Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent anti-inflammatory and anti-tumor activity in several pre-clinical models and has proven both safe and efficacious in clinical trials for advanced gastro-intestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared to cyclophosphamide post-aHSCT (PTCy). Strikingly, we found Minnelide improved survival, weight loss and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score and colonic length. Notably, in addition to decreased donor T cell infiltration early post-HSCT, several regulatory cell populations including Tregs, ILC2s and MDSCs in the colon were increased which together may account for Minnelide’s GVHD suppression post-HSCT. Importantly, Minnelide GVHD prevention was accompanied by preservation of graft-versus-tumor (GVT) activity. As Minnelide possesses anti-AML activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for AML patients undergoing aHSCT.
Sabrina N. Copsel, Vanessa T. Garrido, Henry Barreras, Cameron S. Bader, Brent Pfeiffer, Beatriz Mateo-Victoriano, Dietlinde Wolf, Miguel Gallardo, Sophie Paczesny, Krishna V. Komanduri, Cara L. Benjamin, Alejandro Villarino, Ashok K. Saluja, Robert B. Levy
CD4+Foxp3+ regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127hi Tregs with memory features were found in lymphoid organs, presented in high proportions within long-surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory-like CD127hi Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag–/– hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL-7 to the CD127hi Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory-like CD127hi Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.
Yuanfei Zhao, Leigh Nicholson, Hannah Wang, Yi Wen Qian, Wayne J. Hawthorne, Elvira Jimenez-Vera, Brian S. Gloss, Joey Lai, Adwin Thomas, Yi Vee Chew, Heather Burns, Geoff Y. Zhang, Yuan Min Wang, Natasha M. Rogers, Guoping Zheng, Shounan Yi, Stephen I. Alexander, Philip J. O’Connell, Min Hu
While the function of many leukocytes in transplant biology has been well defined, the role of eosinophils is controversial and remains poorly explored. Conflicting data exist regarding eosinophils’ role in alloimmunity. Due to their prevalence in the lung, and their defined role in other pulmonary pathologies such as asthma, we set out to explore the role of eosinophils in the long-term maintenance of the lung allograft. We noted that depletion of eosinophils results in the generation of donor-specific antibodies. Eosinophil depletion increased memory B cell, plasma cell, and antibody-secreting cell differentiation and resulted in de novo generation of follicular germinal centers. Germinal center formation depended on the expansion of CD4+Foxp3–Bcl6+CXCR5+PD-1+ T follicular helper (Tfh) cells, which increase in number after eosinophil depletion. Mechanistically, we demonstrate that eosinophils prevent Tfh cell generation by acting as the dominant source of IFN-γ in an established lung allograft, thus facilitating Th1 rather than Tfh polarization of naive CD4+ T cells. Our data thus describe what we believe is a unique and previously unknown role for eosinophils in maintaining allograft tolerance and suggest that indiscriminate administration of eosinophil-lytic corticosteroids for treatment of acute cellular rejection may inadvertently promote humoral alloimmunity.
Zhongcheng Mei, May A. Khalil, Yizhan Guo, Dongge Li, Anirban Banerjee, Yuriko Terada, Yuhei Yokoyama, Christina Kratzmeier, Kelly Chen, Lushen Li, Christine L. Lau, Jean-Paul Courneya, Irina G. Luzina, Sergei P. Atamas, Andrew E. Gelman, Daniel Kreisel, Elizabeth A. Jacobsen, Alexander S. Krupnick
Although cold preservation remains the gold standard in organ transplantation, cold stress–induced cellular injury is a significant problem in clinical orthotopic liver transplantation (OLT). Because a recent study showed that cold stress activates ferroptosis, a form of regulated cell death, we investigated whether and how ferroptosis determines OLT outcomes in mice and humans. Treatment with ferroptosis inhibitor (ferrostatin-1) during cold preservation reduced lipid peroxidation (malondialdehyde; MDA), primarily in liver sinusoidal endothelial cells (LSECs), and alleviated ischemia/reperfusion injury in mouse OLT. Similarly, ferrostatin-1 reduced cell death in cold-stressed LSEC cultures. LSECs deficient in nuclear factor erythroid 2-related factor 2 (NRF2), a critical regulator of ferroptosis, were susceptible to cold stress–induced cell death, concomitant with enhanced endoplasmic reticulum (ER) stress and expression of mitochondrial Ca2+ uptake regulator (MICU1). Indeed, supplementing MICU1 inhibitor reduced ER stress, MDA expression, and cell death in NRF2-deficient but not WT LSECs, suggesting NRF2 is a critical regulator of MICU1-mediated ferroptosis. Consistent with murine data, enhanced liver NRF2 expression reduced MDA levels, hepatocellular damage, and incidence of early allograft dysfunction in human OLT recipients. This translational study provides a clinically applicable strategy in which inhibition of ferroptosis during liver cold preservation mitigates OLT injury by protecting LSECs from peritransplant stress via an NRF2-regulatory mechanism.
Hidenobu Kojima, Hirofumi Hirao, Kentaro Kadono, Takahiro Ito, Siyuan Yao, Taylor Torgerson, Kenneth J. Dery, Hiroaki Kitajima, Takahiro Ogawa, Fady M. Kaldas, Douglas G. Farmer, Jerzy W. Kupiec-Weglinski
Adoptive transfer of immune regulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of non-relapse mortality after allogeneic hematopoietic stem-cell transplantation. Mucosal-associated invariant T cells (MAITs) were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAITs in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic-GVHD model. We found that MAIT cells, whether freshly purified or shortly expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAITs strongly delayed GVHD onset and severity when transferred early after PBMC injection, but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels, and increased IL-10 levels. MAITs acted partly in a contact-dependent manner, which likely required direct interaction of their TCR with MR1 induced on host-reactive T cells. These results support the setup of clinical trials using MAITs as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.
Nana Talvard-Balland, Marion Lambert, Mathieu F. Chevalier, Norbert Minet, Marion Salou, Marie Tourret, Armelle Bohineust, Idan Milo, Véronique Parietti, Thomas Yvorra, Gérard Socié, Olivier Lantz, Sophie Caillat-Zucman
Innate immune cells are important in the initiation and potentiation of alloimmunity in transplantation. Immediately upon organ anastomosis and reperfusion, recipient monocytes enter the graft from circulation and differentiate to inflammatory macrophages to promote allograft inflammation. However, factors that drive their differentiation to inflammatory macrophages are not understood. Here, we showed that the receptor tyrosine kinase AXL was a key driver of early intragraft differentiation of recipient infiltrating monocytes to inflammatory macrophages in the presence of allogeneic stimulation and cell-cell contact. In this context, the differentiated inflammatory macrophages were capable of efficient alloantigen presentation and allo-stimulation of T cells of the indirect pathway. Consequently, early and transient AXL inhibition with the pharmacological inhibitor bemcentinib resulted in a profound reduction of initial allograft inflammation and a significant prolongation of allograft survival in a murine heart transplant model. Our results support further investigation of AXL inhibition as part of an induction regimen for transplantation.
Collin Z. Jordan, Matthew Tunbridge, Irma Husain, Hiroki Kitai, Miriam E. Dilts, Olivia K. Fay, Koki Abe, Catherine Xiang, Jean Kwun, Tomokazu Souma, Edward B. Thorp, Xunrong Luo
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
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