Late allograft failure is characterized by cumulative subclinical insults manifesting over many years. Although immunomodulatory therapies targeting host T cells have improved short-term survival rates, rates of chronic allograft loss remain high. We hypothesized that other immune cell types may drive subclinical injury, ultimately leading to graft failure. We collected whole-genome transcriptome profiles from 15 independent cohorts composed of 1,697 biopsy samples to assess the association of an inflammatory macrophage polarization–specific gene signature with subclinical injury. We applied penalized regression to a subset of the data sets and identified a 3-gene inflammatory macrophage–derived signature. We validated discriminatory power of the 3-gene signature in 3 independent renal transplant data sets with mean AUC of 0.91. In a longitudinal cohort, the 3-gene signature strongly correlated with extent of injury and accurately predicted progression of subclinical injury 18 months before clinical manifestation. The 3-gene signature also stratified patients at high risk of graft failure as soon as 15 days after biopsy. We found that the 3-gene signature also distinguished acute rejection (AR) accurately in 3 heart transplant data sets but not in lung transplant. Overall, we identified a parsimonious signature capable of diagnosing AR, recognizing subclinical injury, and risk-stratifying renal transplant patients. Our results strongly suggest that inflammatory macrophages may be a viable therapeutic target to improve long-term outcomes for organ transplantation patients.
Tej D. Azad, Michele Donato, Line Heylen, Andrew B. Liu, Shai S. Shen-Orr, Timothy E. Sweeney, Jonathan Scott Maltzman, Maarten Naesens, Purvesh Khatri
Memory T cells pose a significant problem to successful therapeutic control of unwanted immune responses during autoimmunity and transplantation, as they are differentially controlled by cosignaling receptors such as CD28 and CTLA-4. Treatment with abatacept and belatacept impede CD28 signaling by binding to CD80 and CD86, but they also have the unintended consequence of blocking the ligands for CTLA-4, a process that may inadvertently boost effector responses. Here, we show that a potentially novel anti-CD28 domain antibody (dAb) that selectively blocks CD28 but preserves CTLA-4 coinhibition confers improved allograft survival in sensitized recipients as compared with CTLA-4 Ig. However, both CTLA-4 Ig and anti-CD28 dAb similarly and significantly reduced the accumulation of donor-reactive CD8+ memory T cells, demonstrating that regulation of the expansion of CD8+ memory T cell populations is controlled in part by CD28 signals and is not significantly impacted by CTLA-4. In contrast, selective CD28 blockade was superior to CTLA-4 Ig in inhibiting IFN-γ, TNF, and IL-2 production by CD8+ memory T cells, which in turn resulted in reduced recruitment of innate CD11b+ monocytes into allografts. Importantly, this superiority was CTLA-4 dependent, demonstrating that effector function of CD8+ memory T cells is regulated by the balance of CD28 and CTLA-4 signaling.
Danya Liu, I. Raul Badell, Mandy L. Ford
Lungs allografts have worse long-term survival compared with other organ transplants. This is most likely due to their unique immunoregulation that may not respond to traditional immunosuppression. For example, local NO generation by inducible NOS (iNOS) is critical for lung allograft acceptance but associates with rejection of other solid organs. The source of NO in accepting lung allografts remains unknown. Here, we report that, unlike the case for other pulmonary processes in which myeloid cells control NO generation, recipient-derived eosinophils play a critical and nonredundant role in iNOS-mediated lung allograft acceptance. Depletion of eosinophils reduces NO levels to that of recipients with global deletion of iNOS and leads to a costimulatory blockade–resistant form of rejection. Furthermore, NO production by eosinophils depends on Th1 polarization by inflammatory mediators, such as IFN-γ and TNF-α. Neutralization of such mediators abrogates eosinophil suppressive capacity. Our data point to what we believe to be a unique and previously unrecognized role of eosinophil polarization in mediating allograft tolerance and put into perspective the use of high-dose eosinophil-ablating corticosteroids after lung transplantation.
Oscar Okwudiri Onyema, Yizhan Guo, Qing Wang, Mark H. Stoler, Christine Lau, Kang Li, Christopher Daniel Nazaroff, Xingan Wang, Wenjun Li, Daniel Kreisel, Andrew E. Gelman, James J. Lee, Elizabeth A. Jacobsen, Alexander Sasha Krupnick
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
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