T-CELL TREATMENTS FOR SOLID AND HEMATOLOGICAL TUMORS

Cell-based therapies have demonstrated potency and efficacy as cancer treatment modalities. T cells can be dichotomized by their T cell receptor (TCR) complexes where alpha/beta T cells (95% of T cells) and gamma/delta T cells (+T cells proliferated to clinically significant numbers and ROR1+ tumor cells were effectively targeted and killed by both ROR1-specific CAR+ T cell populations, although ROR1RCD137 were superior to ROR1RCD28 in clearance of leukemia xenografts in vivo. The second specific aim focused on generating bi-specific CD19-specific CAR+ gamma/delta T cells with polyclonal TCRgamma/delta repertoire on CD19+ artificial antigen presenting cells (aAPC). Enhanced cytolysis of CD19+ leukemia was observed by CAR+ gamma/delta T cells compared to CARneg gamma/delta T cells, and leukemia xenografts were significantly reduced compared to control mice in vivo. The third specific aim looked at the broad anti-tumor effects of polyclonal gamma/delta T cells expanded on aAPC without CAR+ T cells, where Vdelta1, Vdelta2, and Vdelta3 populations had naïve, effector memory, and central memory phenotypes and effector function strength in the following order: Vdelta2>Vdelta3>Vdelta1. Polyclonal gamma/delta T cells eliminated ovarian cancer xenografts in vivo and increased survival compared to control mice. Thus, translating these methodologies to clinical trials will provide cancer patients novel, safe, and effective options for their treatment.

Immunologic characteristics of immunogenic variants generated by {\it in vitro\/} treatment of a methylcholanthrene-induced murine fibrosarcoma MCA-F with 1 methyl-3-nitro-1-nitrosoguanidine, 5 -aza-2$\sp\prime$-deoxycytidine, or ultraviolet radiation

This study addresses the questions of whether the frequency of generation and in vivo cross-reactivity of highly immunogenic tumor clones induced in a single parental murine fibrosarcoma cell line MCA-F is more closely related to the agent used to induce the Imm$\sp{+}$ clone or whether these characteristics are independent of the agents used. These questions were addressed by treating the parental tumor cell line MCA-F with UV-B radiation (UV-B), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), or 5-aza-2$\sp\prime$-deoxycytidine (5-azaCdR). The frequency of Imm$\sp{+}$ variant generation was similarly high for the three different agents, suggesting that the frequency of Imm$\sp{+}$ generation was related more closely to the cell line than to the inducing agent used. Cross-reactivity was tested with two Imm$\sp{+}$ clones from each treatment group in a modified immunoprotection assay that selectively engendered antivariant, but not antiparental immunity. Under these conditions each clone, except one, immunized against itself. The MNNG-induced clones engendered stronger antivariant immunity but a weaker variant cross-reactive immunity could also be detected.^ This study also characterized the lymphocyte populations responsible for antivariant and antiparental immunity in vivo. Using the local adoptive transfer assay (LATA) and antibody plus complement depletion of T-cell subsets, we showed that immunity induced by the Imm$\sp{+}$ variants against the parent MCA-F was transferred by the Thy1.2$\sp{+}$, L3T4a$\sp{+}$, Lyt2.1$\sp{-}$ (CD4$\sp{+}$) population, without an apparent contribution by Thy1.2$\sp{+}$, L3T4a$\sp{-}$, Lyt2.1$\sp{+}$ (CD8$\sp{+}$) cells. A role for Lyt2.1$\sp{+}$T lymphocytes in antivariant, but not antiparent immunity was supported by the results of LATA and CTL assays. Immunization with low numbers of viable Imm$\sp{+}$ cells, or with high numbers of non viable Imm$\sp{+}$ cells engendered only antivariant immunity without parental cross-protection. The associative recognition of parental antigens and variant neoantigens resulting in strong antiparent immunity was investigated using somatic cells hybrids of Imm$\sp{+}$ variants of MCA-F and an antigenically distinct tumor MCA-D. An unexpected result of these latter experiments was the expression of a unique tumor-specific antigen by the hybrid cells. These studies demonstrate that the parental tumor-specific antigen and the variant neoantigen must be coexpressed on the cell surface to engender parental cross-protective immunity. (Abstract shortened with permission of author.) ^

Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

The effects of IL -10 producing monocytes on T-cell activation in patients with epithelial ovarian cancer

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites of patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-β2, but not IL-12, IL-1α, or TNF-α, and expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface antigens. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T-cells from the peritoneal cavity of patients with ovarian carcinoma. Cytokine transcripts, including IL-2, GM-CSF, and IFN-γ were detected in T-cells isolated from patient specimens that also contained the IL-10 producing monocytes, although the IFN-γ and IL-2 proteins could not be detected in T-cells co-incubated with the IL-10 producing monocytes in vitro. Additionally, IL-10 producing monocytes co-cultured with autologous T-cells inhibited the proliferation of the T-cells in response to PHA. T-cell proliferation and cytokine protein production could be restored by the addition of neutralizing antibodies to IL-10R and TGF-β to the co-culture system. These results suggested that this subset of monocytes may modulate antitumor immune responses by inhibiting T-cell proliferation and cytokine protein production. Further studies determined that the precursors to the inhibitory monocytes were tumor-associated and only present in the peripheral blood of patients with ovarian cancer and not present in the peripheral blood of healthy donors. These precursors could be induced to the suppressor phenotype by the addition of IL-2 and GM-CSF, two cytokines detected in the peritoneal cavity of ovarian cancer patients. Lastly, it was shown that the suppressor monocytes from the peritoneal cavity of ovarian cancer patients could be differentiated to a non-inhibitory phenotype by the addition of TNF-α and IFN-γ to the culture system. The differentiated monocytes did not produce IL-10, expressed the activation antigens HLA-DR, CD80, and CD86, and were able to stimulate autologous T-cells in vitro. Since a concomitant reduction in immune function is associated with tumor growth and progression, the effects of these monocytes are of considerable importance in the context of tumor immunotherapy. ^

La doble imagen de la moralidad de Augusto

Resumen: Augusto llegó al poder por ser hijo adoptivo de César. Hasta recibir el título de Augusto en el año 27 a. C., fue conocido como G. Iulius Caesar Diui filius. Ser heredero de César tenía que ser algo más que recibir su nombre y sus poderes: también sus cualidades personales debían recordar al Dictador. Por eso, durante la mitad del triunvirato, Augusto intentó emular a su padre adoptivo también en sus cualidades como amante. Su tumultuosa vida afectiva y conyugal, en efecto, se concentra entre los años 44 y 35 a. C., con varios matrimonios o esponsales, y el nacimiento de su única hija. De esta época proceden la mayoría de las historias que –ciento cincuenta años después- contaría Suetonio a partir de una fuente cercana a Marco Antonio. A partir de la sacrosantidad promulgada para Livia en el año 35 a. C., sin embargo, comienza una nueva etapa en la imagen moral de Augusto, centrada en la virtud y la monogamia: una etapa que le llevaría años después a desterrar a su propia hija y, después, a su nieta, por adulterio. De esta doble imagen de Augusto, la primera debida a la emulación de su padre adoptivo, y la segunda debida a una necesidad de justificación ideológica de su régimen frente al de su enemigo Marco Antonio, proceden las noticias aparentemente contradictorias sobre los “vicios privados y públicas virtudes” de Gayo Julio César Augusto.

Tras las huellas de Horacio : Carm.,1,2 y 4, 15

Hor., en Carm., 1, 2 y 4, 15 recoge una serie de referencias a dioses importantes para la familia adoptiva de Octaviano y para el propio gobernante, que han sido tradicionalmente analizadas e interpretadas desde un punto de vista interno: su presencia se explicaría en función de su relación con la gens Iulia. El punto de vista que quiero ofrecer en mi trabajo es complementario a éste: a partir de un análisis externo de las referencias a estos dioses, creo que su presencia se puede explicar en función de los templos en los que "habitaban" en Roma, del lugar donde estos templos se encontraban y de las representaciones icónicas que servían a su culto. Al mismo tiempo, la nueva relación que establezco entre 1,2 y 4, 15 aporta evidencias para una comprensión distinta de la segunda edición de la poesía lírica de Horacio, la que se presentó con el libro 4

Tras las huellas de Horacio : Carm.,1,2 y 4, 15

Hor., en Carm., 1, 2 y 4, 15 recoge una serie de referencias a dioses importantes para la familia adoptiva de Octaviano y para el propio gobernante, que han sido tradicionalmente analizadas e interpretadas desde un punto de vista interno: su presencia se explicaría en función de su relación con la gens Iulia. El punto de vista que quiero ofrecer en mi trabajo es complementario a éste: a partir de un análisis externo de las referencias a estos dioses, creo que su presencia se puede explicar en función de los templos en los que "habitaban" en Roma, del lugar donde estos templos se encontraban y de las representaciones icónicas que servían a su culto. Al mismo tiempo, la nueva relación que establezco entre 1,2 y 4, 15 aporta evidencias para una comprensión distinta de la segunda edición de la poesía lírica de Horacio, la que se presentó con el libro 4

Tras las huellas de Horacio : Carm.,1,2 y 4, 15

Hor., en Carm., 1, 2 y 4, 15 recoge una serie de referencias a dioses importantes para la familia adoptiva de Octaviano y para el propio gobernante, que han sido tradicionalmente analizadas e interpretadas desde un punto de vista interno: su presencia se explicaría en función de su relación con la gens Iulia. El punto de vista que quiero ofrecer en mi trabajo es complementario a éste: a partir de un análisis externo de las referencias a estos dioses, creo que su presencia se puede explicar en función de los templos en los que "habitaban" en Roma, del lugar donde estos templos se encontraban y de las representaciones icónicas que servían a su culto. Al mismo tiempo, la nueva relación que establezco entre 1,2 y 4, 15 aporta evidencias para una comprensión distinta de la segunda edición de la poesía lírica de Horacio, la que se presentó con el libro 4

Diagnosis and Management of Grain-Induced Asthma

Grain-induced asthma is a frequent occupational allergic disease mainly caused by inhalation of cereal flour or powder. The main professions affected are bakers, confectioners, pastry factory workers, millers, farmers, and cereal handlers. This disorder is usually due to an IgE-mediated allergic response to inhalation of cereal flour proteins. The major causative allergens of grain-related asthma are proteins derived from wheat, rye and barley flour, although baking additives, such as fungal α-amylase are also important. This review deals with the current diagnosis and treatment of grain-induced asthma, emphasizing the role of cereal allergens as molecular tools to enhance diagnosis and management of this disorder. Asthma-like symptoms caused by endotoxin exposure among grain workers are beyond the scope of this review. Progress is being made in the characterization of grain and bakery allergens, particularly cereal-derived allergens, as well as in the standardization of allergy tests. Salt-soluble proteins (albumins plus globulins), particularly members of the α-amylase/trypsin inhibitor family, thioredoxins, peroxidase, lipid transfer protein and other soluble enzymes show the strongest IgE reactivities in wheat flour. In addition, prolamins (not extractable by salt solutions) have also been claimed as potential allergens. However, the large variability of IgE-binding patterns of cereal proteins among patients with grain-induced asthma, together with the great differences in the concentrations of potential allergens observed in commercial cereal extracts used for diagnosis, highlight the necessity to standardize and improve the diagnostic tools. Removal from exposure to the offending agents is the cornerstone of the management of grain-induced asthma. The availability of purified allergens should be very helpful for a more refined diagnosis, and new immunomodulatory treatments, including allergen immunotherapy and biological drugs, should aid in the management of patients with this disorder.

ASPECTOS PSICODINÂMICOS E CAPACIDADE MATERNA DE MÃES DE CRIANÇAS ABRIGADAS

Este estudo teve como objetivos investigar aspectos da dinâmica intrapsíquica de mães de crianças institucionalizadas em abrigo por ordem judicial, e identificar recursos defensivos utilizados por essas mães. Para atingir estes objetivos, realizou-se uma investigação clínica com estudo de três casos de mães de crianças abrigadas. Foram utilizados dois instrumentos: a) Roteiro de Entrevista roteiro de temas a serem abordados em uma ou mais entrevistas não diretivas de cunho clínico, a fim de auxiliar na investigação da psicodinâmica destas mães. b) Procedimento de Desenho Estória com Tema técnica projetiva que associa o uso de desenhos com estórias, como forma de explorar livre e dinamicamente os conteúdos da personalidade. A técnica permite o estudo das características formais e estruturais da personalidade, pois tem a particularidade de facilitar a expressão de aspectos inconscientes relacionados a pontos de angústias presentes, focos conflituosos e perturbações emergentes. Estes procedimentos foram realizados nas dependências da instituição (abrigo) onde as crianças estavam hospedadas. Os principais resultados comuns aos três casos foram: Ambigüidade e os Impeditivos de Crescimento a primeira mãe entrevistada ao mesmo tempo ataca a mãe que a abandona (mãe biológica e a mãe adotiva), em busca de uma mãe idealizada. Essa ambigüidade a impede de crescer. Nota-se a mesma tentativa de idealização na segunda mãe estudada que demonstra dificuldade em aceitar a atual situação em que vive e não consegue perceber que a aproximação de sua mãe é por causa da doença que ela adquiriu e não por continência. A terceira e última mãe entrevistada demonstra conteúdos persecutórios diante do abrigamento dos filhos e dificuldade de sentir gratidão. Os mecanismos predominantes que aparecem nos três casos são os de: idealização e regressão a estágios primitivos. Nota-se ainda, depressão, dificuldade de elaboração da posição depressiva. Estas mães não conseguem vivenciar continuamente a realidade psíquica, que implicaria na elaboração da posição depressiva, pois não conseguem fazer, ainda que tentem, uma comparação entre os mundos interno e externo, o que as levariam à uma melhor compreensão das semelhanças e diferenças. De modo que, a figura dos pais (principalmente da mãe) fica cindida entre aterrorizante e idealizada, porém os mecanismos predominantes são suas fantasias que propiciam idealização; identificação projetiva maciça. A persecutoriedade e a culpa, ao mesmo tempo parecem indicar a depressão que pode ser tão forte que levam à intensificação destes sentimentos. Há a presença da inveja que também intensifica as angústias persecutórias, requerendo mecanismos de defesa que violentam as funções psíquicas.(AU)

ASPECTOS PSICODINÂMICOS E CAPACIDADE MATERNA DE MÃES DE CRIANÇAS ABRIGADAS

Este estudo teve como objetivos investigar aspectos da dinâmica intrapsíquica de mães de crianças institucionalizadas em abrigo por ordem judicial, e identificar recursos defensivos utilizados por essas mães. Para atingir estes objetivos, realizou-se uma investigação clínica com estudo de três casos de mães de crianças abrigadas. Foram utilizados dois instrumentos: a) Roteiro de Entrevista roteiro de temas a serem abordados em uma ou mais entrevistas não diretivas de cunho clínico, a fim de auxiliar na investigação da psicodinâmica destas mães. b) Procedimento de Desenho Estória com Tema técnica projetiva que associa o uso de desenhos com estórias, como forma de explorar livre e dinamicamente os conteúdos da personalidade. A técnica permite o estudo das características formais e estruturais da personalidade, pois tem a particularidade de facilitar a expressão de aspectos inconscientes relacionados a pontos de angústias presentes, focos conflituosos e perturbações emergentes. Estes procedimentos foram realizados nas dependências da instituição (abrigo) onde as crianças estavam hospedadas. Os principais resultados comuns aos três casos foram: Ambigüidade e os Impeditivos de Crescimento a primeira mãe entrevistada ao mesmo tempo ataca a mãe que a abandona (mãe biológica e a mãe adotiva), em busca de uma mãe idealizada. Essa ambigüidade a impede de crescer. Nota-se a mesma tentativa de idealização na segunda mãe estudada que demonstra dificuldade em aceitar a atual situação em que vive e não consegue perceber que a aproximação de sua mãe é por causa da doença que ela adquiriu e não por continência. A terceira e última mãe entrevistada demonstra conteúdos persecutórios diante do abrigamento dos filhos e dificuldade de sentir gratidão. Os mecanismos predominantes que aparecem nos três casos são os de: idealização e regressão a estágios primitivos. Nota-se ainda, depressão, dificuldade de elaboração da posição depressiva. Estas mães não conseguem vivenciar continuamente a realidade psíquica, que implicaria na elaboração da posição depressiva, pois não conseguem fazer, ainda que tentem, uma comparação entre os mundos interno e externo, o que as levariam à uma melhor compreensão das semelhanças e diferenças. De modo que, a figura dos pais (principalmente da mãe) fica cindida entre aterrorizante e idealizada, porém os mecanismos predominantes são suas fantasias que propiciam idealização; identificação projetiva maciça. A persecutoriedade e a culpa, ao mesmo tempo parecem indicar a depressão que pode ser tão forte que levam à intensificação destes sentimentos. Há a presença da inveja que também intensifica as angústias persecutórias, requerendo mecanismos de defesa que violentam as funções psíquicas.(AU)

T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function

T helper (Th) cells can be categorized according to their cytokine expression. The differential induction of Th cells expressing Th1 and/or Th2 cytokines is key to the regulation of both protective and pathological immune responses. Cytokines are expressed transiently and there is a lack of stably expressed surface molecules, significant for functionally different types of Th cells. Such molecules are of utmost importance for the analysis and selective functional modulation of Th subsets and will provide new therapeutic strategies for the treatment of allergic or autoimmune diseases. To this end, we have identified potential target genes preferentially expressed in Th2 cells, expressing interleukin (IL)-4, IL-5, and/or IL-10, but not interferon-γ. One such gene, T1/ST2, is expressed stably on both Th2 clones and Th2-polarized cells activated in vivo or in vitro. T1/ST2 expression is independent of induction by IL-4, IL-5, or IL-10. T1/ST2 plays a critical role in Th2 effector function. Administration of either a mAb against T1/ST2 or recombinant T1/ST2 fusion protein attenuates eosinophilic inflammation of the airways and suppresses IL-4 and IL-5 production in vivo following adoptive transfer of Th2 cells.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Generation of CD8+ T cells specific for transporter associated with antigen processing deficient cells

Cells with impaired transporter associated with antigen processing (TAP) function express low levels of cell surface major histocompatibility complex (MHC) class I molecules, and are generally resistant to lysis by MHC class I restricted cytotoxic T lymphocytes (CTLs). Here we report the generation of MHC class I restricted CD8+ CTLs that surprisingly require target cell TAP deficiency for efficient recognition. C57BL/6 (B6) mice immunized with syngenic B7–1 (CD80) expressing TAP-deficient cells generated a potent CTL response against both TAP-deficient RMA-S tumor cells and TAP-deficient Con A blasts, whereas the corresponding TAP-expressing target cells were considerably less susceptible or resistant to lysis. The CTL epitopes recognized were expressed also by the human TAP-deficient cell line T2, transfected with appropriate MHC class I molecules. B6 mice immunized with B7–1-transfected TAP-deficient RMA-S cells were protected from outgrowth of a subsequent RMA-S tumor challenge. These findings are discussed in relation to the biochemical nature of MHC class I dependent CTL epitopes associated with impaired TAP function, as well as implications for immunotherapy and autoimmunity.

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host–tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12–B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 × 107 pfu/mouse) of AdIL12–B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12–B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.