InVivoMAb mouse IgG2a isotype control, unknown specificity

The macrophage checkpoint receptor SIRPα signals against phagocytosis by binding CD47 expressed on all cells - including macrophages. Here, inhibiting cis interactions between SIRPα and CD47 on the same macrophage increases eating approximately the same as inhibiting trans interactions. Antibody blockade of CD47, as pursued in clinical trials against cancer, is applied separately to human-derived macrophages and to red blood cell (RBC) targets for phagocytosis, and both scenarios produce surprisingly similar increases in RBC engulfment. Blockade of both macrophages and targets results in hyper-phagocytosis, and knockdown of macrophage-CD47 likewise increases eating of 'foreign' cells and particles, decreases SIRPα's baseline inhibitory signaling, and linearly increases binding of soluble-CD47 in trans, consistent with cis-trans competition. Many cell types express both SIRPα and CD47, including mouse melanoma B16 cells, and CRISPR-mediated deletions modulate B16 phagocytosis consistent with cis-trans competition. Additionally, soluble SIRPα binding to human-CD47 displayed on Chinese hamster ovary (CHO) cells is suppressed by SIRPα co-display, and atomistic computations confirm SIRPα bends and binds CD47 in cis. Safety and efficacy profiles for CD47-SIRPα blockade might therefore reflect a disruption of both cis and trans interactions. © 2020. Published by The Company of Biologists Ltd.

Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ε chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. T-ALLs are aggressive malignant lymphoid proliferations of T-cell precursors characterized by high relapse rates and poor prognosis, calling for the search for novel therapeutic options. Here, we report that the lineage-specific TCR/CD3 developmental checkpoint controlling cell death in normal T-cell progenitors remains switchable to induce massive tumor cell apoptosis in T-ALL and is amenable to preclinical therapeutic intervention. Cancer Discov; 6(9); 972-85. ©2016 AACR.See related commentary by Lemonnier and Mak, p. 946This article is highlighted in the In This Issue feature, p. 932. ©2016 American Association for Cancer Research.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with limited effective treatment options. Emerging evidence links enriched environment (EE)-induced eustress to PDAC inhibition. However, the underlying mechanisms remain unclear. In this study, we explored the role of gut microbiota in PDAC-suppressive effects of EE. We demonstrated that depletion of gut microbiota with antibiotics abolished EE-induced tumor suppression, while fecal microbiota transplantation (FMT) from EE mice significantly inhibited tumor growth in both subcutaneous and orthotopic PDAC models housed in standard environment. 16S rRNA sequencing revealed that EE enhanced gut microbiota diversity and selectively enriched probiotic Lactobacillus, particularly L. reuteri. Treatment with L. reuteri significantly suppressed PDAC tumor growth and increased natural killer (NK) cell infiltration into the tumor microenvironment. Depletion of NK cells alleviated the anti-tumor effects of L. reuteri, underscoring the essential role of NK cell-mediated immunity in anti-tumor response. Clinical analysis of PDAC patients showed that higher fecal Lactobacillus abundance correlated with improved progression-free and overall survival, further supporting the therapeutic potential of L. reuteri in PDAC. Overall, this study identifies gut microbiota as a systemic regulator of PDAC under psychological stress. Supplementation of psychobiotic Lactobacillus may offer a novel therapeutic strategy for PDAC.

Metabolic reprogramming, particularly upregulated de novo pyrimidine biosynthesis, drives cancer progression and immune evasion. Dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, is a promising therapeutic target, but its inhibitors often face resistance in immune-refractory melanoma, linked to low basal stimulator of interferon genes (STING) expression.

The molecular and functional changes in endothelial cells during disease progression such as cancer have been noted but the mechanism of their activation is still under-studied. Previously we discovered that tumor-derived Oncostatin M induced tumor-associated vascular phenotypes, and the activated endothelial cells in turn promoted tumor progression and metastasis of clear-cell renal cell carcinoma (ccRCC). However, the mechanism of Oncostatin M action remains unknown. Here, we reveal that Oncostatin M signaling triggers specific epigenetic reprogramming of endothelial cells through upregulation of lysine acetyltransferase 6B, leading to increased histone 3 lysine 14 acetylation (H3K14ac) in vitro and in vivo. H3K14ac-modified chromatins upregulate specific gene sets associated with hypoxic response, hyper-angiogenesis, inflammation, and mesenchymal transition. Targeting H3K14ac in endothelial cells by interfering with acetyltransferase 6B function or neutralizing Oncostatin M ameliorates the premalignant hyperplastic phenotypes in the autochthonous ccRCC mouse model and diminishes tumor growth and metastasis in the ccRCC xenograft model.

Prevotella melaninogenica is enriched in the lungs of people with cystic fibrosis (pwCF), yet its functional impact on respiratory tract homeostasis remains incompletely understood. Prior studies identified immune modulatory effects following lung exposure to Prevotella, but the relevance of these findings for CF infections is unknown.

Chemokines and their receptors play a pivotal role in shaping the tumor microenvironment (TME) and modulating immune responses by orchestrating immune cell recruitment, spatial positioning, and facilitating cell-cell interactions. However, the exact mechanisms underlying chemokine signaling across different cell populations within the TME remain poorly understood. In this study, we utilized multiple-omics approaches to explore the relationship between CCR1+ macrophages, CD8+ exhausted T (Tex) cells, and immune checkpoint blockade (ICB) therapy response, as well as the role of chemokine signaling in the formation of CCR1+ macrophage and CD8+ Tex cell niches. We found that CCR1+ macrophages were closely associated with ICB outcomes in melanoma. Additionally, combination therapy with a CCR1 antagonist and anti-PD-1 monoclonal antibody significantly reduced tumor burden in melanoma mouse models, which was attributed to the substantial depletion of CD8+ Tex cells. Further, CCR1+ macrophages were found to co-localize with CD8+ Tex cells in human melanoma tissue, and the CCR1+ macrophage-CD8+ Tex cell niche was correlated with ICB treatment response in mice. Importantly, the CCR1-CCL3 axis was identified as a critical mediator in the formation of this niche. Overall, our study underscores the spatial relationship between CCR1+ macrophages and CD8+ Tex cells in ICB therapy, providing a promising strategy to overcome ICB resistance in melanoma.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation that leads to tissue damage and disability. RA affects approximately 0.5-1% of the global population and is driven by a complex interplay of genetic susceptibility, environmental factors, and immune dysregulation. While biologic and targeted synthetic DMARDs improved RA treatment, they have limitations in efficacy, safety, and accessibility. B-cell-targeting therapies, such as anti-CD20, have shown effectiveness, but only with broad immunosuppression, which can increase infection risk and compromise humoral immunity. Therefore, there is an unmet need for more selective therapeutic strategies that modulate pathogenic immune pathways while preserving protective immune functions. It has been suggested that targeting the BAFF pathway may offer a more favorable therapeutic approach compared to targeting CD20.

Immunotherapy combinations can improve patient outcomes, yet the interactions within the tumor microenvironment (TME) that drive therapeutic synergy are poorly understood. Tumor establishment drives monocyte recruitment and differentiation into tumor-associated macrophages (TAMs), which have essential roles in coordinating immune responses and are thus attractive targets for therapeutic modulation. In a murine model of combination anti-programmed cell death protein 1 (PD-1) and its ligand (anti-PD-L1) checkpoint blockade, tumor control was associated with increased infiltration of CD8+ T cells and M1-like repolarization of TAMs. Live-cell imaging of the tumor microenvironment revealed close contacts between tumor-infiltrating CD8+ T cells and TAMs, in which the extent of the contact interfaces increased with combination immunotherapy. Treatment with anti-PD-L1 was able to increase macrophage expression of pro-inflammatory factors and phagocytic activity, suggesting a role for TAMs in reactivating CD8+ T cells in the TME. However, co-treatment with anti-PD-1 was ultimately necessary for tumor control, indicating the need for combination targeting of the TME.

Influenza A viruses remain a global health threat, yet no universal antibody therapy exists. Clinical programs have centered on neutralizing mAbs, only to be thwarted by strain specificity and rapid viral escape. We instead engineered three non-neutralizing IgG2a mAbs that target distinct, overlapping epitopes within the conserved N terminus of the M2 ectodomain (M2e). Combined at low dose, this "triple M2e-mAb" confers robust prophylactic and therapeutic protection in mice challenged with diverse human and zoonotic IAV strains, including highly pathogenic variants. Therapeutic efficacy depends on Fc-mediated effector activity via FcγRI, FcγRIII, and FcγRIV, rather than in vitro neutralization. Serial passaging in triple M2e-mAb-treated immunocompetent and immunodeficient hosts failed to generate viral escape mutants. Our findings redefine the influenza-specific antibody therapeutic design and support Fc-optimized, non-neutralizing M2e-mAbs as a broadly effective, mutation-resistant, off-the-shelve therapy with direct relevance to human pandemic preparedness.

Lymph nodes (LN) are the staging grounds for antitumor immunity; therefore, their high susceptibility to metastatic colonization is a paradox. Previous studies have suggested that extrinsic tumor-derived factors precondition the draining LN to enable tumor cell survival by promoting a state of immune suppression. In this study, we investigate whether properties of the LN itself may impede its ability to clear metastasizing tumor cells. Using multiple immunocompetent transplant models, we show that LNs possess intrinsic features, independent of preconditioning, which make them an advantageous site for tumor cells to evade T-cell control. Tumor growth in the LN is facilitated by regulatory T cells, which locally suppress the cytolytic capacity of tumor-specific CD8+ T cells by restricting IL2. These findings identify an intrinsic mechanism that contributes to the high rate of LN metastasis in solid tumors.

Dysregulated amino acid metabolism creates cancer-specific vulnerabilities. Neuroblastoma tumors have dysregulated arginine metabolism that renders them sensitive to systemic arginine deprivation. Arginase therapy has been proposed as a therapeutic approach for neuroblastoma treatment and has a favorable safety profile in pediatric cancer patients, however optimal therapeutic combinations remain unexplored.

Acyl coenzyme A binding protein (ACBP encoded by diazepam binding inhibitor DBI) is involved in non-malignant liver diseases. Here, we show that DBI mRNA and circulating ACBP/DBI levels are increased in patients with hepatocellular carcinoma (HCC). We investigated its role in hepatocarcinogenesis in mice, inhibiting ACBP/DBI by three methods: (1) inducible whole-body or liver-specific knockout of DBI, (2) a point mutation of the ACBP/DBI receptor (GABRG2), and (3) induction of autoantibodies neutralizing ACBP/DBI. ACBP/DBI plays a major pro-carcinogenic role in HCC induced by intrahepatic transplantation of HCC cell lines, transgenic co-expression of the two oncogenes Myc and Ctnnb1, and chronic challenge with a Western-style diet together with either carbon tetrachloride (CCl4) or diethylnitrosamine. ACBP/DBI inhibition normalizes HCC-associated gene expression, reducing oncogenic alterations in cell cycle-, immunomodulatory-, and ferroptosis-regulatory genes. ACBP/DBI inhibition increases HCC responses to PD-1 blockade and sensitizes HCC to the therapeutic induction of ferroptosis. Hence, ACBP/DBI constitutes an actionable target involved in HCC pathogenesis.

Radiotherapy (RT) can stimulate anti-cancer T cell responses, and cytokines, notably interleukin-2 (IL-2), are necessary for optimal T cell function and memory. However, timing and IL-2 receptor (IL-2R) bias of IL-2 signals are ill-defined. Using image-guided RT in a mouse colon cancer model, we observed single high-dose (20 Gy) RT transiently upregulated IL-2Rα (CD25) on effector CD8+ T cells, facilitating the use of CD25-biased IL-2 immunotherapy. Timed administration of CD25-biased IL-2 treatment after RT favored intratumoral expansion of CD8+ T cells over regulatory T cells, which resulted in comparable anti-tumor effects as with RT plus IL-2Rβ (CD122)-biased IL-2 immunotherapy. Moreover, intratumoral CD8+ T cells of animals receiving combined IL-2R-biased IL-2 and RT showed reduced markers of exhaustion. These combination treatments affected both primary irradiated and distant non-irradiated tumors and achieved durable responses. We demonstrate that timed IL-2R subunit-biased IL-2 immunotherapy synergizes with single high-dose RT to achieve potent anti-cancer immunity.

CD19-targeted chimeric antigen receptor T cells (CAR-T19) have shown remarkable success in B cell malignancies, but most patients relapse within 1-2 years. Here, we identified interleukin-1 (IL-1) receptor antagonist (IL-1ra) as a mediator of M2-like macrophage-derived inhibition of CAR-T19 in mantle cell lymphoma (MCL), as well as a potential target to enhance CAR-T19 efficacy. In preclinical models that recapitulated interactions between tumor, macrophages, and T cells, we demonstrated that M2-derived IL-1ra impairs IL-1 signaling and functions of CAR-T19. These findings were validated using clinical samples from the ZUMA-2 trial that led to the FDA approval of CAR-T19 in MCL. Single-cell RNA sequencing of CAR-T19 products and baseline myeloid cells indicated downregulated IL-1β production, enriched immunosuppressive phenotypes, and IL-1ra upregulation in the non-responder monocytes, as well as impaired IL-1β signaling and T cell functions in the non-responder CAR-T19 products. Furthermore, our preclinical studies of IL-1β showed enhanced CAR-T antitumor activities. Collectively, these data present a potential role for IL-1 signaling and IL-1ra in CAR-T19 failure.

Metabolic heterogeneity resulting from the intra-tumoral heterogeneity mediates massive adverse outcomes of tumor therapy, including chemotherapeutic resistance, but the mechanisms inside remain largely unknown. Here, we find that the de novo pyrimidine synthesis pathway determines the chemosensitivity. Chemotherapeutic drugs promote the degradation of cytosolic Carbamoyl-phosphate synthetase II, Aspartate transcarbamylase, and Dihydroorotase (CAD), an enzyme that is rate-limiting for pyrimidine synthesis, leading to apoptosis. We also find that CAD needs to be cleaved by caspase-3 on its Asp1371 residue, before its degradation. Overexpressing CAD or mutating Asp1371 to block caspase-3 cleavage confers chemoresistance in xenograft and Cldn18-ATK gastric cancer models. Importantly, mutations related to Asp1371 of CAD are found in tumor samples that failed neoadjuvant chemotherapy and pharmacological targeting of CAD-Asp1371 mutations using RMY-186 ameliorates chemotherapy efficacy. Our work reveals the vulnerability of de novo pyrimidine synthesis during chemotherapy, highlighting CAD as a promising therapeutic target and biomarker.

Strong T cell receptor (TCR) and interleukin (IL)-27 signaling influence type 1 regulatory (Tr1) T cell development, but whether other signals determine their differentiation is unclear. Utilizing Tg4 TCR transgenic mice, we established a model for rapid Tr1 cell induction. A single high dose of [4Y]-MBP peptide drove the differentiation of Il10+ T cells with Tr1 cell mRNA and protein signatures. Kinetic transcriptional and phenotypic analyses revealed that the Tr1 cell module was transient and preceded by Ifng transcription in other CD4+ T cells. Changes in Tr1 cell frequency correlated with altered macrophage activation, while neutralization of interferon (IFN)γ reduced Tr1 cell frequency and the TCR signal strength markers Nur77, inducible T cell costimulator (ICOS), and OX40. Antibody depletion experiments inferred that the relevant source of IFNγ was not natural killer (NK) cell derived. Additionally, blocking IL-27 in combination with IFNγ neutralization additively reduced Tr1 cell frequency in vivo. These findings reveal that IFNγ has a non-redundant role in augmenting Tr1 cell differentiation in vivo.