Antigen-loaded Monocyte Administration and Flt3 Ligand Augment the Antitumor Efficacy of Immune Checkpoint Blockade in a Murine Melanoma Model.

Undifferentiated monocytes can be loaded with tumor antigens (Ag) and administered intravenously to induce antitumor cytotoxic T lymphocyte (CTL) responses. This vaccination strategy exploits an endogenous Ag cross-presentation pathway, where Ag-loaded monocytes (monocyte vaccines) transfer their Ag to resident splenic dendritic cells (DC), which then stimulate robust CD8 + CTL responses. In this study, we investigated whether monocyte vaccination in combination with CDX-301, a DC-expanding cytokine Fms-like tyrosine kinase 3 ligand (Flt3L), could improve the antitumor efficacy of anti-programmed cell death (anti-PD-1) immune checkpoint blockade. We found that Flt3L expanded splenic DC over 40-fold in vivo and doubled the number of circulating Ag-specific T cells when administered before monocyte vaccination in C57BL/6 mice. In addition, OVA-monocyte vaccination combined with either anti-PD-1, anti-programmed cell death ligand 1 (anti-PD-L1), or anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) suppressed subcutaneous B16/F10-OVA tumor growth to a greater extent than checkpoint blockade alone. When administered together, OVA-monocyte vaccination improved the antitumor efficacy of Flt3L and anti-PD-1 in terms of circulating Ag-specific CD8 + T cell frequency and inhibition of subcutaneous B16/F10-OVA tumor growth. To our knowledge, this is the first demonstration that a cancer vaccine strategy and Flt3L can improve the antitumor efficacy of anti-PD-1. The findings presented here warrant further study of how monocyte vaccines can improve Flt3L and immune checkpoint blockade as they enter clinical trials.

Nicotinic stimulation of splenic T cells is protective in endoscopic retrograde cholangiopancreatography-induced acute pancreatitis in mice.

It has previously been shown that current smoking is protective against endoscopic retrograde cholangiopancreatography (ERCP)-induced acute pancreatitis, but the mechanism of this effect was not identified. We tested the hypothesis that nicotine is the active factor in this protection in a mouse model of ERCP. Pretreatment with nicotine dose dependently inhibited acute pancreatitis caused by infusion of ERCP contrast solution into the main pancreatic duct in mice. 3-2,4-Dimethoxybenzylidene anabaseine (GTS-21), a specific partial agonist of the α7 nicotinic cholinergic receptor (α7nAChR), also protected the pancreas against ERCP-induced acute pancreatitis. The effects of GTS-21 were abolished by pretreatment with the nicotinic receptor antagonist mecamylamine. Surgical splenectomy performed 7 days before ERCP-induced pancreatitis blocked the protective effects of GTS-21. Intravenous injection of a crude preparation of total splenocytes prepared from mice pretreated with GTS-21 inhibited ERCP-induced pancreatitis; splenocytes from mice treated with vehicle had no effect. When T cells were removed from the crude GTS-21-treated splenocyte preparation by immunomagnetic separation, the remaining non-T-cell splenocytes did not protect against ERCP-induced acute pancreatitis. We conclude that nicotine protects against ERCP-induced acute pancreatitis and that splenic T cells are required for this effect. Stimulation of α7 nicotinic cholinergic receptors may protect against ERCP-induced acute pancreatitis and may also be a novel approach to therapeutic reversal of ongoing acute pancreatitis.NEW & NOTEWORTHY Epidemiological evidence indicated that acute smoking reduced the risk of endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis, but the mechanism has remained elusive. The current findings indicate the nicotine reduces the severity of ERCP-induced pancreatitis by stimulating a population of splenic T cells that exert a protective effect on the pancreas. These findings raise the possibility that nicotinic agonists might be useful in treating pancreatitis.

Monocytes as a Cellular Vaccine Platform to Induce Antitumor Immunity.

We recently developed a monocyte-based cellular vaccine platform for cancer treatment. In contrast to the traditional utilization of monocytes as precursors to generate dendritic cells (DC) for vaccination purposes, we find that freshly isolated monocytes with no differentiation process can be loaded with tumor antigens (Ag) and trigger robust antitumor cytotoxic T lymphocyte (CTL) responses. In this chapter, we describe methods to prepare, administer, and evaluate murine Ly-6Chi monocyte-based cellular vaccines for their therapeutic efficacy. This includes procedures for isolation, purity determination, Ag loading, administration of bone marrow (BM)-derived monocytes, as well as methods to determine vaccine efficacy through the examination of Ag-specific CD8+ T cell expansion and antitumor responses in murine melanoma models. As a vaccine platform, undifferentiated monocytes can be easily adapted to different tumor models with a multitude of target antigens. The method described here seeks to facilitate preclinical research of monocyte-based vaccination as a strategy for cancer immunotherapy.

Th17 Immunity in the Colon Is Controlled by Two Novel Subsets of Colon-Specific Mononuclear Phagocytes.

Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

Antigen-loaded monocyte administration induces potent therapeutic antitumor T cell responses.

Efficacy of dendritic cell (DC) cancer vaccines is classically thought to depend on their antigen-presenting cell (APC) activity. Studies show, however, that DC vaccine priming of cytotoxic T lymphocytes (CTLs) requires the activity of endogenous DCs, suggesting that exogenous DCs stimulate antitumor immunity by transferring antigens (Ags) to endogenous DCs. Such Ag transfer functions are most commonly ascribed to monocytes, implying that undifferentiated monocytes would function equally well as a vaccine modality and need not be differentiated to DCs to be effective. Here, we used several murine cancer models to test the antitumor efficacy of undifferentiated monocytes loaded with protein or peptide Ag. Intravenously injected monocytes displayed antitumor activity superior to DC vaccines in several cancer models, including aggressive intracranial glioblastoma. Ag-loaded monocytes induced robust CTL responses via Ag transfer to splenic CD8+ DCs in a manner independent of monocyte APC activity. Ag transfer required cell-cell contact and the formation of connexin 43-containing gap junctions between monocytes and DCs. These findings demonstrate the existence of an efficient gap junction-mediated Ag transfer pathway between monocytes and CD8+ DCs and suggest that administration of tumor Ag-loaded undifferentiated monocytes may serve as a simple and efficacious immunotherapy for the treatment of human cancers.

Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients.

After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

Hypernatremic myopathy caused by a hypothalamic mixed germ cell tumor mimicking polymyositis.

Hypernatremic myopathy was rarely reported in the literature and its clinical features have never been well-described. We present a 22-year-old man who had adipsic hypernatremia manifested with progressive proximal muscle weakness and remarkably high creatine kinase level that has never been reported in the cases of hypernatremic myopathy. His initial presentations were similar to that of polymyositis without the evidence of central nervous system dysfunction and hypopituitarism. The serum level of sodium at the beginning of myopathy is the lowest known in the literature. All the clinical presentations in this patient resulted from a hypothalamic mixed germ cell tumor with sub-acute intra-tumoral hemorrhage.