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Dendritic cells ignite adaptive immunity by priming naïve T lymphocytes. Human monocyte-derived dendritic cells (MDDCs) infected withToxoplasma gondiiinduce T-lymphocyte gamma interferon production and may thus activateT. gondii-specific immunity. However, we now demonstrate thatT. gondii-infected MDDCs are poor at activating T lymphocytes and are unable to induce specific cytotoxic T lymphocytes. On the other hand, MDDCs acquiring nonviableT. gondiiantigens directly, or indirectly through captured apoptotic or necrotic cell bodies, induce potent T-lymphocyte activation. T lymphocytes exposed to infected MDDCs are significantly impaired in upregulation of CD69 and CD28, are refractory to activation, and die through contact-dependent apoptosis mediated by an as-yet-unidentified mechanism not requiring Fas, tumor necrosis factor-related apoptosis-inducing ligand, leukocyte function antigen 1, intercellular adhesion molecule 1, tumor necrosis factor alpha, interleukin 10, alpha interferon, gamma interferon, prostaglandins, or reactive nitrogen intermediates. Bystander T lymphocytes that were neither infected nor apoptotic were refractory to activation, suggesting global dysfunction. Immunosuppression and T-lymphocyte unresponsiveness and apoptosis are typical of acuteT. gondiiinfection. Our data suggest that infected dendritic cells contribute to these processes. On the other hand, host cells infected withT. gondiiare resistant to multiple inducers of apoptosis. Thus, regulation of host cell and bystander cell apoptosis by viableT. gondiimay be significant components of a strategy to evade immunity and enhance intracellular parasite survival.

Autologous dendritic cells (DCs) transfected with mRNA encoding prostate-specific antigen (PSA) are able to stimulate potent, T cell–mediated antitumor immune responses in vitro. A phase I trial was performed to evaluate this strategy for safety, feasibility, and efficacy to induce T cell responses against the self-protein PSA in patients with metastatic prostate cancer. In 13 study subjects, escalating doses of PSA mRNA–transfected DCs were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope PSA in six of seven subjects; three patients that could be analyzed exhibited a transient molecular clearance of circulating tumor cells. The demonstration of vaccine safety, successful in vivo induction of PSA-specific immunity, and impact on surrogate clinical endpoints provides a scientific rationale for further clinical investigation of RNA-transfected DCs in the treatment of human cancer.

Background Cancer prevention is a cost-effective alternative to treatment. Oral rapamycin (eRapa) prevents distinct cancers and extends life in mice [1] , making it a candidate broad-spectrum cancer prevention agent. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), has significant immune effects that are unstudied in cancer. We hypothesize that eRapa prevents cancer through improved cancer immune surveillance, which we tested in a carcinogen-induced, inflammation-driven skin cancer model. Methods eRapa was used at 14 ppm microencapsulated rapamycin. Skin cancer. Wild-type C57BL/6 (WT) or syngeneic β δ knockout (KO) and interferon (IFN)- γ KO mice (lacking all T cells or IFN- γ , respectively) got eRapa or control for 1 month, followed once with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), then twice weekly applications with the inflammatory agent 12-O-tetradecanoylphorbol (TPA) for 24 weeks. Flow cytometry assessed cells and IFN- γ . Statistics. Tumors (unpaired t test and 2-way ANOVA) and malignant degeneration (Fischer exact test). Results eRapa reduced benign ( p = .004) and malignant ( p = .03) tumors in WT mice. T cells and IFN- γ mediate cancer immune surveillance so their effects on eRapa cancer prevention were studied. eRapa reduced skin tumors in β δ KO mice lacking all T cells ( p = .04), but not in IFN- γ KO mice ( p = .13), consistent with loss of beneficial eRapa-induced, non-T cell IFN- γ . In support, WT or IFN- γ KO T cell transfer into IFN- γ KO mice did not alter eRapa cancer prevention in this model. In WT mice on DMBA/TPA, eRapa increased IFN- γ producing dermal natural killer (NK) cells ( p γ KO mice, suggesting that eRapa-mediated IFN- γ facilitated local NK cell accumulation. Conclusions eRapa prevents carcinogen and inflammation-induced skin cancer, which involves a non-T cell that could be an NK cell, and is IFN- γ dependent. We also show that eRapa prevents cancer and extends life in distinct mouse cancer models, which paves the way for use of mTOR inhibitors as broad spectrum human cancer prevention agents.

Tregs hinder anti-tumor immunity, and depleting them treats cancers effectively in mice. By contrast, Treg depletion in human trials has limited efficacy. In a phase I trial of advanced stage carcinomas (breast, lung, melanoma, ovarian, bladder), we showed that the IL-2/diphtheria fusion toxin denileukin diftitox (DT) significantly depleted functional CD4+CD25hiFoxp3+ Tregs in blood with improved T cell function (increased Ki-67 and interferon-γ) with minimal effects on other blood mononuclear cells. One patient with stage IV ovarian carcinoma experienced significant reduction of metastatic tumor burden with denileukin diftitox at 12 μg/kg. Additional work showed that Treg depletion effects could last up to 4 weeks, and that weekly DT at 12 μg/kg eventually depleted anti-tumor and tumor-specific CD8+ T cells. Based on these data, we conducted a phase II clinical trial of epithelial ovarian carcinomas FIGO stage III-IV failing standard treatments, using DT as a single agent at 12 μg/kg by intravenous infusion every 3-4 weeks. In this trial, DT significantly depleted blood and tumor microenvironmental Tregs with only grade I-II toxicities, but with minimal clinical efficacy in 28 consecutive patients. This trial was halted for futility according to the Simon 2-stage design. Interferon-α alone does not treat ovarian cancer, but we now show that it significantly improves clinical and immune DT-mediated Treg depletion efficacy in human ovarian cancer. In the ID8 mouse ovarian carcinoma model, DT modestly increased survival and anti-tumor immunity. Interferon-α alone did not affect Treg numbers or function, but enhanced CD8+ T cell anti-tumor immunity. Interferon-α plus DT increased mouse survival significantly over either individual drug. In type I IFNR-/- mice unable to mediate interferon-α signals, interferon-α directly increased adoptively transferred IFNR+CD8+ T cell function independent of CD4+ T cell help. When combined with DT, IFN-α reduced Treg function without further numerical Treg reduction by indirect effects on tumor microenvironmental dendritic cells. In vitro studies identified IFN-α-driven dendritic cell IL-6 as a mechanism for reducing Treg function. When three ovarian cancer patients failed DT alone in the phase II trial, two had clinical and immune benefit by adding pegylated interferon-α2a in a separate trial, with acceptable toxicities. This trial was halted due to a lack of further DT. These studies demonstrate that DT depletes Tregs in distinct human carcinomas but is unlikely to be clinically effective as a single agent. We identified novel IFN-α mechanisms that improve Treg depletion effects using FDA-approved agents that can be rapidly translated. More selective Treg depletion agents and rationale combinations with other agents could improve clinical efficacy further. Citation Format: Suzanne Thibodeaux, Vincent Hurez, Shawna Wall, Srilakshmi Pandeswara, Benjamin Daniel, Aijie Liu, Lishi Sun, Leslie Wood, Weiping Zou, Tyler Curiel. Interferon-α enhances clinical benefits of regulatory T cell depletion in ovarian cancer through direct T cell effects and by inducing bystander IL-6. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-259. doi:10.1158/1538-7445.AM2014-LB-259

Reciprocal regulation of opposing functions characterizes biological systems. We now show that adenovirus-infected plasmacytoid dendritic cells (PDC) inhibit monocyte to myeloid dendritic cell (MDC) differentiation and function, and that adenovirus-infected monocytes inhibit PDC type I interferon secretion. Adenovirus-infected PDC secreted IFN-alpha, beta and omega in an 86:2:1 ratio. PDC type I interferons inhibited MDC differentiation and function (reduced IL-12 secretion, IFN-gamma induction, MLR and CD40 expression, and increased CD1a(+)CD14(+) cells). Type I interferon receptor blocking antibody reversed all PDC effects, and recombinant IFN-alpha, beta or omega replicated all effects, except reduced CD40. Adenovirus-infected monocytes suppressed PDC type I interferon secretion, which was reversed with anti-IL-10 neutralizing antibodies. Exogenous IL-10 suppressed PDC type I interferon secretion without reducing PDC viability. Therefore, monocyte IL-10 regulates PDC type I interferon secretion, and PDC type I interferons inhibit MDC differentiation and function. Such reciprocal regulation of potentially opposing influences may help modulate anti-pathogen immunity.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Monocyte-derived dendritic cells (MDDCs) are the most commonly used type of human DC, but are only rarely prepared from mice largely because the yield is small, and there are other easier methods available to produce mouse DCs. Blood is obtained by cardiac puncture. PBMCs are separated by density gradient centrifugation and plated at 8–10 million cells/well in a six-well plate. The mouse peritoneal cavity harbors a small resident population of myeloid cells that are generally regarded as being macrophages. Peritoneal contents are recovered by gentle lavage. Mature mouse DCs will differentiate from fetal liver progenitor cells using a stromal cell feeder layer, GM-CSF, Flt-3L, and SCF to induce differentiation, followed by GM-CSF plus TNFα to induce maturation. DCs isolated from mouse myeloma or GM-CSF and CD40L-transduced colon carcinoma cells will activate tumor-specific T cells without exogenous antigen loading.

Subgroups of the B cell malignancies are known to be associated with Epstein–Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV- TK ) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV- TK /GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.

Toxoplasma gondii antigens are superantigens in mice. To investigate a superantigen effect in humans, lymphocytes from T. gondii-seronegative subjects were studied for proliferation to T. gondii antigens (TA). Marked cellular proliferation, predominantly of CD4+ lymphocytes, was apparent. TA elicited expansions of Vbeta-bearing lymphocytes in all subjects, but different Vbeta-bearing lymphocytes were expanded in different subjects in both CD4+ and CD8+ subpopulations. Cord blood cells also proliferated to TA. Previously fixed antigen-presenting cells were unable to present TA. Thus, T. gondii appears to produce a molecule(s) that induces polyclonal activation of human T cells and requires antigen processing to mediate this effect. That T. gondii does not appear to behave as a superantigen in humans is important in understanding the pathogenesis of T. gondii infection in immunocompromised hosts and in the design of anti-T. gondii vaccines.

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.

A widely applicable cancer prevention strategy would be a cost-effective approach to lessen the enormous burden of cancer. Enterically-released microencapsulated rapamycin (eRapa) extends lifespan in mice and delays or prevents cancer development, suggesting cancer prevention as a longevity mechanism. Rapamycin acts through inhibition of mechanistic target of rapamycin (mTOR), which has significant immune effects that are surprisingly little studied in cancer treatment or prevention. In naive mice, chronic eRapa significantly reduced Th1 (CXCR3 + ) cell numbers ( p = .03) and T cell interferon (IFN)- γ ( p = .04), increased Th17 (CCR6 + ) cell numbers ( p = .04) and T cell IL-17 ( p = .03), increased T cell IL-22 ( p = .02), and reduced immune suppressing regulatory T cells ( p βδ −/− and IFN- γ −/− mice (lacking all T cells or IFN- γ , respectively) were given eRapa or control, and skin tumors were induced with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) followed by the inflammatory promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). eRapa reduced papilloma size in WT BL6 mice ( p = .02) versus controls, the first demonstration that oral rapamycin prevents inflammation-mediated neoplasia. eRapa also significantly reduced papillomas in βδ −/− mice ( p = .04) and increased papillomas in IFN- γ −/− mice ( p = .05). These data are consistent with the concept that eRapa protects from carcinogen-induced dermal carcinogenesis through a non-T cell, IFN- γ -dependent mechanism, which we suspect includes natural killer cells. We have also shown that eRapa prevents cancer in a variety of mouse cancer models, in which we are further studying immune mechanisms. Immune contributions from mTOR inhibitors in cancer prevention (and treatment) require more attention.

Background: Autophagy is a cellular process that generates energy to assure cell survival in response to insults such as hypoxia, nutrient deprivation or cytotoxic mediators. Autophagy contributes to cancer cell survival as well as drug resistance. Several anticancer agents, including Vorinostat (V) are reported to induce autophagy as a cytoprotective mechanism. In preclinical models, the addition of Hydroxycholoquine (HCQ) to V inhibited autophagy and significantly increased the anticancer effects of V. The objectives of this trial were to determine the MTD of HCQ in combination with V both administered daily (QD) in patients (pts) with advanced solid tumors, to evaluate PK and PD parameters, and test anti-tumor activity. Biomarkers were evaluated in tumors and peripheral blood mononuclear cells (PBMC) to investigate synergistic antitumor activity of V in combination with HCQ. Methods: We used a 3+3 dose escalation design of V at 300-400 mg QD plus HCQ QD at 400-1000 mg in 21 day cycles. Patients with advanced solid tumors and no available treatment with ECOG PS 0-2 and adequate organ function were eligible. Results: 32 patients were enrolled with 27 evaluable for toxicity studies. No dose limiting toxicity (DLT) was observed in cohorts 1 or 2. In cohort 3 (400 mg V/600 mg HCQ), 1/6 pts had DLT of Gr 3 anemia and fatigue. In cohort 4 (400 mg V/800 mg HCQ), 4/8 pts had DLT with Gr 3 fatigue (3 pts) and Gr 2 seizure on C1D8 (1 pt). De-escalation to 400 mg V/600 mg HCQ resulted in no further DLTs, defining the MTD. 10 pts were treated at the MTD. Treatment-related toxicities were Gr 1-2: nausea (11 pts), diarrhea (8), fatigue (6), anorexia (4), weight loss (4), anemia (4), and elevated creatinine (4). Gr 3 toxicities were fatigue (3), anemia, thrombocytopenia and neutropenia (1 each). One pt (renal cell cancer) had a confirmed PR (cohort 2, completed 43 cycles), and 2 pts with colorectal cancer had prolonged SD (>4 cycles). HCQ trough and V plasma concentrations (preliminary) showed no drug interactions compared to previously published data. PD analyses of biomarkers of HCQ and V in PBMCs at baseline compared to weeks 1 and 6 confirmed the results from pre-clinical studies showing accumulation of autophagic vesicles and induction of LC3-II, p21 and cathepsin D. The results of biopsies from 3 patients at MTD demonstrated greater induction of these PD markers in tumor compared to PBMC. Conclusions: The MTD was established as HCQ 600 mg + V 400 mg. Dose-dependent fatigue represented DLT in several patients. Anti-tumor activity was seen in one patient with RCC and 2 patients with CRC had prolonged SD. Based on these data phase II studies are planned in patients with renal and colorectal cancer. Citation Format: Monica M. Mita, Devalingam Mahalingam, John Sarantopoulos, Ravi Amaravadi, Alain Mita, Tyler Curiel, Steffan Nawrocki, Frank Giles, Jennifer Carew. A Phase 1 dose escalation study to establish the maximum tolerated dose (MTD), pharmacokinetic (PK) and pharmacodynamic (PD) parameters of hydroxychloroquine (HCQ) in combination with vorinostat (V) in patients with advanced solid tumors - final results. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1686. doi:10.1158/1538-7445.AM2013-1686

Previous reports from this laboratory have demonstrated that human CD4+ Toxoplasma-specific cytotoxic T cell (CTL) clones generated by stimulation of peripheral blood mononuclear cells with Toxoplasma RH strain antigens also recognized target cells expressing C strain antigens. To extend these observations, additional Toxoplasma isolates were studied. A simple system for assessment of cytotoxicity using T cell lines rather than cloned CTL was used. Stimulation of human peripheral blood mononuclear cells with Toxoplasma RH strain antigens elicited cytotoxic T cell lines specific for target cells expressing antigens derived from many other Toxoplasma strains. Cell lines produced by stimulation with antigens derived from the related, nonpathogenic coccidian Besnoitia jellesoni were also cytotoxic for target cells expressing Toxoplasma antigens. Proliferative responses to many Toxoplasma isolates and to the Toxoplasma p30 protein were also noted.

Although 13 years have passed since identification of human immunodeficiency virus-1 (HIV-1) as the cause of AIDS, we do not yet know how HIV kills its primary target, the T cell that carries the CD4 antigen. We and others have shown an increase in the percentage of apoptotic cells among circulating CD4+ (and CD8+) T cells of HIV-seropositive individuals and an increase in frequency of apoptosis with disease progression. However, it is not known if this apoptosis occurs in infected or uninfected T cells. We show here, using in situ labelling of lymph nodes from HIV-infected children and SIV-infected macaques, that apoptosis occurs predominantly in bystander cells and not in the productively infected cells themselves. These data have implications for pathogenesis and therapy, namely, arguing that rational drug therapy may involve combination agents targeting viral replication in infected cells and apoptosis of uninfected cells.

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, beta-galactosidase (beta-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a beta-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists.

Despite advances in conventional therapy, many lives continue to be lost to common forms of B-cell cancers, including leukemias, lymphomas and multiple myeloma. We propose a novel approach to therapy of such cancers using controlled expression of a diphtheria toxin gene (DT-A) to kill malignant cells. We have previously demonstrated selective killing of various cell types, in vitro and in vivo, by cell-specific, transcriptionally controlled expression of this gene. Organ-specific ablation in otherwise healthy transgenic mice has convincingly demonstrated the exquisite specificity achievable by this technique. In the studies now described, DT-A was delivered in vitro and in vivo using a novel gene delivery system employing DNA physically attached to the exterior of adenovirus. After demonstrating the efficacy of gene delivery to Epstein-Barr virus transformed human B-cells in vitro, in vivo work was performed using a SCID mouse model for B-cell lymphoma, in which protection against tumor was observed. The concepts of tissue-regulated toxin gene therapy, and this novel adenovirus gene delivery system are discussed.

The human immunodeficiency virus (HIV) causes persistent infection of T cells. Chemotherapy for infection in humans may slow HIV-related disease progression, but it does not eradicate virus. Thus, other treatment modalities are warranted. We have previously demonstrated that the human T cell line H9, ordinarily permissive for HIV infection, may be protected against infection with the LAI strain of HIV by intracellular immunization with the gene encoding diphtheria toxin A chain (DT-A) under the control of HIV Tat and Rev. Cloned cells were protected for up to 6 days in vitro. We now report protection against the LAI laboratory isolate for up to 59 days, and against clinical HIV strains of differing phenotypic properties and cell tropisms for up to 59 days. In some cases, protection was complete in that no residual HIV was detected by HIV p24 antigen production, co-culture with parental H9 cells, or the polymerase chain reaction (PCR). CD4+ surface expression of DT-A transduced cloned H9 cells was similar to parental H9 in most cases. These results suggest that toxin gene therapy for HIV infection may ultimately be feasible.

Virus-specific cytotoxic T lymphocytes (CTL) are frequently of the CD8+ surface phenotype, although CTL of the CD4+ surface phenotype have also been described. Published reports of CTL derived from peripheral blood mononuclear cells (PBMC) of individuals infected with human immunodeficiency virus type 1 (HIV-1) have described primarily cells of the CD8+ surface phenotype. However, CD4+ HIV-1 envelope-specific CTL have been reported after in vitro stimulation with HIV-1 envelope protein of peripheral blood cells obtained from HIV-1-seronegative donors, in peripheral blood cells after vaccination of HIV-1-seronegative persons with HIV-1 envelope proteins, and in cerebrospinal fluid cells of HIV-1-infected individuals. Recently, CD4+ HIV-1 gag-specific CTL were also reported. We now report a patient from whom we derived HIV-1 envelope-specific CTL cell lines of the CD4+ surface phenotype. Our cell culture technique did not employ exogenous viral antigenic stimulation, and may therefore yield cells that more closely reflect those in the underlying populations from which they were derived. These CTL did not appear to have the clear human leukocyte antigen (HLA) class II restriction pattern typically seen in CD4-expressing cells and were not functionally inhibited by anti-CD3 antibody. Further work will be required to define the role of CD4+ CTL in the pathogenesis of HIV-1 disease.

Expression of a gene encoding the diphtheria toxin A (DT-A) chain, under the control of human immunodeficiency virus-1 (HIV-1) proteins Tat and Rev, has previously been shown to confer on cells an impaired ability to produce HIV. That work was done in HeLa cell lines that had stably integrated the regulated DT-A gene in a plasmid context. To increase the efficiency with which the HIV-regulated DT-A gene could be introduced into cells, we studied a recombinant, amphotropic murine leukemia virus containing the HIV-regulated DT-A transcription unit. Here we demonstrate that such recombinant retroviruses can be packaged, for both wild-type DT-A and an attenuated version, tox 176. In transient transfection assays, the proviral constructs exhibited similar basal and trans-activated levels of DT-A expression to the parental plasmids. Transduced H9 cells expressed the integrated DT-A gene upon transfection with plasmids encoding Tat and Rev, as assayed by decreased expression of a cotransfected luciferase reporter gene. Furthermore, the transduced H9 cells were substantially impaired in their ability to produce HIV, as demonstrated by p24 assays of culture supernatants following either transfection with an HIV proviral clone or infection with HIV-IIIB. These data demonstrate that basal expression of the regulated DT-A gene has been reduced to a tolerable level, both in packaging cells and transduced H9 cells. The use of HIV-regulated retroviruses encoding the highly lethal DT-A product may eventually be applicable as a gene therapy approach for the acquired immunodeficiency syndrome (AIDS).

Interleukin (IL)-12 is a proinflammatory cytokine generally regarded as a master regulator of type 1 T cell mediated immunity against intracellular pathogens, including T. gondii and cancers. A critical measure of IL-12 activity is its ability to induce IFN-γ production by NK and T cells. Using a transgenic T. gondii expressing ovalbumin (OVA), we show here that by flow cytometry analysis T. gondii-OVA infected BL6 IL-12-/- mice generate OVA-pentamer+CD8+ T cells similar to WT and surprisingly, T. gondii-infected IL-12-/- mice produce more IFN-γ +CD8+ T cells than T. gondii-infected WT mice (avg 9.6% in IL-12-/- vs 3.7% in WT). We confirmed that function of OVA-specific CD8+ T cells in IL-12-/- was comparable to BL6 WT using an in vivo OVA+ target lysis assay. As expected, IFN-γ +CD4+ T cells decreased from an avg 29% to 9% in WT mice compared to IL-12-/- mice respectively when infected with T. gondii. These results demonstrate a novel IL-12-independent pathway to CD8+ CTL that may be less dependent of CD4+ T cell help than previously thought.

Toxoplasma gondii is a medically important, obligate intracellular protozoan parasite that causes significant morbidity and mortality in hosts with defective cellular immunity. Using antisense RNA knockdown, we generated T. gondii tachyzoites expressing 5 - 10-fold less TgMAPK1, a stress-response mitogen-activated protein kinase. In infection, T. gondii converts from the rapidly-dividing tachyzoite to the latent bradyzoite form. We show here that T. gondii tachyzoites with reduced TgMAPK1 display distinctive bradyzoite characteristics, including expressing Bag1 (a bradyzoite-specific antigen) and significantly reducing Sag1 (a tachyzoite-specific antigen). Although knockdown (KD) tachyzoites were dramatically less sensitive to the anti-proliferative effects of IFN-γ than wildtype (WT), these parasites were nonetheless significantly less virulent than WT in BL6 mice, based on: (i) KD parasites achieved only a fraction of the parasite burden in tissues of infected mice relative to control parasites, (ii) mice infected with KD produced substantially less IFN-γ, and (iii) the majority of mice challenged with KD were able to survive lethal challenge. These results show that TgMAPK1 is an important virulence determinant affecting stage conversion, proliferation, and replication. This work was supported by funds from NIH AI060424 and Johnson & Johnson Focused Giving.

We tested Treg function in B7-H1-/- (KO) mice. 6-10 wk male (M) or female (F) KO and M or F wildtype (WT) had equal numbers of CD4+CD25hiFoxP3+ T cells (Tregs) with similar CD25 and Foxp3 MFI. CTLA-4 trended lower in F vs M KO. M and F WT and M KO Tregs suppressed T cell proliferation equally in vitro but F KO Tregs were ~30% less suppressive vs the other 3 groups. Transfer of M KO Tregs fully protected RAG1-/- mice from CD4+RBhi T cell colitis but F KO Tregs did not, with up to 30% weight loss demonstrating poor in vivo Treg function. In M or F WT or KO with subcutaneous B16 melanoma, tumor was different at day 15 in F (159 mm3) vs M (458 mm3, p = 0.04) and F KO vs F WT (593 mm3, p = 0.01). We detected OVA-specific immunity in 0/3 M KO bearing OVA+ B16 vs 3/3 in F KO based on in vivo OT-I cell proliferation. Tregs in pre-pubertal 3-wk F KO had similar phenotype and function vs post-pubertal WT and M KO Tregs. Culture with TGF-β +IL-2 converted 6-wk CD4+CD25- F KO T cells into CD4+CD25hiFoxP3+ T cells with suppression similar to WT, but lower CD25. Thus, F KO T cells can become functional Tregs ex vivo. This previously unknown sex/T cell co-signaling interaction could explain sex-related differences in cancer, autoimmune disease, infection susceptibility and response to hormone antagonists. We hypothesize Treg dysfunction to improve immunity and tumor resistance in KO and estrogen as a B7-H1 co-factor in Treg development. CTLA-4 defects remain undefined. Supported by CA105207, FD003118, and Voelcker and Greehey Trusts

Treg depletion improves anti-tumor immunity but is unlikely to be curative alone. Denileukin diftitox (DT) depletes Tregs in BL6 mice and prolongs survival 11% at 5 µg/mouse weekly starting 2 weeks after challenge with ID8 ovarian cancer. Weekly human interferon (hIFN)-α 20,000 U/day on 4 consecutive days of 7 plus weekly DT survival improved 26% vs DT alone. Neither DT nor DT+hIFN-α improved survival in RAG1-/- mice with ID8, showing a requirement for adaptive immunity. hIFN-α alone reduced in vitro Treg suppression ~15% vs PBS, but did not clearly enhance DT-mediated Treg depletion, or increase T cell activation by CD69 expression. A patient in our clinical trial of DT for ovarian cancer failed, with CA-125 increasing 4.5-fold after 4 monthly cycles. Addition of weekly pegylated IFN-α2a to failed monthly DT improved blood Treg depletion 50% over DT alone and reduced CA-125 by 9-fold, demonstrating immunologic and clinical efficacy. To provide additional mechanistic insight we studied dendritic cell (DC) activation in hIFN-α treatment. In mice with ID8 tumor, hIFN-α significantly increased CD86 expression on myeloid and plasmacytoid DC. IFN-α may thus have an expanded role in treatment of epithelial carcinomas when used with Treg depletion, through enhanced Treg depletion or DC activation among other mechanisms. Supported by FD003118, the Fanny Rippel Foundation, the Voelcker Trust and the Greehey Fund

2506 Background: Denileukin diftitox is a fusion toxin consisting of interleukin-2 genetically fused to diphtheria toxin. It is approved to treat cutaneous T cell lymphoma. Our recent Phase I trial demonstrated that denileukin diftitox depletes human regulatory T cells (Tregs), and is associated with improved measures of T cell immunity and clinical improvements in ovarian cancer. This Phase II trial tests whether denileukin diftitox has clinical activity in the treatment of relapsed epithelial ovarian cancer. Methods: Patients with Stage III or IV ovarian cancer who have failed first-line therapy with optimal surgical debulking and platinum-based chemotherapy were eligible. Denileukin diftitox was given at 12 mcg/kg once monthly. In the Phase II design, patients received no chemotherapy for at least two weeks and no immune or radiation therapy for at least four weeks prior to enrollment. Infusion pretreatment includes antihistamines, acetaminophen and anti-emetics, but no corticosteroid. Results: Six patients (F1,2,4–7) have been treated to date. F1 experienced CA-125 stabilization and approximately 20% reduction in her pelvic mass after two cycles, but was then withdrawn for hypoalbuminemia. F2 experienced CA-125 stabilization after two cycles and remains stable through four cycles. F4 experienced a 20% drop in CA-125 after two cycles, but then progressed. F5 progressed after one cycle. Treatment was recently initiated for F6–7. Existing immune data to date confirm that denileukin diftitox treatment in the Phase II trial is associated with reductions in phenotypic CD4+CD25hi blood Tregs and increased CD3+IFN-γ+ T cells consistent with our Phase I results. Studies of Treg function, T cell cytokine production and CD4+CD25+ T cell FOXP3 content using saved clinical material will be undertaken when full laboratory operations resume. The trial continues to accrue patients. Conclusions: These data suggest that denileukin diftitox depletes Tregs in ovarian cancer. The link between Treg depletion and any observed immune changes or clinical effects is under active investigation, and remains to be determined. No significant financial relationships to disclose.

Despite a compelling logic, tumor immunotherapy has generally failed. Work by us and others suggests that this failure owes in part to tumor-mediated immune dysfunction. CD4+CD25+ regulatory T cells (Tregs) are thought to contribute to this dysfunction. In support, we show that in ovarian cancer patients, Tregs block tumor-specific immunity, promote tumor growth and predict poor survival. We hypothesize that depleting Tregs will improve immunity and improve therapeutic outcomes in cancer patients. Ontak is a recombinant protein fusing the CD25-binding domain of IL-2 to the active domain of diphtheria toxin. It was designed to kill CD4+CD25+ leukemia cells. We tested the hypothesis that Ontak kills CD4+CD25+ Tregs and thus improves immunity in cancer patients. Patients 1-3 (advanced-stage cancer of the ovary, breast or lung) received a single intravenous infusion of 9 μg/kg Ontak. Patient 4 had relapsed stage IIIC ovarian cancer and received a single dose of Ontak at 12 μg/kg. Blood immune cells from before and after treatment were enumerated and cytokines were studied by flow cytometry. T cell suppression was also tested. Prior to infusion, the mean blood CD4+CD25+ cells/mm3 was 126, which was 26.8% of CD4+ T cells. This decreased to a mean of 78 cells/mm3 blood and mean of 19.0% of CD4+ T cells 3-5 days after Ontak. Also, interferon-γ-expressing T cells increased approximately 50% and FOXP3 (a functional marker for Tregs) was reduced after Ontak. In patient 4, CD4+CD25+ cells inhibited T cell proliferation in vitro prior to but not after treatment. Further, a single Ontak-mediated Treg depletion reduced blood CA-125 from 121 to 38 U/mL four weeks later in patient 4, suggesting clinical efficacy. Patient 4 then received 6 additional doses of Ontak at 12 μg/kg with a further decrease in blood CA-125 level to 16 U/mL four weeks after the final dose. Additionally, a PET/CT fusion demonstrated dramatic reduction of metastatic disease. The CA-125 level remains normal two months after the final Ontak dose. Our data demonstrate for the first time that Ontak efficiently depletes Tregs in patients with cancer, and Treg depletion is associated with improved T cell immunity, normalized CA-125 and reduced tumor metastasis. The data may help explain failures of current tumor immunotherapy protocols. It further suggests that Treg depletion or a combination with current immunotherapeutic regimens will be a novel strategy for treating patients with cancer.

Purpose of Study Toxoplasma gondii causes significant morbidity and mortality in immunocompromised patients such as those co-infected with HIV. We have cloned and sequenced two mitogen-activated protein kinase (MAPK) genes which encode novel MAP kinases that are predicted to have distinctly different properties. These predictions are based on examining differences in their primary amino acid sequences and differential gene expression in context with available functional data. Both MAPKs are druggable targets, and we predict that reducing or abolishing their expression in T. gondii will prove to be detrimental to the parasite9s ability to proliferate and/or differentiate. Current experiments are underway in this regard. Methods The T. gondii strain Me-49 genome has been completely sequenced, but individual genes from this avirulent strain are still in the process of being annotated. Using information available at this genomic database (www.toxodb.org), along with our nucleotide sequencing data obtained from the virulent RH strain (acquired using an ABI-7700 automated sequencer), we have identified two MAPK genes, which we have designated tgMAPK-1 and tgMAPK-2. Expression, purification, and functional assays performed on TgMAPK-1 have been previously described (Brumlik et al, 2004). Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out in order to examine the differential expression of both these genes. Results Based on the deduced amino acid sequences encoded by these genes (which share only 33% identity), each MAP kinase has unique characteristics. RT-PCR profiles suggest that although both genes encode stress-response MAPKs, tgMAPK-1 appears to be expressed in the bradyzoite (latent) form while tgMAPK-2 seems to be more constitutively expressed. ClustalW alignment of these MAPKs has been performed, along with a phylogenetic analysis. Strain variations between both MAPKs are also discussed. Conclusions Pyridinylimidazoles designed to block the human stress-response p38 MAPK also inhibit TgMAPK-1 in vitro, block T. gondii replication, and protect T. gondii-infected mice. Both TgMAPK-1 and TgMAPK-2 are druggable targets, although it remains to be seen the effect that pyridinylimidazoles have on TgMAPK-2. Understanding the relationship between TgMAPK-1 and TgMAPK-2 and their ability to mediate stress-response and/or stage-differentiation will be important to advancing treatment strategies for T. gondii-infected patients.

Despite a compelling logic, tumor immunotherapy has generally failed. Work by us and others suggests that this failure owes in part to tumor-mediated immune dysfunction. CD4+CD25+ regulatory T cells (Tregs) are thought to contribute to this dysfunction. In support, we show that in ovarian cancer patients, Tregs block tumor-specific immunity, promote tumor growth and predict poor survival. We hypothesize that depleting Tregs will improve immunity and improve therapeutic outcomes in cancer patients. Ontak is a recombinant protein fusing the CD25-binding domain of IL-2 to the active domain of diphtheria toxin. It was designed to kill CD4+CD25+ leukemia cells. We tested the hypothesis that Ontak kills CD4+CD25+ Tregs and thus improves immunity in cancer patients. Patients 1-3 (advanced-stage cancer of the ovary, breast or lung) received a single intravenous infusion of 9 μg/kg Ontak. Patient 4 had relapsed stage IIIC ovarian cancer and received a single dose of Ontak at 12 μg/kg. Blood immune cells from before and after treatment were enumerated and cytokines were studied by flow cytometry. T cell suppression was also tested. Prior to infusion, the mean blood CD4+CD25+ cells/mm3 was 126, which was 26.8% of CD4+ T cells. This decreased to a mean of 78 cells/mm3 blood and mean of 19.0% of CD4+ T cells 3-5 days after Ontak. Also, interferon-γ-expressing T cells increased approximately 50% and FOXP3 (a functional marker for Tregs) was reduced after Ontak. In patient 4, CD4+CD25+ cells inhibited T cell proliferation in vitro prior to but not after treatment. Further, a single Ontak-mediated Treg depletion reduced blood CA-125 from 121 to 38 U/mL four weeks later in patient 4, suggesting clinical efficacy. Patient 4 then received 6 additional doses of Ontak at 12 μg/kg with a further decrease in blood CA-125 level to 16 U/mL four weeks after the final dose. Additionally, a PET/CT fusion demonstrated dramatic reduction of metastatic disease. The CA-125 level remains normal two months after the final Ontak dose. Our data demonstrate for the first time that Ontak efficiently depletes Tregs in patients with cancer, and Treg depletion is associated with improved T cell immunity, normalized CA-125 and reduced tumor metastasis. The data may help explain failures of current tumor immunotherapy protocols. It further suggests that Treg depletion or a combination with current immunotherapeutic regimens will be a novel strategy for treating patients with cancer.

Background. Whole organ extracorporeal perfusion of a genetically modified humanized (transgenic) pig liver has been proposed as a technology that may sustain patients with severe liver failure while awaiting human liver transplantation. Methods. We report on two cases of successful extracorporeal perfusion of a transgenic pig liver in patients awaiting transplantation for fulminant hepatic failure. The pig livers used were transgenic for human CD55 (decay-accelerating factor) and human CD59. These transgenic modifications are designed to reduce or eliminate the hyperacute rejection inherent in pig-to-primate xenotransplants. We also report on the results of serial surveillance testing for presence of the porcine endogenous retrovirus (PoERV) in these two patients. Results. Extracorporeal perfusion in two patients was performed for 6.5 and 10 hr, respectively, followed by the successful transplantation of a human liver and resultant healthy patients (18 and 5 months later as of this writing). The porcine livers showed evidence of synthetic and secretory function (decreasing protime and bilirubin, bile production). Serial polymerase chain reaction analysis of these patients' peripheral blood mononuclear cells has failed to show presence of PoERV DNA sequences. Conclusions. The CD55/CD59 transgenic porcine liver appears capable of safely bridging a patient to liver transplantation. Human PoERV infection from these livers has yet to be demonstrated.

A gene encoding a 113-amino acid, NH2-terminal fragment of CD4, rsT4.113, was constructed and expressed in Escherichia coli under the control of the tryptophan operon promoter. Following induction, rsT4.113 is produced at 5-10% of total E. coli protein, and it is found in inclusion bodies. The protein is purified in two steps under denaturing and reducing conditions. Solubilized rsT4.113 is first purified on a column of Q-Sepharose to remove low molecular weight contaminants and then purified to greater than 95% homogeneity by gel filtration. Renaturation of rsT4.113 is achieved at approximately 20% yield by dilution and dialysis. High performance liquid chromatography analysis of renatured rsT4.113 reveals a less than 15% contaminant of reduced protein. Purified and renatured rsT4.113 contains epitopes for both OKT4a and Leu3a, anti-CD4 monoclonal antibodies which block CD4-gp 120 association, but lacks measurable affinity toward a nonblocking anti-CD4 monoclonal antibody, OKT4. By comparison to a longer form (375 amino acids) of recombinant soluble T4 produced in mammalian cells that contains the entire extracellular domain, rsT4.113 has a comparable affinity for binding to OKT4a and Leu3a in a radioimmunoassay. Analysis of antiviral activity of rsT4.113 demonstrates that the E. coli-derived protein inhibits human immunodeficiency virus-induced syncytium formation with an IC50 of 5-10 micrograms/ml. These data demonstrate that the human immunodeficiency virus-binding domain of CD4 is localized within the NH2-terminal 113 amino acids of CD4 and is contained within a structure homologous to the kappa variable-like domain of immunoglobulins.