Administration of anti-RhD immunoglobulin (Ig) to decrease maternal alloimmunization (antibody-mediated immune suppression [AMIS]) was a landmark clinical development. However, IgG has potent immune-stimulatory effects in other settings (antibody-mediated immune enhancement [AMIE]). The dominant thinking has been that IgG causes AMIS for antigens on RBCs but AMIE for soluble antigens. However, we have recently reported that IgG against RBC antigens can cause either AMIS or AMIE as a function of an IgG subclass. Recent advances in mechanistic understanding have demonstrated that RBC alloimmunization requires the IFN-α/-β receptor (IFNAR) and is inhibited by the complement C3 protein. Here, we demonstrate the opposite for AMIE of an RBC alloantigen (IFNAR is not required and C3 enhances). RBC clearance, C3 deposition, and antigen modulation all preceded AMIE, and both CD4+ T cells and marginal zone B cells were required. We detected no significant increase in antigen-specific germinal center B cells, consistent with other studies of RBC alloimmunization that show extrafollicular-like responses. To the best of our knowledge, these findings provide the first evidence of an RBC alloimmunization pathway which is IFNAR independent and C3 dependent, thus further advancing our understanding of RBCs as an immunogen and AMIE as a phenomenon.
Arijita Jash, Thomas Pridmore, James B. Collins, Ariel M. Hay, Krystalyn E. Hudson, Chance John Luckey, James C. Zimring
Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP impacts risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2 knockout (Tet2–/–) and floxed control mice (Tet2f/f) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2–/– mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2–/– mice. We delineated the transcriptional landscape of Tet2–/– neutrophils and found that while inflammation-related pathways were upregulated in Tet2–/– neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake and neutrophil extracellular trap (NET) formation by Tet2–/– neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.
Candice Quin, Erica N. DeJong, Elina K. Cook, Yi Zhen Luo, Caitlyn Vlasschaert, Sanathan Sadh, Amy J.M. McNaughton, Marco M. Buttigieg, Jessica A Breznik, Allison E. Kennedy, Kevin Zhao, Jeffrey Mewburn, Kimberly J. Dunham-Snary, Charles C.T. Hindmarch, Alexander G. Bick, Stephen L. Archer, Michael J. Rauh, Dawn M.E. Bowdish
Nuclear factor kappa-B (NFκB) is activated in arrhythmogenic cardiomyopathy (ACM) patient-derived iPSC-cardiac myocytes under basal conditions and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single cell RNA sequencing to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA sequencing and cellular indexing of transcriptomes and epitomes (CITE-seq) studies revealed marked pro-inflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells and fibroblasts in the pathogenesis of ACM.
Stephen P. Chelko, Vinay R. Penna, Morgan Engel, Emily A. Shiel, Ann M. Centner, Waleed Farra, Elisa N. Cannon, Maicon Landim-Vieira, Niccole Schaible, Kory Lavine, Jeffrey E. Saffitz
Gender affirming hormone therapy (GAHT) is often prescribed to transgender (TG) adolescents to alleviate gender dysphoria, but the impact of GAHT on the growing skeleton is unclear. We found GAHT to improve trabecular bone structure via increased bone formation in young male mice and not to affect trabecular structure in female mice. GAHT modified gut microbiome composition in both male and female mice. However, fecal microbiota transfers (FMT) revealed that GAHT-shaped gut microbiome was a communicable regulator of bone structure and turnover in male, but not in female mice. Mediation analysis identified two species of Bacteroides as significant contributors to the skeletal effects of GAHT in male mice, with Bacteroides supplementation phenocopying the effects of GAHT on bone. Bacteroides have the capacity to expand Treg populations in the gut. Accordingly, GAHT expanded intestinal regulatory T cells (Tregs) and stimulated their homing to the bone marrow (BM) in male but not in female mice. Attesting to the functional relevance of Tregs, pharmacological blockade of Treg expansion prevented GAHT-induced bone anabolism. In summary, in male mice GAHT stimulated bone formation and improved trabecular structure by promoting Treg expansion via a microbiome-mediated effect. In female mice GAHT neither improved nor impaired trabecular structure.
Subhashis Pal, Xochitl Morgan, Hamid Y. Dar, Camilo Anthony Gacasan, Sanchiti Patil, Andreea Stoica, Yi-Juan Hu, M. Neale Weitzmann, Rheinallt M. Jones, Roberto Pacifici
In lung, thromboxane A2 (TXA2) activates the TP receptor to induce pro-inflammatory and bronchoconstrictor effects. Thus, TP receptor antagonists and TXA2 synthase inhibitors have been tested as potential asthma therapeutics in humans. Th9 cells play key roles in asthma and regulate the lung immune response to allergens. Herein, we found that TXA2 reduces Th9 cell differentiation during allergic lung inflammation. Th9 cells were decreased ~2-fold and airway hyperresponsiveness was attenuated in lungs of allergic mice treated with TXA2. Naïve CD4+ T cell differentiation to Th9 cells and IL-9 production was inhibited dose-dependently by TXA2 in vitro. TP receptor deficient mice had a ~2-fold increase in numbers of Th9 cells in lungs in vivo after OVA exposure compared to wild type (WT) mice. Naïve CD4+ T cells from TP deficient mice exhibited increased Th9 cell differentiation and IL-9 production in vitro compared to CD4+ T cells from WT mice. TXA2 also suppressed Th2 and enhanced Treg differentiation both in vitro and in vivo. Thus, in contrast to its acute, pro-inflammatory effects, TXA2 also has longer-lasting immunosuppressive effects that attenuate the Th9 differentiation that drives asthma progression. These findings may explain the paradoxical failure of anti-thromboxane therapies in the treatment of asthma.
Hong Li, J. Alyce Bradbury, Matthew L. Edin, Artiom Gruzdev, Huiling Li, Joan P. Graves, Laura M. DeGraff, Fred B. Lih, Chiguang Feng, Erin R. Wolf, Carl D. Bortner, Stephanie J. London, Matthew A. Sparks, Thomas M. Coffman, Darryl C. Zeldin
BACKGROUND. Weakly virulent environmental mycobacteria (EM) can cause severe disease in HLA-DRB1*15:02 or 16:02 adult individuals harboring neutralizing anti-IFN-γ autoantibodies (nAIGAs). The overall prevalence of nAIGA in a general population are unknown as is the the penetrance of nAIGA in HLA-DRB1*15:02 or 16:02 individuals, and the proportion of patients with unexplained, adult-onset EM infections carrying nAIGAs. METHODS. This study analyzed the detection and neutralization of anti-IFN-γ autoantibodies (auto-Abs) from 8,430 healthy individuals of the general population, 257 HLA-DRB1*15:02 or 16:02 carriers, 1,063 patients with autoimmune disease, and 497 patients with unexplained severe disease due to EM. RESULTS. We find that anti-IFN-γ autoantibodies detected in 4,148 of 8,430 healthy individuals (49.2%) from the general population of an unknown HLA-DRB1 genotype are not neutralizing. Moreover, we do not find nAIGAs in 257 individuals carrying HLA-DRB1* 15:02 or 16:02, including 71 individuals with detectable anti-IFN-g autoantibodies (27.6%). Additionally, nAIGA are absent in 1,063 patients with an autoimmune disease. Furthermore, we find only a few other autoantibodies in seven patients with nAIGAs tested. Finally, seven of 497 patients (1.4%) with unexplained severe disease due to EM harbored nAIGA. Yet, nAIGA are absent in the remaining 41 patients who are HLA-DRB1*15:02 or 16:02, the 45 patients with IFN-g autoantibodies, and the five patients with HLA-DRB1*15:02 or 16:02 and IFN-g autoantibodies . CONCLUSION. These findings suggest that nAIGAs are isolated and that their penetrance in HLA-DRB1*15:02 or 16:02 individuals is low, implying that they may be triggered by rare germline or somatic variants. In contrast, the risk of mycobacterial disease in patients with nAIGAs is high, confirming that these nAIGAs are causal of EM disease. FUNDING. The Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, the National Institutes of Health (NIH) (R01AI095983), the National Center for Advancing Translational Sciences (NCATS), the NIH Clinical and Translational Science Award (CTSA) program (UL1 TR001866), and partly by French National Research Agency (ANR).
Jessica N. Peel, Rui Yang, Tom Le Voyer, Adrian Gervais, Jérémie Rosain, Paul Bastard, Anish Behere, Axel Cederholm, Aaron Bodansky, Yoann Seeleuthner, Clément Conil, Jing-Ya Ding, Wei-Te Lei, Lucy Bizien, Camille Soudee, Mélanie Migaud, Masato Ogishi, Ahmad Yatim, Danyel Lee, Jonathan Bohlen, Thomas Perpoint, Laura Perez, Fernando Messina, Roxana Genet, Ludovic Karkowski, Mathieu Blot, Emmanuel Lafont, Laurie Toullec, Claire Goulvestre, Souad Mehlal-Sedkaoui, Jérôme Sallette, Fernando Martin, Anne Puel, Emmanuelle Jouanguy, Mark S. Anderson, Nils Landegren, Pierre Tiberghien, Laurent Abel, Stéphanie Boisson-Dupuis, Jacinta Bustamante, Cheng-Lung Ku, Jean-Laurent Casanova
Antitumor responses of CD8+ T cells are tightly regulated by distinct metabolic fitness. High levels of glutathione (GSH) are observed in the majority of tumors contributing to cancer progression and treatment resistance in part by preventing glutathione peroxidase 4 (GPX4) dependent ferroptosis. Here, we show the necessity of the adenosine A2A receptor (A2AR) signaling and the glutathione (GSH)-GPX4 axis in orchestrating metabolic fitness and survival of functionally competent CD8+ T cells. Activated CD8+ T cells treated ex vivo with simultaneous inhibition of A2AR and lipid peroxidation acquire a superior capacity to proliferate and persist in vivo, demonstrating a translatable means to prevent ferroptosis in adoptive cell therapy (ACT). Additionally, we identify a particular cluster of intratumoral CD8+ T cells expressing a putative gene signature of GSH metabolism (GMGS) in association with clinical response and survival across several human cancers. Our study addresses a key role of GSH-GPX4 and adenosinergic pathways in fine-tuning the metabolic fitness of antitumor CD8+ T cells.
Siqi Chen, Jie Fan, Ping Xie, Jihae Ahn, Michelle Fernandez, Leah K. Billingham, Jason Miska, Jennifer D. Wu, Derek A. Wainwright, Deyu Fang, Jeffrey A. Sosman, Yong Wan, Yi Zhang, Navdeep S. Chandel, Bin Zhang
Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.
Orhan Efe, Rodrigo B. Gassen, Leela Morena, Yoshikazu Ganchiku, Ayman Al Jurdi, Isadora T. Lape, Pedro Ventura-Aguiar, Christian LeGuern, Joren C. Madsen, Zachary Shriver, Gregory J. Babcock, Thiago J. Borges, Leonardo V. Riella
Tissue-intrinsic mechanisms that regulate severity of systemic pathogenic immune-mediated diseases, such as acute graft-versus-host disease (GVHD), remain poorly understood. Following allogeneic hematopoietic stem cell transplantation, autophagy, a cellular stress protective response, is induced in host nonhematopoietic cells. To systematically address the role of autophagy in various host nonhematopoietic tissues, both specific classical target organs of acute GVHD (intestines, liver, and skin) and organs conventionally not known to be targets of GVHD (kidneys and heart), we generated mice with organ-specific knockout of autophagy related 5 (ATG5) to specifically and exclusively inhibit autophagy in the specific organs. When compared with wild-type recipients, animals that lacked ATG5 in the gastrointestinal tract or liver showed significantly greater tissue injury and mortality, while autophagy deficiency in the skin, kidneys, or heart did not affect mortality. Treatment with the systemic autophagy inducer sirolimus only partially mitigated GVHD mortality in intestine-specific autophagy-deficient hosts. Deficiency of autophagy increased MHC class I on the target intestinal epithelial cells, resulting in greater susceptibility to damage by alloreactive T cells. Thus, autophagy is a critical cell-intrinsic protective response that promotes tissue tolerance and regulates GVHD severity.
Katherine Oravecz-Wilson, Emma Lauder, Austin Taylor, Laure Maneix, Jeanine L. Van Nostrand, Yaping Sun, Lu Li, Dongchang Zhao, Chen Liu, Pavan Reddy
Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI). A primary contributor to infection chronicity is an expansion of granulocytic myeloid-derived suppressor cells (G-MDSCs) that are critical for orchestrating the anti-inflammatory biofilm milieu. Single-cell sequencing and bioinformatic metabolic algorithms were used to explore the link between G-MDSC metabolism and S. aureus PJI outcome. Glycolysis and the hypoxia response through hypoxia-inducible factor-1 alpha (HIF-1a) were significantly enriched in G-MDSCs. Interfering with both pathways in vivo, using a 2-deoxyglucose nanopreparation and granulocyte-targeted HIF-1a conditional knockout mice, respectively, attenuated G-MDSC-mediated immunosuppression and reduced bacterial burden in a mouse model of S. aureus PJI. In addition, scRNA-seq analysis of granulocytes from PJI patients also showed an enrichment in glycolysis and hypoxia response genes. These findings support the importance of a glycolysis/HIF-1a axis in promoting G-MDSC anti-inflammatory activity and biofilm persistence during PJI.
Christopher M. Horn, Prabhakar Arumugam, Zachary Van Roy, Cortney E. Heim, Rachel W. Fallet, Blake P. Bertrand, Dhananjay Shinde, Vinai C. Thomas, Svetlana G. Romanova, Tatiana K. Bronich, Curtis W. Hartman, Kevin L. Garvin, Tammy Kielian