Immunology
Abstract
Intestinal acute graft-versus-host disease (aGVHD) is a common life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although tissue-resident memory T (TRM) cells are thought to play a pathophysiological role in animal models of aGVHD, little is known about the role of distinct subsets of TRM cells in human intestinal aGVHD. Herein, we combined multiplex immunohistochemical staining with single-cell RNA sequencing to elucidate the differentiation trajectory, lineage commitment, clonal expansion, and functional properties of distinct CD8+ TRM cell subsets in human intestinal aGVHD. We identified a predominant GZMK+CD8+ TRM subset, characterized by the GZMK and CD49A markers. Intestinal aGVHD was associated with infiltration of GZMK+CD8+ T cells with TRM features, which showed enhanced clonal expansion, IFN signaling pathway–associated proinflammatory pathway expression, and lineage bifurcation differentiation properties. High GZMK+CD8+ TRM subset infiltration was associated with greater human intestinal aGVHD severity and poor prognosis. Together, our studies highlight the importance of the GZMK+CD8+ TRM subset in human intestinal aGVHD, and interest for designing GZMK+CD8+ TRM cell–targeted therapies.
Authors
Yiming Sun, Yutong Xue, Chenyuyao Song, Xinyu Liu, Ruoyang Shao, Zhiping Fan, Ren Lin, Fen Huang, Na Xu, Li Xuan, Min Dai, Jing Sun, Qifa Liu, Hua Jin
Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED/APS-1) is a monogenic autoimmune disorder of impaired central tolerance classically diagnosed by the presence of 2 out of 3 classic triad manifestations: chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency. However, many patients develop non-triad manifestations years earlier, delaying recognition and care. In 2016, we proposed expanded diagnostic criteria incorporating 3 early clinical manifestations — APECED rash, autoimmune enteritis, and enamel hypoplasia — based on observations in 35 North American patients. Here, we provide further support for the clinical utility of these expanded diagnostic criteria in independent cohorts of 57 American and 12 European patients enrolled in a prospective natural history study at the NIH. Across all cohorts, the expanded diagnostic criteria decreased the time to diagnosis by half relative to the classic diagnostic criteria. Patients exhibited an enrichment of early non-endocrine autoimmune manifestations, underscoring disease heterogeneity and the potential for developing organ-specific autoimmunity before endocrine failure. These findings demonstrate the clinical utility of the expanded APECED diagnostic criteria and support the notion that their adoption might enable earlier disease recognition and timely immunomodulatory therapy to improve long-term outcomes.
Authors
Elise M.N. Ferré, Joseph Pechacek, Monica M. Schmitt, Taura Webb, Heather Moorman, Thomas DiMaggio, Princess Barber, Vasielios Oikonomou, Stacey R. Rose, Peter D. Burbelo, Lindsey B. Rosen, Amy P. Hsu, Jennifer Stoddard, Shakuntala Rampertaap, Sergio D. Rosenzweig, Anjali Rai, Maria Teresa Magone, Chantal Cousineau-Krieger, Niki M. Moutsopoulos, Pamela J. Gardner, Heidi H. Kong, Leslie Castelo-Soccio, Ariane Soldatos, Katherine R. Calvo, Meryl Waldman, Behdad Afzali, Stefania Pittaluga, David Kleiner, Steven M. Holland, Kevin Fennelly, Jill Rothschild, Bryce A. Seifert, Magdalena Walkiewicz-Yvon, Theo Heller, Karen Winer, Michail S. Lionakis
Abstract
Systemic sclerosis (SSc) is a rare autoimmune disease characterized by vasculopathy and fibrosis of the skin and internal organs. Individuals with SSc often suffer from chronic acid reflux and dysphagia due to loss of esophageal motility. However, the pathogenesis of esophageal dysmotility in SSc is poorly understood. To determine whether distinct changes in esophageal epithelial cells contribute to esophageal involvement in SSc, we investigated the stratified squamous esophageal epithelium from proximal and distal biopsies using single-cell RNA sequencing (n=306,372 cells) in individuals with SSc compared those with gastroesophageal reflux disease (GERD) and healthy controls. The proportion of epithelial cells in the apical, superficial compartment of the esophageal epithelium was reduced in SSc (9.4% vs 21.6% in HCs). Differential gene expression in SSc was primarily limited to the superficial compartment (3,572 genes vs. 232 in all other compartments, based on pseudobulk analysis), with significant upregulation of extracellular matrix and keratinization genes. These cellular and molecular changes in SSc were highly correlated with those seen in GERD, indicating they were secondary to reflux; however, their magnitudes were more pronounced in the proximal esophagus, suggesting that esophageal dysmotility leads to greater proximal acid exposure, which may contribute to aspiration. SSc-specific gene dysregulation implicated immunoregulatory pathways likely pertinent to pathogenic mechanisms. Ligand-receptor interaction analysis revealed enhanced pro-fibrotic signaling between fibroblasts and epithelial cells in SSc. Cell type localization and SSc-specific changes were confirmed by spatial molecular imaging. By offering a comprehensive view of transcriptional dysregulation at single-cell resolution in human esophageal epithelial cells in SSc compared to GERD and healthy tissue, this work clarifies the state of epithelial cells in SSc-induced esophageal dysfunction.
Authors
Matthew Dapas, Margarette H. Clevenger, Hadijat-Kubura M. Makinde, Tyler Therron, Dustin A. Carlson, Mary Carns, Kathleen Aren, Cenfu Wei, Kainat Mian, Lutfiyya N. Muhammad, Carrie L. Richardson, Parambir S Dulai, Monique Hinchcliff, John E. Pandolfino, Harris R. Perlman, Deborah R. Winter, Marie-Pier Tetreault
Microbiome-Derived Metabolites Shape CD4⁺ T-Cell Differentiation and Immune Aging in HIV-1 Infection
Abstract
The role of aromatic gut-derived bacterial metabolites (GDBMs) in shaping immune cell metabolism and function remains poorly explored. Using ex vivo metabolomic profiling of paired plasma and CD4⁺ T-cells from people living with HIV-1 (PLWH), we identified a network of aromatic GDBMs whose cell-associated abundance, rather than systemic levels, was linked to broad alterations in CD4⁺ T-cell metabolic and functional states. Among these, p-cresol sulfate (PCS) emerged as a mechanistic prototype. Ex vivo flow cytometry and single-cell RNA sequencing of CD4⁺ T-cells stratified by cell-associated PCS levels revealed dose-dependent enrichment of transcriptional programs associated with impaired differentiation, regulatory-like identity, and cellular senescence. In vitro transcriptomic and proteomic analyses of PCS-exposed CD4⁺ T cells demonstrated induction of cell-cycle arrest, mitochondrial dysfunction, and senescence-associated programs, including upregulation of p16 and p21. Integration of these immunometabolic findings with HIV-1 reservoir measurements revealed that CD4⁺ T-cell states defined by cell-associated GDBMs track with intact proviral DNA levels in vivo. These findings define a microbiome-derived axis that reshapes CD4⁺ T-cell metabolism and fate, promotes immune aging in PLWH, and may foster immunometabolic states linked to long-term HIV-1 reservoir persistence.
Authors
Amanda Cabral da Silva, Luke Flantzer, Jaclyn Weinberg, Shuya Kyu, Lisa P. Daley-Bauer, Anyce Godoy, Ana Carolina Santana, Aarthi Talla, Amber Rittgers, Sarah Welbourn, David E. Gordon, Jeffrey A. Tomalka, Vincent C. Marconi, Dean P. Jones, Souheil-Antoine Younes
Abstract
Myeloid-Derived Suppressor Cells (MDSCs) represent a heterogeneous population of immature myeloid cells with potent immunosuppressive capabilities that contribute to viral persistence in chronic infections. However, their direct impact on the latent HIV reservoir remains poorly understood. Here, we report that people with HIV (PWH) exhibit elevated levels of MDSCs with notable immunosuppressive activity. Both granulocytic (G-MDSCs) and monocytic (M-MDSCs) subsets expressing arginase 1 (ARG1) or indoleamine 2,3-dioxygenase (IDO) are increased during treated infection, with low-level viral transcription preferentially associated with the expansion of highly suppressive G-MDSCs. Functional assays revealed that G-MDSCs robustly inhibit HIV reactivation from latent reservoirs. Mechanistically, G-MDSCs mediate this inhibition through a contact-independent mechanism, primarily involving ARG1 activity. Our findings demonstrate the capacity of G-MDSCs to sustain HIV reservoirs, suggesting that targeting these cells could potentiate therapeutic strategies aimed at eliminating HIV reservoirs through viral reactivation.
Authors
Ana Gallego-Cortés, Judith Grau-Expósito, Irene Mota-Gómez, Aleix Benitez-Martinez, Josep Castellvi, Jordi Navarro, Adrian Curran, Joaquin Burgos, Paula Suanzes, Vicenç Falcó, Meritxell Genescà, Maria J. Buzon
Abstract
Authors
Muhammad Atif Rauf, Sanskriti Agarwal, Rebecca R. Baker, Jennifer Steeden, Alfredo Petrosino, Maria Kiliaris, Robert Unwin, Keith Siew, Alan D. Salama, Rhys D.R. Evans
Abstract
Chronic Graft-Versus-Host disease (cGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic transplantation. CGVHD pathophysiology involves cooperation between Tfollicular helper cells (TFH) and germinal center B-cells (GCB), allo- and auto-antibody depositions in cGVHD tissues, and fibrosis. We evaluated human CD19-directed chimeric antigen receptor (CAR19) T-cell therapy in a clinically relevant murine cGVHD model with bronchiolitis obliterans syndrome (BOS). Although CD8 CAR19 T-cells effectively reduced peripheral B-cell and GCB frequencies, pulmonary function was unimproved. In contrast, a single CAR19 CD4 regulatory T-cells (Treg) infusion mitigated ongoing pulmonary disease and modulated germinal centers (GC) associated with reduced TFH frequencies compared to control Tregs but without measurable B-cell depletion. Compared to EGFR Treg infusion, mice receiving CAR19 Tregs exhibited enhanced suppression of B-cell activation, preserved splenic architecture, and provided greater opportunities for interaction with CD19+ B-cells at the B-cell follicle boundary zones. Taken together with the absence of detectable B-cell cytolysis, these findings are most consistent with GC suppression rather than B-cell depletion as the dominant mechanism. Overall, our findings suggest that CAR19 Tregs represent a promising and safe cGVHD/BOS therapeutic strategy, offering immunosuppressive benefits and improved disease outcomes that may be more limited with CD8 CAR19 T-cell treatment.
Authors
Sujeong Jin, Michael C. Zaiken, Cameron McDonald-Hyman, Christina R. Hartigan, Sara Bolivar-Wagers, Jemma H. Larson, Yiyun Peng, Sophia Hani, Megan Riddle, Asim Saha, Angela Panoskaltsis-Mortari, Eun Ko, Yujie Zhao, Rocio Amaro Marquez, Pooja Shree Marri Baskar, Cindy R. Eide, William J. Murphy, Keli L. Hippen, Geoffrey R. Hill, Jakub Tolar, Peter T. Sage, Christopher A. Pennell, Leslie S. Kean, Bruce R. Blazar
Abstract
Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, standard PBMC isolation from blood requires technical expertise and over two hours of onsite processing using Ficoll density gradient separation (‘Ficoll’) for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of immediate onsite whole blood cryopreservation and subsequent batched PBMC isolation in a central laboratory prior to sequencing. We compared multimodal single-cell immune profiling results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical outputs including cell substate fractions, marker genes, and surface protein expression were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis. Capture of T cell receptor transcripts was also comparable across both methods. Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding multimodal single-cell analyses in multicenter studies of sepsis and other diseases.
Authors
Alyssa K. DuBois, Pierre O. Ankomah, Alexis C. Campbell, Renee Hua, Olivia K. Nelson, Christopher A. Zeuthen, M. Kartik Das, Shira Mann, Abigail Mauermann, Blair A. Parry, Nathan I. Shapiro, Michael R. Filbin, Roby P. Bhattacharyya
Abstract
Post-acute sequelae of SARS-CoV-2 (PASC) occurs in subsets of individuals, including those with pre-existing lung disease. To investigate PASC pathogenesis and therapeutics in a chronic bronchitis mouse model (Scnn1b-Tg), Scnn1b-Tg and WT mice were inoculated with a mouse adapted SARS-CoV-2 virus (SARS-CoV-2MA10) and followed for 60 days. Viral titer, histology, immunohistochemistry (IHC), single-cell RNA sequencing, RNA in situ hybridization, and spatial transcriptomic profiling characterized disease pathologies. Scnn1b-Tg mice inoculated with SARS-CoV-2MA10 exhibited lower viral titers and less weight loss than WT mice. Airway epithelia of Scnn1b-Tg mice were less infected than epithelia of WT mice, reflecting increased airway mucus and enhanced epithelial antiviral activities in Scnn1b-Tg mice. However, Scnn1b-Tg mice subsequently exhibited heterogeneous airway and parenchymal disease with elevated Il33 expression characteristic of human eosinophilic pneumonia. Cohorts of infected mice were administered a monoclonal antibody targeting the IL-33 receptor (ST2) or enteral prednisone. Administration of an anti-ST2 monoclonal antibody mitigated development of eosinophilic pneumonia while enteral prednisone suppressed IL33 expression and disease. The eosinophilic pneumonia in Scnn1b-Tg mice after SARS-CoV-2MA10 infection mimics reports of eosinophilic pneumonia in humans post-SARS-CoV-2, suggesting targeting of IL-33 may be beneficial in treating post-viral eosinophilic pneumonia in humans.
Authors
Padraig E. Hawkins, Sarah R. Leist, Hong Dang, Minako Saito, Lisa C. Morton, Rodney C. Gilmore, Stephen A. Schworer, Ella F. Burns, Jason R. Rock, Robert S. Hagan, James J. Pestka, Alexandra Schäfer, Kenichi Okuda, Lauren K. Heine, Jack R. Harkema, Wanda K. O'Neal, Alessandra Livraghi-Butrico, Raymond J. Pickles, Ralph S. Baric, Richard C. Boucher
Abstract
Extracellular vesicles (EVs)-mediated inter-organ communication represents a promising frontier in transplant immunology; however, its role in cardiac allograft rejection remains poorly characterized. We performed proteomic profiling of plasma-derived EVs in a rat heterotopic heart transplantation model and identified a distinct liver-predominant protein signature during acute rejection, with Antithrombin III (ATIII) emerging as a top candidate. Functional validation revealed that pharmacological EV inhibition intensified systemic and intragraft inflammation, whereas adeno-associated virus (AAV)-mediated silencing of hepatic ATIII directly accelerated allograft rejection. Conversely, AAV-mediated hepatocyte-specific ATIII overexpression attenuated rejection pathology, reduced immune cell recruitment, and markedly prolonged median graft survival. This protective effect was achieved without evidence of coagulopathic complications, indicating an immunomodulatory mechanism beyond ATIII’s canonical anticoagulant function. Mechanistically, ATIII overexpression was associated with upregulation of heme oxygenase-1 (HO-1) in the liver and suppression of proinflammatory cytokine expression in the graft. These findings highlight hepatocyte-derived EVs as important mediators of a liver-heart signaling axis in transplant rejection, and further implicate the protein ATIII as a contributor to this axis. Our study reveals a therapeutically targetable liver-heart signaling axis in transplant rejection, whereby enhancing liver-derived ATIII or its downstream pathways (such as HO-1) could attenuate acute cardiac allograft rejection.
Authors
Shiyu Dai, Wei Zhou, Fangyu Chen, Huanyu Zhang, Zhenchun Ji, Xuejing Zong, Wanruo Zhang, Jie Hu, Shumin Jiang, Fei Wang, Zhenya Shen
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