Background: Innate immune activation impacts lung transplant outcomes. Dectin-1 is an innate receptor important for pathogen recognition. We hypothesized that genotypes reducing dectin-1 activity would be associated with infection, graft dysfunction, and death in lung transplant recipients. Methods: We assessed the rs16910526 CLEC7A gene polymorphism Y238X, which results in dectin-1 truncation, in 321 lung allograft recipients at a single institution and in 1,129 lung allograft recipients in the multi-center lung transplant outcomes group (LTOG) cohort. Differences in dectin-1 mRNA, cytokines, protein levels, immunophenotypes, and clinical factors were assessed. Results: Y238X carriers had decreased dectin-1 mRNA expression (P = 0.0001), decreased soluble dectin-1 protein concentrations in BAL (P = 0.008) and plasma (P = 0.04), and decreased monocyte surface dectin-1 (P = 0.01) compared to wild type subjects. Y238X carriers had an increased risk of fungal pathogens (HR 1.17, CI 1.0 – 1.4), an increased risk of graft dysfunction or death (HR 1.6, CI 1.0 – 2.6), as well increased mortality in the UCSF cohort (HR 1.8, CI 1.1 – 3.8) and in the LTOG cohort (HR 1.3, CI 1.1 – 1.6), compared to CLEC7A wildtype subjects. Conclusion: Increased rates of graft dysfunction and death associated with this dectin-1 polymorphism may be amplified by immunosuppression that drives higher fungal burden from compromised pathogen recognition. Funding: Project funding came from the UCSF Nina Ireland Program for Lung Health (NIPLH) Innovative Grant program, award number IK2CX001034 from the Clinical Sciences Research & Development Service of the VA Office of Research and Development, and the Joel D. Cooper Career Development Award from the International Society for Heart and Lung Transplantation.
Daniel R. Calabrese, Ping Wang, Tiffany Chong, Jonathan Hoover, Jonathan P. Singer, Dara Torgerson, Steven R. Hays, Jeffrey A. Golden, Jasleen Kukreja, Daniel Dugger, Jason D. Christie, LTOG investigators, John R. Greenland
Islet transplantation can restore lost glycemic control in type 1 diabetes subjects, but is restricted in its clinical application by limiting supplies of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signalling thresholds. Here we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for approximately 45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon regulatory T cells, was antigen-specific and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signalling, graft hyper-inflammation and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators with no impact on glucose stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signalling thresholds to favour immune tolerance and promotes islet allogeneic survival. Clinically this would allow for reduced immunosuppression and support the use of alternate islet sources.
Nathan W. Zammit, Stacey N. Walters, Karen L. Seeberger, Philip J. O’Connell, Gregory S. Korbutt, Shane T. Grey
B7-H4 is a negative regulatory B7 family member. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4–/– versus WT recipients markedly accelerated GVHD-induced lethality. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. Rapid mortality in B7-H4–/– recipients was associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased T effector function (proliferation, proinflammatory cytokines, cytolytic molecules), and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4–/– versus WT recipients required multiple metabolic pathways, increased extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs), and increased expression of fuel substrate transporters. During GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. B7-H4–/– donor T cells given to WT recipients increased GVHD mortality and had function and biological properties similar to WT T cells from allogeneic B7-H4–/– recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4–/– mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.
Asim Saha, Patricia A. Taylor, Christopher J. Lees, Angela Panoskaltsis-Mortari, Mark J. Osborn, Colby J. Feser, Govindarajan Thangavelu, Wolfgang Melchinger, Yosef Refaeli, Geoffrey R. Hill, David H. Munn, William J. Murphy, Jonathan S. Serody, Ivan Maillard, Katharina Kreymborg, Marcel van den Brink, Chen Dong, Shuyu Huang, Xingxing Zang, James P. Allison, Robert Zeiser, Bruce R. Blazar
Tissue engineering is a promising approach to address organ shortages currently limiting clinical transplantation. “Off-the-shelf” engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells basally express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. We report here that CRISPR/Cas9-mediated dual ablation of β2-microglobulin and CIITA in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.
Jonathan Merola, Melanie Reschke, Richard W. Pierce, Lingfeng Qin, Susann Spindler, Tania Baltazar, Thomas D. Manes, Francesc Lopez-Giraldez, Guangxin Li, Laura G. Bracaglia, Catherine Xie, Nancy Kirkiles-Smith, W. Mark Saltzman, Gregory T. Tietjen, George Tellides, Jordan S. Pober
Steroid-refractory intestinal acute graft-versus-host disease (aGVHD) is a frequently fatal condition with little known about mechanisms driving failed steroid responses in gut mucosa. To uncover novel molecular insights in steroid-refractory aGVHD, we compared gene expression profiles of rectosigmoid biopsies from patients at diagnosis of clinical stage 3-4 lower intestinal aGVHD (N=22), to repeat biopsies when the patients became steroid refractory (N=22), and normal controls (N=10). We also performed single gene analyses of factors associated with tolerance (programmed death ligand-1 [PDL1], indoleamine 2,3 dioxygenase [IDO1], and T cell immunoreceptor with Ig and ITIM domains [TIGIT]) and found that significantly higher expression levels of these aGVHD inhibitory genes (PDL1, IDO1, TIGIT) at aGVHD onset became decreased in the steroid-refractory state. We examined genes triggered by microbial ligands to stimulate gut repair, amphiregulin (AREG) and the aryl hydrocarbon receptor (AhR), and found that both AREG and AhR gene expression levels were increased at aGVHD onset and remained elevated in steroid-refractory aGVHD. We also identified higher expression levels of metallothioneines, metal-binding enzymes induced in stress responses, and M2 macrophage genes in steroid-refractory aGVHD. We observed no differences in T-cell subsets between onset and steroid-refractory aGVHD. Patients with a rapidly fatal course showed greater DNA damage and a distinct microbial signature at aGVHD onset, whereas patients with more prolonged survival exhibited a gene expression profile consistent with activation of Smoothened. Our results extend the paradigm beyond T cell-centric therapies for steroid-refractory GI aGVHD and highlight new mechanisms for therapeutic exploration.
Shernan G. Holtan, Ashraf Shabaneh, Brian C. Betts, Armin Rashidi, Margaret L. MacMillan, Celalettin Ustun, Khalid Amin, Byron P. Vaughn, Justin Howard, Alexander Khoruts, Mukta Arora, Todd E. DeFor, Darrell Johnson, Bruce R. Blazar, Daniel J. Weisdorf, Jinhua Wang
Human islet isolation is a cost-/resource-intensive program generating islets for cell therapy in Type 1 diabetes. However, only a third of cadaveric pancreas get to clinical transplantation due to low quality/number of islets. There is a need to identify biomarker(s) that predict the quality of islets, prior to initiating their isolation. Here, we sequenced transcriptome from 18 human islet preparations stratified into three groups (Gr.1: Best quality/transplantable islets, Gr.2: Intermediary quality, Gr.3: Inferior quality/non-transplantable islets) based on routine measurements including islet purity/viability. Machine-learning algorithms involving penalized regression analyses identified 10 long-non-coding(lnc)RNAs significantly different across all group-wise comparisons (Gr1VsGr2, Gr2vsGr3, Gr1vsGr3). Two variants of Metastasis-Associated Lung Adenocarcinoma Transcript-1(MALAT1) lncRNA were common across all comparisons. We confirmed RNA-seq findings in a “validation set” of 75 human islet preparations. Finally, in 19 pancreas samples, we demonstrate that assessing the levels of MALAT1 variants alone (ROC curve AUC: 0.83) offers highest specificity in predicting post-isolation islet quality and improves the predictive potential for clinical islet transplantation when combined with Edmonton Donor Points/Body Mass Index(BMI)/North American Islet Donor Score(NAIDS). We present this resource of islet-quality-stratified lncRNA transcriptome data and identify MALAT1 as a biomarker that significantly enhances current selection methods for clinical (GMP)-grade islet isolation.
Wilson K.M. Wong, Guozhi Jiang, Anja E. Sørensen, Yi Vee Chew, Cody Lee-Maynard, David Liuwantara, Lindy Williams, Philip O’Connell, Louise T. Dalgaard, Ronald C. Ma, Wayne J. Hawthorne, Mugdha V. Joglekar, Anandwardhan A. Hardikar
BACKGROUND. Acute graft-versus-host disease (aGvHD) is a major factor that limits the successful outcomes of allogeneic hematopoietic cell transplantation (alloHSCT). Currently there are few validated biomarkers that can help predict the risk of aGvHD in clinical settings. METHODS. We performed an integrated metabolomics and transcriptomics study and identified biomarkers that distinguish alloHSCT recipients with aGvHD from alloHSCT recipients without aGvHD in two separate cohorts. RESULTS. Pathway analysis of 38 significantly altered metabolites and 1148 differentially expressed genes uncovered a distinctly altered glycerophospholipid (GPL) metabolism network. Subsequently, we developed an aGvHD risk score (GRS) based on 5 metabolites markers from GPL metabolism to predict the risk of aGvHD. GRS showed a positive predictive value of 92.2% and 89.6% in the training and validation cohorts, respectively. In addition, high GRS was correlated with poor overall survival. Gene expressions of GPL-related lipases were significantly altered in aGvHD samples, leading to dysregulated GPLs. CONCLUSIONS. Using integrative “Omic” analysis, we unraveled a comprehensive view of the molecular perturbations underlying the pathogenesis of aGvHD. Our work represents an initial investigation of a unique metabolic and transcriptomic network that may help identify aGvHD at an early stage and facilitate preemptive therapy. FUNDING. National Natural Science Foundation of China (NSFC; 81530047, 81870143, 81470321, 81770160, 81270567, 81270638, 81573396, 81703674). Shanghai Sailing Program from Science and Technology Commission Shanghai Municipality (17YF1424700). Scholarship from Shanghai Municipal Health and Family Planning Commission (2017BR012). Special Clinical Research in Health Industry in Shanghai (20184Y0054).
Yue Liu, Aijie Huang, Qi Chen, Xiaofei Chen, Yang Fei, Xiaoming Zhao, Weiping Zhang, Zhanying Hong, Zhenyu Zhu, Jianmin Yang, Yifeng Chai, Jianmin Wang, Xiaoxia Hu
Solid organ transplantation can treat end-stage organ failure, but the half-life of transplanted organs colonized with commensals is much shorter than that of sterile organs. Whether organ colonization plays a role in this shorter half-life is not known. We have previously shown that an intact whole-body microbiota can accelerate the kinetics of solid organ allograft rejection in untreated colonized mice when compared to germ-free (GF) or to antibiotic-pre-treated colonized mice, by enhancing the capacity of antigen presenting cells (APCs) to activate graft-reactive T cells. However, the contribution of intestinal versus skin microbiota to these effects was unknown. Here, we demonstrate that colonizing the skin of GF mice with a single commensal, Staphylococcus epidermidis (S. epi), while preventing intestinal colonization with oral vancomycin, was sufficient to accelerate skin graft rejection. Notably, unlike the mechanism by which whole-body microbiota accelerates skin graft rejection, cutaneous S. epi did not enhance the priming of alloreactive T cells in the skin-draining lymph nodes (LNs). Rather, cutaneous S. epi augmented the ability of skin APCs to drive the differentiation of alloreactive T cells. This study reveals that the extra-intestinal donor microbiota can affect transplant outcome and may contribute to the shorter half-life of colonized organs.
Yuk Man Lei, Martin Sepulveda, Luqiu Chen, Ying Wang, Isabella Pirozzolo, Betty Theriault, Anita S. Chong, Yasmine Belkaid, Maria-Luisa Alegre
Acute rejection of human allografts has been viewed mostly through the lens of adaptive immunity, and the intragraft landscape of innate immunity genes has not been characterized in an unbiased fashion. We performed RNA sequencing of 34 kidney allograft biopsy specimens from 34 adult recipients; 16 were categorized as Banff acute T cell–mediated rejection (TCMR) and 18 as normal. Computational analysis of intragraft mRNA transcriptome identified significantly higher abundance of mRNA for pattern recognition receptors in TCMR compared with normal biopsies, as well as increased expression of mRNAs for cytokines, chemokines, interferons, and caspases. Intragraft levels of calcineurin mRNA were higher in TCMR biopsies, suggesting underimmunosuppression compared with normal biopsies. Cell-type-enrichment analysis revealed higher abundance of dendritic cells and macrophages in TCMR biopsies. Damage-associated molecular patterns, the endogenous ligands for pattern recognition receptors, as well markers of DNA damage were higher in TCMR. mRNA expression patterns supported increased calcium flux and indices of endoplasmic, cellular oxidative, and mitochondrial stress were higher in TCMR. Expression of mRNAs in major metabolic pathways was decreased in TCMR. Our global and unbiased transcriptome profiling identified heightened expression of innate immune system genes during an episode of TCMR in human kidney allografts.
Franco B. Mueller, Hua Yang, Michelle Lubetzky, Akanksha Verma, John R. Lee, Darshana M. Dadhania, Jenny Z. Xiang, Steven P. Salvatore, Surya V. Seshan, Vijay K. Sharma, Olivier Elemento, Manikkam Suthanthiran, Thangamani Muthukumar
Despite the accepted notion that granulocytes play a universally destructive role in organ and tissue grafts, it has been recently described that eosinophils can facilitate immunosuppression-mediated acceptance of murine lung allografts. The mechanism of eosinophil-mediated tolerance, or their role in regulating alloimmune responses in the absence of immunosuppression, remains unknown. Using lung transplants in a fully MHC-mismatched BALB/c (H2d) to C57BL/6 (H2b) strain combination, we demonstrate that eosinophils downregulate T cell–mediated immune responses and play a tolerogenic role even in the absence of immunosuppression. We further show that such downregulation depends on PD-L1/PD-1–mediated synapse formation between eosinophils and T cells. We also demonstrate that eosinophils suppress T lymphocyte responses through the inhibition of T cell receptor/CD3 (TCR/CD3) subunit association and signal transduction in an inducible NOS–dependent manner. Increasing local eosinophil concentration, through administration of intratracheal eotaxin and IL-5, can ameliorate alloimmune responses in the lung allograft. Thus, our data indicate that eosinophil mobilization may be utilized as a novel means of lung allograft–specific immunosuppression.
Oscar Okwudiri Onyema, Yizhan Guo, Bayan Mahgoub, Qing Wang, Amir Manafi, Zhongcheng Mei, Anirban Banerjee, Dongge Li, Mark H. Stoler, Melissa T. Zaidi, Adam G. Schrum, Daniel Kreisel, Andrew E. Gelman, Elizabeth A. Jacobsen, Alexander Sasha Krupnick
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