Donor Macrophages Research Articles

Donor Macrophage Pyroptosis Contributed to the Development of aGVHD

Introduction Pyroptosis, a novel form of inflammation-related programmed cell death, often occurs in myeloid cells. Many studies have found that macrophage pyroptosis plays an important role in multiple inflammatory diseases, such as sepsis, liver injury, and autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis (Journal of Autoimmunity, 2018). Acute GVHD (graft versus host disease) is an immune disease involving various inflammatory factors, and macrophage dysfunction was observed in our previous study (Sci China Life Sci, 2020). During the initiation of aGVHD, large amounts of DAMPs and PAMPs are also released by radiotherapy and infection, which may trigger pyroptosis. However, whether macrophages undergo pyroptosis in aGVHD remains unknown and needs further exploration. Methods The peritoneal macrophages from aGVHD mice were collected after transplantation. Scanning electron microscopy was used to observe cell morphology. Flow cytometry, LDH release and PI staining were used to evaluate cell death. Target tissues, including skin, liver, colon and ileum, were obtained after transplantation for histopathologic analysis, immunofluorescence, immunohistochemical staining and Wb tests. Pyroptosis-related inflammatory factors, including IL-1β and IL-18, were evaluated by a BCA assay and immunohistochemistry. Macrophages and CD4 + T cells were isolated from the bone marrow and spleens of C57 mice. Macrophage-specific GSDMD knockdown mice using adeno-associated serotype 9 virus (AAV9) viral vectors and macrophage conditional GSDMD KO mice (GSDMD rlysm) were used to demonstrate the role of macrophage pyroptosis in the development of aGVHD. Intestinal tissues and peripheral blood from patients with aGVHD and without aGVHD were collected to further validate the occurrence of macrophage pyroptosis in aGVHD. Results Scanning electron microscopy images showed the formation of membrane pores in the peritoneal macrophages of aGVHD mice. Moreover, cell death and LDH release were significantly increased in the peritoneal macrophages of aGVHD mice. The number of GSDMD + macrophages and expression levels of IL-1β and IL-18 were significantly increased in the target tissues of aGVHD mice. The expression of GSDMD-N was also significantly increased in the liver tissue of aGVHD mice. These results indicated that aGVHD was accompanied by macrophage pyroptosis. In addition, elevated levels of IL-1β and IL-18 were observed in the serum and colons of aGVHD patients, which further demonstrated the occurrence of macrophage pyroptosis in aGVHD. To determine whether macrophage pyroptosis contributed to the development of aGVHD, we used macrophage-specific GSDMD knockdown mice with AAV9 viral vectors and WT mice as recipients or donors to compare their disease severity and overall survival. Less pathological damage to tumor tissues, lower clinical scores and longer survival were observed in recipients with macrophage-specific GSDMD knockdown in their donors, indicating that macrophage pyroptosis in donor mice promoted the development of aGVHD. Furthermore, similar results were observed in recipients when macrophage-conditioned GSDMD KO mice (GSDMD rlysm) were their donors. Given the important role of CD4 +T cells in the pathogenesis of aGVHD, the effects of macrophage pyroptosis on CD4 + T cells in vitro were further investigated. In vitro, macrophage pyroptosis promoted the proliferation of CD4 + T cells and increased the proportion of CD69 + cells among CD4 +T cells. The proportions of Th1 and Th17 cells were increased, and the proportion of Tregs was decreased after coculturing with pyroptosed macrophages. These results suggested that macrophage pyroptosis might be involved in the development of aGVHD by affecting CD4 +T cells. In addition, a reduced proportion of activated CD4 +T cells, Th1, Th17 cells and an increased proportion of Tregs were observed in recipients when their donors were macrophage-specific GSDMD knockdown or GSDMD rlysm, further demonstrating that macrophage pyroptosis from donors promoted the development of aGVHD by affecting the function of CD4 + T cells. Conclusions In conclusion, macrophage pyroptosis derived from donors were first found in aGVHD, and they might participate in the development of aGVHD by affecting the activation, proliferation and differentiation of CD4 + T cells.

Intestinal Epithelial-Derived IL-34 Regulates Macrophage Polarization in an Apolipoprotein E Dependent Manner and Mitigates the Severity of Graft Versus Host Disease

Acute graft versus host disease (GVHD) of the gastrointestinal (GI) tract is a major complication of allogeneic bone marrow transplantation. While donor T cells are the proximate trigger of GVHD, innate cells also contribute to disease pathogenesis, although the role of these cell populations is not as well defined. Macrophages, for example, are known contributors in the pathophysiology of chronic GVHD, but their role in acute GVHD is not well delineated. Fundamentally, macrophages are dependent upon exposure to m-CSF and IL-34 which serve as critical trophic factors necessary for their development through binding to the CSF-1R. IL-34 is a more recently discovered cytokine produced by macrophages, but whose role in GVHD pathogenesis is unknown. To examine this question, we employed a murine bone marrow transplantation model (B10.BR [H-2k]®B6[H-2b]) and observed that that there was no difference in GVHD-induced lethality when recipients were reconstituted with marrow grafts from wild type (WT) or IL-34 -/- donors. In contrast, recipient IL-34 -/- animals had significantly worse survival compared to WT mice indicating that host IL-34 expression protected animals from lethal GVHD. We observed no difference in the absolute number of donor macrophages in the colons of WT versus IL-34 -/- mice; however, IL-34 -/- recipient mice had a significant increase in the number of inflammatory Ly6C + macrophages, and a corresponding decrease in Ly6C - cells. Single cell RNA sequence (scRNAseq) analysis of donor macrophages from the colons of WT and IL-34 -/- recipients revealed that macrophages from WT animals had increased expression of anti-inflammatory genes (i.e., CD83, ApoE, Selenop, Lyz1 and Mrc1), whereas macrophages from IL-34 -/- recipients had increased expression of pro-inflammatory genes (i.e., Ifitm1, S100A8, S100A9 and GOs2), indicating that absence of host IL-34 skewed donor macrophages towards an inflammatory phenotype. Since the CSF-1R is not expressed on T cells, we sought to determine the effect of IL-34/CSF-1R signaling on proinflammatory T cells in GVHD target organs. scRNAseq analysis of colonic CD4+ T cells revealed that Csf2 (GM-CSF) was the most differentially expressed gene in IL-34 -/- CD4+ T cells. Functionally, this was confirmed by the observation of a significantly increased number of CD4 + GM-CSF+ T cells in the colons of these mice. Notably, there was no difference in proinflammatory CD8 + T cells, nor any effect on T cell phenotype in the liver or lung, demonstrating that the mitigating effect of host IL-34 expression was confined to the GI tract. To determine the cellular source of IL-34 production, we employed IL-34 LacZ reporter mice and showed that β-Gal co-localized with the epithelial marker, E-cadherin, indicating IL-34 is produced primarily by intestinal epithelial cells. Additional studies using chimeric mice in which IL-34 was restricted to either the hematopoietic or non-hematopoietic compartmentrevealed that absence of IL-34 in the non-hematopoietic compartment phenocopied findings observed with global IL-34 -/- animals, confirming that intestinal epithelial cells were the primary source of this cytokine. The administration of exogenous IL-34 prolonged survival, abrogated pathological damage in the colon and resulted in a corresponding decrease in pro-inflammatory macrophages and T cells, demonstrating this pathway could be pharmacologically targeted. Re-analysis of the scRNAseq data on donor macrophages from the colons of WT and IL-34 -/- mice revealed that apolipoprotein E ( Apoe) was the most differentially expressed gene in macrophages from WT GVHD animals. To confirm a functional role for ApoE, mice were transplanted with T cell depleted (TCD) BM from either WT or ApoE -/- mice and treated with IL-34. These studies revealed that the protective effect conferred by IL-34 was abrogated in animals that received TCD ApoE -/- BM grafts, indicating that ApoE expression in donor macrophages was necessary for agonist driven IL-34-mediated regulation of GVHD. Finally, we confirmed these studies have translational relevance by demonstrating IL-34 and Apoe have increased expression in intestinal epithelial cells and macrophages, respectively, in the colons of humans with GVHD. Thus, these results identify IL-34 as a novel intestinal epithelial cell-derived cytokine that regulates macrophage polarization and mitigates GVHD in an ApoE-dependent manner.

Graft-resident donor macrophages functionally regulate transplant outcomes

Graft-resident donor macrophages functionally regulate transplant outcomes

High-density lipoproteins mediate small RNA intercellular communication between dendritic cells and macrophages

HDL are dynamic transporters of diverse molecular cargo and play critical roles in lipid metabolism and inflammation. We have previously reported that HDL transport both host and nonhost small RNAs (sRNA) based on quantitative PCR and sRNA sequencing approaches; however, these methods require RNA isolation steps which have potential biases and may not isolate certain forms of RNA molecules from samples. HDL have also been reported to accept functional sRNAs from donor macrophages and deliver them to recipient endothelial cells; however, using PCR to trace HDL-sRNA intercellular communication has major limitations. The present study aims to overcome these technical barriers and further understand the pathways involved in HDL-mediated bidirectional flux of sRNAs between immune cells. To overcome these technical limitations, SYTO RNASelect, a lipid-penetrating RNA dye, was used to quantify a) overall HDL-sRNA content, b) bidirectional flux of sRNAs between HDL and immune cells, c) HDL-mediated intercellular communication between immune cells, and d) HDL-mediated RNA export changes in disease. Live cell imaging and loss-of-function assays indicate that the endo-lysosomal system plays a critical role in macrophage storage and export of HDL-sRNAs. These results identify HDL as a substantive mediator of intercellular communication between immune cells and demonstrate the importance of endocytosis for recipient cells of HDL-sRNAs. Utilizing a lipid-penetrating RNA-specific fluorescence dye, we were able to both quantify the absolute concentration of sRNAs transported by HDL and characterize HDL-mediated intercellular RNA transport between immune cells.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

IL-1β-dependent extravasation of preexisting lung-restricted autoantibodies during lung transplantation activates complement and mediates primary graft dysfunction.

Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1β–mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective.

Clonal Hematopoiesis of Indeterminate Potential and Kidney Function Decline in the General Population

Clonal hematopoiesis of indeterminate potential (CHIP), defined by the age-related ontogenesis of expanded leukemogenic variants indicative of a genetically distinct clonal leukocyte population, is associated with risk of hematologic malignancy and cardiovascular disease. In experimental models, recapitulation of CHIP promotes kidney interstitial fibrosis with direct tissue infiltration of donor macrophages. We tested the hypothesis that CHIP is associated with kidney function decline in the general population. Cohort study. 12,004 individuals from 3 community-based cohorts in the TOPMed Consortium. CHIP status from whole-genome sequences obtained from DNA extracted from peripheral blood. Risk of 30% decline in estimated glomerular filtration rate (eGFR) and percent eGFR decline per year during the follow-up period. Cox proportional hazards models for 30% eGFR decline end point and generalized estimating equations for annualized relative change in eGFR with meta-analysis. Study-specific estimates were combined using fixed-effect meta-analysis. The median baseline eGFR was 84mL/min/1.73m2. The prevalence of CHIP was 6.6%, 9.0%, and 12.2% in persons aged 50-60, 60-70, and>70 years, respectively. Over a median follow-up period of 8 years, for the 30% eGFR outcome 205 events occurred among 1,002 CHIP carriers (2.1 events per 100 person-years) and 2,041 events in persons without CHIP (1.7 events per 100 person-years). In meta-analysis, CHIP was associated with greater risk of a 30% eGFR decline (17% [95% CI, 1%-36%] higher; P=0.04). Differences were not observed between those with baseline eGFR above or below 60mL/min/1.73m2, of age above or below 60 years, or with or without diabetes. Small number of participants with moderate-to-advanced kidney disease and restricted set of CHIP driver variants. We report an association between CHIP and eGFR decline in 3 general population cohorts without known kidney disease. Further studies are needed to investigate this novel condition and its potential impact among individuals with overt kidney disease.

Donor Macrophages Modulate Rejection After Heart Transplantation.

Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations. Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2+ (C-C chemokine receptor 2) and CCR2- macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection. Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2+ and CCR2- macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2+ and CCR2- macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2- macrophages reduced allograft survival, depletion of donor CCR2+ macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2+ macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival. Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2+ macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart-based interventions.

Donor macrophages influence rejection of transplanted hearts.

Donor macrophages influence rejection of transplanted hearts.

Unveiling the mechanisms of hematopoietic stem cell transplantation: Balancing cell senescence and proliferation in cancer and beyond

In a recent Nature Medicine publication, Sailor etal.1 elucidate the complex mechanism of hematopoietic stem cell transplantation (HSCT) and chemotherapy, highlighting the occurrence of host microglia senescence coupled with donor macrophage engraftment. These findings warrant a more in-depth understanding of HSCT and its adjunctive therapies to enhance current and future treatments for cancer and a plethora of pro-inflammatory disorders.

A Biomaterial Model to Assess the Effects of Age in Vascularization

As humans age, there is an increased risk for developing age-associated diseases. Many of these diseases, such as cardiovascular disease, involve dysfunction in the vasculature. Cardiovascular disease stems from endothelial cell dysfunction and reduction in vascularization. Macrophages, prominent innate immune cells involved in orchestrating inflammation and wound healing, have a significant influence on vascularization. While much recent work has investigated the crosstalk between endothelial cells and macrophages, it is still not well defined. The interactions between the cell types are even less understood in specific disease states such as advanced age. Understanding how age influences macrophage/endothelial cell interaction is essential for understanding cardiovascular disease development in the elderly. In the polyethylene glycol (PEG)-based hydrogel system, we model the effects of age on vascularization by encapsulating endothelial cells, pericytes, and human donor macrophages. We created a biomaterial model system in which macrophages, either from young (<35 years old) or old (>65 years old) donors, interact with the modeled vasculature, termed microvessels. Confocal image analysis of vessel density, vessel length, and branch points were used to quantify microvessel growth depending on the age of the macrophage donor. Alongside this, soluble factor secretion and gene expression were evaluated using ELISA and NanoString to showcase biological mechanisms based on the age of each donor. Endothelial cells cultured with macrophages from old donors have reduced microvessel density. There also is reduced soluble factor secretion by the macrophages from old donors, which likely influenced microvessel growth. Altogether, we establish our PEG-based hydrogel vascular model as a system to evaluate patient-specific cell function as well as proposed mechanisms for how age influences microvessels.

Hematopoietic stem cell transplantation chemotherapy causes microglia senescence and peripheral macrophage engraftment in the brain.

Hematopoietic stem cell transplantation (HSCT) is a therapy used for multiple malignant and nonmalignant diseases, with chemotherapy used for pretransplantation myeloablation. The post-HSCT brain contains peripheral engrafted parenchymal macrophages, despite their absence in the normal brain, with the engraftment mechanism still undefined. Here we show that HSCT chemotherapy broadly disrupts mouse brain regenerative populations, including a permanent loss of adult neurogenesis. Microglial density was halved, causing microglial process expansion, coinciding with indicators of broad senescence. Although microglia expressed cell proliferation markers, they underwent cell cycle arrest in S phase with a majority expressing the senescence and antiapoptotic marker p21. In vivo single-cell tracking of microglia after recovery from chemical depletion showed loss of their regenerative capacity, subsequently replaced with donor macrophages. We propose that HSCT chemotherapy causes microglial senescence with a gradual decrease to a critical microglial density, providing a permissive niche for peripheral macrophage engraftment of the brain.

Calprotectin Expressing Donor-Derived Macrophages Increase in Acute Gastrointestinal Graft-Versus-Host Disease

Acute graft versus host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). GVHD is therefore the main obstacle for a more widespread use of this highly effective and potentially curative therapy. Although donor T cells are believed to be key mediators in the pathogenesis of acute GVHD, recent reports have suggested that monocyte-derived macrophages also contribute. However, data to support a role for macrophages in acute GVHD in the gastrointestinal tract are sparse. Here we performed a spatiotemporal in situ study to determine the presence of donor and recipient macrophage subsets in colon biopsies from allo-HSCT patients with and without GVHD. Our study was a retrospective study examining colon biopsies from 31 allo-HSCT patients (10 females), of which 21 (5 females) had clinical and histologically-verified GVHD. To distinguish host from donor macrophages we examined gender mismatched donors applying a combination of immunostaining and fluorescence in situ hybridization with probes to X and Y chromosomes. The density of colonic mucosal macrophages was significantly increased (P = .0031) in patients with acute GVHD (n = 21) compared with patients without GVHD (n = 10). Most macrophages were of donor origin in both groups; however, in acute GVHD there was a fivefold increase in donor-derived macrophages expressing the antimicrobial protein calprotectin; reminiscent of recently emigrated proinflammatory monocytes. Moreover, colonic macrophages were found in close proximity to both host and donor T cells. Together, our results suggest that donor-derived proinflammatory macrophages are involved in the immunopathology of colonic acute GHVD in humans.

Identification of neurodegeneration indicators and disease progression in metachromatic leukodystrophy using quantitative NMR-based urinary metabolomics.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the arylsulfatase A (ARSA). ARSA deficiency leads to an accumulation of sulfatides primarily in the nervous system ultimately causing demyelination. With evolving therapeutic options, there is an increasing need for indicators to evaluate disease progression. Here, we report targeted metabolic urine profiling of 56 MLD patients including longitudinal sampling, using 1H (proton) nuclear magnetic resonance (NMR) spectroscopy. 1H‐NMR urine spectra of 119 MLD samples and 323 healthy controls were analyzed by an in vitro diagnostics research (IVDr) tool, covering up to 50 endogenous and 100 disease‐related metabolites on a 600‐MHz IVDr NMR spectrometer. Quantitative data reports were analyzed regarding age of onset, clinical course, and therapeutic intervention. The NMR data reveal metabolome changes consistent with a multiorgan affection in MLD patients in comparison to controls. In the MLD cohort, N‐acetylaspartate (NAA) excretion in urine is elevated. Early onset MLD forms show a different metabolic profile suggesting a metabolic shift toward ketogenesis in comparison to late onset MLD and controls. In samples of juvenile MLD patients who stabilize clinically after hematopoietic stem cell transplantation (HSCT), the macrophage activation marker neopterin is elevated. We were able to identify different metabolic patterns reflecting variable organ disturbances in MLD, including brain and energy metabolism and inflammatory processes. We suggest NAA in urine as a quantitative biomarker for neurodegeneration. Intriguingly, elevated neopterin after HSCT supports the hypothesis that competent donor macrophages are crucial for favorable outcome.

Discovery and Preclinical Characterization of CC-95251, an Anti-SIRPα Antibody That Enhances Macrophage-Mediated Phagocytosis of Non-Hodgkin Lymphoma (NHL) Cells When Combined with Rituximab

Introduction: The transmembrane protein CD47 is ubiquitously expressed on normal cells and often overexpressed on cancer cells, including NHL. Binding of CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an anti-phagocytic signal enabling tumor cells to escape the innate immune response. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a pro-phagocytic signal with the use of tumor-opsonizing antibodies. Targeting SIRPα on monocytes and macrophages rather than the ubiquitously expressed CD47 may overcome some toxicities associated with anti-CD47 therapies. Here, we report the discovery and characterization of a fully human anti-SIRPα antibody and its preclinical activity in combination with the opsonizing antibody rituximab in CD20+ diffuse large B-cell lymphoma (DLBCL) cell lines.Methods: A total of ~ 10 10 fully human immunoglobulin G antibodies were screened for binding to the extracellular domain of recombinant human SIRPα using a yeast display platform. Surface plasmon resonance was used to determine CC-95251 binding coverage across SIRPα haplotypes. The ability of CC-95251 to block CD47-SIRPα interaction was measured using Octet ® and Biacore™ assays. We determined the crystal structure of SIRPα in complex with the CC-95251 Fab to characterize its epitope and to define the structural basis for CD47-SIRPα interaction blockade. To identify tumor types likely susceptible to CD47-SIRPα axis disruption, expression levels of CD47-SIRPα and CD163 were assessed in bulk tumor samples using The Cancer Genome Atlas (TCGA) data. The antitumor effects of CC-95251 in combination with rituximab were examined by measuring the percentage of phagocytic macrophages in co-culture experiments of differentiated macrophages and CD20+ DLBCL cell lines (OCI-Ly3, RIVA, Pfeiffer, and Karpas 422). To confirm CC-95251 binding to monocytes, immunophenotyping of peripheral blood mononuclear cells from healthy donors and cynomolgus monkeys was performed using multiparameter flow cytometry. Lastly, pharmacokinetics and hematologic effects were analyzed in cynomolgus monkeys after treatment with 10, 30, or 100 mg/kg CC-95251.Results: Initial screening by yeast display yielded ~ 350 candidates. The top 24 clones were characterized fully and CC-95251 was selected as the lead monoclonal antibody exhibiting high binding affinity across the 6 most prevalent SIRPα human haplotypes. CC-95251 potently blocked CD47-SIRPα binding in a dose-dependent manner, with a concentration of 100 nM inhibiting CD47 binding almost completely. Co-crystallization modeling showed that CC-95251 engages SIRPα in a region overlapping the CD47 binding site, demonstrating a mechanism for CD47-SIRPα blockade.DLBCL was identified as a suitable tumor type for CC-95251 treatment based on CD47-SIRPα expression and macrophage infiltration. Co-culture experiments of donor macrophages and several DLBCL cell lines showed that CC-95251 monotherapy had weak-to-moderate antitumor activity. However, when combined with rituximab, the levels of phagocytic macrophages were markedly increased in a CC-95251 dose-dependent manner, suggesting that inhibition of the CD47-SIRPα anti-phagocytic axis with CC-95251 and activation of pro-phagocytic signaling with rituximab provide an enhanced antitumor effect in DLBCL cell lines.CC-95251 predominantly bound to cells of myeloid origin, including monocytes and, to a lesser extent, myeloid dendritic cells, whereas no binding to natural killer cells was observed. Toxicology studies in cynomolgus monkeys showed safe intravenous delivery of CC-95251 at therapeutic doses, with no evidence of white blood cell, monocyte, lymphocyte, or red blood cell depletion.Conclusions: CC-95251 is a novel, high-affinity, fully human monoclonal anti-SIRPα antibody that blocks the binding of CD47 to SIRPα. When combined with the therapeutic opsonizing antibody rituximab, CC-95251 enhances macrophage phagocytic activity against DLBCL cell lines in co-culture models. These results support the clinical evaluation of CC-95251 + rituximab for relapsed or refractory NHL. A phase 1 dose-escalation and -expansion study of CC-95251 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). DisclosuresChan: Bristol Myers Squibb: Current Employment. Trout: Bristol Myers Squibb: Ended employment in the past 24 months. Mikolon: Bristol Myers Squibb: Current Employment. Adams: Bristol Myers Squibb: Current Employment. Guzman: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Fenalti: Bristol Myers Squibb: Current Employment. Mavrommatis: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Abbasian: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Dearth: Bristol Myers Squibb: Current Employment. Fox: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Sivakumar: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hariharan: Bristol Myers Squibb: Current Employment.

Abstract 119: Donor Macrophages Modulate Cellular Rejection After Heart Transplantation

Heart transplant rejection is common and is associated with significant morbidity and mortality. Current immunosuppressive therapies primarily target recipient T-cells and have a multitude of untoward effects including infections, malignancies, and end-organ damage. Recent studies implicate the roles of antigen presenting cells towards pathogenesis of allograft rejection through recruitment and activation of T-cells. The importance of antigen presenting cell origin, identity, and functional importance remains unknown. Using complimentary imaging and single cell RNA sequencing techniques, we show that donor and recipient monocytes and macrophages co-exist after heart transplantation. These myeloid populations have diverse transcriptional signatures that evolve throughout ongoing rejection. Donor macrophages can be defined ontologically and based on their expression of C-C chemokine receptor 2 (CCR2) and expression of MHC-II. Donor CCR2+ and CCR2- populations can be further defined based on their gene expression profiles, highlighting the marked heterogeneity in the donor macrophage population. Selective depletion of CCR2+ macrophages result in prolonged allograft survival. We use longitudinal single cell RNA sequencing to show that donor CCR2+ and CCR2- macrophages have distinct activation mechanisms such that donor CCR2+ macrophages signal through MyD88/NF-kB. Conditional depletion of MyD88 in donor macrophages recapitulates the donor CCR2+ depletion phenotype. Further interrogation of MyD88 conditionally depleted allografts shows reduced T-cell alloreactivity, holding promise for a potential therapeutic target pathway. Together, we show the molecular identity, diversity, and evolution of donor and recipient monocytes and macrophages as well as the functional relevance and activation pathways of donor macrophages in cardiac allografts.

Modulation of Donor Cardiac Macrophages is Sufficient to Suppress Rejection and Extend Allograft Survival

Purpose Cellular rejection confers significant morbidity and mortality after heart transplant. Current immunosuppression targets recipient immune cells and increases the risk of life-threatening complications. An alternative approach is to target donor immune cells prior to or at the time of transplant. This approach may be advantageous as it avoids consequences of systemic immunosuppression. Macrophages represent the major immune cell in the donor heart. The heart contains 2 distinct macrophage populations defined by their expression of C-C Chemokine Receptor 2 (CCR2+ and CCR2-) with divergent functions. Their roles in the pathophysiology of heart transplant rejection are yet to be explored. Methods We used a murine allogenic heart transplant model (CD45.2 Bl6 donor, CD45.1 Balb/c recipient) with CTLA4-Ig immunosuppression (200µg days 0,2,4,6). Inclusion of congenic and macrophage reporter strains allowed high fidelity separation of donor CCR2- and CCR2+ macrophages from recipient immune cells. Macrophage depletion, cellular imaging, single cell RNA sequencing and conditional knockout studies were employed to investigate the functions and pathways responsible for donor macrophage activation during rejection. Results High resolution imaging demonstrated that donor macrophages are activated following transplant based on increased size, number of projections, and clustering behavior. Selective depletion of donor CCR2- macrophages resulted in accelerated rejection (16d vs 28 d, p Conclusion Donor CCR2- and CCR2+ macrophage have opposing roles in the pathogenesis of allograft rejection. Prevention of donor CCR2+ macrophage activation through inhibition of MyD88 signaling represents a new therapeutic target to prevent or suppress heart transplant rejection.

Macrophage depletion of CMV latently infected donor hearts ameliorates recipient accelerated chronic rejection.

Cytomegalovirus (CMV) infection is linked to acceleration of solid organ transplant vascular sclerosis (TVS) and chronic rejection (CR). Donor latent CMV infection increases cardiac-resident macrophages and T cells leading to increased inflammation, promoting allograft rejection. To investigate the role of cardiac-resident passenger macrophages in CMV-mediated TVS/CR, macrophages were depleted from latently ratCMV (RCMV)-infected donor allografts prior to transplantation. Latently RCMV-infected donor F344 rats were treated with clodronate, PBS, or control liposomes 3days prior to cardiac transplant into RCMV-naïve Lewis recipients. Clodronate treatment significantly increased graft survival from post-operative day (POD)61 to POD84 and decreased TVS at rejection. To determine the kinetics of the effect of clodronate treatment's effect, a time study revealed that clodronate treatment significantly decreased macrophage infiltration into allograft tissues as early as POD14; altered allograft cytokine/chemokine protein levels, fibrosis development, and inflammatory gene expression profiles. These findings support our hypothesis that increased graft survival as a result of allograft passenger macrophage depletion was in part a result of altered immune response kinetics. Depletion of donor macrophages prior to transplant is a strategy to modulate allograft rejection and reduce TVS in the setting of CMV+donors transplanted into CMV naïve recipients.

Research Highlights

Research Highlights

Harnessing Expressed Single Nucleotide Variation and Single Cell RNA Sequencing To Define Immune Cell Chimerism in the Rejecting Kidney Transplant.

In solid organ transplantation, donor-derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient from donor cells. Whole-exome sequencing of donor and recipient DNA and single-cell RNA sequencing (scRNA-seq) of five human kidney transplant biopsy cores distinguished immune cell contributions from both participants. DNA-sequence comparisons used single nucleotide variants (SNVs) identified in the exome sequences across all samples. Analysis of expressed SNVs in the scRNA-seq data set distinguished recipient versus donor origin for all 81,139 cells examined. The leukocyte donor/recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages displayed inflammatory activation whereas donor macrophages demonstrated antigen presentation and complement signaling. Recipient-origin T cells expressed cytotoxic and proinflammatory genes consistent with an effector cell phenotype, whereas donor-origin T cells appeared quiescent, expressing oxidative phosphorylation genes. Finally, both donor and recipient T cell clones within the rejecting kidney suggested lymphoid aggregation. The results indicate that donor-origin macrophages and T cells have distinct transcriptional profiles compared with their recipient counterparts, and that donor macrophages can persist for years post-transplantation. Analysis of single nucleotide variants and their expression in single cells provides a powerful novel approach to accurately define leukocyte chimerism in a complex organ such as a transplanted kidney, coupled with the ability to examine transcriptional profiles at single-cell resolution.