Multifactoral immune modulation potentiates durable remission in multiple models of aggressive malignancy.

Tumors typically lack canonical danger signals required to activate adaptive immunity and also frequently employ substantial immunomodulatory mechanisms that downregulate adaptive responses and contribute to escape from immune surveillance. Given the variety of mechanisms involved in shielding tumors from immune recognition, it is not surprising that single-agent immunomodulatory approaches have been largely unsuccessful in generating durable antitumor responses. Here we report a unique combination of immunomodulatory and cytostatic agents that recondition the tumor microenvironment and eliminate complex and/or poor-prognosis tumor types including the non-immunogenic 4T-1 model of TNBC, the aggressive MOC-2 model of HNSCC, and the high-risk MYCN-amplified model of neuroblastoma. A course of therapy optimized for TNBC cured a majority of tumors in both ectopic and orthotopic settings and eliminated metastatic spread in all animals tested at the highest doses. Immune responses were transferable between therapeutic donor and naïve recipient through adoptive transfer, and a sizeable abscopal effect on distant, untreated lesions could be demonstrated experimentally. Similar results were observed in HNSCC and neuroblastoma models, with characteristic remodeling of the tumor microenvironment documented in all model systems. scRNA-seq analysis implicated upregulation of innate immune responses and antigen presentation in tumor cells and the myeloid cell compartment as critical early events. This analysis also highlighted the potential importance of the autonomic nervous system in the governance of inflammatory processes. The data indicate that the targeting of multiple pathways and mechanisms of action can result in substantial synergistic antitumor effects and suggest follow-up in the neoadjuvant setting may be warranted.

Immunomodulatory proteins from hookworms reduce cardiac inflammation and modulate regulatory responses in a mouse model of chronic Trypanosoma cruzi infection.

Introduction: Hookworms are parasitic helminths that secrete a variety of proteins that induce anti-inflammatory immune responses, stimulating increased CD4 + Foxp3+ regulatory T cells and IL-10 production. Hookworm-derived recombinant proteins AIP-1 and AIP-2 have been shown to reduce inflammation in mouse models of inflammatory bowel disease and inflammatory airway disease by inducing CD4+Foxp3+ cells and IL-10 production. In contrast, chronic infection with the protozoal parasite Trypanosoma cruzi, the causative agent of Chagas disease, leads to chronic inflammation in tissues. Persistence of the parasites in tissues drives chronic low-grade inflammation, with increased infiltration of inflammatory cells into the heart, accompanied by increased production of inflammatory cytokines. There are no current antiparasitic drugs that effectively reduce or prevent chronic myocarditis caused by the onset of Chagas disease, thus new therapies are urgently needed. Therefore, the impact of AIP-1 and AIP-2 on myocarditis was investigated in a mouse model of chronic T. cruzi infection. Methods: Female BALB/c mice infected with bioluminescent T. cruzi H1 strain trypomastigotes for 70 days were treated once daily for 7 days with 1mg/kg AIP-1 or AIP-2 protein by intraperitoneal injection. Control mice were left untreated or treated once daily for 14 days with 25mg/kg aspirin in drinking water. At 84 days of infection, splenocytes, cardiac tissue and serum were collected for evaluation. Results: Treatment with both AIP-1 and AIP-2 proteins significantly reduced cardiac cellular infiltration, and reduced cardiac levels of IFNγ, IL-6 and IL-2. AIP-2 treatment reduced cardiac expression of COX-2. Further, while incubation with AIP-1 and AIP-2 proteins did not induce a significant upregulation of an immunoregulatory phenotype in dendritic cells (DC), there was a modest upregulation of CD11c +CD11b+MHCII+SIRPα+ expression, suggesting a regulatory phenotype. Ex-vivo stimulation of splenocytes from the treatment groups with AIP-1 loaded DC induced reduced levels of cytotoxic and pro-inflammatory T cells, stimulation with AIP-2 loaded DC specifically induced enhanced levels of CD4+CD25+Foxp3+ regulatory T cells among treatment groups. Discussion: All in vivo and in vitro results demonstrate that hookworm-derived AIP-1 and AIP-2 proteins reduce T. cruzi induced cardiac inflammation, possibly through multiple anti-inflammatory mechanisms.

A subset of cytotoxic effector memory T cells enhances CAR T cell efficacy in a model of pancreatic ductal adenocarcinoma.

In humans, the natural killer (NK) cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αß T cell receptor-expressing subset with characteristic specificity for tissue-localized viruses. This subset also displays enhanced cytotoxic and memory phenotypes. Here, we characterized this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161-equivalent CD8+ T cell populations (CD8+NK1.1+) revealed substantial up-regulation of granzymes, perforin, killer lectin-like receptors, and innate signaling molecules in comparison to CD8+NK1.1- T cells. Adoptive transfer of CD8+NK1.1+ cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8+NK1.1- cells. Freshly isolated human CD8+CD61+ T cells exhibited heightened allogeneic killing activity in comparison to CD8+CD61- T cells or total peripheral blood mononuclear cells (PBMCs). To determine whether this subset might improve the antitumor efficacy of CAR T cell therapy against solid tumors, we compared bulk PBMCs, CD8+CD161-, and CD8+CD161+ T cells transduced with a human epidermal growth factor receptor-2 (HER2)-specific CAR construct. In vitro, CD8+CD161+ CAR-transduced T cells killed HER2+ targets faster and with greater efficiency. Similarly, these cells mediated enhanced in vivo antitumor efficacy in xenograft models of HER2+ pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.

A randomized, double-blind, placebo-controlled study of daily cannabidiol for the treatment of canine osteoarthritis pain.

ABSTRACT: Over the last 2 decades, affirmative diagnoses of osteoarthritis (OA) in the United States have tripled due to increasing rates of obesity and an aging population. Hemp-derived cannabidiol (CBD) is the major nontetrahydrocannabinol component of cannabis and has been promoted as a potential treatment for a wide variety of disparate inflammatory conditions. Here, we evaluated CBD for its ability to modulate the production of proinflammatory cytokines in vitro and in murine models of induced inflammation and further validated the ability of a liposomal formulation to increase bioavailability in mice and in humans. Subsequently, the therapeutic potential of both naked and liposomally encapsulated CBD was explored in a 4-week, randomized placebo-controlled, double-blinded study in a spontaneous canine model of OA. In vitro and in mouse models, CBD significantly attenuated the production of proinflammatory cytokines IL-6 and TNF-α while elevating levels of anti-inflammatory IL-10. In the veterinary study, CBD significantly decreased pain and increased mobility in a dose-dependent fashion among animals with an affirmative diagnosis of OA. Liposomal CBD (20 mg/day) was as effective as the highest dose of nonliposomal CBD (50 mg/day) in improving clinical outcomes. Hematocrit, comprehensive metabolic profile, and clinical chemistry indicated no significant detrimental impact of CBD administration over the 4-week analysis period. This study supports the safety and therapeutic potential of hemp-derived CBD for relieving arthritic pain and suggests follow-up investigations in humans are warranted.

MHC class I and II peptide homology regulates the cellular immune response.

Mammalian immune responses are initiated by "danger" signals--immutable molecular structures known as PAMPs. When detected by fixed, germline encoded receptors, pathogen-associated molecular pattern (PAMPs) subsequently inform the polarization of downstream adaptive responses depending upon identity and localization of the PAMP. Here, we report the existence of a completely novel "PAMP" that is not a molecular structure but an antigenic pattern. This pattern--the incidence of peptide epitopes with stretches of 100% sequence identity bound to both dendritic cell (DC) major histocompatibility (MHC) class I and MHC class II--strongly induces TH 1 immune polarization and activation of the cellular immune response. Inherent in the existence of this PAMP is the concomitant existence of a molecular sensor complex with the ability to scan and compare amino acid sequence identities of bound class I and II peptides. We provide substantial evidence implicating the multienzyme aminoacyl-tRNA synthetase (mARS) complex and its AIMp1 structural component as the key constituents of this complex. The results demonstrate a wholly novel mechanism by which T-helper (TH ) polarization is governed and provide critical information for the design of vaccination strategies intended to provoke cell-mediated immunity.