Expanding the use of monoclonal antibody therapy of cancer by using ionising radiation to upregulate antibody targets.

BACKGROUND: Monoclonal antibody (mAb) therapy for the treatment of solid and haematologic malignancies has shown poor response rates as a monotherapy. Furthermore, its use is limited to tumours expressing certain molecular targets. It has been shown that single-dose radiation can induce immunogenic modulation that is characterised by cell-surface phenotypic changes leading to augmented tumour cell/cytotoxic T-cell interaction. METHODS: We examined radiation's ability to upregulate mAb therapy targets. We also used radiation to sensitise tumour cells to antibody-dependent cell-mediated cytotoxicity (ADCC). RESULTS: Radiation significantly increased cell-surface and total protein expression of mAb targets HER2, EGFR, and CD20. Focusing on HER2, targeted by trastuzumab, we observed significant upregulation of HER2 following radiation of 3 out of 3 breast cancer cell lines, one of which was triple negative, as well as in residential stem-cell populations. HER2 upregulation was sustained up to 96 h following radiation exposure and was largely dependent on intracellular reactive oxygen species. Improved ADCC and sensitisation to the antiproliferative effects of trastuzumab demonstrated the functional significance of radiation-induced HER2 upregulation. CONCLUSIONS: We show that single-dose radiation enhances mAb therapy. These findings highlight a mechanism for combining radiation with immunotherapy and expand the patient population that can be treated with targeted therapy.

The embryonic transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to conventional antitumor therapies.

The T-box transcription factor Brachyury, a molecule frequently detected in human cancers but seldom found in normal adult tissue, has recently been characterized as a driver of the epithelial-to-mesenchymal switch of human carcinomas. In the current investigation, we present data demonstrating that in two different human lung carcinoma models expression of Brachyury strongly correlates with increased in vitro resistance to cytotoxic therapies, such as chemotherapy and radiation. We also demonstrate that chemotherapy treatment in vitro selects for tumor cells with high levels of Brachyury and that the degree of resistance to therapy correlates with the level of Brachyury expression. In vitro and in vivo, human lung carcinoma cells with higher levels of Brachyury divide at slower rates than those with lower levels of Brachyury, a phenomenon associated with marked downregulation of cyclin D1, phosphorylated Rb and CDKN1A (p21). Chromatin immunoprecipitation and luciferase reporter assays revealed that Brachyury binds to a half T-box consensus site located within the promoter region of the p21 gene, indicating a potential mechanism for the observed therapeutic resistance associated with Brachyury expression. Finally, we demonstrate that in vivo treatment of tumor xenografts with chemotherapy results in the selective growth of resistant tumors characterized by high levels of Brachyury expression. Altogether, these results suggest that Brachyury expression may attenuate cell cycle progression, enabling tumor cells to become less susceptible to chemotherapy and radiation in human carcinomas.

A triad of costimulatory molecules synergize to amplify T-cell activation in both vector-based and vector-infected dendritic cell vaccines.

The activation of a T cell has been shown to require two signals via molecules present on professional antigen presenting cells: signal 1, via a peptide/MHC complex, and signal 2, via a costimulatory molecule. Here, the role of three costimulatory molecules in the activation of T cells was examined. Poxvirus (vaccinia and avipox) vectors were employed because of their ability to efficiently express multiple genes. Murine cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induced the activation of T cells to a far greater extent than cells infected with vectors expressing any one or two costimulatory molecules. Despite this T-cell "hyperstimulation" using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Results employing the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell to T-cell ratios. Experiments employing a four-gene construct also showed that TRICOM recombinants could enhance antigen-specific T-cell responses in vivo. These studies thus demonstrate the ability of vectors to introduce three costimulatory molecules into cells, thereby activating both CD4+ and CD8+ T-cell populations to levels greater than those achieved with the use of only one or two costimulatory molecules. This new threshold of T-cell activation has broad implications in vaccine design and development. Dendritic cells infected with TRICOM vectors were found to greatly enhance naïve T-cell activation, and peptide-specific T-cell stimulation. In vivo, peptide-pulsed DCs infected with TRICOM vectors induced cytotoxic T lymphocyte activity markedly and significantly greater than peptide-pulsed DCs.

Enhanced activation of rhesus T cells by vectors encoding a triad of costimulatory molecules (B7-1, ICAM-1, LFA-3).

Since the rhesus is often used as a "gatekeeper" model for the evaluation of malaria and simian immunodeficiency virus (SIV)/HIV vaccines, the identification of strategies to enhance the activation of rhesus T cells would potentially aid in the generation of more potent vaccines directed against these infectious agents. Several molecules normally found on the surface of professional human APCs are capable of providing the second signals critical for T cell activation: B7-1 (CD80), ICAM-1 (CD54), and LFA-3 (CD58). With the exception of B7, T cell costimulatory molecules in the rhesus have not been identified. We have recently designed and characterized both recombinant vaccinia and recombinant avipox vectors containing the transgenes for a triad of human T cell costimulatory molecules (B7-1, ICAM-1, LFA-3; designated TRICOM). Here, we demonstrate the enhanced activation of rhesus T cells stimulated with rhesus APCs infected with TRICOM vectors in the presence of signal 1. Infection with TRICOM vectors led to significant improvement of APC capabilities in terms of reduction of the amount of signal 1 needed to activate naive T cells, and reduction in the amount of APCs required to activate T cells using a constant amount of signal 1. Antibody blocking studies demonstrated that each of the three costimulatory molecule transgenes contributed to the enhanced proliferation of T cells. TRICOM-enhanced T cell activation was shown to correspond to increases in type 1 cytokines and a reduced level of apoptosis. TRICOM-infected autologous B cells from rhesus immunized with either an SIV vaccine or a malaria vaccine stimulated significantly greater levels of IFN-gamma in response to specific peptide than stimulation with uninfected autologous B cells or B cells infected with wild-type vector. The ability to augment immune responses using poxvirus-based vaccines containing multiple costimulatory molecule transgenes can now be addressed in the rhesus macaque model.

Synergy of vaccine strategies to amplify antigen-specific immune responses and antitumor effects.

Several different vaccine strategies have been evaluated and combined in an attempt to amplify T-cell responses toward induction of antitumor immunity. The model tumor antigen used was carcinoembryonic antigen (CEA). While initial T-cell activation studies were conducted in conventional mice, combined vaccine strategy studies and antitumor studies were conducted in transgenic mice in which CEA is expressed in normal gastrointestinal tissue and CEA protein is found in sera. The studies reported here demonstrate: (a) A recombinant avipox (fowlpox, rF) vector expressing the signal 1 (CEA) and the B7-1 costimulatory molecule transgenes (designated rF-CEA/B7-1) is more potent in inducing CEA-specific T-cell responses than rF-CEA; one administration of recombinant fowlpox vector expressing CEA and three different costimulatory molecule transgenes (B7-1, ICAM-1, LFA-3, designated rF-CEA/TRICOM) was more potent in inducing CEA-specific T-cell responses than four vaccinations with rF-CEA or two vaccinations with rF-CEA/B7-1. Moreover, up to four vaccinations with rF-CEA/TRICOM induced greater CEA-specific T-cell responses with each vaccination. (b) A diversified prime and boost strategy using a prime with a recombinant vaccinia vector expressing CEA and the triad of costimulatory molecules (designated rV-CEA/TRICOM) and a boost with rF-CEA/TRICOM was more potent in inducing CEA-specific T-cell responses than the repeated use of rF-CEA/TRICOM alone. (c) The addition of granulocyte macrophage colony-stimulating factor (GM-CSF) to the rF-CEA or rF-CEA/TRICOM vaccinations via the simultaneous administration of a rF-GM-CSF vector enhanced CEA-specific T-cell responses. These strategies (TRICOM/diversified prime and boost/GM-CSF) were combined to treat CEA-expressing carcinoma liver metastases in CEA-transgenic mice; vaccination was initiated 14 days posttumor transplant. Antitumor effects in terms of survival and CD8(+) and CD4(+) responses specific for CEA were also observed in this CEA-transgenic mouse model. These studies demonstrate that the use of cytokines and diversified prime and boost regimens can be combined with the use of recombinant vectors expressing signal 1 and multiple costimulatory molecules to further amplify T-cell responses toward more effective vaccine strategies.

Vector-driven hyperexpression of a triad of costimulatory molecules confers enhanced T-cell stimulatory capacity to DC precursors.

Activation of T cells requires at least two signals: signal 1, via the T-cell receptor, and signal 2, in which a costimulatory molecule on the antigen presenting cell (APC) interacts with a ligand on the T cell. Dendritic cells (DCs) are the most potent APCs in part due to their expression of costimulatory molecules. DCs, however, constitute only a minor percentage of APCs in the body, and the in vitro preparation of DCs is both costly and time consuming. The studies reported here demonstrate that one can utilize other APCs, such as bone marrow progenitor cells (BMPCs) and make them markedly more effective as APCs; this was accomplished by their infection with recombinant poxviruses (either the replication-defective avipox or vaccinia), which contain transgenes for a triad of costimulatory molecules (B7-1, ICAM-1 and LFA-3, designated TRICOM). APCs infected with TRICOM vectors are shown to significantly enhance the activation of both naive and effector CD4(+) and CD8(+) T-cell populations. The use of TRICOM vectors in vaccine strategies is discussed.

Enhancing the potency of peptide-pulsed antigen presenting cells by vector-driven hyperexpression of a triad of costimulatory molecules.

Recombinant orthopox vectors (both replication-defective fowlpox [rF], and replication competent vaccinia [rV] have been developed that simultaneously express three T-cell costimulatory molecule transgenes. The constituents of this triad of costimulatory molecules (designated TRICOM) are B7-1, ICAM-1, and LFA-3. We have previously shown that infection of murine dendritic cells (DCs) with TRICOM vectors increases their level of expression of the triad of costimulatory molecules and enhances the efficacy of DCs to activate T cells. While DCs are arguably the most potent antigen presenting cell (APC), limitations clearly exist in their use due to the level of effort and cost for their generation. The studies reported here demonstrate that a generic APC population, murine splenocytes, can be made markedly more efficient as APCs by infection with either rF-TRICOM or rV-TRICOM vectors. Infection of splenocytes with either TRICOM vector led to significant improvement of APC capabilities in terms of: (a) enhancement of mixed lymphocyte reactions; (b) a reduction in the amount of signal 1 to activate naive T cells; and (c) a reduction in the amount of APCs required to activate T cells using a constant amount of signal 1. TRICOM-enhanced T-cell activation was shown to correspond to increases in type-1 cytokines and a reduced level of apoptosis, compared with T cells activated with uninfected or control vector-infected splenocytes. In vitro and in vivo experiments compared DCs with TRICOM-infected splenocytes. Infection of splenocytes with TRICOM vectors markedly enhanced their ability to activate T cells to levels approaching that of DCs. These studies thus demonstrate for the first time that an abundant and accessible population of APCs obtainable without lengthy culture or the use of costly exogenous cytokines (in contrast to that of DCs) can be made more potent as APCs with the use of vectors that express a triad of costimulatory molecules.

PMA-treated K-562 leukemia cells mediate a TH2-specific expansion of CD4+ T cells in vitro.

Highly enriched preparations of human CD3+CD4+ T-lymphocytes were stimulated with mitogen or OKT3 to determine the capacity of K-562 cells to function as accessory cells. Phorbol 12-myristate 13-acetate (PMA)-treated K-562 cells were induced to differentiate along the megakaryocytic lineage and could supplant monocyte-accessory cell function. Intracytoplasmic analysis of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) established that IL-4, and not IFN-gamma, was preferentially produced by the activated lymphocytes. This polarized stimulation is compatible with a type 2 or humoral immune response of purified T cells co-cultured with differentiated K-562 cells in vitro, and may have implications in immunoregulation due to disease progression.

Enhanced activation of T cells by dendritic cells engineered to hyperexpress a triad of costimulatory molecules.

BACKGROUND: Activation and proliferation of T cells are essential for a successful cellular immune response to an antigen. Antigen-presenting cells (APCs) activate T cells through a two-signal mechanism. The first signal is antigen specific and causes T cells to enter the cell cycle. The second signal involves a costimulatory molecule that interacts with a ligand on the T-cell surface and leads to T-cell cytokine production and their proliferation. Dendritic cells express several costimulatory molecules and are believed to be the most potent APCs. Two recombinant poxvirus vectors (replication-defective avipox [fowlpox; rF] and a replication-competent vaccinia [rV]) have been engineered to express a triad of costimulatory molecules (B7-1, intercellular adhesion molecule-1, and leukocyte function-associated antigen-3; designated TRICOM). This study was designed to determine if dendritic cells infected with these vectors would have an enhanced capacity to stimulate T-cell responses. METHODS: Murine dendritic cells (of both intermediate maturity and full maturity) were infected with rF-TRICOM or rV-TRICOM and were used in vitro to stimulate naive T cells with the use of a pharmacologic agent as signal 1, to stimulate T cells in allospecific mixed lymphocyte cultures, and to stimulate CD8(+) T cells specific for a peptide from the ovalbumin (OVA) protein. In addition, dendritic cells infected with TRICOM vectors were pulsed with OVA peptide and used to vaccinate mice to examine T-cell responses in vivo. All statistical tests were two-sided. RESULTS: Dendritic cells infected with either rF-TRICOM or rV-TRICOM were found to greatly enhance naive T-cell activation (P<.001), allogeneic responses of T cells (P<.001), and peptide-specific T-cell stimulation in vitro (P<.001). Peptide-pulsed dendritic cells infected with rF-TRICOM or rV-TRICOM induced cytotoxic T-lymphocyte activity in vivo to a markedly greater extent than peptide-pulsed dendritic cells (P =.001 in both). CONCLUSIONS: The ability of dendritic cells to activate both naive and effector T cells in vitro and in vivo can be enhanced with the use of poxvirus vectors that potentiate the hyperexpression of a triad of costimulatory molecules. Use of either rF-TRICOM or rV-TRICOM vectors significantly improved the efficacy of dendritic cells in priming specific immune responses. These studies have implications in vaccine strategies for both cancer and infectious diseases.

Vaccination with a recombinant vaccinia vaccine containing the B7-1 co-stimulatory molecule causes no significant toxicity and enhances T cell-mediated cytotoxicity.

B7-1 is a co-stimulatory molecule that provides a second signal for T-cell activation. Several studies have demonstrated that vaccination with a vector containing genes encoding B7-1 and an antigen appears to be efficacious at promoting immune responsiveness to the antigen. To evaluate the safety of such a protocol and determine the effect of the B7-1 vector on immune responsiveness, female C57BL/6 mice were administered Wyeth wild-type vaccinia virus (V-WT) or V-WT containing the gene for B7-1 (rV-B7-1) as a single s. c. injection or 3 monthly s.c. injections. Immunologic parameters were evaluated in half of the mice and general toxicity in the other half. Immunologic end points included determination of splenic lymphocyte phenotypes, mitogen-induced T- and B-cell proliferation, T-cell proliferation in response to alloantigens, cell-mediated cytotoxicity (CMC), natural killer cell activity and serum anti-nuclear antibody (ANA) titers. No significant signs of general toxicity were noted. The primary immunologic effect was an increase in the ability of spleen cells to lyse allogeneic targets and to proliferate in response to allogeneic stimulation. Numbers of splenic CD8(+) cells were also increased. These effects were more pronounced after 3 vaccinations than after a single vaccination. Minimal differences in ANA were observed between mice immunized with V-WT and rV-B7-1. In addition, no serum antibodies against B7-1 were detected in any mice. The data suggest that vaccination with rV-B7-1 augments CMC with minimal toxicity.

Strategies in the development of recombinant vaccines for colon cancer.

A new era involving the evaluation of recombinant vaccines for colon cancer has begun with the concurrent emergence of insights and technologies in the fields of molecular biology and immunology. These advances include (I) the identification and cloning of an array of genes associated with the neoplastic process, such as oncogenes, suppressor genes, genes encoding oncofetal antigens, and tissue lineage determinants; (2) the development of a variety of viral and bacterial vectors to deliver and present gene products; (3) the identification of numerous T-cell costimulatory molecules and the knowledge of their mode of action; (4) the cloning and analysis of the modes of action of an array of cytokines and other immunomodulatory molecules; and (5) a more sophisticated knowledge of the mode(s) of antigen presentation and T-cell activation.

A triad of costimulatory molecules synergize to amplify T-cell activation.

The activation of a T cell has been shown to require two signals via molecules present on professional antigen-presenting cells: signal 1, via a peptide/MHC complex; and signal 2, via a costimulatory molecule. Here, the role of three costimulatory molecules in the activation of T cells was examined. Poxvirus (vaccinia and avipox) vectors were used because of their ability to efficiently express multiple genes. Murine cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induced the activation of T cells to a far greater extent than cells infected with any one or two costimulatory molecules. Despite this T-cell "hyperstimulation" using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Results using the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell:T-cell ratios. Experiments using a four-gene construct also showed that TRICOM recombinants can enhance antigen-specific T-cell responses in vivo. These studies thus demonstrate for the first time the ability of vectors to introduce three costimulatory molecules into cells, thereby activating both CD4+ and CD8+ T-cell populations to levels greater than those achieved with the use of only one or two costimulatory molecules. This new threshold of T-cell activation has broad implications in vaccine design and development.

The diversity of T-cell co-stimulation in the induction of antitumor immunity.

T-cell activation has now been shown to require at least two signals. The first signal is antigen-specific, is delivered through the T-cell receptor (TCR) via the peptide/major histocompatibility complex (MHC), and causes the T cell to enter the cell cycle. The second, or co-stimulatory, signal is required for cytokine production and proliferation, and is mediated through ligand interaction on the surface of the T cell. This chapter deals with: 1) comparative studies on the use of a dual-gene construct of a recombinant vaccinia (rV) vector containing a tumor-associated antigen (TAA) gene and a co-stimulatory molecule gene vs the use of admixtures of rV-TAA and rV containing the co-stimulatory molecule to induce anti-tumor immunity; 2) the use of an admixture of vaccinia viruses containing a TAA gene and the B7-1 co-stimulatory molecule gene to induce a therapeutic response in a lung metastasis tumor model; 3) the antitumor efficacy of whole-tumor-cell vaccines in which the B7-1 co-stimulatory molecule is expressed in a tumor-cell vaccine via a vaccinia vs a retroviral vector; 4) the use of recombinant poxviruses containing the genes for the co-stimulatory molecules ICAM-1 or LFA-3 to induce antitumor immunity; and 5) the use of poxvirus vectors containing a triad of co-stimulatory molecules (B7-1, ICAM-1 and LFA-3) that synergize to enhance both CD4+ and CD8+ T-cell responses to a new threshold.

Comparative studies of a retrovirus versus a poxvirus vector in whole tumor-cell vaccines.

A number of experimental and clinical studies have used retroviral vectors to express transgenes in whole tumor-cell vaccines. Recently, poxvirus vectors such as vaccinia or avipox have been used toward this goal. The studies reported here compare for the first time the use of a retroviral vector versus a poxvirus vector (vaccinia) in whole tumor-cell vaccines. The transgene used was the T-cell costimulatory molecule B7-1, and the tumor was the weakly or nonimmunogenic MC38 murine colon adenocarcinoma. Recombinant retrovirus (R-B7) and the recombinant vaccinia (V-B7) induced equivalent expression of B7 on the surface of the carcinoma cell. Using live whole-tumor cells as vaccine, cells transduced via recombinant retrovirus (MC38/R-B7) and recombinant vaccinia (MC38/V-B7) equally induced protection against challenge by native MC38 cells 14 days later. Upon rechallenge with native MC38 cells 40 days later, however, the MC38/R-B7 vaccine was shown to be less effective than the MC38/V-B7 vaccine. Similar results were obtained when the tumor cells were irradiated prior to administration. When comparative studies were conducted in which X-irradiated tumor-cell vaccines were given to mice bearing experimental lung metastases, the MC38/V-B7 vaccine was shown to be significantly (P = 0.0351) more effective than the MC38/R-B7 vaccine. Additional studies were carried out in mice that had received vaccinia virus previously. Again, the X-irradiated MC38/V-B7 vaccine was statistically (P = 0.024) more effective than the MC38/R-B7 vaccine in the elimination of metastases. When the naïve and vaccinia-immune mice for each vaccination group were combined for meta-analysis (n = 16), the MC38/V-B7 was significantly more effective than the MC38/R-B7 in the treatment of pulmonary metastases (P = 0.0014) in this model. These studies thus demonstrate for the first time that a whole tumor-cell vaccine (either live or X-irradiated) containing a vaccinia transgene is at least as efficient, and sometimes more efficient, in inducing antitumor effects compared with the same vaccine using a retrovirus to express the transgene. The implications for the clinical applications of such approaches are discussed.

Anti-tumor immunity elicited by a recombinant vaccinia virus expressing CD70 (CD27L).

CD70, a ligand of the T cell costimulatory receptor CD27, is expressed mainly on activated B cells and has been shown to increase cytotoxic activity and proliferation of preferentially unprimed T cells. Reported herein is the construction of a recombinant vaccinia virus encoding CD70 (designated rV-CD70) and a demonstration of its biological effect on naive T cells in vitro and in vivo. In a whole tumor cell vaccine model, the growth of CD70-negative murine colon adenocarcinoma (MC38) tumor cells infected with rV-CD70 (multiplicity of infection [MOI] of 0.1) and transplanted into syngeneic C57BL/6 mice was inhibited completely while control tumors infected with wild-type vaccinia grew rapidly and killed mice within 3-5 weeks. Tumor-free mice previously immunized with rV-CD70-infected tumors were partially protected against rechallenge with wild-type tumors, demonstrating the induction of systemic anti-tumor immunity. In addition, immunization of C57BL/6 mice with rV-CD70 admixed with vaccinia virus encoding carcinoembryonic antigen (rV-CEA) was superior to treatment with rV-CEA alone in inducing CEA-specific lymphoproliferative T cell responses and reducing growth of murine colon carcinomas transduced with CEA. These studies demonstrate for the first time the potential utility of a recombinant vaccinia virus expressing CD70 to enhance T cell responses and mediate anti-tumor immunity.

Induction of anti-tumor immunity elicited by tumor cells expressing a murine LFA-3 analog via a recombinant vaccinia virus.

T cell activation requires binding of the T cell receptor to the major histocompatibility molecule-peptide complex in the presence of adhesion and/or costimulatory molecules such as B7-1 (CD80), B7-2 (CD86), ICAM-1 (CD54), and LFA-3 [corrected]. The major ligand of CD2 is CD48, the murine analog of human leukocyte function-associated antigen 3 (LFA-3). To determine the effect of LFA-3 expression on the immunogenicity of tumor cells, we constructed a recombinant vaccinia virus containing the murine LFA-3 gene (designated rV-LFA-3). rV-LFA-3 was shown to be functional in vitro in terms of expression of LFA-3, T cell proliferation, adhesion, and cytotoxicity. Subcutaneous inoculation of rV-LFA-3-infected murine colon adenocarcinoma tumor cells (MC38) into immunocompetent syngeneic C57BL/6 mice resulted in complete lack of tumor growth. Inoculation of MC38 cells infected with equal doses of control wild-type vaccinia virus resulted in tumor growth in all animals. In addition, partial immunological protection was demonstrated against subsequent challenge with uninfected parental tumor cells up to 56 days after vaccination with rV-LFA-3-infected cells. Anti-tumor memory was also demonstrated by using gamma-irradiated MC38 cells and cells from another carcinoma model (CT26). These studies demonstrate that expression of LFA-3 via a poxvirus vector can be used to induce anti-tumor immunity.

The use of combination vaccinia vaccines and dual-gene vaccinia vaccines to enhance antigen-specific T-cell immunity via T-cell costimulation.

Several recombinant vaccinia viruses are currently being evaluated to induce antigen-specific immunity to a variety of infectious disease agents and tumor associated antigens. T-cell costimulation is extremely important in enhancing T-cell responses, and recombinant vaccines have now been shown to be effective vectors to express a range of these molecules. Both combination vaccines (an admixture of a recombinant vaccinia virus expressing a specific target antigen and a recombinant vaccinia virus expressing a costimulatory molecule) and dual-gene vaccines expressing both transgenes on the same vector have been shown capable of effectively enhancing antigen-specific responses via T-cell costimulation. In this report, we compare for the first time the use of both types of approaches to enhance antigen-specific T-cell responses, and we demonstrate the importance of route of vaccine administration and vaccine dose in attaining optimal T-cell responses. These studies should have direct bearing on the design of vaccine clinical trials for infectious agents and/or tumor associated antigens, in which T-cell costimulatory molecules will be employed to enhance antigen-specific T-cell responses via the use of either combination or dual-gene vaccinia vaccines.

Differential regulation of interleukin-1alpha and interleukin-1beta in K-562 cells.

Interleukin (IL)-1alpha and IL-1beta are encoded by two separate genes, but both function as comitogens for lymphocyte activation. In this study, we observed K-562 cells to express constitutively mRNA for IL-1alpha, although IL-1alpha was not detected in the growth-conditioned medium (GCM). However, IL-1beta mRNA was not expressed unless the cells had been treated with phorbol myristate acetate (PMA). Both IL-1alpha and IL-1beta were detected in the GCM after the cells had been cultured with PMA, suggesting that IL-1 elaboration required PMA treatment. The K-562 cells treated with PMA differentiated to the myeloblastic stage, as observed by nuclear morphologic properties by electron microscopy. PMA treatment induced de novo expression of CD61 or gpIIIa, a marker associated with megakaryoblasts. These results showed that although K-562 cells constitutively expressed IL-1alpha mRNA, PMA treatment was required for secretion. On the other hand, both the expression and secretion of IL-1beta required treatment with PMA. This study showed that K-562 cells treated with PMA differentiated to the myeloblastic stage and expressed and secreted IL-1alpha and IL-1beta.