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.

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.

Genetic Adjuvantation of a Cell-Based Therapeutic Vaccine for Amelioration of Chagasic Cardiomyopathy.

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a leading cause of heart disease ("chagasic cardiomyopathy") in Latin America, disproportionately affecting people in resource-poor areas. The efficacy of currently approved pharmaceutical treatments is limited mainly to acute infection, and there are no effective treatments for the chronic phase of the disease. Preclinical models of Chagas disease have demonstrated that antigen-specific CD8+ gamma interferon (IFN-γ)-positive T-cell responses are essential for reducing parasite burdens, increasing survival, and decreasing cardiac pathology in both the acute and chronic phases of Chagas disease. In the present study, we developed a genetically adjuvanted, dendritic cell-based immunotherapeutic for acute Chagas disease in an attempt to delay or prevent the cardiac complications that eventually result from chronic T. cruzi infection. Dendritic cells transduced with the adjuvant, an adenoviral vector encoding a dominant negative isoform of Src homology region 2 domain-containing tyrosine phosphatase 1 (SHP-1) along with the T. cruzi Tc24 antigen and trans-sialidase antigen 1 (TSA1), induced significant numbers of antigen-specific CD8+ IFN-γ-positive cells following injection into BALB/c mice. A vaccine platform transduced with the adenoviral vector and loaded in tandem with the recombinant protein reduced parasite burdens by 76% to >99% in comparison to a variety of different controls and significantly reduced cardiac pathology in a BALB/c mouse model of live Chagas disease. Although no statistical differences in overall survival rates among cohorts were observed, the data suggest that immunotherapeutic strategies for the treatment of acute Chagas disease are feasible and that this approach may warrant further study.

Modeling dendritic cell vaccination for influenza prophylaxis: potential applications for niche populations.

BACKGROUND: Cancer patients can exhibit negligible responses to prophylactic vaccinations, including influenza vaccination. To help address this issue, we developed in vitro and in vivo models of dendritic cell (DC) immunotherapy for the prevention of influenza virus infection. METHODS: Human cord blood (CB)-derived or mouse splenocyte-derived DCs were loaded with purified recombinant hemagglutinin (rHA). T-cell responses to HA-loaded CB-derived DCs were determined by ELISpot. Protective efficacy was determined by vaccination of BALB/c mice with a single injection of 10(6) autologous DCs. DC migration to peripheral lymphoid organs was verified by carboxyfluorescein succinimidyl ester staining, and HA-specific antibody titers were determined by enzyme-linked immunosorbent assay. Mice were then challenged intranasally with BALB/c-adapted A/New Caledonia influenza virus derived from four consecutive lung pool passages. Antigen-presenting cell (APC) dysfunction was modeled using the MAFIA transgenic system, in which the Csf1r promoter conditionally drives AP20178-inducible Fas. RESULTS: CB-derived human DCs were able to generate de novo T-cell responses against rHA, as determined by a system of rigorous controls. Mice vaccinated intraperitoneally developed HA titers detectable at serum dilutions of >1:1000. HA seroconverters survived virus challenge, whereas unvaccinated controls and vaccinated nonseroconverters lost weight and died. Furthermore, use of a model of APC-specific immunosuppression revealed that DC vaccination could generate HA-specific antibody titers under conditions in which protein vaccination could not. CONCLUSIONS: The model demonstrates that DC immunotherapy for the prevention of influenza is feasible, and studies are underway to determine whether populations of immunosuppressed individuals might ultimately benefit from the procedure.

The mARS complex: a critical mediator of immune regulation and homeostasis.

Over the course of evolution, many proteins have undergone adaptive structural changes to meet the increasing homeostatic regulatory demands of multicellularity. Aminoacyl tRNA synthetases (aaRS), enzymes that catalyze the attachment of each amino acid to its cognate tRNA, are such proteins that have acquired new domains and motifs that enable non-canonical functions. Through these new domains and motifs, aaRS can assemble into large, multi-subunit complexes that enhance the efficiency of many biological functions. Moreover, because the complexity of multi-aminoacyl tRNA synthetase (mARS) complexes increases with the corresponding complexity of higher eukaryotes, a contribution to regulation of homeostatic functions in multicellular organisms is hypothesized. While mARS complexes in lower eukaryotes may enhance efficiency of aminoacylation, little evidence exists to support a similar role in chordates or other higher eukaryotes. Rather, mARS complexes are reported to regulate multiple and variegated cellular processes that include angiogenesis, apoptosis, inflammation, anaphylaxis, and metabolism. Because all such processes are critical components of immune homeostasis, it is important to understand the role of mARS complexes in immune regulation. Here we provide a conceptual analysis of the current understanding of mARS complex dynamics and emerging mARS complex roles in immune regulation, the increased understanding of which should reveal therapeutic targets in immunity and immune-mediated disease.

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.

CD8+CD161+ T-Cells: Cytotoxic Memory Cells With High Therapeutic Potential.

NK1.1 and its human homolog CD161 are expressed on NK cells, subsets of CD4+ and CD8+ T cells, and NKT cells. While the expression of NK1.1 is thought to be inhibitory to NK cell function, it is reported to play both costimulatory and coinhibitory roles in T-cells. CD161 has been extensively studied and characterized on subsets of T-cells that are MR1-restricted, IL-17 producing CD4+ (TH17 MAIT cells) and CD8+ T cells (Tc17 cells). Non-MAIT, MR1-independent CD161-expressing T-cells also exist and are characterized as generally effector memory cells with a stem cell like phenotype. Gene expression analysis of this enigmatic subset indicates a significant enhancement in the expression of cytotoxic granzyme molecules and innate like stress receptors in CD8+NK1.1+/CD8+CD161+ cells in comparison to CD8+ cells that do not express NK1.1 or CD161. First identified and studied in the context of viral infection, the role of CD8+CD161+ T-cells, especially in the context of tumor immunology, is still poorly understood. In this review, the functional characteristics of the CD161-expressing CD8+ T cell subset with respect to gene expression profile, cytotoxicity, and tissue homing properties are discussed, and application of this subset to immune responses against infectious disease and cancer is considered.

Beyond T-Cells: Functional Characterization of CTLA-4 Expression in Immune and Non-Immune Cell Types.

The immune response consists of a finely-tuned program, the activation of which must be coupled with inhibitory mechanisms whenever initiated. This ensures tight control of beneficial anti-pathogen and anti-tumor responses while preserving tissue integrity, promoting tissue repair, and safeguarding against autoimmunity. A cogent example of this binary response is in the mobilization of co-stimulatory and co-inhibitory signaling in regulating the strength and type of a T-cell response. Of particular importance is the costimulatory molecule CD28 which is countered by CTLA-4. While the role of CD28 in the immune response has been thoroughly elucidated, many aspects of CTLA-4 biology remain controversial. The expression of CD28 is largely constrained to constitutive expression in T-cells and as such, teasing out its function has been somewhat simplified by a limited and specific expression profile. The expression of CTLA-4, on the other hand, while reported predominantly in T-cells, has also been described on a diverse repertoire of cells within both lymphoid and myeloid lineages as well as on the surface of tumors. Nonetheless, the function of CTLA-4 has been mostly described within the context of T-cell biology. The focus on T-cell biology may be a direct result of the high degree of amino acid sequence homology and the co-expression pattern of CD28 and CTLA-4, which initially led to the discovery of CTLA-4 as a counter receptor to CD28 (for which a T-cell-activating role had already been described). Furthermore, observations of the outsized role of CTLA-4 in Treg-mediated immune suppression and the striking phenotype of T-cell hyperproliferation and resultant disease in CTLA-4-/- mice contribute to an appropriate T-cell-centric focus in the study of CTLA-4. Complete elucidation of CTLA-4 biology, however, may require a more nuanced understanding of its role in a context other than that of T-cells. This makes particular sense in light of the remarkable, yet limited utility of anti-CTLA-4 antibodies in the treatment of cancers and of CTLA-4-Ig in autoimmune disorders like rheumatoid arthritis. By fully deducing the biology of CTLA-4-regulated immune homeostasis, bottlenecks that hinder the widespread applicability of CTLA-4-based immunotherapies can be resolved.

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.

AIMp1 Potentiates TH1 Polarization and Is Critical for Effective Antitumor and Antiviral Immunity.

Dendritic cells (DCs) must integrate a broad array of environmental cues to exact control over downstream immune responses including TH polarization. The multienzyme aminoacyl-tRNA synthetase complex component AIMp1/p43 responds to cellular stress and exerts pro-inflammatory functions; however, a role for DC-expressed AIMp1 in TH polarization has not previously been shown. Here, we demonstrate that the absence of AIMp1 in bone marrow-derived DC (BMDC) significantly impairs cytokine and costimulatory molecule expression, p38 MAPK signaling, and TH1 polarization of cocultured T-cells while significantly dysregulating immune-related gene expression. These deficits resulted in significantly compromised BMDC vaccine-mediated protection against melanoma. AIMp1 within the host was also critical for innate and adaptive antiviral immunity against influenza virus infection in vivo. Cancer patients with AIMp1 expression levels in the highest tertiles exhibited a 70% survival advantage at 15-year postdiagnosis as determined by bioinformatics analysis of nearly 9,000 primary human tumor samples in The Cancer Genome Atlas database. These data establish the importance of AIMp1 for the effective governance of antitumor and antiviral immune responses.

Chemo-immunotherapy mediates durable cure of orthotopic KrasG12D/p53-/- pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States, exhibiting a five-year overall survival (OS) of only 7% despite aggressive standard of care. Recent advances in immunotherapy suggest potential application of immune-based treatment approaches to PDAC. To explore this concept further, we treated orthotopically established K-rasG12D/p53-/- PDAC tumors with gemcitabine and a cell-based vaccine previously shown to generate durable cell-mediated (TH1) immunity. Tumor progression was monitored by IVIS. The results indicated that the combination of chemotherapy and dendritic cell (DC) vaccination was effective in eliminating tumor, preventing metastasis and recurrence, and significantly enhancing OS. No animal that received the combination therapy relapsed, while mice that received gemcitabine-only or vaccine-only regimens relapsed and progressed. Analysis of circulating PBMC demonstrated that mice receiving the combination therapy exhibited significantly elevated levels of CD8+IFNγ+CCR7+NK1.1+ T-cells with significantly reduced levels of exhausted GITR+CD8+ T-cells after the cessation of treatment. Retro-orbital tumor re-challenge of surviving animals at six-months post-treatment demonstrated durable antitumor immunity only among mice that had received the combination therapy. CD8+ splenocytes derived from surviving mice that had received the combination therapy were sorted into NK1.1pos and NK1.1neg populations and adoptively transferred into naive recipients. Transfer of only 1,500 CD8+NK1.1pos T-cells was sufficient to mediate tumor rejection whereas transfer of 1,500 CD8+NK1.1neg T-cells imparted only minimal effects. The data suggest that addition of a TH1 DC vaccine regimen as an adjuvant to existing therapies can mediate eradication of tumors and offer durable protection against PDAC.

Dendritic Cell-Secreted Cytotoxic T-Lymphocyte-Associated Protein-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.

The remarkable functional plasticity of professional antigen-presenting cells (APCs) allows the adaptive immune system to respond specifically to an incredibly diverse array of potential pathogenic insults; nonetheless, the specific molecular effectors and mechanisms that underpin this plasticity remain poorly characterized. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is one of the most well-known and well-studied members of the B7 superfamily and negatively regulates T cell responses by a variety of known mechanisms. Although CTLA-4 is thought to be expressed almost exclusively among lymphoid lineage hematopoietic cells, a few reports have indicated that nonlymphoid APCs can also express the CTLA-4 mRNA transcript and that transcript levels can be regulated by external stimuli. In this study, we substantially build upon these critical observations, definitively demonstrating that mature myeloid lineage dendritic cells (DC) express significant levels of intracellular CTLA-4 that they constitutively secrete in microvesicular structures. CTLA-4(+) microvesicles can competitively bind B7 costimulatory molecules on bystander DC, resulting in downregulation of B7 surface expression with significant functional consequences for downstream CD8(+) T-cell responses. Hence, the data indicate a previously unknown role for DC-derived CTLA-4 in immune cell functional plasticity and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.

A dicistronic gene pair within a cluster of "EF-hand" protein genes in the genomes of Drosophila species.

Androcam is a Drosophila melanogaster calmodulin-related protein that functions specifically in the testis. We show that the Acam gene is part of a cluster of three intronless genes arranged in a head-to-tail manner. The additional genes also encode calmodulin-related proteins with testis-specific transcription. Acam and the 5'-most gene (gene1) generate monocistronic transcripts. Surprisingly, the central gene (gene2) is transcribed only as a dicistronic transcript with Acam. A similar cluster is found in D. yakuba. In D. pseudoobscura, the cluster contains four genes: two Acam-type genes downstream of a single gene related to both gene1 and gene2 and a fourth weakly related gene. Nevertheless, the D. pseudoobscura cluster also generates a dicistronic transcript from a gene pair analogous to the gene2-Acam pair. A cotranscribed gene1/2-Acam gene pair may be the founding feature of this locus. Although Acam protein is present in D. melanogaster and D. pseudoobscura testes, cognate proteins for the gene1/2-type ORFs are not detectable by immunoblotting and mass spectrometry techniques.