Assessing rejection-related disease in kidney transplant biopsies based on archetypal analysis of molecular phenotypes

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Abstract

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.

Authors

Jeff Reeve, Georg A. Böhmig, Farsad Eskandary, Gunilla Einecke, Carmen Lefaucheur, Alexandre Loupy, Philip F. Halloran, the MMDx-Kidney study group

T cell progenitor therapy–facilitated thymopoiesis depends upon thymic input and continued thymic microenvironment interaction

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Abstract

Infusion of in vitro–derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant aids the recovery of the thymus damaged by total body irradiation. To understand the interaction between proTs and the thymic microenvironment, WT mice were lethally irradiated and given T cell–deficient (Rag1-/-) marrow with WT in vitro–generated proTs, limiting mature T cell development to infused proTs. ProTs within the host thymus led to a significant increase in thymic epithelial cells (TECs) by day 21 after transplant, increasing actively cycling TECs. Upon thymus egress (day 28), proT TEC effects were lost, suggesting that continued signaling from proTs is required to sustain TEC cycling and cellularity. Thymocytes increased significantly by day 21, followed by a significant improvement in mature T cell numbers in the periphery by day 35. This protective surge was temporary, receding by day 60. Double-negative 2 (DN2) proTs selectively increased thymocyte number, while DN3 proTs preferentially increased TECs and T cells in the spleen that persisted at day 60. These findings highlight the importance of the interaction between proTs and TECs in the proliferation and survival of TECs and that the maturation stage of proTs has unique effects on thymopoiesis and peripheral T cell recovery.

Authors

Michelle J. Smith, Dawn K. Reichenbach, Sarah L. Parker, Megan J. Riddle, Jason Mitchell, Kevin C. Osum, Mahmood Mohtashami, Heather E. Stefanski, Brian T. Fife, Avinash Bhandoola, Kristin A. Hogquist, Georg A. Holländer, Juan Carlos Zúñiga-Pflücker, Jakub Tolar, Bruce R. Blazar

Ceramide synthesis regulates T cell activity and GVHD development

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Abstract

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy for a variety of hematologic malignances, yet its efficacy is impeded by the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production, and migration of alloreactive donor T cells. Hence, strategies to limit GVHD are highly desirable. Ceramides are known to contribute to inflammation and autoimmunity. However, their involvement in T-cell responses to alloantigens is undefined. In the current study, we specifically characterized the role of ceramide synthase 6 (CerS6) after allo-HCT using genetic and pharmacologic approaches. We found that CerS6 was required for optimal T cell activation, proliferation, and cytokine production in response to alloantigen and for subsequent induction of GVHD. However, CerS6 was partially dispensable for the T cell–mediated antileukemia effect. At the molecular level, CerS6 was required for efficient TCR signal transduction, including tyrosine phosphorylation, ZAP-70 activation, and PKCθ/TCR colocalization. Impaired generation of C16-ceramide was responsible for diminished allogeneic T cell responses. Furthermore, targeting CerS6 using a specific inhibitor significantly reduced T cell activation in mouse and human T cells in vitro. Our study provides a rationale for targeting CerS6 to control GVHD, which would enhance the efficacy of allo-HCT as an immunotherapy for hematologic malignancies in the clinic.

Authors

M. Hanief Sofi, Jessica Heinrichs, Mohammed Dany, Hung Nguyen, Min Dai, David Bastian, Steven Schutt, Yongxia Wu, Anusara Daenthanasanmak, Salih Gencer, Aleksandra Zivkovic, Zdzislaw Szulc, Holger Stark, Chen Liu, Ying-Jun Chang, Besim Ogretmen, Xue-Zhong Yu

CTLA4-Ig in combination with FTY720 promotes allograft survival in sensitized recipients

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Abstract

Despite recent evidence of improved graft outcomes and safety, the high incidence of early acute cellular rejection with belatacept, a high-affinity CTLA4-Ig, has limited its use in clinical transplantation. Here we define how the incomplete control of endogenous donor-reactive memory T cells results in belatacept-resistant rejection in an experimental model of BALB/c.2W-OVA donor heart transplantation into C57BL/6 recipients presensitized to donor splenocytes. These sensitized mice harbored modestly elevated numbers of endogenous donor-specific memory T cells and alloantibodies compared with naive recipients. Continuous CTLA4-Ig treatment was unexpectedly efficacious at inhibiting endogenous graft-reactive T cell expansion but was unable to inhibit late CD4⁺ and CD8⁺ T cell infiltration into the allografts, and rejection was observed in 50% of recipients by day 35 after transplantation. When CTLA4-Ig was combined with the sphingosine 1-phosphate receptor-1 (S1PR1) functional antagonist FTY720, alloantibody production was inhibited and donor-specific IFN-γ–producing T cells were reduced to levels approaching nonsensitized tolerant recipients. Late T cell recruitment into the graft was also restrained, and graft survival improved with this combination therapy. These observations suggest that a rational strategy consisting of inhibiting memory T cell expansion and trafficking into the allograft with CTLA4-Ig and FTY720 can promote allograft survival in allosensitized recipients.

Authors

Stella H. Khiew, Jinghui Yang, James S. Young, Jianjun Chen, Qiang Wang, Dengping Yin, Vinh Vu, Michelle L. Miller, Roger Sciammas, Maria-Luisa Alegre, Anita S. Chong

Models of Lung Transplant Research: a consensus statement from the National Heart, Lung, and Blood Institute workshop

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Abstract

Lung transplantation, a cure for a number of end-stage lung diseases, continues to have the worst long-term outcomes when compared with other solid organ transplants. Preclinical modeling of the most common and serious lung transplantation complications are essential to better understand and mitigate the pathophysiological processes that lead to these complications. Various animal and in vitro models of lung transplant complications now exist and each of these models has unique strengths. However, significant issues, such as the required technical expertise as well as the robustness and clinical usefulness of these models, remain to be overcome or clarified. The National Heart, Lung, and Blood Institute (NHLBI) convened a workshop in March 2016 to review the state of preclinical science addressing the three most important complications of lung transplantation: primary graft dysfunction (PGD), acute rejection (AR), and chronic lung allograft dysfunction (CLAD). In addition, the participants of the workshop were tasked to make consensus recommendations on the best use of these complimentary models to close our knowledge gaps in PGD, AR, and CLAD. Their reviews and recommendations are summarized in this report. Furthermore, the participants outlined opportunities to collaborate and directions to accelerate research using these preclinical models.

Authors

Vibha N. Lama, John A. Belperio, Jason D. Christie, Souheil El-Chemaly, Michael C. Fishbein, Andrew E. Gelman, Wayne W. Hancock, Shaf Keshavjee, Daniel Kreisel, Victor E. Laubach, Mark R. Looney, John F. McDyer, Thalachallour Mohanakumar, Rebecca A. Shilling, Angela Panoskaltsis-Mortari, David S. Wilkes, Jerry P. Eu, Mark R. Nicolls

Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

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Abstract

Better identification of severe acute graft-versus-host disease (GvHD) may improve the outcome of this life-threatening complication of allogeneic hematopoietic stem cell transplantation. GvHD induces tissue damage and the release of damage-associated molecular pattern (DAMP) molecules. Here, we analyzed GvHD patients (n = 39) to show that serum heat shock protein glycoprotein 96 (Gp96) could be such a DAMP molecule. We demonstrate that serum Gp96 increases in gastrointestinal GvHD patients and its level correlates with disease severity. An increase in Gp96 serum level was also observed in a mouse model of acute GvHD. This model was used to identify complement C3 as a main partner of Gp96 in the serum. Our biolayer interferometry, yeast two-hybrid and in silico modeling data allowed us to determine that Gp96 binds to a complement C3 fragment encompassing amino acids 749–954, a functional complement C3 hot spot important for binding of different regulators. Accordingly, in vitro experiments with purified proteins demonstrate that Gp96 downregulates several complement C3 functions. Finally, experimental induction of GvHD in complement C3–deficient mice confirms the link between Gp96 and complement C3 in the serum and with the severity of the disease.

Authors

Antoine Seignez, Anne-Laure Joly, Killian Chaumonnot, Adonis Hazoumé, Michel Sanka, Guillaume Marcion, Christophe Boudesco, Arlette Hammann, Renaud Seigneuric, Gaetan Jégo, Patrick Ducoroy, Patrice Delarue, Patrick Senet, Cristina Castilla-Llorente, Eric Solary, Marie-Agnès Durey, Marie-Thérèse Rubio, Olivier Hermine, Evelyne Kohli, Carmen Garrido

Interruption of OX40L signaling prevents costimulation blockade–resistant allograft rejection

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Abstract

The potential of costimulation blockade to serve as a novel transplant immunosuppression strategy has been explored for over 20 years, culminating in the recent clinical approval of belatacept for renal transplant patients. Despite improving long-term graft function and survival compared with calcineurin inhibitors, clinical acceptance of belatacept has been hindered by elevated rates of acute rejection. We examined the signaling pathways required to activate costimulation blockade–resistant alloreactive T cells and identified the OX40/OX40L secondary costimulatory pathway as a promising target. We next sought to improve the clinical efficacy of traditional costimulation blockade using belatacept by coupling it with anti-OX40L. Using a murine transplant model, we demonstrate that combined blockade enhances the suppression of alloreactive T cell proliferation and effector functions including both cytokine release and cytotoxic degranulation. We also show that anti-OX40L may be particularly useful in targeting alloreactive memory T cell responses that are relatively unaffected by traditional costimulation blockade regimens. Finally, we translated this therapy to a clinically relevant nonhuman primate renal transplant model, validating the efficacy of this regimen in a potentially novel steroid- and calcineurin inhibitor–free immunosuppression regimen.

Authors

William H. Kitchens, Ying Dong, David V. Mathews, Cynthia P. Breeden, Elizabeth Strobert, Maria E. Fuentes, Christian P. Larsen, Mandy L. Ford, Andrew B. Adams

Loss of immune homeostasis dictates SHIV rebound after stem-cell transplantation

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Abstract

The conditioning regimen used as part of the Berlin patient’s hematopoietic cell transplant likely contributed to his eradication of HIV infection. We studied the impact of conditioning in simian-human immunodeficiency virus–infected (SHIV-infected) macaques suppressed by combination antiretroviral therapy (cART). The conditioning regimen resulted in a dramatic, but incomplete depletion of CD4⁺ and CD8⁺ T cells and CD20⁺ B cells, increased T cell activation and exhaustion, and a significant loss of SHIV-specific Abs. The disrupted T cell homeostasis and markers of microbial translocation positively correlated with an increased viral rebound after cART interruption. Quantitative viral outgrowth and Tat/rev–induced limiting dilution assays showed that the size of the latent SHIV reservoir did not correlate with viral rebound. These findings identify perturbations of the immune system as a mechanism for the failure of autologous transplantation to eradicate HIV. Thus, transplantation strategies may be improved by incorporating immune modulators to prevent disrupted homeostasis, and gene therapy to protect transplanted cells.

Authors

Christopher W. Peterson, Clarisse Benne, Patricia Polacino, Jasbir Kaur, Cristina E. McAllister, Abdelali Filali-Mouhim, Willi Obenza, Tiffany A. Pecor, Meei-Li Huang, Audrey Baldessari, Robert D. Murnane, Ann E. Woolfrey, Keith R. Jerome, Shiu-Lok Hu, Nichole R. Klatt, Stephen DeRosa, Rafick P. Sékaly, Hans-Peter Kiem

An early-biomarker algorithm predicts lethal graft-versus-host disease and survival

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Abstract

BACKGROUND. No laboratory test can predict the risk of nonrelapse mortality (NRM) or severe graft-versus-host disease (GVHD) after hematopoietic cellular transplantation (HCT) prior to the onset of GVHD symptoms.

METHODS. Patient blood samples on day 7 after HCT were obtained from a multicenter set of 1,287 patients, and 620 samples were assigned to a training set. We measured the concentrations of 4 GVHD biomarkers (ST2, REG3α, TNFR1, and IL-2Rα) and used them to model 6-month NRM using rigorous cross-validation strategies to identify the best algorithm that defined 2 distinct risk groups. We then applied the final algorithm in an independent test set (n = 309) and validation set (n = 358).

RESULTS. A 2-biomarker model using ST2 and REG3α concentrations identified patients with a cumulative incidence of 6-month NRM of 28% in the high-risk group and 7% in the low-risk group (P < 0.001). The algorithm performed equally well in the test set (33% vs. 7%, P < 0.001) and the multicenter validation set (26% vs. 10%, P < 0.001). Sixteen percent, 17%, and 20% of patients were at high risk in the training, test, and validation sets, respectively. GVHD-related mortality was greater in high-risk patients (18% vs. 4%, P < 0.001), as was severe gastrointestinal GVHD (17% vs. 8%, P < 0.001). The same algorithm can be successfully adapted to define 3 distinct risk groups at GVHD onset.

CONCLUSION. A biomarker algorithm based on a blood sample taken 7 days after HCT can consistently identify a group of patients at high risk for lethal GVHD and NRM.

FUNDING. The National Cancer Institute, American Cancer Society, and the Doris Duke Charitable Foundation.

Authors

Matthew J. Hartwell, Umut Özbek, Ernst Holler, Anne S. Renteria, Hannah Major-Monfried, Pavan Reddy, Mina Aziz, William J. Hogan, Francis Ayuk, Yvonne A. Efebera, Elizabeth O. Hexner, Udomsak Bunworasate, Muna Qayed, Rainer Ordemann, Matthias Wölfl, Stephan Mielke, Attaphol Pawarode, Yi-Bin Chen, Steven Devine, Andrew C. Harris, Madan Jagasia, Carrie L. Kitko, Mark R. Litzow, Nicolaus Kröger, Franco Locatelli, George Morales, Ryotaro Nakamura, Ran Reshef, Wolf Rösler, Daniela Weber, Kitsada Wudhikarn, Gregory A. Yanik, John E. Levine, James L.M. Ferrara

Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells

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Abstract

Rat and human CD4⁺ and CD8⁺ Tregs expressing low levels of CD45RC have strong immunoregulatory properties. We describe here that human CD45 isoforms are nonredundant and identify distinct subsets of cells. We show that CD45RC is not expressed by CD4⁺ and CD8⁺ Foxp3⁺ Tregs, while CD45RA/RB/RO are. Transient administration of a monoclonal antibody (mAb) targeting CD45RC in a rat cardiac allotransplantation model induced transplant tolerance associated with inhibition of allogeneic humoral responses but maintained primary and memory responses against cognate antigens. Anti-CD45RC mAb induced rapid death of CD45RC^high T cells through intrinsic cell signaling but preserved and potentiated CD4⁺ and CD8⁺ CD45RC^low/– Tregs, which are able to adoptively transfer donor-specific tolerance to grafted recipients. Anti-CD45RC treatment results in distinct transcriptional signature of CD4⁺ and CD8⁺ CD45RC^low/– Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-host disease (GVHD) in immune-humanized NSG mice. Thus, short-term anti-CD45RC is a potent therapeutic candidate to induce transplantation tolerance in human.

Authors

Elodie Picarda, Séverine Bézie, Laetitia Boucault, Elodie Autrusseau, Stéphanie Kilens, Dimitri Meistermann, Bernard Martinet, Véronique Daguin, Audrey Donnart, Eric Charpentier, Laurent David, Ignacio Anegon, Carole Guillonneau