Chronic lung allograft dysfunction (CLAD) is a major complication after lung transplantation that results from a complex interplay of innate inflammatory and alloimmune factors, culminating in parenchymal and/or obliterative airway fibrosis. Excessive IL-17A signaling and chronic inflammation have been recognized as key factors in these pathological processes. Herein, we developed a model of repeated airway inflammation in mouse minor alloantigen-mismatched single-lung transplantation. Repeated intratracheal LPS instillations augmented pulmonary IL-17A expression. LPS also increased acute rejection, airway epithelial damage, and obliterative airway fibrosis, similar to human explanted lung allografts with antecedent episodes of airway infection. We then investigated the role of donor and recipient IL-17 receptor A (IL-17RA) in this context. Donor IL-17RA deficiency significantly attenuated acute rejection and CLAD features, whereas recipient IL-17RA deficiency only slightly reduced airway obliteration in LPS allografts. IL-17RA immunofluorescence positive staining was greater in human CLAD lungs compared with control human lung specimens, with localization to fibroblasts and myofibroblasts, which was also seen in mouse LPS allografts. Taken together, repeated airway inflammation after lung transplantation caused local airway epithelial damage, with persistent elevation of IL-17A and IL-17RA expression and particular involvement of IL-17RA on donor structural cells in development of fibrosis.
Tatsuaki Watanabe, Stephen C. Juvet, Gregory Berra, Jan Havlin, Wenshan Zhong, Kristen Boonstra, Tina Daigneault, Miho Horie, Chihiro Konoeda, Grace Teskey, Zehong Guan, David M. Hwang, Mingyao Liu, Shaf Keshavjee, Tereza Martinu
Regulatory T cells (Treg) have potential for the treatment of autoimmune diseases and graft rejection. Antigen-specificity and functional stability are considered to be critical for their therapeutic efficacy. In this study, expansion of human Treg in the presence of porcine PBMC (Xn-Treg) allowed the selection of a distinct Treg subset, co-expressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared to their unsorted and HLA-DA+CD27+ double positive (DP) cell depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-Enriched Xn-Treg expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal MLR. They also had less methylated Treg-specific demethylated region (TSDR) of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD-SCID IL2 receptor γ–/– (NSG) mice with HLA-DR+CD27+ DP-Enriched Xn-Treg in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Treg and upregulated expression of Treg functional genes, IL10 and CTLA4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-Enriched Xn-Treg expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Treg with potential as a Treg-based therapy.
Xiaoqian Ma, Lu Cao, Martina Raneri, Hannah Wang, Qi Cao, Yuanfei Zhao, Naiara G. Bediaga, Gaetano Naselli, Leonard C. Harrison, Wayne J. Hawthorne, Min Hu, Shounan Yi, Philip J. O’Connell
Follicular helper T (Tfh) cells have been implicated in controlling rejection after allogeneic kidney transplantation, but the precise subsets, origins, and functions of Tfh cells in this process have not been fully characterized. Here we show that a subset of effector Tfh cells marked by previous IL-21 production is potently induced during allogeneic kidney transplantation and is inhibited by immunosuppressive agents. Single-cell RNA-Seq revealed that these lymph node (LN) effector Tfh cells have transcriptional and clonal overlap with IL-21–producing kidney-infiltrating Tfh cells, implicating common origins and developmental trajectories. To investigate the precise functions of IL-21–producing effector Tfh cells in LNs and allografts, we used a mouse model to selectively eliminate these cells and assessed allogeneic B cell clonal dynamics using a single B cell culture system. We found that IL-21–producing effector Tfh cells were essential for transplant rejection by regulating donor-specific germinal center B cell clonal dynamics both systemically in the draining LN and locally within kidney grafts. Thus, IL-21–producing effector Tfh cells have multifaceted roles in Ab-mediated rejection after kidney transplantation by promoting B cell alloimmunity.
Hengcheng Zhang, Cecilia B. Cavazzoni, Manuel A. Podestà, Elsa D. Bechu, Garyfallia Ralli, Pragya Chandrakar, Jeong-Mi Lee, Ismail Sayin, Stefan G. Tullius, Reza Abdi, Anita S. Chong, Bruce R. Blazar, Peter T. Sage
Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independent of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.
Jesse Santos, Ping Wang, Avishai Shemesh, Fengchun Liu, Tasha Tsao, Oscar A. Aguilar, Simon J. Cleary, Jonathan P. Singer, Ying Gao, Steven R. Hays, Jeffrey Golden, Lorriana E. Leard, Mary Ellen Kleinhenz, Nicholas A. Kolaitis, Rupal J. Shah, Aida Venado, Jasleen Kukreja, S. Sam Weigt, John A. Belperio, Lewis L. Lanier, Mark R. Looney, John R. Greenland, Daniel R. Calabrese
Tissue-resident lymphocytes (TRLs) are critical for local protection against viral pathogens in peripheral tissue. However, it is unclear if TRLs perform a similar role in transplanted organs under chronic immunosuppressed conditions. The present study aimed to characterize the TRL compartment in human kidney transplant nephrectomies and examine its potential role in antiviral immunity. The TRL compartment of kidney transplants contained diverse innate(-like) and adaptive TRL populations expressing the canonical residency markers CD69, CD103, and CD49a. Chimerism of donor and recipient cells was present in 43% of kidney transplants and occurred in all TRL subpopulations. Paired single-cell transcriptome and T-cell receptor (TCR) sequencing showed that donor and recipient tissue-resident memory T (TRM) cells exhibit striking similarities in their transcriptomic profiles and share numerous TCR clonotypes predicted to target viral pathogens. Virus dextramer staining further confirmed that CD8 TRM cells of both donor and recipient origin cexpress TCRs with specificities against common viruses, including CMV, EBV, BK polyomavirus, and influenza A. Overall, the study results demonstrate that a diverse population of TRLs resides in kidney transplants and offer compelling evidence that TRM cells of both donor and recipient origin reside within this TRL population and may contribute to local protection against viral pathogens.
Daphne M. Hullegie-Peelen, Hector Tejeda Mora, Dennis A. Hesselink, ERIC M.J. BINDELS, Thierry Paulus Pierre van den Bosch, Marian C. Clahsen-van Groningen, Marjolein Dieterich, Sebastiaan Heidt, Robert C. Minnee, Georges M.G.M. Verjans, Martin J. Hoogduijn, Carla C. Baan
Regulatory T cells (Tregs) expressing Chimeric Antigen Receptors (CARs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs) and donor-specific antibodies. We generated Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, PD1 and GITR, but not 41BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no co-stimulatory domain. These data revealed that exogenous co-stimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti-HLA-A2 alloantibodies. This study reveals a new dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.
Isaac Rosado-Sánchez, Manjurul Haque, Kevin Salim, Madeleine Speck, Vivian C.W. Fung, Dominic A. Boardman, Majid Mojibian, Giorgio Raimondi, Megan K. Levings
Posttransplant cyclophosphamide (PTCy) is associated with a low incidence of chronic graft-versus-host disease (cGVHD) following hematopoietic stem cell (HSC) transplantation. Previous studies have shown the important roles of B cell immunity in cGVHD development. Here, we investigated the long-term reconstitution of B lymphopoiesis after PTCy using murine models. We first demonstrated that the immune homeostatic abnormality leading to cGVHD is characterized by an initial increase in effector T cells in the bone marrow and subsequent B and Treg cytopenia. PTCy, but not cyclosporine A or rapamycin, inhibits the initial alloreactive T cell response, which restores intra-bone marrow B lymphogenesis with a concomitant vigorous increase in Tregs. This leads to profound changes in posttransplant B cell homeostasis, including decreased B cell activating factors, increased transitional and regulatory B cells, and decreased germinal center B cells. To identify the cells responsible for PTCy-induced B cell tolerance, we selectively depleted Treg populations that were graft or HSC derived using DEREG mice. Deletion of either Treg population without PTCy resulted in critical B cytopenia. PTCy rescued B lymphopoiesis from graft-derived Treg deletion. In contrast, the negative effect of HSC-derived Treg deletion could not be overcome by PTCy, indicating that HSC-derived Tregs are essential for maintaining favorable B lymphopoiesis following PTCy. These findings define the mechanisms by which PTCy restores homeostasis of the B cell lineage and reestablishes immune tolerance.
Yuichi Sumii, Takumi Kondo, Shuntaro Ikegawa, Takuya Fukumi, Miki Iwamoto, Midori Filiz Nishimura, Hiroyuki Sugiura, Yasuhisa Sando, Makoto Nakamura, Yusuke Meguri, Takashi Matsushita, Naoki Tanimine, Maiko Kimura, Noboru Asada, Daisuke Ennishi, Yoshinobu Maeda, Ken-ichi Matsuoka
BACKGROUND. Currently, no laboratory tests exist to stratify for the risk of developing sinusoidal obstruction syndrome (SOS), an early endothelial complication after hematopoietic cell transplantation (HCT). Risk biomarkers of SOS have not been verified in a prospective cohort accounting for differences between practices across institutions. Herein, we aimed to define risk groups for SOS occurrence using three proteins: L-Ficolin, Hyaluronic Acid (HA), and Stimulation-2 (ST2). METHODS. Between 2017 to 2021, we prospectively accrued 80 pediatric patients across 4 US centers. Biomarkers were tested by ELISA blind to patient groupings and associated with SOS incidence at day 35 post-HCT, and overall survival (OS) at day 100 post-HCT. Cutpoints were identified using retrospective cohorts and applied to the prospective cohort. RESULTS. Combination of the three biomarkers measured at day 3 post-HCT in the prospective cohort provided 80% (95%CI, 55-100%) sensitivity and 73% (95%CI, 62-83%) specificity for risk of SOS occurrence. Patients with low L-Ficolin were 9 times (95%CI 3-32) more likely to develop SOS, while patients with high HA and ST2 were 6.5 (95%CI 1.9-22.0) and 5.5 (95%CI 2.3-13.1) times more likely to develop SOS. These three markers also predicted worse day 100 OS [L-Ficolin: HR, 10.0 (95%CI 2.2-45.1), P=0.0002; HA: HR, 4.1 (95%CI 1.0-16.4), P=0.031; ST2: HR, 3.9 (95%CI 0.9-16.4), P=0.04]. CONCLUSION. L-Ficolin, HA, and ST2 levels measured as early as three days post-HCT improved risk stratification for SOS occurrence and OS and may guide risk-adapted preemptive therapy. TRIAL REGISTRATION. ClinicalTrials.gov NCT03132337. FUNDING. NICHD P50HD090215, R01HD074587, NCI R01CA168814 and NHLBI K24HL156896.
Yan Han, Alan Bidgoli, Brittany P. DePriest, Alejandra Méndez, Khadijeh Bijangi-Vishehsaraei, Evelio D. Perez-Albuerne, Robert A. Krance, Jamie Renbarger, Jodi L. Skiles, Sung W. Choi, Hao Liu, Sophie Paczesny
Chronic lung allograft dysfunction (CLAD) is the leading cause of death in lung transplant recipients. CLAD is characterized clinically by a persistent decline in pulmonary function and histologically by the development of airway-centered fibrosis known as bronchiolitis obliterans. There are no approved therapies to treat CLAD, and the mechanisms underlying its development remain poorly understood. We performed single-cell RNA-Seq and spatial transcriptomic analysis of explanted tissues from human lung recipients with CLAD, and we performed independent validation studies to identify an important role of Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling in airway epithelial cells that contributes to airway-specific alloimmune injury. Specifically, we established that activation of JAK-STAT signaling leads to upregulation of major histocompatibility complex 1 (MHC-I) in airway basal cells, an important airway epithelial progenitor population, which leads to cytotoxic T cell–mediated basal cell death. This study provides mechanistic insight into the cell-to-cell interactions driving airway-centric alloimmune injury in CLAD, suggesting a potentially novel therapeutic strategy for CLAD prevention or treatment.
Aaditya Khatri, Jamie L. Todd, Fran L. Kelly, Andrew Nagler, Zhicheng Ji, Vaibhav Jain, Simon G. Gregory, Kent J. Weinhold, Scott M. Palmer
Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium–induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.
Erica Dander, Paola Vinci, Stefania Vetrano, Camilla Recordati, Rocco Piazza, Grazia Fazio, Donatella Bardelli, Mattia Bugatti, Francesca Sozio, Andrea Piontini, Sonia Bonanomi, Luca Bertola, Elena Tassistro, Maria Grazia Valsecchi, Stefano Calza, William Vermi, Andrea Biondi, Annalisa Del Prete, Silvano Sozzani, Giovanna D’Amico
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