Here, we demonstrate that electroporation-enhanced immunization with a rationally designed HPV DNA vaccine (GX-188E), preferentially targeting HPV antigens to dendritic cells, elicits a significant E6/E7-specific IFN-γ-producing T-cell response in all nine cervical intraepithelial neoplasia 3 (CIN3) patients. Importantly, eight out of nine patients exhibit an enhanced polyfunctional HPV-specific CD8 T-cell response as shown by an increase in cytolytic activity, proliferative capacity and secretion of effector molecules. Notably, seven out of nine patients display complete regression of their lesions and viral clearance within 36 weeks of follow up. GX-188E administration does not elicit serious vaccine-associated adverse events at all administered doses. These findings indicate that the magnitude of systemic polyfunctional CD8 T-cell response is the main contributing factor for histological, cytological and virological responses, providing valuable insights into the design of therapeutic vaccines for effectively treating persistent infections and cancers in humans.

Owing to its tumor tropism and prolonged transgene expression, mesenchymal stem cell (MSC) has been considered as an ideal delivery vehicle for cancer gene therapies or therapeutic vaccines. In this study, we demonstrated that intratumoral (i.t.) injection of MSCs expressing modified interleukin-12 (MSCs/IL-12M) exhibited stronger tumor-specific T-cell responses and antitumor effects as well as more sustained expressions of IL-12 and interferon (IFN)-γ in both sera and tumor sites than did IL-12M-expressing adenovirus (rAd/IL-12M) in mice bearing both solid and metastatic tumors. Subcutaneous (s.c.) injection of MSCs/IL-12M at contralateral site of tumor exhibited similar levels of serum IL-12 and IFN-γ as i.t. injection, but much weaker antitumor effects in both B16F10 melanoma and TC-1 cervical cancer models than i.t. injection. Although intravenous (i.v.) injection elicited earlier peak serum levels of cytokines, it induced weaker tumor-specific T-cell responses and antitumor effects than i.t. injection, indicating that serum cytokine levels are not surrogate indicators of antitumor effects. Taken together, these results indicated that MSC is more efficient than adenovirus as a cytokine gene delivery vehicle and that i.t. injection of MSCs/IL-12M is the best approach to induce strong tumor-specific T-cell responses that correlate with anti-metastatic effects as well as inhibition of solid tumor growth, although MSCs themselves have an ability to migrate into the tumor site. In addition, MSCs/IL-12M embedded in Matrigel (MSCs/IL-12M/Matrigel) exhibited significant antitumor effects even in immunodeficient mice such as SCID and BNX mice lacking T, B and natural killer (NK) cells, but not in IFN-γ knockout mice. Our findings provide an optimal approach for designing an efficient clinical protocol of MSC-based cytokine gene therapy to induce strong tumor-specific T-cell responses and therapeutic anticancer efficacy.

IL-12p70 plays a pivotal role in regulating the Th1/Th2 balance in the initial stage of immune responses. In contrast, IL-12p40, which is produced excess over IL-12p70, has been known to down-regulate IL-12p70-mediated responses by acting as an antagonist. To investigate in vivo function of IL-12p40, RH7777 rat hepatoma cells were engineered to inducibly express mouse IL-12p40 under the tight control of doxycycline (dox). In the absence of dox, s.c. injection of these cells into syngeneic rat was shown to generate tumors. However, the induction of IL-12p40 by dox was sufficient for inhibiting tumor formation, as well as for tumor regression. Immunohistochemical analysis showed that macrophages, but not CD4+ T, CD8+ T, and NK cells, were predominantly recruited into tumor sites as early as 3 days after IL-12p40 induction. These results were further supported by the observation that IL-12p40, but not C-terminal deletion mutants by more than 5 amino acids, was able to chemoattract peritoneal macrophages in vitro, suggesting that IL-12p40, when produced in a large excess over IL-12p70 in vivo, can initially amplify the immune responses against tumors by directly recruiting macrophages. Our findings indicate that IL-12p40 may function as an effector molecule as well as an antagonist of IL-12p70.

Persistent infection with hepatitis C virus (HCV) is associated with the development of liver cirrhosis and hepatocellular carcinoma. To examine the oncogenic potential of the HCV core gene product, primary rat embryo fibroblasts (REFs) were transfected with the core gene in the presence or absence of the H-rasoncogene. In contrast to a previous report (R. B. Ray, L. M. Lagging, K. Meyer, and R. Ray, J. Virol. 70:4438–4443, 1996), HCV core proteins from two different genotypes (type 1a and type 1b) were not found to transform REFs to tumorigenic phenotype in cooperation with the H-rasoncogene, although the core protein was successfully expressed 20 days after transfection. In addition, REFs transfected with E1A- but not core-expressing plasmid showed the phenotype of immortalized cells when selected with G418. The biological activity was confirmed by observing the transcription activation from two viral promoters, Rous sarcoma virus long terminal repeat and simian virus 40 promoter, which are known to be activated by the core protein from HCV-1 isolate. In contrast to the result with primary cells, the Rat-1 cell line, stably expressing HCV core protein, exhibited focus formation, anchorage-independent growth, and tumor formation in nude mice. HCV core protein was able to induce the transformation of Rat-1 cells with various efficiencies depending on the expression level of the core protein. These results indicate that HCV core protein has an oncogenic potential to transform the Rat-1 cell line but is not sufficient to either immortalize primary REFs by itself or transform primary cells in conjunction with the H-rasoncogene.

Objective: Programmed death-ligand 1 (PD-L1) has been shown to negatively regulate immune responses via its interaction with PD-1 receptor. In this study, we investigated the effects of PD-L1-Fc treatment on intestinal inflammation using two murine models of inflammatory colitis induced by dextran sulfate sodium (DSS) and T-cell transfer. Design: The anti-colitis effect of adenovirus expressing Fc-conjugated PD-L1 (Ad/PD-L1-Fc) and recombinant PD-L1-Fc protein was evaluated in DSS-treated wild-type and Rag-1 knockout (KO) mice. We examined differentiation of T-helper cells, frequency of innate immune cells, and cytokine production by dendritic cells (DCs) in the colon from DSS-treated mice after PD-L1-Fc administration. In Rag-1 KO mice reconstituted with CD4 CD45RB^high T cells, we assessed the treatment effect of PD-L1-Fc protein on the development of colitis. Results: Administration of Ad/PD-L1-Fc significantly ameliorated DSS-induced colitis, which was accompanied by diminished frequency of interleukin (IL)-17A-producing CD4 T cells and increased interferon-γ-producing CD4 T cells in the colon of DSS-fed mice. The anti-colitic effect of PD-L1-Fc treatment was also observed in DSS-treated Rag-1 KO mice, indicating lymphoid cell independency. PD-L1-Fc modulated cytokine production by colonic DCs and the effect was dependent on PD-1 expression. Furthermore, PD-L1-Fc protein could significantly reduce the severity of colitis in CD4 CD45RB^high T-cell-transferred Rag-1 KO mice. Conclusions: Based on the protective effect of PD-L1-Fc against DSS-induced and T-cell-induced colitis, our results suggest that PD-1-mediated inhibitory signals have a crucial role in limiting the development of colonic inflammation. This implicates that PD-L1-Fc may provide a novel therapeutic approach to treat inflammatory bowel disease.

T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo , as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. IMPORTANCE Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.