Tregs hold great promise as a cellular therapy for multiple immunologically mediated diseases, given their ability to control immune responses. The success of such strategies depends on the expansion of healthy, suppressive Tregs ex vivo and in vivo following the transfer. In clinical studies, levels of transferred Tregs decline sharply in the blood within a few days of the transfer. Tregs have a high rate of apoptosis. Here, we describe a new mechanism of Treg self-inflicted damage. We show that granzymes A and -B (GrA and GrB), which are highly upregulated in human Tregs upon stimulation, leak out of cytotoxic granules to induce cleavage of cytoplasmic and nuclear substrates, precipitating apoptosis in target cells. GrA and GrB substrates were protected from cleavage by inhibiting granzyme activity in vitro. Additionally, we show — by using cytometry by time of flight (CYTOF) — an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed an activated phenotype but were significantly more apoptotic than non–GrB expressing Tregs. This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.
Esilida Sula Karreci, Siawosh K. Eskandari, Farokh Dotiwala, Sujit K. Routray, Ahmed T. Kurdi, Jean Pierre Assaker, Pavlo Luckyanchykov, Albana B. Mihali, Omar Maarouf, Thiago J. Borges, Abdullah Alkhudhayri, Kruti R. Patel, Amr Radwan, Irene Ghobrial, Martina McGrath, Anil Chandraker, Leonardo V. Riella, Wassim Elyaman, Reza Abdi, Judy Lieberman, Jamil Azzi
It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker–tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemically distinguishable variant of the marker human placental alkaline phosphatase (ALPP). In this dual transgenic system, donor lines ubiquitously express WT, heat-resistant ALPP protein, whereas recipient lines express a heat-labile ALPP mutant (ALPPE451G) resulting from a single amino acid substitution. Tolerance of recipient lines to ALPP-expressing cells and tissues was verified by skin transplantation. Using this model, we show that intraarticularly injected MSC contribute to regeneration of articular cartilage in full-thickness cartilage defects mainly via a nonprogenitor-mediated mechanism.
Daniela Zwolanek, María Satué, Verena Proell, José R. Godoy, Kathrin I. Odörfer, Magdalena Flicker, Sigrid C. Hoffmann, Thomas Rülicke, Reinhold G. Erben
Cellular therapies based on permanent genetic modification of conventional T cells have emerged as a promising strategy for cancer. However, it remains unknown if modification of T cell subsets, such as Tregs, could be useful in other settings, such as allograft transplantation. Here, we use a modular system based on a chimeric antigen receptor (CAR) that binds covalently modified mAbs to control Treg activation in vivo. Transient expression of this mAb-directed CAR (mAbCAR) in Tregs permitted Treg targeting to specific tissue sites and mitigated allograft responses, such as graft-versus-host disease. mAbCAR Tregs targeted to MHC class I proteins on allografts prolonged islet allograft survival and also prolonged the survival of secondary skin grafts specifically matched to the original islet allograft. Thus, transient genetic modification to produce mAbCAR T cells led to durable immune modulation, suggesting therapeutic targeting strategies for controlling alloreactivity in settings such as organ or tissue transplantation.
Antonio Pierini, Bettina P. Iliopoulou, Heshan Peiris, Magdiel Pérez-Cruz, Jeanette Baker, Katie Hsu, Xueying Gu, Ping-Ping Zheng, Tom Erkers, Sai-Wen Tang, William Strober, Maite Alvarez, Aaron Ring, Andrea Velardi, Robert S. Negrin, Seung K. Kim, Everett H. Meyer
T cells are central to the detrimental alloresponses that develop in autoimmunity and transplantation, with CD28 costimulatory signals being key to T cell activation and proliferation. CTLA4-Ig molecules that bind CD80/86 and inhibit CD28 costimulation offer an alternative immunosuppressive treatment, free from some of the chronic toxicities associated with calcineurin inhibition. However, CD80/86 blockade by CTLA4-Ig also results in the loss of coinhibitory CTLA4 signals that are critical to the regulation of T cell activation. Here, we show that a nonactivating monovalent anti-CD28 that spares CTLA4 signaling is an effective immunosuppressant in a clinically relevant humanized mouse transplant model. We demonstrate that selective CD28 blockade prolongs human skin allograft survival through a mechanism that includes a reduction in the cellular graft infiltrate. Critically, selective CD28 blockade promotes Treg function in vivo and synergizes with adoptive Treg therapy to promote transplant survival. In contrast to CTLA4-Ig treatment, selective CD28 blockade promotes regulation of alloimmune responses and facilitates Treg-based cellular therapy.
Masaaki Zaitsu, Fadi Issa, Joanna Hester, Bernard Vanhove, Kathryn J. Wood
The role of negative regulators or suppressors of the damage-associated molecular pattern–mediated (DAMP-mediated) stimulation of innate immune responses is being increasingly appreciated. However, the presence and function of suppressors of DAMP-mediated effects on T cells, and whether they can be targeted to mitigate T cell–dependent immunopathology remain unknown. Sialic acid–binding immunoglobulin-like lectin G (Siglec-G) is a negative regulator of DAMP-mediated responses in innate immune cells, but its T cell–autonomous role is unknown. Utilizing loss-of-function–based (genetic knockout) and gain-of-function–based (agonist) approaches, we demonstrate that in the presence of certain DAMPs, Siglec-G suppressed in vitro and in vivo T cell responses. We also demonstrate that its T cell–autonomous role is critical for modulating the severity of the T cell–mediated immunopathology, graft-versus-host disease (GVHD). Enhancing the Siglec-G signaling in donor T cells with its agonist, a CD24Fc fusion protein, ameliorated GVHD while preserving sufficient graft-versus-tumor (GVT) effects in vivo. Collectively, these data demonstrate that Siglec-G is a potentially novel negative regulator of T cell responses, which can be targeted to mitigate GVHD.
Tomomi Toubai, Corinne Rossi, Katherine Oravecz-Wilson, Cynthia Zajac, Chen Liu, Thomas Braun, Hideaki Fujiwara, Julia Wu, Yaping Sun, Stuart Brabbs, Hiroya Tamaki, John Magenau, Pang Zheng, Yang Liu, Pavan Reddy
Rejection affects greater than 80% of face transplants, yet no diagnostic criteria for antibody-mediated rejection (AMR) following face transplantation have been established. Given that different treatment strategies are required to address AMR and T cell–mediated rejection (TCMR), there is a critical need to delineate the features that can differentiate these two alloimmune responses. Here, we report the longitudinal immunological examination of what we believe to be the first and only highly sensitized recipient of a crossmatch-positive face transplant up to 4 years following transplantation. We conducted gene expression profiling on allograft biopsies collected during suspected AMR and TCMR episodes as well as during 5 nonrejection time points. Our data suggest that there are distinctive molecular features in AMR, characterized by overexpression of endothelial-associated genes, including ICAM1, VCAM1, and SELE. Although our findings are limited to a single patient, these findings highlight the potential importance of developing and implementing molecular markers to differentiate AMR from TCMR to guide clinical management. Furthermore, our case illustrates that molecular assessment of allograft biopsies offers the potential for new insights into the mechanisms underlying rejection. Finally, our medium-term outcomes demonstrate that face transplantation in a highly sensitized patient with a positive preoperative crossmatch is feasible and manageable.
Thet Su Win, Naoka Murakami, Thiago J. Borges, Anil Chandraker, George Murphy, Christine Lian, Victor Barrera, Shannan Ho Sui, David Schoenfeld, Jessica Teague, Ericka Bueno, Stefan G. Tullius, Bohdan Pomahac, Rachael A. Clark, Leonardo V. Riella
Adiponectin is a pleiotropic cytokine with diverse immunomodulatory effects on macrophages and lymphocytes. In the current paradigm, lymphocytes and macrophages respond to adiponectin that is produced by adipocytes and other parenchymal cells. Using a model of chronic arterial inflammation in cardiac transplants, we found that T cells derived from the recipient migrate to the heart and produce adiponectin locally. The evidence that T cells produce significant amounts of adiponectin is based on 3 experimental approaches. First, CD4+ T cells isolated from the blood and spleen after cardiac transplantation express mRNA for adiponectin. Second, reconstitution of T cell–deficient recipients with transgenic CD4+ T cells that express receptors for donor antigens results in arterial infiltrates containing T cells and increased mRNA expression for adiponectin in cardiac transplants. Third, CD4+ T cells isolated from the allograft secrete adiponectin in vitro. Taken together, these data indicate that adiponectin-competent cells originating in the recipient migrate into the transplant. Establishing T cells as a source of adiponectin provides a new dimension, to our knowledge, to the modulatory effects of adiponectin on immune responses.
Sreedevi Danturti, Karen S. Keslar, Leah R. Steinhoff, Ran Fan, Nina Dvorina, Anna Valujskikh, Robert L. Fairchild, William M. Baldwin III
Chronic graft-versus-host disease (cGvHD) remains a major complication of allogeneic stem cell transplantation requiring novel therapies. CD146 and CCR5 are expressed by activated T cells and associated with increased T cell migration capacity and Th17 polarization. We performed a multiparametric flow cytometry analysis in a cohort of 40 HSCT patients together with a cGvHD murine model to understand the role of CD146-expressing subsets. We observed an increased frequency of CD146+ CD4 T cells in the 20 patients with active cGvHD with enhanced RORγt expression. This Th17-prone subset was enriched for cells coexpressing CD146 and CCR5 that harbor mixed Th1/Th17 features and were more frequent in cGvHD patients. Utilizing a murine cGvHD model with bronchiolitis obliterans (BO), we observed that donor T cells from CD146-deficient mice versus those from WT mice caused significantly reduced pulmonary cGvHD. Reduced cGvHD was not the result of failed germinal center B cell or T follicular helper cell generation. Instead, CD146-deficient T cells had significantly lower pulmonary macrophage infiltration and T cell CCR5, IL-17, and IFN-γ coexpression, suggesting defective pulmonary end-organ effector mechanisms. We, thus, evaluated the effect of TMP778, a small-molecule RORγt activity inhibitor. TMP778 markedly alleviated cGvHD in murine models similarly to agents targeting the Th17 pathway, such as STAT3 inhibitor or IL-17–blocking antibody. Our data suggest CD146-expressing T cells as a cGvHD biomarker and suggest that targeting the Th17 pathway may represent a promising therapy for cGvHD.
Edouard Forcade, Katelyn Paz, Ryan Flynn, Brad Griesenauer, Tohti Amet, Wei Li, Liangyi Liu, Giorgos Bakoyannis, Di Jiang, Hong Wei Chu, Mercedes Lobera, Jianfei Yang, David S. Wilkes, Jing Du, Kate Gartlan, Geoffrey R. Hill, Kelli P.A. MacDonald, Eduardo L. Espada, Patrick Blanco, Jonathan S. Serody, John Koreth, Corey S. Cutler, Joseph H. Antin, Robert J. Soiffer, Jerome Ritz, Sophie Paczesny, Bruce R. Blazar
Hematopoietic stem cell transplantation (HSCT) offers a cure for cancers that are refractory to chemotherapy and radiation. Most HSCT recipients develop chronic graft-versus-host disease (cGVHD), a systemic alloimmune attack on host organs. Diagnosis is based on clinical signs and symptoms, as biopsies are risky. T cells are central to the biology of cGVHD. We found that a low Treg/CD4+ T effector memory (Tem) ratio in circulation, lymphoid, and target organs identified early and established mouse cGVHD. Using deuterated water labeling to measure multicompartment in vivo kinetics of these subsets, we show robust Tem and Treg proliferation in lymphoid and target organs, while Tregs undergo apoptosis in target organs. Since deuterium enrichment into DNA serves as a proxy for cell proliferation, we developed a whole-body clinically relevant deuterium MRI approach to nonradioactively detect cGVHD and potentially allow imaging of other diseases characterized by rapidly proliferating cells.
Nataliya P. Buxbaum, Donald E. Farthing, Natella Maglakelidze, Martin Lizak, Hellmut Merkle, Andrea C. Carpenter, Brittany U. Oliver, Veena Kapoor, Ehydel Castro, Gregory A. Swan, Liliane M. dos Santos, Nicolas J. Bouladoux, Catherine V. Bare, Francis A. Flomerfelt, Michael A. Eckhaus, William G. Telford, Yasmine Belkaid, Remy J. Bosselut, Ronald E. Gress
Conventional histologic diagnosis of rejection in kidney transplants has limited repeatability due to its inherent requirement for subjective assessment of lesions, in a rule-based system that does not acknowledge diagnostic uncertainty. Molecular phenotyping affords opportunities for increased precision and improved disease classification to address the limitations of conventional histologic diagnostic systems and quantify levels of uncertainty. Microarray data from 1,208 kidney transplant biopsies were collected prospectively from 13 centers. Cross-validated classifier scores predicting the presence of antibody-mediated rejection (ABMR), T cell–mediated rejection (TCMR), and 5 related histologic lesions were generated using supervised machine learning methods. These scores were used as input for archetypal analysis, an unsupervised method similar to cluster analysis, to examine the distribution of molecular phenotypes related to rejection. Six archetypes were generated: no rejection, TCMR, 3 associated with ABMR (early-stage, fully developed, and late-stage), and mixed rejection (TCMR plus early-stage ABMR). Each biopsy was assigned 6 scores, one for each archetype, representing a probabilistic assessment of that biopsy based on its rejection-related molecular properties. Viewed as clusters, the archetypes were similar to existing histologic Banff categories, but there was 32% disagreement, much of it probably reflecting the “noise” in the current histologic assessment system. Graft survival was lowest for fully developed and late-stage ABMR, and it was better predicted by molecular archetype scores than histologic diagnoses. The results provide a system for precision molecular assessment of biopsies and a new standard for recalibrating conventional diagnostic systems.
Jeff Reeve, Georg A. Böhmig, Farsad Eskandary, Gunilla Einecke, Carmen Lefaucheur, Alexandre Loupy, Philip F. Halloran, the MMDx-Kidney study group
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