Engineered CD47 protects T cells for enhanced antitumour immunity.

Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.

P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi.

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of invading agents by phagocytes. In the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through mimicry at the mammalian anti-phagocytic "don't eat me" signaling axis between CD47 (ligand) and SIRPα (receptor). We identified a protein, P66, on the surface of Borrelia burgdorferi that, like CD47, is necessary and sufficient to bind the macrophage receptor SIRPα. Expression of the gene encoding the protein is required for bacteria to bind SIRPα or a high-affinity CD47 reagent. Genetic deletion of p66 increases phagocytosis by macrophages. Blockade of P66 during infection promotes clearance of the bacteria. This study demonstrates that mimicry of the mammalian anti-phagocytic protein CD47 by B. burgdorferi inhibits macrophage-mediated bacterial clearance. Such a mechanism has broad implications for understanding of host-pathogen interactions and expands the function of the established innate immune checkpoint receptor SIRPα. Moreover, this report reveals P66 as a novel therapeutic target in the treatment of Lyme Disease.

Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung.

Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.

Transient receptor potential vanilloid 4 channel inhibition attenuates lung ischemia-reperfusion injury in a porcine lung transplant model.

OBJECTIVE: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel important in many physiological and pathophysiological processes, including pulmonary disease. Using a murine model, we previously demonstrated that TRPV4 mediates lung ischemia-reperfusion injury, the major cause of primary graft dysfunction after transplant. The current study tests the hypothesis that treatment with a TRPV4 inhibitor will attenuate lung ischemia-reperfusion injury in a clinically relevant porcine lung transplant model. METHODS: A porcine left-lung transplant model was used. Animals were randomized to 2 treatment groups (n = 5/group): vehicle or GSK2193874 (selective TRPV4 inhibitor). Donor lungs underwent 30 minutes of warm ischemia and 24 hours of cold preservation before left lung allotransplantation and 4 hours of reperfusion. Vehicle or GSK2193874 (1 mg/kg) was administered to the recipient as a systemic infusion after recipient lung explant. Lung function, injury, and inflammatory biomarkers were compared. RESULTS: After transplant, left lung oxygenation was significantly improved in the TRPV4 inhibitor group after 3 and 4 hours of reperfusion. Lung histology scores and edema were significantly improved, and neutrophil infiltration was significantly reduced in the TRPV4 inhibitor group. TRPV4 inhibitor-treated recipients had significantly reduced expression of interleukin-8, high mobility group box 1, P-selectin, and tight junction proteins (occludin, claudin-5, and zonula occludens-1) in bronchoalveolar lavage fluid as well as reduced angiopoietin-2 in plasma, all indicative of preservation of endothelial barrier function. CONCLUSIONS: Treatment of lung transplant recipients with TRPV4 inhibitor significantly improves lung function and attenuates ischemia-reperfusion injury. Thus, selective TRPV4 inhibition may be a promising therapeutic strategy to prevent primary graft dysfunction after transplant.

Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

SCGB1D2 inhibits growth of Borrelia burgdorferi and affects susceptibility to Lyme disease.

Lyme disease is a tick-borne disease caused by bacteria of the genus Borrelia. The host factors that modulate susceptibility for Lyme disease have remained mostly unknown. Using epidemiological and genetic data from FinnGen and Estonian Biobank, we identify two previously known variants and an unknown common missense variant at the gene encoding for Secretoglobin family 1D member 2 (SCGB1D2) protein that increases the susceptibility for Lyme disease. Using live Borrelia burgdorferi (Bb) we find that recombinant reference SCGB1D2 protein inhibits the growth of Bb in vitro more efficiently than the recombinant protein with SCGB1D2 P53L deleterious missense variant. Finally, using an in vivo murine infection model we show that recombinant SCGB1D2 prevents infection by Borrelia in vivo. Together, these data suggest that SCGB1D2 is a host defense factor present in the skin, sweat, and other secretions which protects against Bb infection and opens an exciting therapeutic avenue for Lyme disease.

Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity.

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.

CXCR2 inhibition in G-MDSCs enhances CD47 blockade for melanoma tumor cell clearance.

The use of colony-stimulating factor-1 receptor (CSF1R) inhibitors has been widely explored as a strategy for cancer immunotherapy due to their robust depletion of tumor-associated macrophages (TAMs). While CSF1R blockade effectively eliminates TAMs from the solid tumor microenvironment, its clinical efficacy is limited. Here, we use an inducible CSF1R knockout model to investigate the persistence of tumor progression in the absence of TAMs. We find increased frequencies of granulocytic myeloid-derived suppressor cells (G-MDSCs) in the bone marrow, throughout circulation, and in the tumor following CSF1R deletion and loss of TAMs. We find that G-MDSCs are capable of suppressing macrophage phagocytosis, and the elimination of G-MDSCs through CXCR2 inhibition increases macrophage capacity for tumor cell clearance. Further, we find that combination therapy of CXCR2 inhibition and CD47 blockade synergize to elicit a significant anti-tumor response. These findings reveal G-MDSCs as key drivers of tumor immunosuppression and demonstrate their inhibition as a potent strategy to increase macrophage phagocytosis and enhance the anti-tumor efficacy of CD47 blockade in B16-F10 melanoma.

UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas.

Neuroendocrine carcinomas, such as neuroendocrine prostate cancer and small-cell lung cancer, commonly have a poor prognosis and limited therapeutic options. We report that ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is elevated in tissues and plasma from patients with neuroendocrine carcinomas. Loss of UCHL1 decreases tumor growth and inhibits metastasis of these malignancies. UCHL1 maintains neuroendocrine differentiation and promotes cancer progression by regulating nucleoporin, POM121, and p53. UCHL1 binds, deubiquitinates, and stabilizes POM121 to regulate POM121-associated nuclear transport of E2F1 and c-MYC. Treatment with the UCHL1 inhibitor LDN-57444 slows tumor growth and metastasis across neuroendocrine carcinomas. The combination of UCHL1 inhibitors with cisplatin, the standard of care used for neuroendocrine carcinomas, significantly delays tumor growth in pre-clinical settings. Our study reveals mechanisms of UCHL1 function in regulating the progression of neuroendocrine carcinomas and identifies UCHL1 as a therapeutic target and potential molecular indicator for diagnosing and monitoring treatment responses in these malignancies.

SIRPα controls CD47-dependent platelet clearance in mice and humans.

Over the last decade, more data has revealed that increased surface expression of the "don't eat me" CD47 protein on cancer cells plays a role in immune evasion and tumor progression, with CD47 blockade emerging as a new therapy in immuno-oncology. CD47 is critical in regulating cell homeostasis and clearance, as binding of CD47 to the inhibitory receptor SIRPα can prevent phagocytosis and macrophage-mediated cell clearance. The purpose of this study was to examine the role of the CD47-SIRPα signal in platelet homeostasis and clearance. Therapeutic reagents targeting the CD47-SIRPα axis are very promising for treatment of hematologic malignancies and solid tumors, but lead to transient anemia or thrombocytopenia in a subset of patients. We found that platelet homeostatic clearance is regulated through the CD47-SIRPα axis and that therapeutic blockade to disrupt this interaction in mice and in humans has a significant impact on platelet levels. Furthermore, we identified genetic variations at the SIRPA locus that impact platelet levels in humans such that higher SIRPA gene expression is associated with higher platelet levels. SIRPA expression at either end of the normal range may affect clinical outcomes of treatment with anti-CD47 therapy.

Prospective isolation of neural stem and progenitor cells from the developing human brain.

Prospective isolation of defined cell types is critical for the functional study of stem cells, especially in primary human tissues. Here, we present a protocol for purifying 10 transcriptomically and functionally distinct neural stem and progenitor cell types from the developing human brain using fluorescence-activated cell sorting. We describe steps for tissue dissociation, staining, and cell sorting as well as downstream functional experiments for measuring clonogenicity, differentiation, and engraftment potential of purified populations. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1.

Immune Surveillance of Acute Myeloid Leukemia Is Mediated by HLA-Presented Antigens on Leukemia Progenitor Cells.

Therapy-resistant leukemia stem and progenitor cells (LSC) are a main cause of acute myeloid leukemia (AML) relapse. LSC-targeting therapies may thus improve outcome of patients with AML. Here we demonstrate that LSCs present HLA-restricted antigens that induce T-cell responses allowing for immune surveillance of AML. Using a mass spectrometry-based immunopeptidomics approach, we characterized the antigenic landscape of patient LSCs and identified AML- and AML/LSC-associated HLA-presented antigens absent from normal tissues comprising nonmutated peptides, cryptic neoepitopes, and neoepitopes of common AML driver mutations of NPM1 and IDH2. Functional relevance of shared AML/LSC antigens is illustrated by presence of their cognizant memory T cells in patients. Antigen-specific T-cell recognition and HLA class II immunopeptidome diversity correlated with clinical outcome. Together, these antigens shared among AML and LSCs represent prime targets for T cell-based therapies with potential of eliminating residual LSCs in patients with AML. SIGNIFICANCE: The elimination of therapy-resistant leukemia stem and progenitor cells (LSC) remains a major challenge in the treatment of AML. This study identifies and functionally validates LSC-associated HLA class I and HLA class II-presented antigens, paving the way to the development of LSC-directed T cell-based immunotherapeutic approaches for patients with AML. See related commentary by Ritz, p. 430 . This article is featured in Selected Articles from This Issue, p. 419.

Unplanned postoperative catheterization during admission for coronary artery bypass grafting is neither cheap nor benign, but may rescue patients.

OBJECTIVE: Literature describing outcomes of myocardial ischemia after coronary artery bypass grafting is sparse. We hypothesized these patients had more complications and incurred higher costs of care. METHODS: Using adult cardiac surgery data and cardiac catheterization (CathPCI) data from the Virginia Cardiac Services Quality Initiative, we identified patients who underwent unplanned cardiac catheterization after coronary artery bypass grafting from 2018 to 2021. Adult cardiac surgery data were matched to CathPCI data examining earliest in-hospital catheterization. Patients not requiring catheterization served as the control group. RESULTS: We identified 10,597 patients who underwent isolated coronary artery bypass grafting, of whom 41 of 10,597 underwent unplanned cardiac catheterization. A total of 21 of 41 patients (51%) received percutaneous coronary intervention, most commonly for non-ST-elevation myocardial infarction (n = 7, 33%) and ST-elevation myocardial infarction (n = 6, 29%). Postoperative cardiac arrest occurred in 14 patients (40%). In patients who underwent percutaneous coronary intervention, 14 (67%) had a single lesion, 4 (19%) had 2 lesions, and 3 (14%) had 3 lesions. The left anterior descending artery (38%) was the most frequently intervened upon vessel. Patients who underwent catheterization were more likely to require balloon pump support (26% vs 11%), to have prolonged ventilation (57% vs 20%), to have renal failure (17% vs 7.1%), and to undergo reintubation (37% vs 3.8%, all P < .04). There was no statistical difference in operative mortality (4.9% vs 2.3%, P = .2) or failure to rescue (4.9% vs 1.6%, P = .14). Total costs were higher in patients who underwent unplanned catheterization ($81,293 vs $37,011, P < .001). CONCLUSIONS: Unplanned catheterization after coronary artery bypass grafting is infrequent but associated with more complications and a higher cost of care. Therefore, determination of an association with operative mortality in patients with suspected ischemia after coronary artery bypass grafting requires additional study.

Effect of Cardiopulmonary Bypass on SARS-CoV-2 Vaccination Antibody Levels.

Background Adults undergoing heart surgery are particularly vulnerable to respiratory complications, including COVID-19. Immunization can significantly reduce this risk; however, the effect of cardiopulmonary bypass (CPB) on immunization status is unknown. We sought to evaluate the effect of CPB on COVID-19 vaccination antibody concentration after cardiac surgery. Methods and Results This prospective observational clinical trial evaluated adult participants undergoing cardiac surgery requiring CPB at a single institution. All participants received a full primary COVID-19 vaccination series before CPB. SARS-CoV-2 spike protein-specific antibody concentrations were measured before CPB (pre-CPB measurement), 24 hours following CPB (postoperative day 1 measurement), and approximately 1 month following their procedure. Relationships between demographic or surgical variables and change in antibody concentration were assessed via linear regression. A total of 77 participants were enrolled in the study and underwent surgery. Among all participants, mean antibody concentration was significantly decreased on postoperative day 1, relative to pre-CPB levels (-2091 AU/mL, P<0.001). Antibody concentration increased between postoperative day 1and 1 month post CPB measurement (2465 AU/mL, P=0.015). Importantly, no significant difference was observed between pre-CPB and 1 month post CPB concentrations (P=0.983). Two participants (2.63%) developed symptomatic COVID-19 pneumonia postoperatively; 1 case of postoperative COVID-19 pneumonia resulted in mortality (1.3%). Conclusions COVID-19 vaccine antibody concentrations were significantly reduced in the short-term following CPB but returned to pre-CPB levels within 1 month. One case of postoperative COVID 19 pneumonia-specific mortality was observed. These findings suggest the need for heightened precautions in the perioperative period for cardiac surgery patients.

The human placenta exhibits a unique transcriptomic void.

The human placenta exhibits a unique genomic architecture with an unexpectedly high mutation burden and many uniquely expressed genes. The aim of this study is to identify transcripts that are uniquely absent or depleted in the placenta. Here, we show that 40 of 46 of the other organs have no selectively depleted transcripts and that, of the remaining six, the liver has the largest number, with 26. In contrast, the term placenta has 762 depleted transcripts. Gene Ontology analysis of this depleted set highlighted multiple pathways reflecting known unique elements of placental physiology. For example, transcripts associated with neuronal function are in the depleted set-as expected given the lack of placental innervation. However, this demonstrated overrepresentation of genes involved in mitochondrial function (p = 5.8 × 10-10), including PGC-1α, the master regulator of mitochondrial biogenesis, and genes involved in polyamine metabolism (p = 2.1 × 10-4).

Postdoctoral National Institutes of Health F32 Grants: Broken Pipeline in the Development of Surgeon-Scientists.

OBJECTIVE: We examined trainees in surgery and internal medicine who received National Institutes of Health (NIH) F32 postdoctoral awards to determine their success rates in obtaining future NIH funding. BACKGROUND: Trainees participate in dedicated research years during residency (surgery) and fellowship (internal medicine). They can obtain an NIH F32 grant to fund their research time and have structured mentorship. METHODS: We collected NIH F32 grants (1992-2021) for Surgery Departments and Internal Medicine Departments from NIH RePORTER, an online database of NIH grants. Nonsurgeons and noninternal medicine physicians were excluded. We collected demographic information on each recipient, including gender, current specialty, leadership positions, graduate degrees, and any future NIH grants they received. A Mann-Whitney U test was used for continuous variables, and a χ 2 test was utilized to analyze categorical variables. An alpha value of 0.05 was used to determine significance. RESULTS: We identified 269 surgeons and 735 internal medicine trainees who received F32 grants. A total of 48 surgeons (17.8%) and 339 internal medicine trainees (50.2%) received future NIH funding ( P < 0.0001). Similarly, 24 surgeons (8.9%) and 145 internal medicine trainees (19.7%) received an R01 in the future ( P < 0.0001). Surgeons who received F32 grants were more likely to be department chair or division chiefs ( P =0.0055 and P < 0.0001). CONCLUSIONS: Surgery trainees who obtain NIH F32 grants during dedicated research years are less likely to receive any form of NIH funding in the future compared with their internal medicine colleagues who received F32 grants.

A Combination of Distinct Vascular Stem/Progenitor Cells for Neovascularization and Ischemic Rescue.

BACKGROUND: Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia. METHODS: The ActinCreER/R26VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions. RESULTS: Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice. CONCLUSIONS: These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

Multiple Forms of Neural Cell Death in the Cyclical Brain Degeneration of A Colonial Chordate.

Human neuronal loss occurs through different cellular mechanisms, mainly studied in vitro. Here, we characterized neuronal death in B. schlosseri, a marine colonial tunicate that shares substantial genomic homology with mammals and has a life history in which controlled neurodegeneration happens simultaneously in the brains of adult zooids during a cyclical phase named takeover. Using an ultrastructural and transcriptomic approach, we described neuronal death forms in adult zooids before and during the takeover phase while comparing adult zooids in takeover with their buds where brains are refining their structure. At takeover, we found in neurons clear morphologic signs of apoptosis (i.e., chromatin condensation, lobed nuclei), necrosis (swollen cytoplasm) and autophagy (autophagosomes, autolysosomes and degradative multilamellar bodies). These results were confirmed by transcriptomic analyses that highlighted the specific genes involved in these cell death pathways. Moreover, the presence of tubulovesicular structures in the brain medulla alongside the over-expression of prion disease genes in late cycle suggested a cell-to-cell, prion-like propagation recalling the conformational disorders typical of some human neurodegenerative diseases. We suggest that improved understanding of how neuronal alterations are regulated in the repeated degeneration-regeneration program of B. schlosseri may yield mechanistic insights relevant to the study of human neurodegenerative diseases.