The role of epithelial cell injury in kidney transplant outcomes – a cross-sectional study

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Abstract

We defined injury-induced transcriptome states in 4502 kidney transplant biopsies taken 1 day to 45 years post-transplant using genome-wide microarrays. Injury was measured by injury-induced gene sets and classifiers previously developed in transplants. In principal component analysis, PC1 correlated with both acute and chronic kidney injury and related inflammation, and PC2 with time post-transplant. PC3 was a novel dimension that correlated with epithelial remodeling pathways. Both PC1 and PC3 correlated with reduced survival, PC1 effects strongly increasing with time whereas PC3 effects being time-independent. In this model, we studied the expression of genes annotated in native kidneys in epithelial cells with failed repair: 12 “New” gene sets previously defined in single nucleus RNA sequencing of native kidneys with AKI (Genome Med.14(1):103). The “New4” gene sets reflecting epithelial-mesenchymal transition (EMT) correlated with injury PC1, lower eGFR, higher donor age, and future failure as strongly as any gene sets previously derived in transplants, independent of nephron segment of origin and graft rejection. These results suggest that there are two distinct dimensions in kidney transplant response to injury: PC1, AKI-induced changes, failed repair, and inflammation; and PC3, a response involving epithelial remodeling without inflammation. Increasing kidney age amplifies PC1 and particularly PC3.

Authors

Philip F. Halloran, Jessica Chang, Martina Mackova, Katelynn Madill-Thomsen, Enver Akalin, Tarek Alhamad, Sanjiv Anand, Miha Arnol, Rajendra Baliga, Mirosław Banasik, Christopher Blosser, Georg Böhmig, Daniel Brennan, Jonathan Bromberg, Klemens Budde, Andrzej Chamienia, Kevin Chow, Michał Ciszek, Declan de Freitas, Dominika Dęborska-Materkowska, Alicja Debska-Ślizień, Arjang Djamali, Leszek Domański, Magdalena Durlik, Gunilla Einecke, Farsad Eskandary, Richard Fatica, Iman Francis, Justyna Fryc, John Gill, Jagbir Gill, Maciej Glyda, Sita Gourishankar, Marta Gryczman, Gaurav Gupta, Petra Hruba, Peter Hughes, Arskarapurk Jittirat, Zeljka Jurekovic, Layla Kamal, Mahmoud Kamel, Sam Kant, Nika Kojc, Joanna Konopa, James Lan, Roslyn Mannon, Arthur Matas, Joanna Mazurkiewicz, Marius Miglinas, Thomas Mueller, Marek Myślak, Seth Narins, Beata Naumnik, Anita Patel, Agnieszka Perkowska-Ptasińska, Michael Picton, Grzegorz Piecha, Emilio Poggio, Silvie Rajnochová Bloudíčková, Thomas Schachtner, Soroush Shojai, Majid Sikosana, Janka Slatinská, Katarzyna Smykal-Jankowiak, Željka Veceric Haler, Ondrej Viklicky, Ksenija Vucur, Matthew R. Weir, Andrzej Wiecek, Zbigniew Włodarczyk, Harold Yang, Ziad Zaky, Patrick T. Gauthier, Christian Hinze

CAR Treg synergy with anti-CD154 promotes infectious tolerance and dictates allogeneic heart transplant acceptance

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Abstract

Successful allograft-specific tolerance induction would eliminate the need for daily immunosuppression and improve posttransplant quality of life. Adoptive cell therapy with regulatory T cells expressing donor-specific chimeric antigen receptors (CAR Tregs) is a promising strategy but, as monotherapy, cannot prolong survival with allografts with multiple MHC mismatches. Using an HLA-A2–transgenic haplo-mismatched heart transplantation model in immunocompetent C57BL/6 recipients, we showed that HLA-A2–specific CAR (A2.CAR) Tregs were able to synergize with a low dose of anti-CD154 to enhance graft survival. Using haplo-mismatched grafts expressing the 2W-OVA transgene and tetramer-based tracking of 2W- and OVA-specific T cells, we showed that in mice with accepted grafts, A2.CAR Tregs inhibited donor-specific T cell, B cell, and antibody responses and promoted a substantial increase in endogenous FOXP3+ Tregs with indirect donor specificity. By contrast, in mice where A2.CAR Tregs failed to prolong graft survival, FOXP3– A2.CAR T cells preferentially accumulated in rejecting allografts, and endogenous donor-specific responses were not controlled. This study therefore provides evidence for synergy between A2.CAR Tregs and CD154 blockade to promote infectious tolerance in immunocompetent recipients of haplo-mismatched heart grafts and defines features of A2.CAR Tregs when they fail to reshape host immunity toward allograft tolerance.

Authors

Samarth S. Durgam, Isaac Rosado-Sánchez, Dengping Yin, Madeleine Speck, Majid Mojibian, Ismail Sayin, Grace E. Hynes, Maria-Luisa Alegre, Megan K. Levings, Anita S. Chong

IFN-γ and donor leukocyte infusions for relapsed myeloblastic malignancies after allogeneic hematopoietic stem cell transplantation

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Abstract

BACKGROUND. The graft-vs-leukemia (GVL) effect contributes to the efficacy of allogeneic stem cell transplantation (alloSCT). However, relapse, indicative of GVL failure, is the greatest single cause of treatment failure. Based on preclinical data showing that IFN-γ is important to sensitize myeloblasts to alloreactive T cells, we performed a phase I trial of IFN-γ combined with donor leukocyte infusions (DLI) in myeloblastic malignancies that relapsed post-HLA-matched alloSCT. METHODS. Patients with relapsed acute myeloid leukemia or myelodysplastic syndrome after alloSCT were eligible. Patients self-administered IFN-γ for 4 weeks (cohort 1) or 1 week (cohort 2), followed by DLI and concurrent IFN-γ for a total of 12 weeks. Bone marrow samples were analyzed by single-cell RNA sequencing (scRNAseq) to assess in vivo responses to IFN-γ by malignant myeloblasts. RESULTS. IFN-γ monotherapy was well tolerated by all subjects (n=7). Treatment-related toxicities after DLI included: grade I-II graft-versus-host disease (n=5), immune effector cell-associated neurotoxicity syndrome (n=2), and idiopathic pulmonary syndrome (n=1), all of which resolved with corticosteroids. Four of 6 DLI recipients achieved minimal residual disease-negative complete remissions and full donor hematopoietic recovery. Median overall survival was 579 days (range, 97-906) in responders. ScRNAseq confirmed in vivo activation of IFN-γ response pathway in hematopoietic stem cell-like or myeloid progenitor cells after IFN-γ in analyzed samples. CONCLUSIONS. IFN-γ was safe and well tolerated in this phase I study of IFN-γ for relapsed AML/MDS post-alloSCT, with a promising efficacy signal when combined with DLI. Larger studies are needed to formally test the efficacy of this approach. TRIAL RESGISTRATION. ClinicalTrials.gov NCT04628338. FUNDING. The research was supported by The UPMC Hillman Cancer Center Cancer Immunology and Immunotherapy Program (CIIP) Pilot Award and Cure Within Reach: Drug Repurposing Clinical Trials to Impact Blood Cancers. Recombinant IFN-gamma (Actimmune®) was donated by Horizon Therapeutics.

Authors

Sawa Ito, Emily Geramita, Kedwin Ventura, Biswas Neupane, Shruti Bhise, Erika M. Moore, Scott Furlan, Warren D. Shlomchik

NKG2D blockade impairs tissue-resident memory T cell accumulation and reduces chronic lung allograft dysfunction

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Abstract

Chronic lung allograft dysfunction (CLAD) substantially limits long-term survival following lung transplantation. To identify potential targets for CLAD prevention, T cells from explanted CLAD lungs and lung-draining lymph nodes, as well as diseased and nondiseased controls were isolated and single-cell RNA sequencing and TCR sequencing were performed. TCR sequencing revealed a clonally expanded population of CD8+ tissue-resident memory T cells (TRMs) with high cytotoxic potential, including upregulation of KLRK1, encoding the co-receptor NKG2D. These cytotoxic CD8+ TRMs accumulated around the CLAD airways and had a 100-fold increase in clonal overlap with lung-draining lymph nodes when compared with non-CLAD lungs. Using a murine model of orthotopic lung transplantation, we confirmed that cytotoxic CD8+ TRM accumulation was due to chronic rejection and not transplantation alone. Furthermore, blocking NKG2D in vivo attenuated the airway remodeling following transplantation and diminished airway accumulation of CD8+ T cells. Our findings support NKG2D as a potential therapeutic target for CLAD, affecting cytotoxic CD8+ TRM accumulation.

Authors

Kaveh Moghbeli, Madeline A. Lipp, Marta Bueno, Andrew Craig, Michelle Rojas, Minahal Abbas, Zachary I. Lakkis, Byron Chuan, John Sembrat, Kentaro Noda, Daniel J. Kass, Kong Chen, Li Fan, Tim Oury, Zihe Zhou, Xingan Wang, John F. McDyer, Oliver Eickelberg, Mark E. Snyder

Orchestrated response from heterogenous fibroblast subsets contributes to repair from surgery-induced stress after airway reconstruction

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Abstract

Surgery of the tracheobronchial tree carries high morbidity, with over half of the complications occurring at the anastomosis. Although fibroblasts are crucial in airway wound healing, the underlying cellular and molecular mechanisms in airway reconstruction remain unknown. We hypothesized that airway reconstruction initiates a surgery-induced stress (SIS) response, altering fibroblast communication within airway tissues. Using single-cell RNAseq, we analyzed native and reconstructed airways and identified five fibroblast subpopulations, each with distinct spatial distributions across anastomotic, submucosal, perichondrial, and paratracheal areas. During homeostasis, Adventitial and Airway fibroblasts (Adventitial Fb and Airway Fb, respectively) maintained tissue structure and created cellular niches by regulating ECM turnover. Under SIS, Perichondrial fibroblasts (PC-Fb) exhibited chondroprogenitor-like gene signatures, and Immune-recruiting fibroblasts (IR-Fb) facilitated cell infiltration. Cthrc1 activated fibroblasts (Cthrc1+ Fb), mainly derived from Adventitial Fb, primarily contributed to fibrotic scar formation and collagen production, mediated by TGFβ. Furthermore, repeated SIS created an imbalance in fibroblast states favoring emergence of CTHRC1+ Fb and leading to impaired fibroblasts-basal cell crosstalk. Collectively, these data identify PC, IR, and Cthrc1+ Fb as a signaling hub, with SIS emerging as a mechanism initiating airway remodeling after reconstruction that, if not controlled, may lead to complications such as stenosis or anastomotic breakdown.

Authors

Jazmin Calyeca, Zakarie Hussein, Zheng Hong Tan, Lumei Liu, Sayali Dharmadhikari, Kimberly M. Shontz, Tatyana A. Vetter, Christopher K. Breuer, Susan D. Reynolds, Tendy Chiang

High-dimensional analysis of NK cells in kidney transplantation uncovers subsets associated with antibody-independent graft dysfunction

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Abstract

Natural Killer (NK) cells respond to diseased and allogeneic cells through NKG2A/HLA-E or Killer-cell Immunoglobulin-like receptor (KIR)/HLA-ABC interactions. Correlations between HLA/KIR disparities and kidney transplant pathology suggest an antibody-independent pathogenic role for NK cells in transplantation, but mechanisms remain unclear. Using CyTOF to characterize recipient peripheral NK cell phenotypes and function, we observed diverse NK cell subsets amongst participants that responded heterogeneously to allo-stimulators. NKG2A+/KIR+ NK cells responded more vigorously than other subsets, and this heightened response persisted post-kidney-transplant despite immunosuppression. In test and validation sets from two clinical trials, pre-transplant donor-induced release of cytotoxicity mediator, Ksp37, by NKG2A+ NK cells correlated with reduced long-term allograft function. Separate analyses showed Ksp37 gene expression in allograft biopsies lacking histological rejection correlated with death censored graft loss. Our findings support an antibody-independent role for NK cells in transplant injury and support further testing of pre-transplant, donor-reactive, NK cell-produced Ksp37 as a risk-assessing, transplantation biomarker.

Authors

Dan Fu Ruan, Miguel Fribourg, Yuko Yuki, Yeon-Hwa Park, Maureen P. Martin, Haocheng Yu, Geoffrey C. Kelly, Brian H. Lee, Ronaldo M. de Real, Rachel Lee, Daniel Geanon, Seunghee Kim-Schulze, Nicholas Chun, Paolo Cravedi, Mary Carrington, Peter S. Heeger, Amir Horowitz

MerTK-dependent efferocytosis by monocytic-MDSCs mediates resolution of post-lung transplant ischemia-reperfusion injury

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Abstract

Lung transplantation (LTx) outcomes are impeded by ischemia-reperfusion injury (IRI) and subsequent chronic lung allograft dysfunction (CLAD). We examined the undefined role of MerTK (receptor Mer tyrosine kinase) on monocytic myeloid-derived suppressor cells (M-MDSCs) in efferocytosis to facilitate resolution of lung IRI. Single-cell RNA sequencing of lung tissue and bronchoalveolar lavage (BAL) from post-LTx patients were analyzed. Murine lung hilar ligation and allogeneic orthotopic LTx models of IRI were used with Balb/c (WT), Cebpb-/- (MDSC-deficient), Mertk-/- or MerTK-CR (cleavage resistant) mice. A significant downregulation in MerTK-related efferocytosis genes in M-MDSC populations of CLAD patients was observed compared to healthy subjects. In the murine IRI model, significant increase in M-MDSCs, MerTK expression, efferocytosis and attenuation of lung dysfunction was observed in WT mice during injury resolution that was absent in Cebpb-/- and Mertk-/- mice. Adoptive transfer of M-MDSCs in Cebpb-/- mice significantly attenuated lung dysfunction and inflammation. Additionally, in a murine orthotopic LTx model, increases in M-MDSCs were associated with resolution of lung IRI in the transplant recipients. In vitro studies demonstrated the ability of M-MDSCs to efferocytose apoptotic neutrophils in a MerTK-dependent manner. Our results suggest that MerTK-dependent efferocytosis by M-MDSCs can substantially contribute to the resolution of post-LTx IRI.

Authors

Victoria Leroy, Denny Joseph Manual Kollareth, Zhenxiao Tu, Jeff Arni C. Valisno, Makena Woolet-Stockton, Biplab K. Saha, Amir M. Emtiazjoo, Mindaugas Rackauskas, Lyle L. Moldawer, Philip A. Efron, Guoshuai Cai, Carl Atkinson, Gilbert R. Upchurch, Jr., Ashish K. Sharma

Cytomegalovirus immunity in high-risk liver transplant recipients following preemptive antiviral therapy vs. prophylaxis

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Abstract

Cytomegalovirus (CMV)-specific T-cells, NK cells, and neutralizing antibodies (nAb) were assessed in a randomized trial of CMV prevention with preemptive antiviral therapy (PET) vs. prophylactic antiviral therapy (PRO) in donor seropositive/recipient seronegative (D+R-) liver transplant recipients (LTxR), at 100 days (end of intervention), and at 6 and 12 months post-transplant. The PET group had significantly increased numbers of circulating polyfunctional T-cells, NK cells, and nAb compared to the PRO group at day 100 and several CMV immune parameters remained significantly higher by 12 months post-transplant. Among PET recipients, preceding CMV viremia (vs. no preceding viremia) was associated with significantly higher levels of most CMV immune parameters at day 100. Higher numbers of CMV-specific polyfunctional T-cells and NKG2C+ NK cells at day 100 were associated with a decreased incidence of CMV disease in multivariable Cox regression. The strongest associations with protection against CMV disease were with increased numbers of CMV-specific polyfunctional CD4 T-cells, CD3negCD56dimCD57negNKG2Cpos, and CD3negCD56dimCD57posNKG2Cpos NK cells. PET is superior to PRO for CMV disease prevention by allowing low-level CMV replication and associated antigen exposure that is promptly controlled by antiviral therapy and facilitates enhanced CMV protective immunity in D+R- LTxR.

Authors

Danniel Zamora, Sayan Dasgupta, Terry Stevens-Ayers, Bradley Edmison, Drew J. Winston, Raymund R. Razonable, Aneesh K. Mehta, G. Marshall Lyon, Michael Boeckh, Nina Singh, David M. Koelle, Ajit P. Limaye

Human gut microbiota-reactive DP8a regulatory T cells prevent acute graft-versus-host disease in a CD73-dependent manner

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Abstract

Graft-versus-host disease (GvHD) is a life-threatening complication frequently occurring following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Since gut microbiota and regulatory T cells (Tregs) are believed to play roles in GvHD prevention, we investigated whether DP8a Tregs, which we have previously described to harbor a TCR-specificity for the gut commensal Faecalibacterium prausnitzii, could protect against GvHD, thereby linking microbiota and its effect on GvHD. We observed a decrease in CD73+ DP8α Treg frequency in allo-HSCT patients at 1-month post-transplantation, which was associated with aGvHD development at 1-month post-transplantation, as compared to aGvHD-free patients, without being correlated to hematological disease’s relapse. Importantly, CD73 activity was shown to be critical for DP8αTreg suppressive function. Moreover, the frequency of host-reactive DP8α Tregs was also lower in aGvHD patients, as compared to aGvHD-free patients, which could embody a protective mechanism responsible for the maintenance of these cell subset in GvHD-free patients. We also showed that human DP8α Tregs protected mice against xeno-GvHD through limiting deleterious inflammation and preserving gut integrity. Altogether, these results demonstrated that human DP8α Tregs mediate aGvHD prevention in a CD73-dependent manner, likely through host-reactivity, advocating for the use of these cells for the development of innovative therapeutic strategies to preclude aGvHD-related inflammation.

Authors

Emmanuelle Godefroy, Patrice Chevallier, Fabienne Haspot, Caroline Vignes, Véronique Daguin, Sylvia Lambot, Margaux Verdon, Margaux De Seilhac, Valentin Letailleur, Anne Jarry, Annabelle Pédron, Thierry Guillaume, Pierre Peterlin, Alice Garnier, Marie-Anne Vibet, Maxence Mougon, Amandine Bourgeois, Maxime Jullien, Francine Jotereau, Frédéric Altare

Lymphotoxin β receptor and tertiary lymphoid organs shape acute and chronic allograft rejection

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Abstract

Solid organ transplantation remains the life-saving treatment for end-stage organ failure, but chronic rejection remains a major obstacle to long-term allograft outcomes and has not improved substantially. Tertiary lymphoid organs (TLO) are ectopic lymphoid structures that form under conditions of chronic inflammation, and evidence from human transplantation suggests that TLO regularly form in allografts undergoing chronic rejection. In this study, we utilized a mouse renal transplantation model and manipulation of the lymphotoxin alpha (LTα) – lymphotoxin beta receptor (LTβR) pathway, which is essential for TLO formation, to define the role of TLO in transplantation. We showed that intragraft TLO are sufficient to activate the alloimmune response and mediate graft rejection in a model where the only lymphoid organs are TLO in the allograft. When transplanted to recipients with a normal set of secondary lymphoid organs, the presence of graft TLO or LTα overexpression accelerated rejection. If the LTβR pathway was disrupted in the donor graft, TLO formation was abrogated, and graft survival prolonged. Intravital microscopy of renal TLO demonstrated that local T and B cell activation in TLOs is similar to that observed in secondary lymphoid organs. In summary, we demonstrated that immune activation in TLO contributes to local immune responses, leading to earlier allograft failure. TLO and the LTαβ-LTβR pathway are therefore prime targets to limit local immune responses and prevent allograft rejection. These findings are applicable to other diseases such as autoimmunity or tumors, where either limiting or boosting local immune responses is beneficial and improves disease outcomes.

Authors

Gang Zhang, Neda Feizi, Daqiang Zhao, Latha Halesha, Amanda L. Williams, Parmjeet S. Randhawa, Khodor I. Abou-Daya, Martin H. Oberbarnscheidt