Search

CD39 (ENTPD1) is a key enzyme responsible for degradation of extracellular ATP and is upregulated in the tumor microenvironment (TME). Extracellular ATP accumulates in the TME from tissue damage and immunogenic cell death, potentially initiating proinflammatory responses that are reduced by the enzymatic activity of CD39. Degradation of ATP by CD39 and other ectonucleotidases (e.g., CD73) results in extracellular adenosine accumulation, constituting an important mechanism for tumor immune escape, angiogenesis induction, and metastasis. Thus, inhibiting CD39 enzymatic activity can inhibit tumor growth by converting a suppressive TME to a proinflammatory environment. SRF617 is an investigational, anti-CD39, fully human IgG4 Ab that binds to human CD39 with nanomolar affinity and potently inhibits its ATPase activity. In vitro functional assays using primary human immune cells demonstrate that inhibiting CD39 enhances T-cell proliferation, dendritic cell maturation/activation, and release of IL-1β and IL-18 from macrophages. In vivo, SRF617 has significant single-agent antitumor activity in human cell line–derived xenograft models that express CD39. Pharmacodynamic studies demonstrate that target engagement of CD39 by SRF617 in the TME inhibits ATPase activity, inducing proinflammatory mechanistic changes in tumor-infiltrating leukocytes. Syngeneic tumor studies using human CD39 knock-in mice show that SRF617 can modulate CD39 levels on immune cells in vivo and can penetrate the TME of an orthotopic tumor, leading to increased CD8+ T-cell infiltration. Targeting CD39 is an attractive approach for treating cancer, and, as such, the properties of SRF617 make it an excellent drug development candidate.

FOXP3+ regulatory T (Treg) cells play a crucial role in orchestrating immune responses across several tissues, including the tumor microenvironment (TME). The dominant immune regulatory activity of Treg cells is evidenced, both clinically and preclinically, by the profound inflammatory dysregulation that arises in their absence. Leveraging controlled depletion of Treg cells in tumor tissue while sparing Treg cells in healthy tissue may be achieved through antibody-mediated targeting of the C-C chemokine receptor 8 (CCR8). CCR8 is a seven transmembrane G-coupled chemokine receptor protein whose expression is predominantly upregulated on Treg cells in several types of tumors compared with peripheral blood. Importantly, depletion of CCR8-expressing Treg cells in tumors provides a novel pathway to immunotherapy that is mechanistically independent of checkpoint inhibition (CPI). Using single-cell transcriptome and flow cytometry analyses, we evaluated the pharmacodynamic changes after treatment with anti-CCR8 antibodies to elucidate the effect of tumor Treg cell-targeting therapies on the downstream mechanisms of antitumor immunity within the TME. SRF114, a highly selective human anti-CCR8 afucosylated antibody, and an anti-mouse CCR8 antibody were utilized to preferentially deplete CCR8+ Treg cells in human CCR8 knock-in (hCCR8KI) or wild-type mice, respectively. Treatment with SRF114 or anti-mouse CCR8 antibody resulted in robust monotherapy activity in models susceptible to CPI. Moreover, pronounced monotherapy activity was also seen in checkpoint-resistant model(s), highlighting a mechanistically CPI-independent role for intratumoral Treg cell depletion to elicit potent antitumor activity. Treatment of tumor-bearing mice with anti-CCR8 antibodies resulted in a significant reduction of tumor-associated Treg cells, while peripheral Treg cells remained unchanged. Analysis of bulk tumor lysates showed that anti-CCR8 therapy elevated levels of pro-inflammatory cytokines and pro-angiogenic and chemoattracting factors. Evaluation of effector T cells demonstrated that anti-CCR8 therapy drives proliferation of CD8+ T cells and an increase in intracellular cytokine production of IFNγ, TNFα, and granzyme A. Additionally, the tumor myeloid compartment was strongly affected by anti-CCR8 therapy. Multiple myeloid cell subsets had increased levels of co-stimulatory molecules and expression of PD-L1. Altogether, selective depletion of intratumoral CCR8+ Treg cells results in robust antitumor activity by reshaping the TME toward a more pro-inflammatory milieu, providing an orthogonal approach to CPI for eliciting antitumor immunity and a strong rationale for evaluating SRF114 as a therapeutic agent for the treatment of cancer. SRF114 is currently in Phase 1 clinical studies. Citation Format: Marisella Panduro, Yue Ren, Ricard Masia, Yu Yang, Andrew C. Lake, Vito J. Palombella, Jonathan A. Hill, James F. Mohan. Depletion of CCR8+ tumor Treg cells with SRF114 or anti-CCR8 therapy promotes robust antitumor activity and reshapes the tumor microenvironment toward a more pro-inflammatory milieu. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5125.

SUMMARY Coordinate control of T cell proliferation, survival, and differentiation are essential for host protection from pathogens and cancer. Long-lived memory cells, whose precursors are formed during the initial immunological insult, provide protection from future encounters, and their generation is the goal of many vaccination strategies. microRNAs (miRNAs) are key nodes in regulatory networks that shape effective T cell responses through the fine-tuning of thousands of genes. Here, using compound conditional mutant mice to eliminate miR-15/16 family miRNAs in T cells, we show that miR-15/16 restrict T cell cycle, survival, and memory T cell differentiation. High throughput sequencing of RNA isolated by cross-linking immuno-precipitation of AGO2 combined with gene expression analysis in miR-15/16-deficient T cells indicates that these effects are mediated through the direct inhibition of an extensive network of target genes within pathways critical to cell cycle, survival, and memory., In Brief Coordinate control of T cell proliferation, survival, and differentiation are essential for effective cell-mediated adaptive immunity. Gagnon et al. define roles for the miR-15/16 family of microRNAs in restricting T cell cycle and long-lived memory T cell accumulation through the direct inhibition of a very large network of target mRNAs., Graphical Abstract

Summary: The phosphatidylinositol 3-kinase (PI3K) signaling cascade downstream of the B cell receptor (BCR) signalosome is essential for B cell maturation. Proper signaling strength is maintained through the PI3K negative regulator phosphatase and tensin homolog (PTEN). Although a role for microRNA (miRNA)-dependent control of the PTEN-PI3K axis has been described, the contribution of individual miRNAs to the regulation of this crucial signaling modality in mature B lymphocytes remains to be elucidated. Our analyses reveal that ablation of miR-29 specifically in B lymphocytes results in an increase in PTEN expression and dampening of the PI3K pathway in mature B cells. This dysregulation has a profound impact on the survival of B lymphocytes and results in increased class switch recombination and decreased plasma cell differentiation. Furthermore, we demonstrate that ablation of one copy of Pten is sufficient to ameliorate the phenotypes associated with miR-29 loss. Our data suggest a critical role for the miR-29-PTEN-PI3K regulatory axis in mature B lymphocytes.

BackgroundThe purine nucleotide adenosine plays an important role in dampening both the innate and adaptive arms of the immune system. In contrast, extracellular adenosine triphosphate (ATP), which can be generated at high levels due to cell stress, initiates proinflammatory responses. Extracellular ATP can be produced in cancerous tumors, and its conversion to adenosine monophosphate by CD39 (ENTPD1) and subsequent degradation to free adenosine by CD73 (NT5E) in the tumor microenvironment (TME) is a major contributor to tumor immune evasion. Accordingly, CD39 serves as an important immune regulator. CD39 is highly expressed in the TME of various tumor types, including lung, prostate, colon, ovarian, and pancreatic cancers, making CD39 an attractive target for immune-based anticancer therapy.SRF617 is a fully human anti-CD39 antibody designed to target human CD39 and inhibit its enzymatic activity. SRF617 provides a dual mechanism of immune activation by increasing inflammatory responses through accumulation of free ATP and alleviating immune suppression by reducing adenosine production. In this way, SRF617 shifts the TME to a proinflammatory state leading to reactivation of the immune system and antitumor function.MethodsA pharmacokinetic/pharmacodynamic profile was determined in CT26 tumor-bearing human CD39 knock-in (hCD39 KI) mice by measuring plasma SRF617 concentration, peripheral blood target occupancy (TO), and ATP hydrolysis activity in frozen tissue sections. Wild-type mice bearing subcutaneous (s.c.) KPC1245 tumors were treated with anti-murine CD39 antibody, alone or in combination with gemcitabine.ResultsCirculating SRF617 was observed in the plasma after intravenous injection of CT26 tumor-bearing hCD39 KI mice, which correlated with CD39-bound SRF617 on B cells. The presence of SRF617 correlated with a decrease in ATPase activity and CD39 expression in tissues. In the KPC tumor model, a combination effect was observed for anti-CD39 treatment with gemcitabine. Treatment with anti-CD39 antibody resulted in an increase in CD8+ T cells in the TME as measured by flow cytometry.ConclusionsSRF617 treatment of hCD39 KI mice led to reduction of ATPase activity in the TME that correlated with peripheral TO and plasma concentration. Anti-CD39 treatment alone or in combination with gemcitabine led to tumor growth inhibition and increased tumor-infiltrating lymphocytes as measured by flow cytometry in s.c. KPC tumors. These studies demonstrate the therapeutic potential of targeting CD39 for the treatment of cancer. SRF617 is currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04336098) and in combination with chemotherapy in patients with pancreatic cancer.

CD112R (PVRIG) is a recently identified inhibitory receptor of the PVR gene family, which includes TIGIT, CD96, and CD226, that is expressed on NK cells and T cells. CD112R suppresses immune cell activation through its association with the cell adhesion molecule CD112 (PVRL2), a ligand that it competes for with the activating receptor CD226. CD112 binding to CD112R induces downstream signaling via recruitment of SHP-1, SHP-2, and SHIP-1 phosphatases to the ITIM in the cytoplasmic tail, resulting in dampened effector cell activation. As CD112 is commonly upregulated on cancer cells and is also expressed on tumor infiltrating myeloid cells, it was hypothesized that disruption of the CD112:CD112R interaction would promote anti-tumor immune responses. Phenotypic screening of a panel of antibodies generated against CD112R led to the identification of SRF813, a high affinity, fully human CD112R antibody that blocks the interaction of CD112R with CD112. To explore the therapeutic potential of CD112R blockade, the ability of SRF813 to increase immune cell activation was evaluated in human peripheral blood mononuclear cells. In vitro assays showed that SRF813 treatment enhanced NK cell activation in response to multiple tumor cell lines. This activation was characterized by upregulation of several cell surface markers, including 4-1BB and ICAM-1, in addition to increased cytokine production and degranulation. The activity of SRF813 was highly dependent on the antibody isotype and Fc receptor engagement. Mechanistically, the enhanced immunologic responses observed in NK cells treated with SRF813 could be reversed in the presence of a CD226 blocking antibody, confirming the role of CD112R in regulating CD226 mediated signaling. Blockade of CD112R in mouse syngeneic tumor models using a murine surrogate of SRF813 demonstrated reduced tumor growth with an accompanying increase in TIL activation following treatment. In the CT-26 tumor model, anti-CD112R treatment resulted in complete tumor regression in a subset of treated mice. These mice rapidly rejected tumors upon re-challenge, signifying the development of an immunologic anti-tumor memory response in these animals. Additional depletion studies highlighted that the anti-tumor efficacy was dependent on both CD8 T cells and NK cells. Moreover, the combination of anti-CD112R with PD-1 blockade led to greater inhibition of tumor growth than either treatment alone. Collectively, these preclinical data demonstrated that CD112R is a negative regulator of immune responses and that CD112R blockade can potentiate anti-tumor responses in cancers that express CD112. Citation Format: Marisella Panduro, Roy M. Dornbrook, Kshama A. Doshi, Jing Hua, Jamie Strand, Vito J. Palombella, Pamela M. Holland, Jonathan A. Hill, James F. Mohan. SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity [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 4548.

Normal repair of skeletal muscle requires local expansion of a special population of Foxp3(+)CD4(+) regulatory T (Treg) cells. Such cells failed to accumulate in acutely injured muscle of old mice, known to undergo ineffectual repair. This defect reflected reduced recruitment of Treg cells to injured muscle, as well as less proliferation and retention therein. Interleukin-33 (IL-33) regulated muscle Treg cell homeostasis in young mice, and its administration to old mice ameliorated their deficits in Treg cell accumulation and muscle regeneration. The major IL-33-expressing cells in skeletal muscle displayed a constellation of markers diagnostic of fibro/adipogenic progenitor cells and were often associated with neural structures, including nerve fibers, nerve bundles, and muscle spindles, which are stretch-sensitive mechanoreceptors important for proprioception. IL-33(+) cells were more frequent after muscle injury and were reduced in old mice. IL-33 is well situated to relay signals between the nervous and immune systems within the muscle context.

Skeletal muscle relies on its regenerative capacity to recover after acute injury. Immune-system cells, notably macrophages and regulatory T cells, play critical roles during muscle regeneration. This study addressed the impact of regulatory T cells on macrophages during muscle repair. In a mouse model of acute injury, regulatory T cells controlled the composition and phenotype of muscle macrophages during muscle repair by limiting production of the inflammatory cytokine, interferon-γ, produced by natural killer and effector T cells. Thus, we uncovered an interferon-γ–centered regulatory loop that can be further explored as a gateway to improved muscle therapies.

Skeletal muscle regeneration is a highly orchestrated process that depends on multiple immune-system cell types, notably macrophages (MFs) and Foxp3+CD4+ regulatory T (Treg) cells. This study addressed how Treg cells rein in MFs during regeneration of murine muscle after acute injury with cardiotoxin. We first delineated and characterized two subsets of MFs according to their expression of major histocompatibility complex class II (MHCII) molecules, i.e., their ability to present antigens. Then, we assessed the impact of Treg cells on these MF subsets by punctually depleting Foxp3+ cells during the regenerative process. Treg cells controlled both the accumulation and phenotype of the two types of MFs. Their absence after injury promoted IFN-γ production, primarily by NK and effector T cells, which ultimately resulted in MF dysregulation and increased inflammation and fibrosis, pointing to compromised muscle repair. Thus, we uncovered an IFN-γ-centered regulatory layer by which Treg cells keep MFs in check and dampen inflammation during regeneration of skeletal muscle.

Coordinate control of T cell proliferation, survival, and differentiation are essential for host protection from pathogens and cancer. Long-lived memory cells, whose precursors are formed during the initial immunological insult, provide protection from future encounters and their generation is the goal of many vaccination strategies. microRNAs are key nodes in regulatory networks that shape effective T cell responses through the fine-tuning of thousands of genes. Here, using new compound conditional mutant mice to eliminate miR-15/16 family miRNAs in T cells, we show that miR-15/16 restrict T cell cycle, survival, and memory T cell differentiation. High throughput sequencing of RNA isolated by cross-linking immunoprecipitation of AGO2 combined with gene expression analysis in miR-15/16 deficient T cells indicate that these effects are mediated through the direct inhibition of an extensive network of known and novel target genes within pathways critical to cell cycle, survival, and memory. This study highlights the important role that miRNAs play in shaping the global gene expression program that is required for T cell cycle, survival, and the proper formation of memory cells.

MicroRNAs (miRNAs) exert powerful effects on immunological function by tuning networks of target genes that orchestrate cell activity. We sought to identify miRNAs and miRNA-regulated pathways that control the type 2 helper T cell (TH2 cell) responses that drive pathogenic inflammation in asthma. Profiling miRNA expression in human airway-infiltrating T cells revealed elevated expression of the miRNA miR-19a in asthma. Modulating miR-19 activity altered TH2 cytokine production in both human and mouse T cells, and TH2 cell responses were markedly impaired in cells lacking the entire miR-17∼92 cluster. miR-19 promoted TH2 cytokine production and amplified inflammatory signaling by direct targeting of the inositol phosphatase PTEN, the signaling inhibitor SOCS1 and the deubiquitinase A20. Thus, upregulation of miR-19a in asthma may be an indicator and a cause of increased TH2 cytokine production in the airways.

The immune system is responsible for defending an organism against the myriad of microbial invaders it constantly confronts. It has become increasingly clear that the immune system has a second major function: the maintenance of organismal homeostasis. Foxp3+CD4+ regulatory T cells (Tregs) are important contributors to both of these critical activities, defense being the primary purview of Tregs circulating through lymphoid organs, and homeostasis ensured mainly by their counterparts residing in parenchymal tissues. This review focuses on so-called tissue Tregs. We first survey existing information on the phenotype, function, sustaining factors, and human equivalents of the three best-characterized tissue-Treg populations—those operating in visceral adipose tissue, skeletal muscle, and the colonic lamina propria. We then attempt to distill general principles from this body of work—as concerns the provenance, local adaptation, molecular sustenance, and targets of action of tissue Tregs, in particular.

SummaryFollicular helper T cells (Tfh cells) are the major producers of interleukin-4 (IL-4) in secondary lymphoid organs where humoral immune responses develop. Il4 regulation in Tfh cells appears distinct from the classical T helper 2 (Th2) cell pathway, but the underlying molecular mechanisms remain largely unknown. We found that hypersensitivity site V (HS V; also known as CNS2), a 3′ enhancer in the Il4 locus, is essential for IL-4 production by Tfh cells. Mice lacking HS V display marked defects in type 2 humoral immune responses, as evidenced by abrogated IgE and sharply reduced IgG1 production in vivo. In contrast, effector Th2 cells that are involved in tissue responses were far less dependent on HS V. HS V facilitated removal of repressive chromatin marks during Th2 and Tfh cell differentiation and increased accessibility of the Il4 promoter. Thus, Tfh and Th2 cells utilize distinct but overlapping molecular mechanisms to regulate Il4, a finding with important implications for understanding the molecular basis of allergic diseases.

Antigen-mediated cross-linking of Immunoglobulin E (IgE) bound to mast cells/basophils via FcɛRI, the high affinity IgE Fc-receptor, is a well-known trigger of allergy. In humans, but not mice, dendritic cells (DCs) also express FcɛRI that is constitutively occupied with IgE. In contrast to mast cells/basophils, the consequences of IgE/FcɛRI signals for DC function remain poorly understood. We show that humanized mice that express FcɛRI on DCs carry IgE like non-allergic humans and do not develop spontaneous allergies. Antigen-specific IgE/FcɛRI cross-linking fails to induce maturation or production of inflammatory mediators in human DCs and FcɛRI-humanized DCs. Furthermore, conferring expression of FcɛRI to DCs decreases the severity of food allergy and asthma in disease-relevant models suggesting anti-inflammatory IgE/FcɛRI signals. Consistent with the improved clinical parameters in vivo, antigen-specific IgE/FcɛRI cross-linking on papain or lipopolysaccharide-stimulated DCs inhibits the production of pro-inflammatory cytokines and chemokines. Migration assays confirm that the IgE-dependent decrease in cytokine production results in diminished recruitment of mast cell progenitors; providing a mechanistic explanation for the reduced mast cell-dependent allergic phenotype observed in FcɛRI-humanized mice. Our study demonstrates a novel immune regulatory function of IgE and proposes that DC-intrinsic IgE signals serve as a feedback mechanism to restrain allergic tissue inflammation.

Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here, we show that mice deficient in Eri1, a conserved 3′-to- 5′ exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK-cell development and maturation. Eri1 -/- NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow (BM) and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse CMV (MCMV) infection. Ly49H + NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1 -/- T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1 -/- T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK-cell development and antiviral immunity. © 2012 by The American Society of Hematology.

Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here we show that mice deficient in Eri1, a conserved 3’-to-5’ exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK cell development and maturation. Eri1-/- NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Furthermore, Ly49H+ NK cells deficient in Eri1 failed to expand efficiently during mouse cytomegalovirus (MCMV) infection. Consequently, Eri1 was required for immune-mediated control of MCMV. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1-/- T cells. Thus mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK cell development and anti-viral immunity.