Activation Of Immune Effector Cells Research Articles

MDNA11 is a long-acting IL-2 superkine that potentiates durable immune response in mouse tumor models and non-human primates.

e14546 Background: The efficacy and safety of recombinant human IL-2 (rhIL-2; Proleukin) to treat certain cancers is limited by a short half-life, marked toxicity and selective high affinity binding to IL2Ra over IL2Rb, resulting in preferential activation of suppressive Tregs. In contrast, MDNA11 has been engineered as a long-acting IL-2 superkine with high affinity IL2Rb receptor selectivity, resulting in preferential anti-cancer effector immune cell activation. Methods: MDNA11 was characterized in both in vitro and in vivo studies including assessment of receptor binding kinetics using BLI/Octet, receptor-mediated signaling in human PMBCs, efficacy in syngeneic mouse tumor models including memory response, as well as safety and PK/PD assessments in non-human primates (NHP). Results: Unlike rhIL-2, MDNA11 does not bind to human IL2Ra but demonstrates a 30-fold higher affinity binding to human IL2Rb. This selectivity resulted in enhanced in vitro STAT5 signaling in human NK and resting CD8 T cells with diminished signaling in Tregs; validation studies in humanized mice are ongoing. In CT26 and MC38 syngeneic tumor models, MDNA11 demonstrates potent and durable efficacy as monotherapy following a Q1W dose schedule for 2 weeks. Synergy with anti-PD1 and anti-CTLA4 immune checkpoint inhibitors (ICIs) was observed and a robust immune memory response developed in all mice with complete tumor clearance. These mice were protected against relapse and tumor re-challenges for up to 8 months without any further treatment, and showed the presence of antigen-specific CD8 T cells. In binding studies with IL-2 receptors of different species, MDNA11 showed highly similar affinity towards human and cynomolgus IL2Rb, confirming the latter as a highly relevant model for toxicology study. MDNA11 was well tolerated in cynomolgus monkeys up to 0.6 mg/kg, while inducing durable (≥10 days) proliferation and expansion of NK and CD8 T cells. Effects on Tregs were minimal and there was no eosinophilia and hypotension (associated with vascular leak syndrome). At high doses of MDNA11, the most common clinical observations were transient loss of appetite and diarrhea. There was modest increase in levels of IFNg and TNFa, but no sign of cytokine release syndrome. Dosing did not trigger development of anti-drug antibodies or histopathologic evidence of pulmonary edema (a major IL-2 induced toxicity). Conclusions: MDNA11 is a long-acting IL-2 superkine that exhibits robust efficacy in mouse tumor models as a single agent and was synergistic in combination with ICIs (anti-CTLA4 and anti-PD1). In NHP, MDNA11 demonstrates selective immune effector cell activation and a favorable safety profile. These data constitute a strong framework for the design of a pivotal GLP toxicology study to further support the planned clinical study of MDNA11 either as a single agent or in combination with ICIs.

Metabolites in the Tumor Microenvironment Reprogram Functions of Immune Effector Cells Through Epigenetic Modifications.

Cellular metabolism of both cancer and immune cells in the acidic, hypoxic, and nutrient-depleted tumor microenvironment (TME) has attracted increasing attention in recent years. Accumulating evidence has shown that cancer cells in TME could outcompete immune cells for nutrients and at the same time, producing inhibitory products that suppress immune effector cell functions. Recent progress revealed that metabolites in the TME could dysregulate gene expression patterns in the differentiation, proliferation, and activation of immune effector cells by interfering with the epigenetic programs and signal transduction networks. Nevertheless, encouraging studies indicated that metabolic plasticity and heterogeneity between cancer and immune effector cells could provide us the opportunity to discover and target the metabolic vulnerabilities of cancer cells while potentiating the anti-tumor functions of immune effector cells. In this review, we will discuss the metabolic impacts on the immune effector cells in TME and explore the therapeutic opportunities for metabolically enhanced immunotherapy.

Interplay between C-type lectin receptors and microRNAs in cellular homeostasis and immune response.

C-type lectin receptors (CLRs) belong to the family of pattern recognition receptors (PRRs). They have a critical role to play in the regulation of a range of physiological functions including development, respiration, angiogenesis, inflammation, and immunity. CLRs can recognize distinct and conserved exogenous pathogen-associated as well as endogenous damage-associated molecular patterns. These interactions set off downstream signaling cascades, leading to the production of inflammatory mediators, activation of effector immune cells as well as regulation of the developmental and physiological homeostasis. CLR signaling must be tightly controlled to circumvent the excessive inflammatory burden and to maintain the cellular homeostasis. Recently, MicroRNAs (miRNAs) have been shown to be important regulators of expression of CLRs and their downstream signaling. The delicate balance between miRNAs and CLRs seems crucial in almost all aspects of multicellular life. Any dysregulations in the miRNA-CLR axes may lead to tumorigenesis or inflammatory diseases. Here, we present an overview of the current understanding of the central role of miRNAs in the regulation of CLR expression, profoundly impacting upon homeostasis and immunity, and thus, development of therapeutics against immune disorders.

Engineered Memory-like NK Cars Targeting a Neoepitope Derived from Intracellular NPM1c Exhibit Potent Activity and Specificity Against Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) continues to be a major therapeutic challenge. There is an emerging need to develop less toxic and more effective targeted therapies. Natural Killer (NK) cells possess many of the key attributes critical for effective cancer therapies- "born to kill" but without apparent risk of graft versus host disease, cytokine release syndrome, or neurotoxicity. Furthermore, their intrinsic propensity to target myeloid blasts makes them particularly attractive for AML. Despite promising clinical results in blood cancer, the development of NK cell-based therapy remains challenging mostly due to NK cells' short lifespan, inadequate proliferation and lack of specific tumor targeting. Here, we utilized a new approach to arm NK cells for adoptive immunotherapy based on innate cell memory. Chimeric antigen receptors (CARs) significantly enhance anti-tumor specificity and activity of immune effector cells. Our innovative CAR-NK cells target a tumor-specific neoepitope in AML and harness potent function pathways in their design to enhance efficacy and minimize toxicity. Methods: 1. Mutated NPM1c as a CAR Target in AML. Most CAR-T cell therapies target tumor-associated antigens (TAAs), which could lead to on-target/off-tumor toxicity as well as tumor resistance. One way to overcome these drawbacks is to target tumor-specific oncogenic driver mutations. The four-nucleotide duplication in nucleophosmin, referred to as NPM1c, is a driver oncogene mutation in about 35% of AML. The mutation creates a neoepitope that is presented by the most common HLA-A2 allele. Using yeast surface display, we have isolated a human single-chain variable fragment (scFv) that specifically binds to the NPM1c epitope-HLA-A2 complex, but not HLA-A2 alone or HLA-A2 loaded with control peptides. 2. Cytokine-Induced Memory-Like (CIML) NK Cells as a CAR Platform. CIML NK cells can provide a unique platform for development of NK cell CARs based on the favorable safety profile, increased proliferation, prolonged persistence and enhanced anti-leukemia function that we have observed in pre-clinical models (Romee et al, Blood 2012) and in patients (Romee et al, Science Trans Med 2016) treated with un-modified CIML NK cells. 3. Efficient Gene Editing in Primary NK Cells. We have overcome the transduction block in primary human and mouse NK cells by utilizing an unconventional pseudotyped lentivirus based on a unique protein with high expression on CIML NK cells. Results: 1. Engineered CAR-T cells with the isolated scFv exhibit potent cytotoxicity both in vitro and in vivo against NPM1c+HLA-A2+ leukemia cells (OCI-AML3) and primary AML blasts, but not NPM1c-HLA-A2+ leukemia cells (OCI-AML2) or HLA-A2- tumor cells (PC-3). 2. The in vivo anti-leukemia efficacy of anti-NPM1c CAR-T cells was however transient (overall survival extended from 28 to 42 days, median survival extended from 21 to 37 days, compared with the control mice adoptively transferred with untraduced T cells), with unneglectable toxicity. 3. Utilizing an unconventional pseudotyped lentivirus to transduce CIML NK cells from healthy donor blood (n = 5 donors), we have successfully generated anti-NPM1c CAR-NK cells with high transduction efficiency (using MOI = 10: transduction rate mean 48%, range 32% to 65%; compared with 2%, range 0.8% to 4.5% for the conventional approach with VSVG pseudotyped lentivirus). 4. Harnessing key cytokine pathways in the CAR design substantially promoted CAR-NK cell survival (indicated by the enhanced cell viability from 29.7% to 75.2%) and proliferation (marked by the increased levels of ki-67 from 60.2% to 94.5%). 5. Anti-NPM1c CAR significantly promoted anti-tumor function (represented by CD107a, IFN-gamma) and tumor-specific killing (measured by annexin V and 7-AAD) of CIML NK cells against AML with NPM1c oncogene (OCI-AML3). 6. Dual-armed CIML NK cells with CAR and cytokine signaling exhibited optimal specificity and sustainability against AML targets. Conclusion: These results demonstrate that the innovative CAR-CIML NK cells could be developed as an efficient cellular immunotherapy for treating NPM1c+HLA-A2+ AML with potentially reduced on-target/off-tumor toxicity and tumor resistance. Our study should drive novel conception and design of CAR-NK cell therapies against myeloid malignancies in the clinic. Figure Disclosures Ritz: Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy.

Nanomaterial-Based Tumor Photothermal Immunotherapy.

In recent years, photothermal therapy (PTT) particularly nanomaterial-based PTT is a promising therapeutic modality and technique for cancer tumor ablation. In addition to killing tumor cells directly through heat, PTT also can induce immunogenic cell death (ICD) to activate the whole-body anti-tumor immune response, including the redistribution and activation of immune effector cells, the expression and secretion of cytokines and the transformation of memory T lymphocytes. When used in combination with immunotherapy, the efficacy of nanomaterial-based PTT can be improved. This article summarized the mechanism of nanomaterial-based PTT against cancer and how nanomaterial-based PTT impacts the tumor microenvironment and induces an immune response. Moreover, we reviewed recent advances of nanomaterial-based photothermal immunotherapy and discussed challenges and future outlook.

Destructive Roles of Fibroblast-like Synoviocytes in Chronic Inflammation and Joint Damage in Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLSs) are important non-immune cells located mostly in the inner layer of the synovium. Indeed, these cells are specialized mesenchymal cells, implicated in collagen homeostasis of the articular joint and provide extracellular matrix (ECM) materials for cartilage and contribute to joint destruction via multiple mechanisms. RA FLS interactions with immune and non-immune cells lead to the development and organization of tertiary structures such as ectopic lymphoid-like structures (ELSs), tertiary lymphoid organs (TLOs), and secretion of proinflammatory cytokines. The interaction of RA FLS cells with immune and non-immune cells leads to stimulation and activation of effector immune cells. Pathological role of RA FLS cells has been reported for many years, while molecular and cellular mechanisms are not completely understood yet. In this review, we tried to summarize the latest findings about the role of FLS cells in ELS formation, joint destruction, interactions with immune and non-immune cells, as well as potential therapeutic options in rheumatoid arthritis (RA) treatment. Our study revealed data about interactions between RA FLS and immune/non-immune cells as well as the role of RA FLS cells in joint damage, ELS formation, and neoangiogenesis, which provide useful informationfor developing new approaches for RA treatment.

Foxp3+ Regulatory T Cells Inhibit CCl4-Induced Liver Inflammation and Fibrosis by Regulating Tissue Cellular Immunity.

Foxp3+ regulatory T (Treg) cells are pivotal in maintaining immunological self-tolerance and tissue homeostasis; however, it remains unclear how tissue Treg cells respond to liver injury and regulate chronic inflammation, which can cause liver fibrosis. We report here that hepatic Treg cells play a critical role in preventing liver pathology by suppressing inflammatory cellular immunity that can promote liver damage and fibrosis. Chronic liver inflammation induced by injections of carbon tetrachloride (CCl4) led to preferential expansion of hepatic Treg cells that prevented liver fibrosis. In contrast, depletion of Treg cells in the CCl4-induced liver fibrosis model exacerbated the severity of liver pathology. Treg depletion unleashed tissue cellular immunity and drove the activation and expansion of the pro-fibrotic IL-4-producing T helper 2 cells, as well as CCR2high Ly-6Chigh inflammatory monocytes/macrophages in the inflamed liver. Although Treg expression of amphiregulin plays a key role in tissue remodeling and repair in various inflammation models, amphiregulin from hepatic Treg cells, the largest producer among liver immune cells, was dispensable for maintaining liver homeostasis and preventing liver fibrosis during CCl4-induced chronic inflammation. Our results indicate that Treg cells control chronic liver inflammation and fibrosis by regulating the aberrant activation and functions of immune effector cells. Harnessing Treg functions, which effectively regulate tissue cellular immunity, may be a therapeutic strategy for preventing and treating liver fibrosis.

Putting into Perspective the Future of Cancer Vaccines: Targeted Immunotherapy

Pre-clinical models and human clinical trials have confirmed the ability of cancer vaccines to induce immune responses that are tumour-specific and, in some cases, associated with clinical response. However, cancer vaccines as a targeted immunotherapy strategy have not yet come of age. So, why the discordance after so much research has been invested in cancer vaccines? There are several reasons for this that include: limited tumour immunogenicity (limited targeted antigen expression, antigen tolerance); antigenic heterogeneity in tumours; heterogeneity of individual immune responses; multiple mechanisms associated with suppressed functional activity of immune effector cells, the underlying rationale for the use of immune checkpoint inhibitors; and immune system exhaustion. The success of checkpoint therapy has refocussed investigations into defining relationships between tumours and host immune systems, appreciating the mechanisms by which tumour cells escape immune surveillance and reinforcing recognition of the potential of vaccines in the treatment and prevention of cancer. Recent developments in cancer immunotherapies, together with associated technologies, for instance, the unparalleled achievements by immune checkpoint inhibitors and neo-antigen identification tools, may foster potential improvements in cancer vaccines for the treatment of malignancies.

Abstract 539: Novel engineered toxin bodies targeting SLAMF7 (CS1)

Abstract Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. Three ETBs are in clinical development (MT-3724 targeting CD20, MT-5111 targeting HER2, and TAK-169 targeting CD38). The novel mechanism of action has potential benefit in different indications including in the relapsed setting, when disease has progressed after chemotherapies and other targeted therapies, and additionally may be able to be combined with standard of care. Additional ETBs are being developed that target other cell surface receptors for solid and hematological malignancies, including SLAMF7 (CS1). SLAMF7 is a clinically validated target of monoclonal antibody therapy. Although clinical responses to the monotherapy were not observed, patients treated with combination therapy using standard of care agents that are known to stimulate immune effector cell activity have improved responses. Thus, a SLAMF7 targeted agent that can act independent of effector cells by directly killing tumor cells has the potential to provide broader benefit to patients. ETBs targeting SLAMF7 harbor the catalytic activity of the SLTA subunit and can effectively and specifically kill multiple myeloma cell lines expressing the target protein in vitro. The coupling of specific targeting to myeloma cells with potent and direct cell kill activity has the potential to deplete malignant cells without reliance on effector functions of the patient immune system, such as antibody or complement dependent cellular cytotoxicity or phagocytosis, and could be beneficial when disease progresses post antibody therapy. Additionally, the novel mechanism of action of an ETB also has potential to be combined with standard of care therapies and/ or to be effective in the relapsed or refractory setting. Citation Format: Aimee Iberg, Garrett L. Cornelison, Caleigh Howard, Garrett L. Robinson, Jay Zhao, Hilario J. Ramos, Erin K. Willert. Novel engineered toxin bodies targeting SLAMF7 (CS1) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 539.

MTOR sustains inflammatory response in celiac disease

Celiac disease (CD) is an enteropathy triggered by the ingestion of gluten proteins in genetically predisposed individuals and characterized by excessive activation of effector immune cells and enhanced production of inflammatory cytokines. However, factors/mechanisms that amplify the ongoing mucosal inflammation in CD are not fully understood. In this study, we assessed whether mammalian target of Rapamycin (mTOR), a pathway that combines intra- and extra-cellular signals and acts as a central regulator for the metabolism, growth, and function of immune and non-immune cells, sustains CD-associated immune response. Our findings indicate that expression of phosphorylated (p)/active form of mTOR is increased in protein lysates of duodenal biopsy samples taken from patients with active CD (ACD) as compared to normal controls. In ACD, activation of mTOR occurs mainly in the epithelial compartment and associates with enhanced expression of p-4EBP, a downstream target of mTOR complex (mTORC)1, while expression of p-Rictor, a component of mTORC2, is not increased. Stimulation of mucosal explants of inactive CD patients with pepsin-trypsin-digested (PT)-gliadin or IFN-γ/IL-21, two cytokines produced in CD by gluten-specific T cells, increases p-4EBP expression. Consistently, blockade of such cytokines in cultures of ACD mucosal explants reduces p-4EBP. Finally, we show that inhibition of mTORC1 with rapamycin in ACD mucosal explants reduces p-4EBP and production of IL-15, a master cytokine produced by epithelial cells in this disorder. Our data suggest that ACD inflammation is marked by activation of mTORC1 in the epithelial compartment.

Immunomagnetic separation in LAK generation in patients with breast cancer.

e15220 Background: The purpose of the study was to evaluate the effect of IL-2 and INF-γ on the proliferation and phenotype of lymphocytes in patients with breast cancer (BC) after T-regs removal from the pool in vitro. Methods: Lymphocytes were isolated from the blood of 11 patients with stage II BC, T-regs were removed by immunomagnetic separation, and samples were cultured for 6 days with cytokines: IL-2 - 0.1/1 μg, INF-γ - 10 IU and their combinations. Results: The level of NK cells in the samples with IL-2 was maintained throughout the experiment, with IFN-γ - gradually decreased, and in the control (samples without stimulation) it decreased sharply. In experimental samples (IL-2, IL-2+IFN-γ), the amount of NKT remained at the initial level by day 6, and in the control it decreased by 2 times (p < 0.05). The number of CD335+ NK cells with high cytotoxicity against target cells increased after 3 days in the test samples with IL-2 by 5.2 times, and after 6 days - by 11.6 times in comparison with the control (p < 0.05). On day 6, the level of double-positive T cells increased by 1.8 times in co-culturing with IL-2+INF-γ (1 μg/10 IU), (p < 0.05). The percentage of double-negative T cells increased in the dynamics of cultivation from days 1 to 6, in some cases statistically significantly (IL-2 - 1 μg: by 2 times; IL-2+INF-γ - 0.1 μg/10 IU: by 1.8 times; p < 0.05). The percentage of CD4+CD38+ cells on day 6 of incubation with IL-2 at both doses increased by 1.8-2 times compared with the control, and CD8+CD38+ by 4-6 times. The percentage of CD4+HLA-DR+ cells after 6 days of cultivation with IL-2 increased by 6 times, and CD8+HLA-DR+ by 8-9 times. Cultivation with INF-γ reduced the stimulating effect, and in some cases it did not appear. The combined effect of IL-2 and INF-γ on day 6 led to an increase in the proportion of CD4+CD25+CD127dim cells by 1.8 times in comparison with the control (p < 0.05), and when exposed to INF-γ only, the number of these cells remained at the control levels. Conclusions: INF-γ is not an effective inducer of activation of immune effector cells, while the effect of IL-2, both alone and in combination with INF-γ, leads to a significant increase in the proportion of cytotoxic cells, as well as to activation of the T cell unit, including CD4 + CD25 + CD127dim.

Intratumoral use of self-delivering RNAi to reprogram the tumor microenvironment and boost the antitumor response.

e15206 Background: Immunotherapy targeting immune checkpoints has shown significant benefit in many cancers. However, systemic delivery can result in severe auto-immune toxicities, the risk increasing with the combination of checkpoint inhibiting antibodies. Local delivery of immunotherapies allows for multiple combination therapies, while potentially preventing significant systemic toxicities. One approach is to use self-delivering RNAi technology, INTASYL™, which has been shown to be safe and effective in clinical applications following local administration. Here we show the use of the INTASYL platform as a checkpoint inhibiting immunotherapy for intratumoral applications, through a series of preclinical in vivo studies. Methods: INTASYL compounds were made against mouse PD-1, TIGIT, PD-L1 and BRD4. INTASYL comprises an asymmetric duplex, a small duplex region (≤ 15 base pairs), a single-stranded phosphorothioate tail and chemical modifications to confer stability resulting in efficient cellular uptake allowing in situ delivery. The efficacy and immunomodulation of these INTASYL compounds was evaluated in the Hepa1-6 and CT26 murine syngeneic tumor models following intratumoral administration. Tumor volumes were recorded throughout the study. Tumor-Infiltrating-Lymphocytes (TILs) were isolated twenty-four hours post the last dose and examined for gene silencing by RT-qPCR. Tumor microenvironment (TME) and T cell activation were analyzed by flow cytometry. Results: Results showed dose-dependent attenuated tumor growth compared to control groups. Relevant changes in the TME included an increase of CD8+ T cells and an increase of activation markers on TILs. Tumor growth reduction correlated with silencing of target mRNA and immune effector cell levels and activation. Conclusions: These studies demonstrate the feasibility of using INTASYL compounds intratumorally to overcome the immunosuppressive TME, resulting in changes in T cell composition and activation, and inhibition of tumor growth. Together, these novel findings support using intratumoral INTASYL as a viable approach to immunotherapy and warrants further investigation in patients.

DKK2 blockage-mediated immunotherapy enhances anti-angiogenic therapy of Kras mutated colorectal cancer

There are limited options for targeted therapies for colorectal cancer (CRC). Anti-EGFR therapy is limited to CRC without KRAS mutations. Even worse, most of CRC are refractory to currently immune checkpoint blockade. DKK2, which is upregulated in CRC, was recently found to suppress host immune responses, and its blockage effectively impeded tumor progression in benign genetic CRC models in our previous study. Here, our recent study demonstrated that in human CRC tumor samples expressing high levels of DKK2, DKK2 blockade caused stronger activation of tumor infiltrating CD8+ T cells in ex vivo culture. Intriguingly, we observed a correlation of high DKK2 expression with increased lymph node metastasis prevalence in these CRC patients as well. Furthermore, in a mouse genetic CRC model with mutations in APC and KRAS, which more closely mimics advanced human CRC, we confirmed the tumor inhibitory effect of DKK2 blockade, which significantly retarded tumor progression and extended survival, with increased immune effector cell activation and reduced angiogenesis. Based on this, we performed a combined administration of DKK2 blockade with sub-optimal anti-VEGFR treatment and observed a synergetic effect on suppressing tumor angiogenesis and progression, as well as extending survival, better than those of every single therapy. Thus, this study provides further evidence for the potential therapeutic application of DKK2 blockade in the clinical treatment of human CRC.

Platelets are critical for survival and tissue integrity during murine pulmonary Aspergillus fumigatus infection.

Beyond their canonical roles in hemostasis and thrombosis, platelets function in the innate immune response by interacting directly with pathogens and by regulating the recruitment and activation of immune effector cells. Thrombocytopenia often coincides with neutropenia in patients with hematologic malignancies and in allogeneic hematopoietic cell transplant recipients, patient groups at high risk for invasive fungal infections. While neutropenia is well established as a major clinical risk factor for invasive fungal infections, the role of platelets in host defense against human fungal pathogens remains understudied. Here, we examined the role of platelets in murine Aspergillus fumigatus infection using two complementary approaches to induce thrombocytopenia without concurrent neutropenia. Thrombocytopenic mice were highly susceptible to A. fumigatus challenge and rapidly succumbed to infection. Although platelets regulated early conidial phagocytosis by neutrophils in a spleen tyrosine kinase (Syk)-dependent manner, platelet-regulated conidial phagocytosis was dispensable for host survival. Instead, our data indicated that platelets primarily function to maintain hemostasis and lung integrity in response to exposed fungal antigens, since thrombocytopenic mice exhibited severe hemorrhage into the airways in response to fungal challenge in the absence of overt angioinvasion. Challenge with swollen, heat-killed, conidia was lethal in thrombocytopenic hosts and could be reversed by platelet transfusion, consistent with the model that fungus-induced inflammation in platelet-depleted mice was sufficient to induce lethal hemorrhage. These data provide new insights into the role of platelets in the anti-Aspergillus host response and expand their role to host defense against filamentous molds.

New Insights about Regulatory T Cells Distribution and Function with Exercise: The Role of Immunometabolism.

A lack of physical activity is linked to the development of many chronic diseases through a chronic low-grade inflammation state. It is now well accepted that the immune system plays a central role in the development of several chronic diseases, including insulin resistance, type 2 diabetes, atherosclerosis, heart failure and certain types of cancer. Exercise elicits a strong anti-inflammatory response independently of weight loss and can be a useful non-pharmacologic strategy to counteract the low-grade inflammation. The CD4+CD25+CD127- FoxP3+ Regulatory T (Treg) cells are a unique subset of helper T-cells, which regulate immune response and establish self-tolerance through the secretion of immunoregulatory cytokines, such as IL-10 and TGF-β, and the suppression of the function and activity of many immune effector cells (including monocytes/macrophages, dendritic cells, CD4+ and CD8+ T cells, and Natural Killers). The metabolic phenotype of Tregs are regulated by the transcription factor Foxp3, providing flexibility in fuel choice, but a preference for higher fatty acid oxidation. In this review, we focus on the mechanisms by which exercise - both acute and chronic - exerts its antiinflammatory effects through Treg cells mobilization. Furthermore, we discuss the implications of immunometabolic changes during exercise for the modulation of Treg phenotype and its immunosuppressive function. This narrative review focuses on the current knowledge regarding the role of Treg cells in the context of acute and chronic exercise using data from observational and experimental studies. Emerging evidence suggests that the immunomodulatory effects of exercise are mediated by the ability of exercise to adjust and improve Tregs number and function.

Emerging Potential of Exosomes in Regenerative Medicine for Temporomandibular Joint Osteoarthritis.

Exosomes are nanosized vesicles (30–140 nm) of endocytic origin that play important roles in regenerative medicine. They are derived from cell membranes during endocytic internalization and stabilize in biological fluids such as blood and synovia. Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease, which, in addition to chronic pain, is characterized by progressive cartilage breakdown, condylar bone remodeling, and synovitis. However, traditional clinical treatments have limited symptom- and structure-modifying effects to restore damaged cartilage and other TMJ tissues. This is due to the limited self-healing capacity of condylar cartilage. Recently, stem-cell-derived exosomes have been studied as an alternative therapeutic approach to tissue repair and regeneration. It is known that trophic regulation of mesenchymal stem cells (MSCs) has anti-inflammatory and immunomodulatory effects under pathological conditions, and research on MSC-derived exosomes is rapidly accumulating. MSC-derived exosomes mimic the major therapeutic effects of MSCs. They affect the activity of immune effector cells and possess multilineage differentiation potential, including chondrogenic and osteogenic differentiation. Furthermore, exosomes are capable of regenerating cartilage or osseous compartments and restoring injured tissues and can treat dysfunction and pain caused by TMJ OA. In this review, we looked at the uniqueness of TMJ, the pathogenesis of TMJ OA, and the potential role of MSC-derived exosomes for TMJ cartilage and bone regeneration.

BI-1206, a Monoclonal Antibody Against Fcγriib, Showed Superior Anti-Tumor Activity in an Ibrutinib-Venetoclax Dual Resistant PDX Model in Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) is a rare but aggressive non-Hodgkin lymphoma (NHL). CD20 antibodies (e.g. rituximab), BTK inhibitors (e.g. ibrutinib and acalabrutinib), and BCL-2 inhibitors (e.g. venetoclax) alone or in combination have shown great anti-MCL efficacy. However, primary and acquired resistance to one or multiple therapies commonly occurs, resulting in poor clinical outcome. Overcoming such mechanisms of resistance holds promise to significantly improve survival, meeting a significant medical need for patients with refractory/relapsed MCL. Recent studies showed Fc gamma receptors (FcγRs) play important roles in controlling therapeutic efficacy. FcgRIIB (CD32B), the inhibitory FcγR, negatively controls antibody efficacy through distinct inhibitory mechanisms in immune effector cells and lymphoma cells, respectively. When expressed on leukemic or lymphoma cells, FcgRIIB promotes rituximab internalization and removal from the tumor cell surface, resulting in reduced immune effector cell activation and ultimately decreased in vivo therapeutic efficacy. We recently developed antagonistic antibodies to FcgRIIB and demonstrated that these antibodies blocked rituximab internalization and helped prevent and overcome rituximab resistance in a PDX model of CLL. In this study, we investigated the expression of FcgRIIB in MCL cell lines and primary patient MCL samples, and we assessed the in vivo efficacy of BI-1206, a monoclonal antibody against FcgRIIB, in MCL PDX models. Methods: Flow cytometry analysis was performed to examine the cell surface expression of FcgRIIB in MCL cell lines (n=8) and primary patient MCL samples (n=27). An orthotopic patient-derived xenograft (PDX) model was established from a MCL patient with dual resistance to ibrutinib-venetoclax. In the first mouse cohort, single-agent ibrutinib, venetoclax, or vehicle control were administrated in mice carrying the orthotopic PDX model to assess their in vivo efficacies. In the second mouse cohort, mice were treated with vehicle, single agent BI1206, rituximab plus lenalidomide, or a combination of BI-1206, rituximab, and lenalidomide (triple combination) to assess their in vivo efficacies in the same PDX model. Results: Flow cytometry analysis showed that all 8 MCL cell lines and all 27 primary patient MCL samples expressed high levels of FcgRIIB, highlighting the potential importance of FcgRIIB on the control of therapeutic efficacy in MCL. In the first mouse cohort, we validated the ibrutinib and venetoclax resistance in the PDX model established from a MCL patient with resistance to both therapies. In the second mouse cohort, single agent BI-1206 (10 mg/kg, twice a week) potently diminished PDX growth in spleen, liver, bone marrow, and peripheral blood (Figure 1). Treatment with rituximab (10 mg/kg, twice per week) plus lenalidomide (50mg/kg, daily) or the triple combination showed similar potency (Figure 1). To investigate whether BI-1206 mediates boosting of rituximab-based targeted drug therapies, and/or overcoming of resistance to such therapies, a follow-up experiment with revised treatment setting using the same PDX model or an alternative CD20/FcγRIIb co-expressing PDX model is currently under investigation. Conclusions: All MCL cell lines and all primary MCL cells tested highly express FcγRIIb on the tumor cell surface. Single agent BI-1206 has potent anti-MCL activity in the FcγRIIb-expressing MCL PDX model to overcome ibrutinib-venetoclax dual resistance. Our data suggests FcγRIIb may be an important target for anti-MCL therapies. Disclosures Wang: Guidepoint Global: Consultancy; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; BioInvent: Consultancy, Research Funding; Aviara: Research Funding; Juno Therapeutics: Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding; Dava Oncology: Honoraria.

Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells.

Mesenchymal stem cells (MSC) have been shown to improve wound healing and suppress inflammatory immune responses. Newer research also indicates that MSC exhibit antimicrobial activity, although the mechanisms underlying this activity have not been fully elucidated. Therefore, we conducted in vitro and in vivo studies to examine the ability of resting and activated MSC to kill bacteria, including multidrug resistant strains. We investigated direct bacterial killing mechanisms and the interaction of MSC with host innate immune responses to infection. In addition, the activity of MSC against chronic bacterial infections was investigated in a mouse biofilm infection model. We found that MSC exhibited high levels of spontaneous direct bactericidal activity in vitro. Moreover, soluble factors secreted by MSC inhibited Staphylococcus aureus biofilm formation in vitro and disrupted the growth of established biofilms. Secreted factors from MSC also elicited synergistic killing of drug‐resistant bacteria when combined with several major classes of antibiotics. Other studies demonstrated interactions of activated MSC with host innate immune responses, including triggering of neutrophil extracellular trap formation and increased phagocytosis of bacteria. Finally, activated MSC administered systemically to mice with established S. aureus biofilm infections significantly reduced bacterial numbers at the wound site and improved wound healing when combined with antibiotic therapy. These results indicate that MSC generate multiple direct and indirect, immunologically mediated antimicrobial activities that combine to help eliminate chronic bacterial infections when the cells are administered therapeutically.

New Insights into Immunotherapy Strategies for Treating Autoimmune Diabetes.

Type 1 diabetes mellitus (T1D) is an autoimmune illness that affects millions of patients worldwide. The main characteristic of this disease is the destruction of pancreatic insulin-producing beta cells that occurs due to the aberrant activation of different immune effector cells. Currently, T1D is treated by lifelong administration of novel versions of insulin that have been developed recently; however, new approaches that could address the underlying mechanisms responsible for beta cell destruction have been extensively investigated. The strategies based on immunotherapies have recently been incorporated into a panel of existing treatments for T1D, in order to block T-cell responses against beta cell antigens that are very common during the onset and development of T1D. However, a complete preservation of beta cell mass as well as insulin independency is still elusive. As a result, there is no existing T1D targeted immunotherapy able to replace standard insulin administration. Presently, a number of novel therapy strategies are pursuing the goals of beta cell protection and normoglycemia. In the present review we explore the current state of immunotherapy in T1D by highlighting the most important studies in this field, and envision novel strategies that could be used to treat T1D in the future.

FcγRIIIa chromatography to enrich a-fucosylated glycoforms and assess the potency of glycoengineered therapeutic antibodies

Therapeutic antibodies can elicit an immune response through different mechanisms, either cell independent via complement activation (CDC) or through activation of immune-effector cells (such as macrophages and NK cells). After target binding, the Fc part of the antibody will interact with Fc receptors on the surface of effector cells, leading to activation and lysis of the target cells by a mechanism called antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC of an antibody can be increased by modifying the carbohydrates on the Fc part. If the fucose on the first N-acetylglucosamine is absent, the affinity for the FcγRIIIa is increased and the ADCC enhanced. We describe the development of a chromatography method that is based on the differential affinity of the Fc receptor FcγRIIIa (high affinity V158 variant) for fucosylated and a-fucosylated antibodies. Immobilized FcγRIIIa can be used for the separation of immunoglobulins carrying these glycosylation variants for both, analytical and preparative purposes. The biological activity and fucose content of three pools enriched for fully fucosylated, mono-fucosylated or a-fucosylated carbohydrates could be characterized. Mono-fucosylated and a-fucosylated immunoglobulins have the same enhanced biological activity compared to fully fucosylated IgGs. A direct, label- and modification-free analytical method for screening of IgGs from culture supernatant was developed and was amenable to high-throughput screening. Clones producing antibodies with a high content of a-fucosylated oligosaccharides could be successfully selected.