Kyung Ho Kang

Depletion of platelets enhances therapeutic antitumor effects generated by therapeutic DNA vaccine

It has been well established that platelets are associated with tumor progression and metastasis as they can support angiogenesis and prevent hemorrhage. As such, platelets may be an ideal target for enhancing anticancer immunotherapies. Thus, we hypothesized that inducing thrombocytopenia would increase the leakiness of tumor vasculature thereby facilitating the effective delivery of antigen-specific T cells generated by DNA vaccination to tumor loci in order to induce CD8+ T cell-mediated tumor killing. We have previously developed a therapeutic HPV DNA vaccine encoding calreticulin (CRT) linked to HPV-16 E7 antigen (CRT/E7) to enhance therapeutic DNA vaccine potency. In the current study, we explore the employment of a platelet-depleting antibody or heparin in combination with CRT/E7 DNA vaccine in a mouse model for its potential to enhance E7-specific CD8+ T cell immune responses as well as antitumor effects against E7-expressing tumors. We induced severe acute thrombocytopenia in mice bearing subcutaneous TC-1 tumors. TC-1 tumor cells originate from the mouse lung and express the HPV-16 E7 tumor-specific antigen. In a TC-1 mouse tumor model, platelet depletion increased E7-specific CD8+ T cells in the peripheral blood and reduced the growth of the tumor mass following CRT/E7 DNA vaccination. Next, we isolated platelet-rich plasma from tumor-bearing and naive mice. When TC-1 tumor cells were coincubated with E7-specific CD8+ T cells under platelet-rich plasma and found that E7-specific CD8+ T cells had decreased IFN-γ secretion in the presence of platelet-rich plasma from tumor-bearing mice compared to that of naive mice. Furthermore, when incubated with E7-specific CD8+ T cells and platelets, TC-1 cells were killed significantly less efficiently compared to those incubated with E7-specific CD8+ T cells alone. Importantly, we found that GFP-tagged TC-1 cells incubated with platelets were coated by the platelets, which were subsequently activated (CD26P+). Taken together, these data suggest that platelet depletion increases tumor-specific CD8+ T cells and enhances their tumor killing effects. Furthermore, This study provides proof in principle that platelets directly inhibit CD8+ T cells from killing tumor cells. In conclusion, platelet depletion or heparin therapy can stimulate antitumor immune responses and this treatment methodology may be applicable to a variety of cancers.

Cryoablation therapy stimulates the antigen-specific T cell immune responses generated by therapeutic HPV DNA vaccine.

Antigen-specific DNA vaccine immunotherapy is an attractive approach for the treatment of cancer since it has the potency to specifically eradicate systemic tumors without damaging the normal cells. However, DNA vaccine shows insufficient immune response for antigen presenting cells and has minimal clinical efficacies. Cryoablation therapy is the therapeutic application of extreme cold for localized destruction of living tissue. It can develop cancer cell necrosis and release damage-associated molecular patterns (DAMPs) in tumor microenvironment. According to other reports, cryotherapy has immunogenic potential. Therefore, we explored the employment of cryoablation in combination with HPV E7 specific CRT/E7 DNA vaccine in a preclinical mouse model. For in vivo experiment, the mice were divided into four groups: group 1 received no treatment after the E7-expressing TC-1 tumor challenge, group 2 was treated with cryoablation therapy, group 3 was immunized with the CRT/E7 DNA vaccine, and group 4 was both immunized and received cryoablation therapy. Mice were monitored for E7-specific CD8 (+) T cell immune responses, myeloid-derived suppressor cells (MDSCs) and antitumor effects. In vitro assay, TC-1 cells were incubated in deep freezing and thawed for determination of HMGB1 from the supernatant. The combination therapy of cryoablation and CRT/E7 DNA vaccination significantly inhibited tumor growth and increased E7-specific CD8+ T cells in spleen compared to other treatment groups. Furthermore, the immunosuppressive MDSCs in tumor mass were significantly decreased in combination therapy group. We found that treatment with deep freezing and thawing led to up-regulation of HMGB1 in TC-1 tumor cells, rendering the tumor cells more susceptible to APCs and E7-specific CD8+ T cell-mediated killing. In conclusion, cryoablation therapy can enhance therapeutic HPV DNA vaccine potency in generating improved antigen-specific immune responses and antitumor effects through the DAMP signal. These findings have important implications for future clinical translation.

Abstract LB-114: The roles of COX-2 inhibitor in regulatory T cells and Th17 cells’ tumor immune regulation

Background: COX-2 and its metabolite, PGE2, affect tumorigenesis and tumor-induced immune suppression. In the field of tumor immunology, regulatory T cells (Tregs) and Th17 cells are emerging as important targets. The presence of Tregs might be important for inducing T-cell suppression and thus allowing tumor growth. Th17 cells play an active role in inflammation and autoimmune diseases. These cell types have reciprocal roles in inflammatory conditions. However, very little is known about the roles of the COX-2 inhibitor in Tregs and Th17 cells in tumor development. Therefore, we investigated the effects of COX-2 inhibitors on Treg and Th17 cell activation in a tumor model. Methods: In vitro assay, to evaluate the effects of COX-2 on the proliferation of Tregs and Th17 cells, CD4+CD62L+ naive T cells were incubated in the presence of TGF-s, with or without IL-6, and PGE2 or a COX-2 inhibitor (celecoxib) was then added. Through Western blotting, we assessed Foxp3 and the transcription factor retinoic acid-related orphan receptor (ROR)-, which were recently described essential for differentiation of Tregs and Th17 cells, respectively. In vivo assay, twenty mice were randomized into a group of normal control, Lewis lung cancer cells (3LL) inoculated control, and celecoxib 10 or 100mg/kg/day treated 3LL inoculated mice groups. The tumor mass and spleen of each mouse were removed for isolation of splenocytes and tumor infiltrating lymphocytes (TIL) for FACS analysis, real time PCR and Western blotting. Results: When CD4+CD62L+ naive T cells were stimulated under Treg-promoting condition (TFG-s only), the expression of Foxp3 increased. When naive T cells were stimulated under Th17-promoting conditions, TGF-s and IL-6 induced significant ROR- expression and IL-17 secretion. When naive T cells were treated with PGE2 and TGF-s, the expression of Foxp3 increased. These increased expressions were decreased in the presence of celecoxib. We assessed the effects of COX-2 on IL-17 production in TGF-s- and IL-6-stimulated naive T cells. We found that IL-17 production was increased with PGE 2 and decreased with celecoxib treatment, in a dose-dependent manner. The expressions of Foxp3 and ROR-γ in the tumor mass were decreased in the celecoxib treated mice groups. FACS analysis demonstrated a decline in the percentage of CD4 + IL17 + and CD4 + CD25 + in the celecoxib treated groups. Conclusion : The results of this study show that PGE2 increases the differentiation of Tregs and Th17 cells, while a COX-2 inhibitor inhibits the differentiation of both Tregs and Th17 cells. Although Tregs and Th17 cells have reciprocal roles, the COX-2 inhibitor decreases the differentiation of both Treg and Th17 cells. This mode of immunoregulatory action by a COX-2 inhibitor requires further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-114.