Obliterative bronchiolitis (OB) is a poorly understood airway disease characterized by the generation of fibrotic bronchiolar occlusions. In the lung transplant setting, OB is a pathological manifestation of bronchiolitis obliterans syndrome (BOS), which is a major impediment to long-term recipient survival. Club cells play a key role in bronchiolar epithelial repair, but whether they promote lung transplant tolerance through preventing OB remains unclear. We determined if OB occurs in mouse orthotopic lung transplants following conditional transgene-targeted club cell depletion. In syngeneic lung transplants club cell depletion leads to transient epithelial injury followed by rapid club cell-mediated repair. In contrast, allogeneic lung transplants develop severe OB lesions and poorly regenerate club cells despite immunosuppression treatment. Lung allograft club cell ablation also triggers the recognition of alloantigens, and pulmonary restricted self-antigens reported associated with BOS development. However, CD8+ T cell depletion restores club cell reparative responses and prevents OB. In addition, ex-vivo analysis reveals a specific role for alloantigen-primed effector CD8+ T cells in preventing club cell proliferation and maintenance. Taken together, we demonstrate a vital role for club cells in maintaining lung transplant tolerance and propose a new model to identify the underlying mechanisms of OB.
Zhiyi Liu, Fuyi Liao, Davide Scozzi, Yuka Furuya, Kaitlyn N. Pugh, Ramsey R. Hachem, Delphine L. Chen, Marlene Cano, Jonathan M. Green, Alexander S. Krupnick, Daniel Kreisel, Anne-Karina T. Perl, Howard J. Huang, Steven L. Brody, Andrew E. Gelman
Antibody-mediated lymphoablation is used in solid organ and stem cell transplantation and autoimmunity. Using murine anti-thymocyte globulin (mATG) in a mouse model of heart transplantation, we previously reported that the homeostatic recovery of CD8+ T cells requires help from depletion-resistant memory CD4+ T cells delivered through CD40-expressing B cells. This study investigated the mechanisms by which B cells mediate CD8+ T cell proliferation in lymphopenic hosts. While CD8+ T cell recovery required MHC class I expression in the host, the reconstitution occurred independently of MHC class I, MHC class II, or CD80/CD86 expression on B cells. mATG lymphoablation upregulated the B cell expression of several cytokine genes, including IL-15 and IL-27, in a CD4-dependent manner. Neither treatment with anti-CD122 mAb nor the use of IL-15Rα–/– recipients altered CD8+ T cell recovery after mATG treatment, indicating that IL-15 may be dispensable for T cell proliferation in our model. Instead, IL-27 neutralization or the use of IL-27Rα–/– CD8+ T cells inhibited CD8+ T cell proliferation and altered the phenotype and cytokine profile of reconstituted CD8+ T cells. Our findings uncover what we believe is a novel role of IL-27 in lymphopenia-induced CD8+ T cell proliferation and suggest that targeting B cell–derived cytokines may increase the efficacy of lymphoablation and improve transplant outcomes.
Katayoun Ayasoufi, Daniel B. Zwick, Ran Fan, Suheyla Hasgur, Michael Nicosia, Victoria Gorbacheva, Karen S. Keslar, Booki Min, Robert L. Fairchild, Anna Valujskikh
Chimeric antigen receptor (CAR) technology can be used to engineer the antigen-specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from non-human antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain Fv derived from a mouse antibody, we developed a panel of 20 humanized (h)A2-CARs. Systematic testing demonstrated variations in expression, ability to bind HLA-A*02:01, and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen-specificity of CARs, revealing that humanization reduced HLA-A cross reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs which can be used to engineer specificity and homing of therapeutic Tregs.
Nicholas A.J. Dawson, Caroline Lamarche, Romy E. Hoeppli, Peter Bergqvist, Vivian Fung, Emma McIver, Qing Huang, Jana Gillies, Madeleine Speck, Paul C. Orban, Jonathan W. Bush, Majid Mojibian, Megan K. Levings
Lung transplantation (LTx) is the only therapeutic option for many patients with chronic lung disease. However, long-term survival after LTx is severely compromised by chronic rejection (chronic lung allograft dysfunction [CLAD]), which affects 50% of recipients after 5 years. The underlying mechanisms for CLAD are poorly understood, largely due to a lack of clinically relevant animal models, but lymphocytic bronchiolitis is an early sign of CLAD. Here, we report that lymphocytic bronchiolitis occurs early in a long-term murine orthotopic LTx model, based on a single mismatch (grafts from HLA-A2:B6–knockin donors transplanted into B6 recipients). Lymphocytic bronchiolitis is followed by formation of B cell–dependent lymphoid follicles that induce adjacent bronchial epithelial cell dysfunction in a spatiotemporal fashion. B cell deficiency using recipient μMT–/– mice prevented intrapulmonary lymphoid follicle formation and lymphocytic bronchiolitis. Importantly, selective inhibition of the follicle-organizing receptor EBI2, using genetic deletion or pharmacologic inhibition, prevented functional and histological deterioration of mismatched lung grafts. In sum, we provided what we believe to be a mouse model of chronic rejection and lymphocytic bronchiolitis after LTx and identified intrapulmonary lymphoid follicle formation as a target for pharmacological intervention of long-term allograft dysfunction after LTx.
Natalia F. Smirnova, Thomas M. Conlon, Carmela Morrone, Peter Dorfmuller, Marc Humbert, Georgios Stathopoulos, Stephan Umkehrer, Franz Pfeiffer, Ali Ö. Yildirim, Oliver Eickelberg
Soluble STimulation-2 (ST2) is increased during graft-versus-host disease (GVHD) while regulatory T cells (Tregs) that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3- T cells and Tregs sorted from different Foxp3 reporter mice indicated that ST2+Tregs isolated from GVHD mice were thymus-derived and predominantly intestine localized. ST2-/- Tregs transplantation was associated with reduced total intestinal Tregs frequency and activation. ST2-/- vs wild-type intestinal Tregs transcriptomes showed decreased Treg functional markers and reciprocally, increased Rorc expression. Rorc-/- T cells transplantation enhanced the frequency and function of intestinal ST2+Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2-producing type-1 and increased IL-4+IL-10+ producing type-2 T cells. Cotransfer of ST2+Tregs sorted from Rorc-/- mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2+KLRG1+ Tregs and decreased type-1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33 stimulated Tregs (TregIL-33) expressed higher amphiregulin, displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or TregIL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of TregIL-33. Our data show an inversely expression of ST2 and RORγt in intestinal Tregs, and TregIL-33 is a potential cellular therapy avenue for preventing GVHD.
Jinfeng Yang, Abdulraouf Ramadan, Dawn K. Reichenbach, Michael Loschi, Jilu Zhang, Brad Griesenauer, Hong Liu, Keli L. Hippen, Bruce R. Blazar, Sophie Paczesny
BACKGROUND. Human cytomegalovirus (CMV) reactivation is a common occurrence early after transplant and is associated with heterogeneous NK cell subset expansion. These adaptive NK cell expansions are highly variable between recipients, with respect to magnitude and relative frequencies of adaptive NK cell subsets. METHODS. To gain insight into the factors that influence adaptive NK cell expansion from a CMV naive graft source, we performed a high-resolution NK cell and CD8+ T cell phenotypic analysis of 215 patients with hematological malignancies that were transplanted with 2 partially HLA matched CMV negative umbilical cord blood units. RESULTS. We found that adaptive NK cells were significantly higher in recipients who received nonmyeloablative conditioning (NMAC) relative to myeloablative conditioning (MAC), and high CMV neutralizing antibody titers correlated with the degree of adaptive NK cell expansion. The frequencies of adaptive NK cell subsets (defined by NKG2C, FcεRγ, EAT-2, and SYK expression) that reconstitute from donor hematopoietic progenitor cells largely matched the frequencies observed in the NK cell compartment of the recipient prior to conditioning, suggesting that host — as well as viral reactivation factors — may determine the phenotypic diversification after transplant. Additionally, multivariable analyses show that higher adaptive NK cell expansion associated with better disease-free survival. CONCLUSIONS. Our findings provide important insights into adaptive NK cell reconstitution after transplant and support a role for adaptive NK cells in promoting better clinical outcomes. FUNDING. The NIH and the National Marrow Donor Program.
Frank Cichocki, Emily Taras, Flavia Chiuppesi, John E. Wagner, Bruce R. Blazar, Claudio Brunstein, Xianghua Luo, Don J. Diamond, Sarah Cooley, Daniel J. Weisdorf, Jeffrey S. Miller
CD4+ follicular helper T (Tfh) cells are specialized providers of T cell help to B cells and can function as pathogenic mediators of murine antibody-dependent chronic graft-versus-host disease (GvHD). Using a parent→F1 model of lupus-like chronic GvHD, in which Tfh cell and germinal center (GC) B cell differentiation occurs over 14 days, we demonstrate that absence of CD4+ T cell–expressed C5a receptor 1 (C5ar1) or pharmacological C5aR1 blockade abrogated generation/expansion of Tfh cells, GC B cells, and autoantibodies. In a Tfh cell–dependent model of chronic GvHD manifested by bronchiolitis obliterans syndrome (BOS), C5aR1 antagonism initiated in mice with established disease ameliorated BOS and abolished the associated differentiation of Tfh and GC B cells. Guided by RNA-sequencing data, mechanistic studies performed using murine and human T cells showed that C5aR1 signaling amplifies IL-6–dependent expression of the transcription factor c-MAF and the cytokine IL-21 via phosphorylating phosphokinase B (AKT) and activating the mammalian target of rapamycin (mTOR). In addition to linking C5aR1-initiated signaling to Tfh cell differentiation, our findings suggest that C5aR1 may be a useful therapeutic target for prevention and/or treatment of individuals with Tfh cell–dependent diseases, including those chronic GvHD patients who have anti-host reactive antibodies.
Divya A. Verghese, Nicholas Chun, Katelyn Paz, Miguel Fribourg, Trent M. Woodruff, Ryan Flynn, Yuan Hu, Huabao Xiong, Weijia Zhang, Zhengzi Yi, Jing Du, Bruce R. Blazar, Peter S. Heeger
Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT). DCs play critical roles in GVHD induction. Modulating autophagy represents a promising therapeutic strategy for the treatment of immunological diseases. Complement receptors C3aR/C5aR expressed on DCs regulate immune responses by translating extracellular signals into intracellular activity. In the current study, we found that C3aR/C5aR deficiency enhanced ceramide-dependent lethal mitophagy (CDLM) in DCs. Cotransfer of host-type C3aR–/–/C5aR–/– DCs in the recipients significantly improved GVHD outcome after allogeneic HCT, primarily through enhancing CDLM in DCs. C3aR/C5aR deficiency in the host hematopoietic compartment significantly reduced GVHD severity via impairing Th1 differentiation and donor T cell glycolytic activity while enhancing Treg generation. Prophylactic treatment with C3aR/C5aR antagonists effectively alleviated GVHD while maintaining the graft-versus-leukemia (GVL) effect. Altogether, we demonstrate that inhibiting C3aR/C5aR induces lethal mitophagy in DCs, which represents a potential therapeutic approach to control GVHD while preserving the GVL effect.
Hung Nguyen, Sandeepkumar Kuril, David Bastian, Jisun Kim, Mengmeng Zhang, Silvia G. Vaena, Mohammed Dany, Min Dai, Jessica Lauren Heinrichs, Anusara Daenthanasanmak, Supinya Iamsawat, Steven Schutt, Jianing Fu, Yongxia Wu, David P. Fairlie, Carl Atkinson, Besim Ogretmen, Stephen Tomlinson, Xue-Zhong Yu
The analysis and validation of flow cytometry–based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability <20% and interclass correlation coefficients >0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman’s correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.
Sabine Ivison, Mehrnoush Malek, Rosa V. Garcia, Raewyn Broady, Anne Halpin, Manon Richaud, Rollin F. Brant, Szu-I Wang, Mathieu Goupil, Qingdong Guan, Peter Ashton, Jason Warren, Amr Rajab, Simon Urschel, Deepali Kumar, Mathias Streitz, Birgit Sawitzki, Stephan Schlickeiser, Janetta J. Bijl, Donna A. Wall, Jean-Sebastien Delisle, Lori J. West, Ryan R. Brinkman, Megan K. Levings
BACKGROUND. The molecular understanding of the progression from acute to chronic organ injury is limited. Ischemia/reperfusion injury (IRI) triggered during kidney transplantation can contribute to progressive allograft dysfunction. METHODS. Protocol biopsies (n = 163) were obtained from 42 kidney allografts at 4 time points after transplantation. RNA sequencing–mediated (RNA-seq–mediated) transcriptional profiling and machine learning computational approaches were employed to analyze the molecular responses to IRI and to identify shared and divergent transcriptional trajectories associated with distinct clinical outcomes. The data were compared with the response to IRI in a mouse model of the acute to chronic kidney injury transition. RESULTS. In the first hours after reperfusion, all patients exhibited a similar transcriptional program under the control of immediate-early response genes. In the following months, we identified 2 main transcriptional trajectories leading to kidney recovery or to sustained injury with associated fibrosis and renal dysfunction. The molecular map generated by this computational approach highlighted early markers of kidney disease progression and delineated transcriptional programs associated with the transition to chronic injury. The characterization of a similar process in a mouse IRI model extended the relevance of our findings beyond transplantation. CONCLUSIONS. The integration of multiple transcriptomes from serial biopsies with advanced computational algorithms overcame the analytical hurdles related to variability between individuals and identified shared transcriptional elements of kidney disease progression in humans, which may prove as useful predictors of disease progression following kidney transplantation and kidney injury. This generally applicable approach opens the way for an unbiased analysis of human disease progression. FUNDING. The study was supported by the California Institute for Regenerative Medicine and by the Swiss National Science Foundation.
Pietro E. Cippà, Bo Sun, Jing Liu, Liang Chen, Maarten Naesens, Andrew P. McMahon
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