Although tumors naturally prime adaptive immune responses, tolerance may limit the capacity to control progression and can compromise effectiveness of immune-based therapies for cancer. Post-proline cleaving enzymes (PPCE) modulate protein function through N-terminal dipeptide cleavage and inhibition of these enzymes has been shown to have anti-tumor activity. We investigated the mechanism by which Val-boroPro, a boronic dipeptide that inhibits post-proline cleaving enzymes, mediates tumor regression and tested whether this agent could serve as a novel immune adjuvant to dendritic cell vaccines in two different murine syngeneic murine tumors. In mice challenged with MB49, which expresses the HY antigen complex, T cell responses primed by the tumor with and without Val-boroPro were measured using interferon gamma ELISPOT. Antibody depletion and gene-deficient mice were used to establish the immune cell subsets required for tumor regression. We demonstrate that Val-boroPro mediates tumor eradication by accelerating the expansion of tumor-specific T cells. Interestingly, T cells primed by tumor during Val-boroPro treatment demonstrate increased capacity to reject tumors following adoptive transfer without further treatment of the recipient. Val-boroPro -mediated tumor regression requires dendritic cells and is associated with enhanced trafficking of dendritic cells to tumor draining lymph nodes. Finally, dendritic cell vaccination combined with Val-boroPro treatment results in complete regression of established tumors. Our findings demonstrate that Val-boroPro has antitumor activity and a novel mechanism of action that involves more robust DC trafficking with earlier priming of T cells. Finally, we show that Val-boroPro has potent adjuvant properties resulting in an effective therapeutic vaccine.

Extracorporeal photopheresis (ECP) is emerging as a therapy for graft-versus-host-disease (GVHD), but the full mechanism of action and the impact on immunity have not been fully established. After murine minor histocompatibility antigen-mismatched bone marrow transplant (alloBMT), co-infusion of ECP-treated splenocytes with T cell-replete bone marrow attenuated GVHD irrespective of the donor strain of the ECP-treated splenocytes, and was associated with increased numbers of regulatory T cells. Co-culture of myeloid dendritic cells (mDC) with ECP-treated splenocytes resulted in increased interleukin (IL)-10 production after sub-maximal stimulation with lipopolysaccharide. Furthermore, male mDCs exposed to ECP-treated splenocytes were less potent at inducing CD8+ HY-responses when used as a vaccine in vivo. The efficacy of ECP-treated splenocytes was enhanced when administered just prior to delayed donor lymphocyte infusion (DLI) following T cell depleted alloBMT, allowing for the administration of sufficient numbers of T cells to respond to mDC vaccination in the absence of a thymus. Finally, the therapeutic effect of ECP-treated splenocytes were lost in recipients of IL-10 deficient bone marrow. We demonstrate that ECP-treated splenocytes attenuate GVHD irrespective of the source of ECP-treated cells via a mechanism that likely involves modulation of DCs, and requires IL-10 produced by bone marrow-derived cells. Importantly, attenuation of GVHD by ECP-treated splenocytes permits DLI-dependent responses to DC vaccines following alloBMT.

Current multimodality therapy consisting of surgery, chemotherapy and radiation will fail in approximately 40% of patients with pediatric sarcomas and results in substantial long-term morbidity in those who are cured. Immunotherapeutic regimens for the treatment of solid tumors typically generate antigen-specific responses too weak to overcome considerable tumor burden and tumor suppressive mechanisms and are in need of adjuvant assistance. Previous work suggests that inhibitors of DASH (Dipeptidyl peptidase IV activity and/or structural homologues) enzymes can mediate tumor regression via immune-mediated mechanisms. Here we demonstrate that the DASH inhibitor, ARI-4175, can induce regression and eradication of well-established solid tumors, both as a single agent and as an adjuvant to a dendritic cell (DC) vaccine and adoptive cell therapy (ACT) in mice implanted with the M3-9-M rhabdomyosarcoma (RMS) cell line. Treatment with effective doses of ARI-4175 correlated with recruitment of myeloid (CD11b+) cells, particularly myeloid dendritic cells (DCs), to secondary lymphoid tissues and with reduced frequency of intratumoral monocytic (CD11b+Ly6-ChiLy6-Glo) myeloid-derived suppressor cells. In immunocompetent mice, combining ARI-4175 with a DC vaccine or ACT with tumor-primed T cells produced significant improvements in tumor responses against well-established M3-9-M tumors. In M3-9-M-bearing immunodeficient (Rag1-/-) mice, ACT combined with ARI-4175 produced greater tumor responses and significantly improved survival compared to either treatment alone. These studies warrant the clinical investigation of ARI-4175 for treatment of sarcomas and other malignancies particularly as an adjuvant to tumor vaccines and ACT.

Body axes and germ layers evolve at gastrulation, and in mammals are driven by many genes; however, what orchestrates the genetic pathways during gastrulation remains elusive. Previously, we presented evidence that microRNA-17 (miRNA-17) family members, miR-17-5p, miR-20a, miR-93, and miR-106a were differentially expressed in mouse embryos and functioned to control differentiation of the stem cell population. Here, we identify function(s) that these miRNAs have during gastrulation. Fluorescent in situ hybridization miRNA probes reveal that these miRNAs are localized at the mid/posterior primitive streak (ps) in distinct populations of primitive ectoderm, mesendoderm, and mesoderm. Seven different miRNA prediction algorithms are identified in silico bone morphogenic protein receptor 2 (Bmpr2) as a target of these miRNAs. Bmpr2 is a member of the TGFβ pathway and invokes stage-specific changes during gastrulation. Recently, Bmpr2 was shown regulating cytoskeletal dynamics, cell movement, and invasion. Our previous and current data led to a hypothesis by which members of the miR-17 family influence gastrulation by suppressing Bmpr2 expression at the primitive streak. This suppression influences fate decisions of cells by affecting genes downstream of BMPR2 as well as mesoderm invasion through regulation of actin dynamics.

Key Points • STAT12/2 BM prevents GVHD induced by delayed donor lymphocyte infusion via the expansion of CD92 Siglec H hi pDCs, which are low producers of IFNa and IL-12.• pDCs recovered from STAT1 2/2 BM chimeras show increased expression of S100A8, S100A9, and STAT3.Selective targeting of non-T cells, including antigen-presenting cells (APCs), is a potential strategy to prevent graft-versus-host-disease (GVHD) but to maintain graft-versus-tumor (GVT) effects. Because type I and II interferons signal through signal transducer and activator of transcription-1 (STAT1), and contribute to activation of APCs after allogeneic bone marrow transplant (alloBMT), we examined whether the absence of STAT1 in donor APCs could prevent GVHD while preserving immune competence. 2/2 pDCs that were isolated after alloBMT showed increased gene expression of S100A8 and S100A9, and transplantation of S100A9 2/2 BM reduced GVHD-free survival. Finally, elevated STAT3 was found in STAT1 2/2 pDCs isolated after alloBMT. We conclude that interfering with interferon signaling in APCs such as pDCs provides a novel approach to regulate the GVHD/GVT axis. (Blood. 2014;124(12):1976-1986