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Purpose: Whole tumor lysates are promising antigen sources for dendritic cell (DC) therapy as they contain many relevant immunogenic epitopes to help prevent tumor escape. Two common methods of tumor lysate preparations are freeze-thaw processing and UVB irradiation to induce necrosis and apoptosis, respectively. Hypochlorous acid (HOCl) oxidation is a new method for inducing primary necrosis and enhancing the immunogenicity of tumor cells., Experimental Design: We compared the ability of DCs to engulf three different tumor lysate preparations, produce T-helper 1 (TH1)-priming cytokines and chemokines, stimulate mixed leukocyte reactions (MLR), and finally elicit T-cell responses capable of controlling tumor growth in vivo., Results: We showed that DCs engulfed HOCl-oxidized lysate most efficiently stimulated robust MLRs, and elicited strong tumor-specific IFN-γ secretions in autologous T cells. These DCs produced the highest levels of TH1-priming cytokines and chemokines, including interleukin (IL)-12. Mice vaccinated with HOCl-oxidized ID8-ova lysate-pulsed DCs developed T-cell responses that effectively controlled tumor growth. Safety, immunogenicity of autologous DCs pulsed with HOCl-oxidized autologous tumor lysate (OCDC vaccine), clinical efficacy, and progression-free survival (PFS) were evaluated in a pilot study of five subjects with recurrent ovarian cancer. OCDC vaccination produced few grade 1 toxicities and elicited potent T-cell responses against known ovarian tumor antigens. Circulating regulatory T cells and serum IL-10 were also reduced. Two subjects experienced durable PFS of 24 months or more after OCDC., Conclusions: This is the first study showing the potential efficacy of a DC vaccine pulsed with HOCl-oxidized tumor lysate, a novel approach in preparing DC vaccine that is potentially applicable to many cancers., (©2013 AACR.)

Background: HER-2/neu overexpression plays a critical role in breast cancer development, and its expression in ductal carcinoma in situ (DCIS) is associated with development of invasive breast cancer. A vaccine targeting HER-2/neu expression in DCIS may initiate immunity against invasive cancer., Methods: A HER-2/neu dendritic cell vaccine was administered to 27 patients with HER-2/neu-overexpressing DCIS. The HER-2/neu vaccine was administered before surgical resection, and pre- and postvaccination analysis was conducted to assess clinical results., Results: At surgery, 5 of 27 (18.5%) vaccinated subjects had no evidence of remaining disease, whereas among 22 subjects with residual DCIS, HER-2/neu expression was eradicated in 11 (50%). When comparing estrogen receptor (ER)(neg) with ER(pos) DCIS lesions, vaccination was more effective in hormone-independent DCIS. After vaccination, no residual DCIS was found in 40% of ER(neg) subjects compared with 5.9% in ER(pos) subjects. Sustained HER-2/neu expression was found in 10% of ER(neg) subjects compared with 47.1% in ER(pos) subjects (P = .04). Postvaccination phenotypes were significantly different between ER(pos) and ER(neg) subjects (P = .01), with 7 of 16 (43.8%) initially presenting with ER(pos) HER-2/neu(pos) luminal B phenotype finishing with the ER(pos) HER-2/neu(neg) luminal A phenotype, and 3 of 6 (50%) with the ER(neg) HER-2/neu(pos) phenotype changing to the ER(neg) HER-2/neu(neg) phenotype., Conclusions: Results suggest that vaccination against HER-2/neu is safe and well tolerated and induces decline and/or eradication of HER-2/neu expression. These findings warrant further exploration of HER-2/neu vaccination in estrogen-independent breast cancer and highlight the need to target additional tumor-associated antigens and pathways., (Copyright © 2012 American Cancer Society.)

Purpose: In spite of increased rates of complete response to initial chemotherapy, most patients with advanced ovarian cancer relapse and succumb to progressive disease. Immunotherapy may have potential for consolidation therapy., Experimental Design: This randomized open-label phase I/II trial evaluated responses of patients with advanced ovarian cancer in remission for vaccination with monocyte-derived dendritic cells (DC) loaded with Her2/neu, hTERT, and PADRE peptides, with or without low-dose intravenous cyclophosphamide. All patients also received pneumococcal vaccine and were randomized to cyclophosphamide 2 days prior to first vaccination. Blood samples were analyzed by ELISPOT and flow cytometry., Results: Of 11 patients, 2 recurred during vaccination. Nine received all 4 doses: 3 patients recurred at 6, 17, and 26 months, respectively, and 6 have no evidence of disease at 36 months. No grade 3/4 vaccine-related toxicities were noted. The 3-year overall survival was 90%. Patients receiving cyclophosphamide showed a non-significant improvement in survival over controls. Patients receiving cyclophosphamide had a transient reduction in neutrophils, but no change in total lymphocytes or regulatory T cells. Modest T-cell responses to Her2/neu and hTERT were seen post-vaccine by IFN-γ ELISPOT. Patients demonstrated below normal responses to the diphtheria conjugate protein CRM197, a component of the pneumococcal vaccine., Conclusions: In this setting, peptide-loaded DC vaccination elicits modest immune responses, but survival is promising. Pneumococcal vaccination revealed substantial immune suppression, even in patients in remission. Rational design of consolidative strategies for ovarian cancer will need to overcome tolerance and immunosuppression.

Twenty-seven patients with HER-2/neu overexpressing ductal carcinoma in situ of the breast were enrolled in a neoadjuvant immunization trial for safety and immunogenicity of DC1-polarized dendritic cells (DC1) pulsed with 6 HER-2/neu promiscuous major histocompatibility complex class II-binding peptides and 2 additional human leukocyte antigen (HLA)-A2.1 class I-binding peptides. DC1 were generated with interferon-γ and a special clinical-grade bacterial endotoxin (lipopolysaccharide) and administered directly into groin lymph nodes 4 times at weekly intervals before scheduled surgical resection of ductal carcinoma in situ. Patients were monitored for the induction of new or enhanced antipeptide reactivity by interferon-γ ELISPOT and enzyme-linked immunosorbentassays performed on Th cells obtained from peripheral blood or excised sentinel lymph nodes. Responses by cytotoxic T lymphocyte against HLA-A2.1-binding peptides were measured using peptide-pulsed T2 target cells or HER-2/neu-expressing or nonexpressing tumor cell lines. DC1 showed surface phenotype indistinct from "gold standard" inflammatory cocktail-activated DC, but displayed a number of distinguishing functional characteristics including the secretion of soluble factors and enhanced "killer DC" capacity against tumor cells in vitro. Postimmunization, we observed sensitization of Th cells to at least 1 class II peptide in 22 of 25 (88%; 95% exact confidence interval, 68.8%-97.5%) evaluable patients, whereas 11 of 13 (84.6%; 95% exact confidence interval, 64%-99.8%) HLA-A2.1 patients were successfully sensitized to class I peptides. Perhaps most importantly, anti-HER-2/neu peptide responses were observed up to 52-month postimmunization. These data show that even in the presence of early breast cancer such DC1 are potent inducers of durable type I-polarized immunity, suggesting potential clinical value for development of cancer immunotherapy.

Background: Dendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines., Methods: Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1) culture media; 2) culture surface; 3) duration of activating DCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma; 4) method of DC harvest; and 5) cryomedia and final DC product formulation., Results: DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning(®) tissue-culture treated surface and Corning(®) ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC) Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs), and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and replated in fresh media., Conclusions: This study examined criteria including DC phenotype, viability, IL-12p70 production and the ability to stimulate MLR as metrics of whole oxidized tumor lysate-pulsed DC immunogenicity and functionality. Development and optimization of this unique method is now being tested in a clinical trial of autologous oxidized tumor lysate-pulsed DC in clinical-scale in recurrent ovarian, primary peritoneal or fallopian tube cancer (NCT01132014).

Immunotherapy using regulatory T cells (Treg) has been proposed, yet cellular and molecular mechanisms of human Tregs remain incompletely characterized. Here, we demonstrate that human Tregs promote the generation of myeloid dendritic cells (DC) with reduced capacity to stimulate effector T cell responses. In a model of xenogeneic graft-versus-host disease (GVHD), allogeneic human DC conditioned with Tregs suppressed human T cell activation and completely abrogated posttransplant lethality. Tregs induced programmed death ligand-1 (PD-L1) expression on Treg-conditioned DC; subsequently, Treg-conditioned DC induced PD-L1 expression in vivo on effector T cells. PD-L1 blockade reversed Treg-conditioned DC function in vitro and in vivo, thereby demonstrating that human Tregs can promote immune suppression via DC modulation through PD-L1 up-regulation. This identification of a human Treg downstream cellular effector (DC) and molecular mechanism (PD-L1) will facilitate the rational design of clinical trials to modulate alloreactivity., Competing Interests: The authors have declared that no competing interests exist.

Overexpression of HER-2/neu (c-erbB2) is associated with increased risk of recurrent disease in ductal carcinoma in situ (DCIS) and a poorer prognosis in node-positive breast cancer. We therefore examined the early immunotherapeutic targeting of HER-2/neu in DCIS. Before surgical resection, HER-2/neu(pos) DCIS patients (n = 13) received 4 weekly vaccinations of dendritic cells pulsed with HER-2/neu HLA class I and II peptides. The vaccine dendritic cells were activated in vitro with IFN-gamma and bacterial lipopolysaccharide to become highly polarized DC1-type dendritic cells that secrete high levels of interleukin-12p70 (IL-12p70). Intranodal delivery of dendritic cells supplied both antigenic stimulation and a synchronized preconditioned burst of IL-12p70 production directly to the anatomic site of T-cell sensitization. Before vaccination, many subjects possessed HER-2/neu-HLA-A2 tetramer-staining CD8(pos) T cells that expressed low levels of CD28 and high levels of the inhibitory B7 ligand CTLA-4, but this ratio inverted after vaccination. The vaccinated subjects also showed high rates of peptide-specific sensitization for both IFN-gamma-secreting CD4(pos) (85%) and CD8(pos) (80%) T cells, with recognition of antigenically relevant breast cancer lines, accumulation of T and B lymphocytes in the breast, and induction of complement-dependent, tumor-lytic antibodies. Seven of 11 evaluable patients also showed markedly decreased HER-2/neu expression in surgical tumor specimens, often with measurable decreases in residual DCIS, suggesting an active process of "immunoediting" for HER-2/neu-expressing tumor cells following vaccination. DC1 vaccination strategies may therefore have potential for both the prevention and the treatment of early breast cancer.

CD4(+)CD25(+) T-regulatory (Treg) cells have been shown to critically regulate self- and allograft tolerance in several model systems. Studies of human Treg cells have been restricted by the small number present in peripheral blood and their naturally hypoproliferative state. To better characterize Treg suppressor cell function, we determined methods for the isolation and expansion of these cells. Stringent magnetic microbead-based purification was required for potent suppressor cell line generation. Culture stimulation with cell-sized Dynabeads coated with anti-CD3 and anti-CD28 monoclonal antibodies, CD4(+) feeder cells, and interleukin 2, provided for marked expansion in cell number (100-fold), with retention and enhancement of suppressor function. The potent Treg cell lines suppressed proliferation in dendritic cell-driven allo-mixed lymphocyte reaction (MLR) cultures by more than 90%. The Treg-derived suppressor cells functioned early in allo-MLR because expression of activation antigens and accumulation of cytokines was nearly completely prevented. Importantly, cultured Treg cells also suppressed activated and matured dendritic cell-driven responses. These results demonstrate that short-term suppressor cell lines can be generated, and they can express a very potent suppressive activity. This approach will enable more detailed biologic studies of Treg cells and facilitate the evaluation of cultured Treg cells as a novel form of immunosuppressive therapy.

Graft versus host disease (GVHD) is triggered by host antigen-presenting cells (APCs) that activate donor T cells to proliferate and differentiate, but which APC-activated donor T-cell subsets mediate GVHD versus beneficial antitumor effects is not known. Using a CD8(+) T cell-dependent mouse model of human GVHD, we found that host dendritic cell (DC)-induced CD44(hi)CD8(+) effector/memory T cells were functionally defective in inducing GVHD, whereas CD44(lo)CD8(+) naive phenotype T cells were extremely potent GVHD inducers. Depletion of CD44(lo)CD8(+) T cells from host DC-stimulated T cells before transplantation prevented GVHD without impairing their antitumor activity in vivo. Compared with CD44(lo)CD8(+) T cells, CD44(hi)CD8(+) T cells expressed high levels of Fas and were efficiently deleted in vivo following transplantation. These results suggest that ex vivo allogeneic DC stimulation of donor CD8(+) T cells may be useful for the prevention of GVHD and for optimizing antitumor therapies in vivo.

Purpose: The use of mature dendritic cells (DCs) presenting tumor-associated antigens (TAAs) to trigger tumor-specific T cells in vivo or in vitro represents a promising approach for cancer immunotherapy. We hypothesized that tumor antigens, mostly unidentified, are present on ovarian tumor cells and that mature DCs could be used to generate tumor-specific responses in unprimed patients. We also sought to measure preexisting antitumor immunity in patients with advanced ovarian cancer., Experimental Design: Autologous DCs from 10 patients with ovarian cancer were pulsed with killed autologous primary tumors as a source of TAAs. DCs were then cultured in the presence of tumor necrosis factor alpha + TRANCE (tumor necrosis factor-related activation-induced cytokine) to induce maturation. Mature TAA-pulsed DCs were used in vitro to stimulate tumor-specific peripheral blood T cells., Results: TRANCE and CD40 ligand were effective at maturing DCs. T-cell lines were generated in vitro that were capable of secreting IFN-gamma in response to autologous tumor. These tumor-specific T cells were MHC class I restricted. The frequency of tumor-specific T cells in uncultured cells from malignant ascites fluid and peripheral blood was measured in the same patients., Conclusions: IFN-gamma-secreting tumor-specific T cells were demonstrated at baseline in uncultured T cells from some unvaccinated ovarian cancer patients; however, the T cells could not kill autologous tumor. These data demonstrate that mature DCs presenting tumor antigens from engulfed autologous tumors can be used to augment antitumor immunity in vitro in patients with epithelial ovarian cancer. The results support the feasibility of therapeutic vaccination of ovarian cancer patients.

Preclinical data are reported that support a human immunodeficiency virus (HIV) vaccine strategy using recombinant canarypox-HIV vectors (ALVAC-HIV) to load human dendritic cells (DCs) with HIV antigens. Clinical-grade DCs were infected with good manufacturing practice-grade ALVAC-HIV vaccine constructs. ALVAC infection, HIV gene expression, and DC viability and function were monitored by use of immunohistochemistry, flow cytometry, blastogenesis assays, antigen-specific interferon (IFN)-gamma enzyme-linked immunospot assay, and enzyme-linked immunosorbent assay protein detection. The vaccines infected both immature and mature DCs, and intracellular HIV-1 Gag protein was detected within hours. ALVAC-HIV induced DC maturation that was mediated by tumor necrosis factor-alpha and induced DC apoptosis that was directly related to the length of vaccine exposure. Of importance, the infected DCs remained functional in T cell stimulation assays and induced HIV antigen-specific CD8(+) T cell production of IFN-gamma from cells of HIV-1-infected individuals. These data support an ongoing HIV vaccine trial comparing conventional vaccine delivery routes with ex vivo vaccine-loaded autologous DCs for immunogenicity in HIV-1-uninfected volunteers.

Novel strategies for the therapy of recurrent ovarian cancer are warranted. We report a study of a combinatorial approach encompassing dendritic cell (DC)-based autologous whole tumor vaccination and anti-angiogenesis therapy, followed by the adoptive transfer of autologous vaccine-primed CD3/CD28-co-stimulated lymphocytes. Recurrent ovarian cancer patients for whom tumor lysate was available from prior cytoreductive surgery underwent conditioning with intravenous bevacizumab and oral metronomic cyclophosphamide, sequentially followed by (1) bevacizumab plus vaccination with DCs pulsed with autologous tumor cell lysate supernatants, (2) lymphodepletion and (3) transfer of 5 × 109 autologous vaccine-primed T-cells in combination with the vaccine. Feasibility, safety as well as immunological and clinical efficacy were evaluated. Six subjects received this vaccination. Therapy was feasible, well tolerated, and elicited antitumor immune responses in four subjects, who also experienced clinical benefits. Of these, three patients with residual measurable disease received outpatient lymphodepletion and adoptive T-cell transfer, which was well tolerated and resulted in a durable reduction of circulating regulatory T cells and increased CD8+ lymphocyte counts. The vaccine-induced restoration of antitumor immunity was achieved in two subjects, who also demonstrated clinical benefits, including one complete response. Our findings indicate that combinatorial cellular immunotherapy for the treatment of recurrent ovarian cancer is well tolerated and warrants further investigation. Several modifications of this approach can be envisioned to optimize immunological and clinical outcomes. [ABSTRACT FROM AUTHOR]