor-specific cytolytic T lymphocytes for experimental therapy in mice with large tumor burdens

Cytotoxic T cells recognize tumor Ags and destroy cancer cells in vitro. Adoptive transfer studies with transgenic T cells specific for tumor Ags have demonstrated that CTL are effective only in mice with small tumor burdens and thus appear to have limited potential in cancer immunotherapy. Here we used transgenic mice that express the TCR specific for an unmutated tumor Ag P1A and multiple lineages of P1A-expressing tumors to address this critical issue. We found that local costimulation, either by expression of B7-1 on the tumor cells or by local administration of anti-CD28 mAb 37N, reinvigorated the function of CTL specific for the tumor Ag, as it substantially increased the efficacy of CTL therapy for mice with large tumor burdens. Our study suggests that CTL-based immunotherapy can be manipulated to deal with large tumors.     

The rationale and practice of CTL therapy against cancer in the mouse and human

It is essential to investigate and elucidate the immune response especially T cell response to either syngeneic or autologous tumor for establishing a rational immunotherapy of cancer. We reported that major immune effector cells capable of inducing tumor regression are cytotoxic T lymphocytes (CTL). We found that there are at least two distinct CTL subsets directed to syngeneic tumor. One CTL subset which is selectively induced by syngeneic solid tumor is independent from CD4 positive helper T cells but requires a soluble factor (s) released from macrophage-like accessory cells designated killer T cell activating factor (KAF) in its induction and generation directed to the homologous tumor. The other CTL subset which is usually induced by syngeneic tumor of hematocytic origin is dependent on CD4 positive helper T cells in its induction. On the basis of our findings regarding the induction and activation mechanism of CTL to syngeneic tumors in the mouse, we have investigated the mechanisms of human CTL generation to autochthonous tumor in peripheral blood mononuclear cells of cancer patients. It was found that the nature of human CTL and its generation to autochthonous tumor are similar to those of murine CTL to syngeneic solid tumor. We are now establishing a rational cancer specific immunotherapy utilizing intravenous passive cell transfer of in vitro activated CTL to autochthonous tumor into an original cancer patient.     

Study of HLA class I restriction and the directed antigens of cytotoxic T lymphocytes at the tumor sites of ovarian cancer

The molecular basis of T-cell-mediated recognition of ovarian cancer cells remains to be fully addressed. In this study we investigated HLA class I restriction and directed antigens of cytotoxic T lymphocytes (CTL) at the sites of ovarian cancer. Three HLA-class-I-restricted CTL lines were established from the tumor sites of ovarian cancer by culturing tumor-infiltrating lymphocytes or tumor-associated ascitic lymphocytes with interleukin-2: (1) HLA-A2402-restricted and ovarian-adenocarcinoma-specific CTL, (2) HLA-A2-restricted CTL recognizing histologically different cancers, and (3) HLA-B52-restricted and ovarian-cancer-specific CTL. HLA-A0201, HLA-A0206 and HLA-A0207 tumor cells were lysed by the HLA-A2-restricted CTL. HLA-B52 restriction of the third CTL line was confirmed by the transfection of HLA-B5201 cDNA into the tumor cells. The HLA-A2-restricted CTL recognized the SART-1, but not the MAGE-1 or MAGE-3 antigen. These results may facilitate a better understanding of the molecular basis of tumor-specific immunity at the tumor site of ovarian cancer. Received: 30 December 1998 / Accepted: 2 March 1999     

Antitumor immunity and cellular cancer therapies

The identification of tumor specific antigens has provided important advance in tumor immunology. It is now established that specific cytotoxic T lymphocytes (CTL) and natural killer cells infiltrate tumor tissues and are effector cells able to control tumor growth. However, such a natural antitumor immunity has limited effects in cancer patients. Failure of host defenses against tumor is consecutive to several mechanisms which are becoming targets to design new immunotherapeutic approaches. CTL are critical components of the immune response to human tumors and induction of strong CTL responses is the goal of most current vaccine strategies. Effectiveness of cytokine therapy, cancer vaccines and injection of cells improving cellular immunity have been established in tumor grafted murine models. Clinical trials are underway. To day, interest is particularly focused on cell therapy: injected cells are either "ready to use" effector cells (lymphocytes) or antigen presenting cells able to induce a protective immune reaction in vivo (dendritic cells). The challenge ahead lie in the careful optimization of the most promising strategies in clinical situation.     

Intratumoral electroporation of IL-12 cDNA eradicates established melanomas by Trp2180–188-specific CD8+ CTLs in a perforin/granzyme-mediated and IFN-γ-dependent manner: application of Trp2180–188 peptides

Intratumoral electroporation (IT-EP) with IL-12 cDNA (IT-EP/IL12) can lead to the eradication of established B16 melanoma tumors in mice. Here, we explore the immunological mechanism of the antitumor effects generated by this therapy. The results show that IT-EP/IL12 applied only once resulted in eradication in 70% animals with large established B16 tumors. Tumor eradication required the participation of CD8+ T cells, but not CD4+ T cells and NK cells. IT-EP/IL12 induced antigen-specific CD8+ T cell responses against the immunodominant Trp2(180-188) epitope and generated a systemic response, resulting in significant therapeutic effects against distal, untreated tumors. The therapeutic effect of IT-EP/IL12 was absent in perforin-deficient mice, indicating that tumor elimination occurred through conventional perforin/granzyme lysis by CTLs. Moreover, this therapy induced some degree of immunological memory that protected approximately one-third of the cured mice against a subsequent tumor challenge. Moreover, antitumor efficacy and long-term protection against B16 were significantly improved by concurrent Trp2 peptide immunization through more induction of Ag-specific CTL responses and more attraction of IFN-γ-expressing CD8+ T cells into tumor sites. The antitumor effect of IT-EP/IL12 required the participation of IFN-γ, which was shown to induce MHC class I expression on B16 cells and increase the lytic activity of the CD8+ CTL generated by IT-EP/IL12. The results from these animal studies may help in the development of IT-EP/IL12 for cancer patients.     

Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells

The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient's tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.     

Targeting an anti-viral CD8+ T cell response to a growing tumor facilitates its rejection

 We demonstrate in a murine model that targeting an anti-viral T cell response to a growing tumor facilitates priming of a tumor-associated antigen (TAA)-specific, rejecting T cell response. Murine P815 mastocytoma cells grow aggressively in a syngeneic host. Transfected P815/S cells (expressing the hepatitis B surface antigen, HBsAg) also grow as subcutaneous tumors, but occasional ‘spontaneous’ rejections after transient growth are observed. Growth of P815/S tumors (but not of P815 tumors) is efficiently suppressed by a CD8⁺ cytotoxic T lymphocyte (CTL)-dependent immune mechanism in mice primed to HBsAg by DNA–immunization. In hosts immunized against HBsAg by DNA vaccination, HBsAg-specific CTL are generated. This specific CTL reactivity was targeted to s.c.-growing P815 tumors by intra tumor injections of either HBsAg-encoding plasmid DNA or viable P815/S cells; this treatment led to tumor rejection in 70–80% of the tumor-bearing animals. All rejecting animals showed a CD8⁺ CTL-dependent resistance to subsequent challenges by native, non-transfected P815 tumors. Targeting an established anti-viral (‘strong’) CTL response to a growing tumor hence is an efficient strategy to facilitate priming of a rejecting CTL response against (‘weak’) TAA in this system. Received: 18 December 1996 / Accepted: 6 February 1997     

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.     

Dendritic Cell-Based Immunotherapy for Solid Tumors

As a treatment for solid tumors, dendritic cell (DC)-based immunotherapy has not been as effective as expected. Here, we review the reasons underlying the limitations of DC-based immunotherapy for solid tumors and ask what can be done to improve immune cell-based cancer therapies. Several reports show that, rather than a lack of immune induction, the limited efficacy of DC-based immunotherapy in cases of renal cell carcinoma (RCC) likely results from inhibition of immune responses by tumor-secreted TGF-β and an increase in the number of regulatory T (Treg) cells in and around the solid tumor. Indeed, unlike DC therapy for solid tumors, cytotoxic T lymphocyte (CTL) responses induced by DC therapy inhibit tumor recurrence after surgery; CTL responses also limit tumor metastasis induced by additional tumor-challenge in RCC tumor-bearing mice. Here, we discuss the mechanisms underlying the poor efficacy of DC-based therapy for solid tumors and stress the need for new and improved DC immunotherapies and/or combination therapies with killer cells to treat resistant solid tumors.     

A cyclophilin B gene encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific CTLs.

We have studied Ags recognized by HLA class I-restricted CTLs established from tumor site to better understand the molecular basis of tumor immunology. HLA-A24-restricted and tumor-specific CTLs established from T cells infiltrating into lung adenocarcinoma recognized the two antigenic peptides encoded by a cyclophilin B gene, a family of genes for cyclophilins involved in T cell activation. These two cyclophilin B peptides at positions 84-92 and 91-99 induced HLA-A24-restricted CTL activity against tumor cells in PBMCs of leukemia patients, but not in epithelial cancer patients or in healthy donors. In contrast, the modified peptides at position 2 from phenylalanine to tyrosine, which had more than 10 times higher binding affinities to HLA-A24 molecules, could induce HLA-A24-restricted CTL activity against tumor cells in PBMCs from leukemia patients, epithelial cancer patients, or healthy donors. PHA-activated normal T cells were resistant to lysis by the CTL line or by these peptide-induced CTLs. These results indicate that a cyclophilin B gene encodes antigenic epitopes recognized by CTLs at the tumor site, although T cells in peripheral blood (except for those from leukemia patients) are immunologically tolerant to the cyclophilin B. These peptides might be applicable for use in specific immunotherapy of leukemia patients or that of epithelial cancer patients.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

Sufficiency of the CD8+ T cell lineage to mount an effective tumoricidal response to syngeneic tumor-bearing novel class I MHC antigens

The origins of "help" in rejection of syngeneic tumors by the CD8 T cell lineage was examined with a model tumor inappropriately expressing novel class I MHC and subject to cytolytic T cell (CTL)-mediated rejection. The requirement for CD4+ Th cells to induce CD8+ CTL effectors in vivo was investigated by using C3H mice selectively depleted of either CD4+ or CD8+ T cells. Rejection of the tumor was vigorous and indistinguishable from normal mice after depletion of CD4+ T cells in vivo. In contrast, in CD8+ T cell-depleted mice tumors grew progressively, confirming that T cells of the CD8+ lineage are required for a tumoricidal immune response, and cells of this lineage are sufficient for a primary response. Taken together, these results demonstrate that, in the absence of CD4+ T cells in vivo, unprimed cells of the CD8+ lineage are fully competent to mount an effective CTL immune response to syngeneic cells expressing novel class I Ag, consistent with the concept that only T cells with class I recognition specificity may be required to satisfy the need for both help and effector functions in the response.     

Colon carcinoma cells induce CXCL11-dependent migration of CXCR3-expressing cytotoxic T lymphocytes in organotypic culture

Adoptive immunotherapy of cancer patients with cytolytic T lymphocytes (CTL) has been hampered by the inability of the CTL to home into tumors in vivo. Chemokines can attract T lymphocytes to the tumor site, as demonstrated in animal models, but the role of chemokines in T-lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a colon carcinoma (CC) patient’s CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic CC culture. CTL migration was mediated by chemokine receptor CXCR3 expressed by the CTL and CXCL11 chemokine secreted by the tumor cells. Excess CXCL11 or antibodies to CXCL11 or CXCR3 inhibited migration of CTL to tumor cells. T cell and tumor cell analyses for CXCR3 and CXCL11 expression, respectively, in ten additional CC samples, may suggest their involvement in other CC patients. Our studies, together with previous studies indicating angiostatic activity of CXCL11, suggest that CXCL11 may be useful as an immunotherapeutic agent for cancer patients when transduced into tumor cells or fused to tumor antigen-specific Ab.     

CpG-conjugated apoptotic tumor cells elicit potent tumor-specific immunity

The primary goal of cancer immunotherapy is to elicit an immune response capable of eradicating established tumors and preventing tumor metastasis. One strategy to achieve this goal utilizes whole killed tumor cells as the primary immunogen. Killed tumor cells provide a comprehensive source of tumor-associated antigens (TAAs), thereby eliminating the need to identify individual antigens. Unfortunately, killed tumor cells tend to be poorly immunogenic. To overcome this limitation, we covalently conjugated immunostimulatory CpG oligodeoxynucleotides (ODN) to apoptotic tumor cells and examined their ability to induce TAA-specific immune responses. Results indicate that CpG conjugation enhances the uptake of cell-based vaccines by dendritic cells (DCs), up-regulates co-stimulatory molecule expression, and promotes the production of immunostimulatory cytokines. Vaccination with CpG-conjugated tumor cells triggers the expansion of tumor-specific cytotoxic T lymphocytes (CTL) that reduce the growth of established tumors and prevents their metastatic spread. Thus, conjugating CpG ODN to cell-based tumor vaccines is an important step toward improving cancer immunotherapy.     

T cell-mediated tumor rejection displays diverse dependence upon perforin and IFN-gamma mechanisms that cannot be predicted from in vitro T cell characteristics

Experimental pulmonary metastases have been successfully treated by adoptive transfer of tumor-sensitized T cells from perforin knockout (KO) or Fas/APO-1 ligand(KO) mice, suggesting a prominent role for secretion of cytokines such as IFN-gamma. In the present study we confirmed that rejection of established methylcholanthrene-205 (MCA-205) pulmonary metastases displayed a requirement for T cell IFN-gamma expression. However, this requirement could be obviated by transferring larger numbers of tumor-sensitized IFN-gamma (KO) T cells or by immunosensitizing sublethal irradiation (500 rad) of the host before adoptive therapy. Extrapulmonary tumors (MCA-205 s.c. and intracranial) that required adjunct sublethal irradiation for treatment efficacy also displayed no requirement for host or T cell expression of IFN-gamma. Nonetheless, rejection of MCA-205 s.c. tumors and i.p. EL-4 tumors, but not MCA-205 pulmonary or intracranial tumors, displayed a significant requirement for T cell perforin expression (i.e., CTL participation). The capacity of T cells to lyse tumor targets and secrete IFN-gamma in vitro before adoptive transfer was nonpredictive of the roles of these activities in subsequent tumor rejection. Adoptive therapy studies employing KO mice are therefore indispensable for revealing a diversity of tumor rejection mechanisms that may lack in vitro correlation due to delays in their induction. Seemingly contradictory KO data from different studies are reconciled by the capacity of anti-tumor T cells to rely on alternative mechanisms when treated in larger numbers, the variable participation of CTL at different anatomic locations of tumor, and the apparent capacity of sublethal irradiation to provide a therapeutic alternative to host or T cell IFN-gamma production.     

Tumor progression despite efficient tumor antigen cross-presentation and effective "arming" of tumor antigen-specific CTL

To determine whether APC function or "arming" of CTL for lytic function are the points at which Ags from a nonimmunogenic tumor fail to induce an effective immune response, we established a murine tumor model that expressed intracellular OVA and selected a clone (cOVA-9) that remained susceptible to lysis by specific CD8(+) T cells throughout tumor growth. Viable cOVA-9 tumor cells grew in normal mice at a rate similar to the parental tumor, and vaccination with irradiated cOVA-9 cells did not induce protection against itself or the parental line, confirming its nonimmunogenic status. In vivo evaluation during tumor growth demonstrated persisting tumor Ag cross-presentation accompanied by the generation of potent, specific CTL which were detectable when tumors were barely palpable. Despite the presence of highly active CTL in the tumor-draining lymph nodes, there was no apparent lysis of tumor-associated APC. These data show that tumor-draining APC are not dysfunctional with regard to two crucial processes, in vivo tumor Ag cross-presentation and specific CTL arming, and that failure to prevent tumor growth is not in the induction phase, but in the effector phase and occurs within the tumor itself before the tumor matrix is established.     

Targeting the primary tumor to generate CTL for the effective eradication of spontaneous metastases

Metastatic disease is the major cause of morbidity and mortality in cancer. Although surgery, chemotherapy, or radiation can often control primary tumor growth, successful eradication of disseminated metastases remains rare. We have now tested whether direct targeting tumor tissues to generate antitumor immune response before surgical excision produces sufficient CTL against micrometastases. One unsolved problem is whether such response allows coming CTL to be educated and then exit the tumor site. Another unsolved problem is whether these CTL can then patrol and effectively eliminate spontaneously metastasized tumor cells in the periphery. In this study, we have shown that adenovirus-expressing TNFSF14 [LIGHT (name derived from homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes); Ad-LIGHT] inoculated directly into primary 4T1 tumor, a highly aggressive, spontaneously metastasizing mammary carcinoma, followed by surgical removal of the primary tumor can eradicate established and disseminated metastatic tumor cells in the peripheral tissues. Furthermore, we clearly show with a fibrosarcoma model Ag104L(d) that local treatment can generate plenty of tumor-specific CTL that exit the primary tumor and infiltrate distal tumors to completely eradicate distal tumors. Therefore, targeting the primary tumor with Ad-LIGHT before surgical excision is a new strategy to elicit better immune response for the eradication of spontaneous metastases.     

Protective immunity against disparate tumors is mediated by a nonpolymorphic MHC class I molecule

Current peptide-based immunotherapies for treatment of model cancers target tumor Ags bound by the classical MHC class I (class Ia) molecules. The extensive polymorphism of class Ia loci greatly limits the effectiveness of these approaches. We demonstrate in this study that the murine nonpolymorphic, nonclassical MHC class I (class Ib) molecule Q9 (Qa-2) promotes potent immune responses against multiple syngeneic tumors. We have previously shown that ectopic expression of Q9 on the surface of class Ia-negative B78H1 melanoma led to efficient CTL-mediated rejection of this tumor. In this study, we report that surface-expressed Q9 on 3LLA9F1 Lewis lung carcinoma and RMA T cell lymphoma also induces potent antitumor CTL responses. Importantly, CTL harvested from animals surviving the initial challenge with Q9-positive 3LLA9F1, RMA, or B78H1 tumors recognized and killed their cognate tumors as well as the other cancer lines. Furthermore, immunization with Q9-expressing 3LLA9F1 or RMA tumor cells established immunological memory that enhanced protection against subsequent challenge with a weakly immunogenic, Q9-bearing melanoma variant. Collectively, the generation of cross-reactive CTL capable of eliminating multiple disparate Q9-expressing tumors suggests that this nonpolymorphic MHC class I molecule serves as a restriction element for a shared tumor Ag(s) common to lung carcinoma, T cell lymphoma, and melanoma.     

In vitro generation of cytotoxic lymphocytes against radiation and radiation leukemia virus-induced tumors

Summary Cytotoxic T lymphocytes (CTL) to syngeneic radiation- or radiation leukemia virus (RadLV)-induced tumors were generated in vitro in mixed lymphocytetumor cultures (MLTC) using splenocytes of mice primed in vivo with inactivated tumor cells. Effective sensitization was obtained with virus-producer cell lines, while cells of a virus-nonproducer line did not sensitize.The CTL could lyse syngeneic, but not allogeneic, tumor cells of established lines producing C-type virus and therefore expressing membrane-associated viral antigenicity.Susceptibility of primary leukemias to cell-mediated lysis could not be tested due to a very high spontaneous ⁵¹ Cr release shortly after labeling. In a cold target competition assay, however, the RadLV-induced, but not the X-radiation-induced primary tumor cells inhibited the cytotoxic reactivity. This inhibition was correlated with the level of viral antigen expression on the inhibiting cells, which was high in the RadLV-induced and low in the radiation-induced primary tumors.These results suggest that antitumor CTL generated under conventional MLTC conditions are largely stimulated by and directed at virus-related antigens not necessarily associated with the malignant state of the cell.     

Vaccination with mRNAs encoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efficiently primes CTL responses, but is insufficient to overcome tolerance to a model tumor/self antigen

Immunization of mice with dendritic cells transfected ex vivo with tumor-associated antigen (TAA)-encoding mRNA primes cytotoxic T lymphocytes (CTL) that mediate tumor rejection. Here we investigated whether direct injection of TAA mRNA, encapsulated in cationic liposomes, could function similarly as cancer immunotherapy. Intradermal and intravenous injection of ovalbumin (OVA) mRNA generated specific CTL activity and inhibited the growth of OVA-expressing tumors. Vaccination studies with DNA have demonstrated that co-administration of antigen (Ag)- and cytokine-encoding plasmids potentiate the T cell response; in analogous fashion, the inclusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA enhanced OVA-specific cytotoxicity. The ability of this GM-CSF-augmented mRNA vaccine to treat an established spontaneous tumor was evaluated in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse, using the SV40 large T Ag (TAg) as a model tumor/self Ag. Repeated vaccination elicited vigorous TAg-specific CTL activity in nontransgenic mice, but tumor-bearing TRAMP mice remained tolerant. Adoptive transfer of naïve splenocytes into TRAMP mice prior to the first vaccination restored TAg reactivity, and slowed tumor progression. The data from this study suggests that vaccination with TAA mRNA is a simple and effective means of priming antitumor CTL, and that immunogenicity of the vaccine can be augmented by co-delivery of GM-CSF mRNA. Nonetheless, limitations of such vaccines in overcoming tolerance to tumor/self Ag may mandate prior or simultaneous reconstitution of the autoreactive T cell repertoire for this form of immunization to be effective.