Search

Supplementary Figures 1-6

legends for sup figures 1-6

Myeloid cell receptor tyrosine kinases TYRO3, AXL, and MERTK and their ligands, GAS6 and PROTEIN S, physiologically suppress innate immune responses, including in the tumor microenvironment. Here, we showed that myeloid-derived suppressor cells (MDSC) dramatically upregulated TYRO3, AXL, and MERTK and their ligands [monocytic MDSCs (M-MDSC)>20-fold, polymorphonuclear MDSCs (PMN-MDSC)>15-fold] in tumor-bearing mice. MDSCs from tumor-bearing Mertk−/−, Axl−/−, and Tyro3−/− mice exhibited diminished suppressive enzymatic capabilities, displayed deficits in T-cell suppression, and migrated poorly to tumor-draining lymph nodes. In coimplantation experiments using TYRO3−/−, AXL−/−, and MERTK−/− MDSCs, we showed the absence of these RTKs reversed the protumorigenic properties of MDSCs in vivo. Consistent with these findings, in vivo pharmacologic TYRO3, AXL, and MERTK inhibition diminished MDSC suppressive capability, slowed tumor growth, increased CD8+ T-cell infiltration, and augmented anti–PD-1 checkpoint inhibitor immunotherapy. Mechanistically, MERTK regulated MDSC suppression and differentiation in part through regulation of STAT3 serine phosphorylation and nuclear localization. Analysis of metastatic melanoma patients demonstrated an enrichment of circulating MERTK+ and TYRO3+ M-MDSCs, PMN-MDSCs, and early-stage MDSCs (e-MDSC) relative to these MDSC populations in healthy controls. These studies demonstrated that TYRO3, AXL, and MERTK control MDSC functionality and serve as promising pharmacologic targets for regulating MDSC-mediated immune suppression in cancer patients.

Type 1 diabetes (T1D) is a consequence of autoimmune-mediated destruction of pancreatic β-cells, but the leading causes for this process remain elusive. We revealed that Ca2+-independent phospholipase A2β (iPLA2β) , which hydrolyzes membrane phospholipids at the sn-2 position to generate bioactive lipids, modulates polarization of macrophages (MΦ) . Several of the iPLA2β-derived lipid signals (iDLs) are proinflammatory and can initiate immune cell infiltration and β-cell damage. Recently, we showed that production of MΦ- proinflammatory lipids (i.e., PGs, DHETs, LTB4) from spontaneous-T1D prone nonobese diabetic mice (NOD) is high during the pre-diabetic phase. Herein, we examined the impact of bone marrow-derived macrophages (BMD-MΦ) -iDLs on T1D development by generating NOD mice with a conditional reduction in MΦ-iPLA2β (NOD.cMΦ iPLA2 𝛽; -/- and NOD.cMΦ iPLA2 𝛽; +/- ) . Our findings reveal that (1) BMD-MΦ from NOD.cMΦiPLA2 𝛽; -/- or NOD.cMΦiPLA2 𝛽; +/- are skewed towards an M2 (anti-inflammatory) phenotype, in comparison with NOD BMD-MΦ (2) production of proinflammatory lipids (PGs, DHETs, and TXB2) is significantly decreased in BMD-MΦ from NOD.cMΦiPLA2 𝛽; -/- and NOD.cMΦiPLA2 𝛽; +/- (3) selective inhibition of PGE2 or DHETs signaling inhibits NOD BMD-MΦ polarization towards a proinflammatory phenotype M1 (4) selective decrease of iPLA2β in MΦ significantly reduces leukocytes infiltration into the pancreas of NOD mice (5) pancreata from NOD.cMΦiPLA2 𝛽; -/- and NOD.cMΦiPLA2 𝛽; +/- mice have significantly higher M2 macrophages and less activated T cells (6) importantly, selective decrease of iPLA2β in MΦ (NOD.cMΦiPLA2 𝛽; -/- and NOD.cMΦiPLA2 𝛽; +/- ) significantly reduces T1D incidence in NOD mice. The overall reduced macrophages-proinflammatory lipids is associated with increased M2 macrophages and less T1D diabetes pathogenesis in selective decrease of MΦ-iPLA2β NOD mice. And inhibition of selective macrophages-derived iDLs production can be targeted to counter T1D. Disclosure A.Almutairi: None. T.White: None. D.Stephenson: None. Y.Gai: None. E.Ubil: None. C.Chalfant: None. S.Ramanadham: None. Funding NIH/NIAID R21 (AI 146743) NIH/NIDDK (R01DK110292) Cell, Developmental, & Integrative Biology (CDIB) , the University of Alabama at BirminghamUAB Comprehensive Diabetes CenterKing Saud bin Abdulaziz University for Health Sciences/ Saudi Arabian Cultural Mission

Macrophages are a specialized class of innate immune cells with multifaceted roles in modulation of the inflammatory response, homeostasis, and wound healing. While developmentally derived or originating from circulating monocytes, naïve macrophages can adopt a spectrum of context-dependent activation states ranging from pro-inflammatory (classically activated, M1) to pro-wound healing (alternatively activated, M2). Tumors are known to exploit macrophage polarization states to foster a tumor-permissive milieu, particularly by skewing macrophages toward a pro-tumor (M2) phenotype. These pro-tumoral macrophages can support cancer progression by several mechanisms including immune suppression, growth factor production, promotion of angiogenesis and tissue remodeling. By preventing the adoption of this pro-tumor phenotype or reprogramming these macrophages to a more pro-inflammatory state, it may be possible to inhibit tumor growth. Here, we describe types of tumor-derived signaling that facilitate macrophage reprogramming, including paracrine signaling and activation of innate immune checkpoints. We also describe intervention strategies targeting macrophage plasticity to limit disease progression and address their implications in cancer chemo- and immunotherapy.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 µm polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 µm barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Myeloid cell receptor tyrosine kinases TYRO3, AXL, and MERTK and their ligands, GAS6 and PROTEIN S, physiologically suppress innate immune responses, including in the tumor microenvironment. Here, we showed that myeloid-derived suppressor cells (MDSC) dramatically upregulated TYRO3, AXL, and MERTK and their ligands [monocytic MDSCs (M-MDSC)>20-fold, polymorphonuclear MDSCs (PMN-MDSC)>15-fold] in tumor-bearing mice. MDSCs from tumor-bearing Mertk−/−, Axl−/−, and Tyro3−/− mice exhibited diminished suppressive enzymatic capabilities, displayed deficits in T-cell suppression, and migrated poorly to tumor-draining lymph nodes. In coimplantation experiments using TYRO3−/−, AXL−/−, and MERTK−/− MDSCs, we showed the absence of these RTKs reversed the protumorigenic properties of MDSCs in vivo. Consistent with these findings, in vivo pharmacologic TYRO3, AXL, and MERTK inhibition diminished MDSC suppressive capability, slowed tumor growth, increased CD8+ T-cell infiltration, and augmented anti–PD-1 checkpoint inhibitor immunotherapy. Mechanistically, MERTK regulated MDSC suppression and differentiation in part through regulation of STAT3 serine phosphorylation and nuclear localization. Analysis of metastatic melanoma patients demonstrated an enrichment of circulating MERTK+ and TYRO3+ M-MDSCs, PMN-MDSCs, and early-stage MDSCs (e-MDSC) relative to these MDSC populations in healthy controls. These studies demonstrated that TYRO3, AXL, and MERTK control MDSC functionality and serve as promising pharmacologic targets for regulating MDSC-mediated immune suppression in cancer patients.

The development of adaptive immune checkpoint inhibitors, which prevent tumor-mediated inactivation of lymphocytes, has drastically improved outcomes for advanced cancer patients. However, only 20-30% of patients benefit from these treatments and those that do often present moderate toxicity and/or later relapse. After discovering a novel innate immune checkpoint, we have identified a therapeutic strategy to restore the tumor-suppressed immune response during chemotherapy. Here, we show that multiple tumor types (including melanoma, lung, breast and pancreatic cancers) co-cultured with murine primary macrophages suppress the expression of the essential Toll-like Receptor (TLR) signal-transducing adapter protein MyD88 by 57-81% +/- 2-5% (mean +/- SEM). In the absence of MyD88, macrophages are unable to respond to TLR stimulating Damage Associated Molecular Patterns (DAMPs) released by dying tumor cells after chemotherapy, leading to as much as 97% +/- 6% reduction in pro-inflammatory gene expression in our co-culture models. Tumors exert this suppression by activating the macrophage Mer receptor, inducing recruitment of a phosphatase, known as PTP1b, and Stat1 to Mer. Increasing association between PTP1b and Stat1 leads to reduced Stat1 phosphorylation and translocation to the nucleus as well as a downstream reduction in MyD88 expression. Treatment with a pharmacological inhibitor of PTP1b (BVT948) blocks the association of Mer, PTP1b and Stat1, restoring MyD88 expression and responsiveness to chemotherapy-released DAMPs. In chemotherapy-resistant murine syngeneic melanoma models, including B16F10 and GEMM6 (Braf V600E, Pten-/-), we show that combination therapy with BVT948 and Cisplatin or Vemurafenib, respectively, leads to a 47% reduction in tumor growth rates. Treating chemo- and immuno-therapy resistant Lewis Lung Cancer (LLC) tumor-bearing mice with combination BVT948/Cisplatin therapy leads to a similar reduction in tumor growth that is magnified with the addition of adaptive immune checkpoint blockade. In summary, we have described a novel mechanism of cancer-mediated immune suppression that dramatically reduces the immune response during chemotherapy. PTP1b inhibition could represent a novel therapeutic strategy to improve the effectiveness of conventional chemotherapy in patients with advanced malignancies. Citation Format: Nestor Prieto-Dominguez, Eric S. Ubil. Restoring the tumor-suppressed immune response during chemotherapy by targeting Mer:PTP1b interactions [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO018.

Tyro3, Axl, Mer (TAM) receptor tyrosine kinases reduce inflammatory, innate immune responses. We demonstrate that tumor-secreted protein S (Pros1), a Mer/Tyro3 ligand, decreased macrophage M1 cytokine expression in vitro and in vivo. In contrast, tumor cells with CRISPR-based deletion of Pros1 failed to inhibit M1 polarization. Tumor cell-associated Pros1 action was abrogated in macrophages from Mer- and Tyro3- but not Axl-KO mice. In addition, several other murine and human tumor cell lines suppressed macrophage M1 cytokine expression induced by IFN-γ and LPS. Investigation of the suppressive pathway demonstrated a role for PTP1b complexing with Mer. Substantiating the role of PTP1b, M1 cytokine suppression was also lost in macrophages from PTP1b-KO mice. Mice bearing Pros1-deficient tumors showed increased innate and adaptive immune infiltration, as well as increased median survival. TAM activation can also inhibit TLR-mediated M1 polarization. Treatment with resiquimod, a TLR7/8 agonist, did not improve survival in mice bearing Pros1-secreting tumors but doubled survival for Pros1-deleted tumors. The tumor-derived Pros1 immune suppressive system, like PD-L1, was cytokine responsive, with IFN-γ inducing Pros1 transcription and secretion. Inhibition of Pros1/TAM interaction represents a potential novel strategy to block tumor-derived immune suppression.

Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial-cell-like phenotype after acute ischaemic cardiac injury. Fibroblast-derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal-to-endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.

The mammalian heart exhibits poor regenerative capacity after acute ischemic injury and heals primarily by fibrosis. Recently, several groups have demonstrated that cardiac fibroblasts can be reprogrammed to adopt myogenic fates using exogenous transcription factors. However, the ability of cardiac fibroblasts to adopt specific cellular fates in the absence of exogenous factors is unclear. Here, we demonstrate that a subset of cardiac fibroblasts adopt endothelial characteristics after ischemic cardiac injury in the absence of any added factors. Using mice harboring genetically labeled fibroblasts (Col1a2CreERT:R26RTdTomato), we show that 34 +/- 3% (mean, SEM) of labeled cardiac fibroblasts in the injury border zone express endothelial markers such as VE-cadherin. Fibroblast derived endothelial cells comprised 25 +/- 2% of total and 8 +/- 2% of luminal endothelial cells at the border zone 3 days after injury. To better understand fibroblast-endothelial reprogramming we subjected cardiac fibroblasts to cellular stress (serum starvation) and found that they formed tubes on Matrigel and up-regulated endothelial specific genes (e.g. VE-cadherin, Flk1, Flt1) 6-20 fold. We show that reprogramming of fibroblasts to endothelial like cells ex vivo is p53 dependent. Inhibiting p53 activity by pharmacological means (Pifithrin-α) or genetic deletion in fibroblasts (Col1a2CreERT:p53fl/fl) led to a 94% decrease in Matrigel tube formation and 90% reduction in endothelial gene expression. Moreover, we observed that p53 levels in cardiac fibroblasts were more than 10-fold higher at the injury border zone using semi-quantitative immunofluorescent staining. Injection of a p53 activator after injury doubled p53 levels in cardiac fibroblasts and increased the rate of fibroblast-endothelial reprogramming by 43%. Enhanced fibroblast-endothelial reprogramming was also associated with decreased collagen deposition 3 days post injury. In summary, we show that cardiac fibroblasts are able to adopt endothelial cell like fates both in vivo and ex vivo in a p53 dependent manner. Manipulation of fibroblast to endothelial reprogramming could represent a novel therapeutic strategy to increase post infarct angiogenesis and enhance function in the injured heart.