Antibody-mediated rejection (AMR) is a principal cause of acute and chronic failure of lung allografts. However, mechanisms mediating this oftentimes fatal complication are poorly understood. Here, we show that Foxp3+ T cells formed aggregates in rejection-free human lung grafts and accumulated within induced bronchus-associated lymphoid tissue (BALT) of tolerant mouse lungs. Using a retransplantation model, we show that selective depletion of graft-resident Foxp3+ T lymphocytes resulted in the generation of donor-specific antibodies (DSA) and AMR, which was associated with complement deposition and destruction of airway epithelium. AMR was dependent on graft infiltration by B and T cells. Depletion of graft-resident Foxp3+ T lymphocytes resulted in prolonged interactions between B and CD4+ T cells within transplanted lungs, which was dependent on CXCR5-CXCL13. Blockade of CXCL13 as well as inhibition of the CD40 ligand and the ICOS ligand suppressed DSA production and prevented AMR. Thus, we have shown that regulatory Foxp3+ T cells residing within BALT of tolerant pulmonary allografts function to suppress B cell activation, a finding that challenges the prevailing view that regulation of humoral responses occurs peripherally. As pulmonary AMR is largely refractory to current immunosuppression, our findings provide a platform for developing therapies that target local immune responses.
Wenjun Li, Jason M. Gauthier, Ryuji Higashikubo, Hsi-Min Hsiao, Satona Tanaka, Linh Vuong, Jon H. Ritter, Alice Y. Tong, Brian W. Wong, Ramsey R. Hachem, Varun Puri, Ankit Bharat, Alexander S. Krupnick, Chyi S. Hsieh, William M. Baldwin III, Francine L. Kelly, Scott M. Palmer, Andrew E. Gelman, Daniel Kreisel
Transplantation with autologous hematopoietic progenitors remains an important consolidation treatment for multiple myeloma (MM) patients and is thought to prolong disease plateau-phase by providing intensive cytoreduction. However, transplantation induces inflammation in the context of profound lymphodepletion that may cause hitherto unexpected immunological effects. We developed preclinical models of bone marrow transplantation (BMT) for MM using Vk*MYC myeloma-bearing recipients and donors that were myeloma-naïve or were myeloma-experienced to simulate autologous transplantation. Surprisingly, we demonstrate broad induction of T cell-dependent myeloma control, most efficiently from memory T cells within myeloma-experienced grafts, but also through priming of naïve T cells after BMT. CD8+ T cells from mice with controlled myeloma had a distinct TCR repertoire and higher clonotype overlap relative to myeloma-free BMT recipients. Furthermore, T cell-dependent myeloma control could be adoptively transferred to secondary recipients, and was myeloma clone-specific. Interestingly, donor-derived IL-17A acted directly on myeloma cells expressing the IL-17-receptor to induce a transcriptional landscape that promoted tumor growth and immune escape. Conversely, donor IFNγ secretion and signaling was critical to protective immunity, and was profoundly augmented by CD137 agonists. These data provide new insights into the mechanisms of action of transplantation in myeloma and suggests rational approaches to improving clinical outcome.
Slavica Vuckovic, Simone A. Minnie, David Smith, Kate H. Gartlan, Thomas S. Watkins, Kate A. Markey, Pamela Mukhopadhyay, Camille Guillerey, Rachel D. Kuns, Kelly R. Locke, Antonia L. Pritchard, Peter A. Johansson, Antiopi Varelias, Ping Zhang, Nicholas D. Huntington, Nicola Waddell, Marta Chesi, John J. Miles, Mark J. Smyth, Geoffrey R. Hill
The targeted delivery of therapeutic drugs to lymph nodes (LNs) provides an unprecedented opportunity to improve the outcomes of transplantation and immune-mediated diseases. The high endothelial venule is a specialized segment of LN vasculature that uniquely expresses peripheral node addressin (PNAd) molecules. PNAd is recognized by MECA79 mAb. We previously generated a MECA79 mAb–coated microparticle (MP) that carries tacrolimus. Although this MP trafficked to LNs, it demonstrated limited therapeutic efficacy in our transplant model. Here, we have synthesized a nanoparticle (NP) as a carrier of anti-CD3, and optimized the conjugation strategy to coat the NP surface with MECA79 mAb (MECA79-anti-CD3-NP) to enhance LN accumulation. As compared with nonconjugated NPs, a significantly higher quantity of MECA79-NPs accumulated in the draining lymph node (DLN). Many MECA79-NPs underwent internalization by T cells and dendritic cells within the LNs. Short-term treatment of murine cardiac allograft recipients with MECA79-anti-CD3-NP resulted in significantly prolonged allograft survival in comparison with the control groups. Prolonged graft survival following treatment with MECA79-anti-CD3-NP was characterized by a significant increase in intragraft and DLN Treg populations. Treg depletion abrogated the prolongation of heart allograft survival. We believe this targeted approach of drug delivery could redefine the methods of administering immune therapeutics in transplantation.
Baharak Bahmani, Mayuko Uehara, Liwei Jiang, Farideh Ordikhani, Naima Banouni, Takaharu Ichimura, Zhabiz Solhjou, Georg J. Furtmüller, Gerald Brandacher, David Alvarez, Ulrich H. von Andrian, Kenji Uchimura, Qiaobing Xu, Ishaan Vohra, Osman A. Yilmam, Yousef Haik, Jamil Azzi, Vivek Kasinath, Jonathan S. Bromberg, Martina M. McGrath, Reza Abdi
Interrupting T cell costimulatory signals as a strategy to control undesired immune responses, such as occur in autoimmunity or transplantation, has the potential to alleviate many of the unwanted side effects associated with current immunosuppressive therapies. Belatacept, a high-affinity version of CTLA4-Ig that blocks ligand ligation to CD28, has been approved for use in kidney transplant recipients. Despite the long-term benefits associated with its use, such as improved renal function and lower cardiovascular risk, a subset of patients treated with belatacept experience elevated rates of acute T cell–mediated rejection, tempering enthusiasm for its use. Here we demonstrate that costimulation-independent T cell alloreactivity relies on signaling through CD122, the shared IL-2 and IL-15 receptor β-chain. Combined costimulatory and CD122 blockade improved survival of transplanted tissue in mice and nonhuman primates by controlling proliferation and effector function of CD8+ T cells. The high-affinity IL-2 receptor was dispensable for memory CD8+ T cell responses, whereas signaling through CD122 as a component of the high-affinity IL-15 receptor was critical for costimulation-independent memory CD8+ T cell recall, distinguishing specific roles for IL-2 and IL-15 in T cell activation. These studies outline a novel approach for clinical optimization of costimulatory blockade strategies in transplantation by targeting CD122.
David V. Mathews, Ying Dong, Laura B. Higginbotham, Steven C. Kim, Cynthia P. Breeden, Elizabeth A. Stobert, Joseph Jenkins, J. Yun Tso, Christian P. Larsen, Andrew B. Adams
Regulatory T-cells (Treg) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T-cells, where protein kinase C-θ (PKC-θ) localizes to the contact-point between T-cells and antigen-presenting cells, in human and mouse Treg, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho-target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Treg with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Treg were significantly better than controls in suppressing alloreactive T-cell priming in graft-versus-host disease, and graft-versus-host disease lethality, using a complete MHC mismatch mouse model of acute graft-versus-host disease (C57BL/6 donor in to BALB/c host). Interestingly, vimentin disruption augmented suppressor function of PKC-θ-deficient mouse Treg. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrated that vimentin is a key metabolic and functional controller of Treg activity, and provide proof-of-principle that disrupting vimentin is a feasible, translationally relevant method to enhance Treg potency.
Cameron McDonald-Hyman, James T. Muller, Michael Loschi, Govindarajan Thangavelu, Asim Saha, Sudha Kumari, Dawn K. Reichenbach, Michelle J. Smith, Guoan Zhang, Brent H. Koehn, Jiqiang Lin, Jason S. Mitchell, Brian T. Fife, Angela Panoskaltsis-Mortari, Colby J. Feser, Andrew Kemal Kirchmeier, Mark J. Osborn, Keli L. Hippen, Ameeta Kelekar, Jonathan S. Serody, Laurence A. Turka, David H. Munn, Hongbo Chi, Thomas A. Neubert, Michael L. Dustin, Bruce R. Blazar
Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract remains the major cause of morbidity and non-relapse mortality after bone marrow transplantation (BMT). The Paneth cell protein, regenerating islet-derived 3-alpha (REG3α), is a biomarker specific for GI GVHD. REG3α serum levels rose in the systematic circulation as GVHD progressively destroyed Paneth cells and reduced GI epithelial barrier function. Paradoxically, GVHD suppressed intestinal REG3γ (the mouse homologue of human REG3α), and the absence of REG3γ in BMT recipients intensified GVHD but did not change the composition of the microbiome. IL-22 administration restored REG3γ production and prevented apoptosis of both intestinal stem cells (ISCs) and Paneth cells, but this protection was completely abrogated in Reg3g−/− mice. In vitro, addition of REG3α reduced the apoptosis of colonic cell lines. Strategies that increase intestinal REG3α/γ to promote crypt regeneration may offer a novel, non-immunosuppressive approach for GVHD and perhaps for other diseases involving the ISC niche such as inflammatory bowel disease.
Dongchang Zhao, Yeung-Hyen Kim, Seihwan Jeong, Joel K. Greenson, Mohammed S. Chaudhry, Matthias Hoepting, Erik R. Anderson, Marcel R.M. van den Brink, Jonathan U. Peled, Antonio L.C. Gomes, Ann E. Slingerland, Michael J. Donovan, Andrew C. Harris, John E. Levine, Umut Özbek, Lora V. Hooper, Thaddeus S. Stappenbeck, Aaron M. Ver Heul, Ta-Chiang Liu, Pavan Reddy, James L.M. Ferrara
Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab′, in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4-Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/sirolimus recipients in the setting of sepsis and a paralyzed INF-γ response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant.
Benjamin K. Watkins, Victor Tkachev, Scott N. Furlan, Daniel J. Hunt, Kayla Betz, Alison Yu, Melanie Brown, Nicolas Poirier, Hengqi Betty Zheng, Agne Taraseviciute, Lucrezia Colonna, Caroline Mary, Gilles Blancho, Jean-Paul Soulillou, Angela Panoskaltsis-Mortari, Prachi Sharma, Anapatricia Garcia, Elizabeth Strobert, Kelly Hamby, Aneesah Garrett, Taylor Deane, Bruce R. Blazar, Bernard Vanhove, Leslie S. Kean
Purinergic receptor-7 (P2X7R) signaling controls Th17 and Th1 generation/differentiation, while NOD-like receptor P3 (NLRP3) acts as a Th2 transcriptional factor. Here, we demonstrated the existence of a P2X7R/NLRP3 pathway in T cells that is dysregulated by a P2X7R intracellular region loss-of-function mutation, leading to NLRP3 displacement and to excessive Th17 generation due to abrogation of the NLRP3-mediated Th2 program. This ultimately resulted in poor outcomes in cardiac-transplanted patients carrying the mutant allele, who showed abnormal Th17 generation. Transient NLRP3 silencing in nonmutant T cells or overexpression in mutant T cells normalized the Th profile. Interestingly, IL-17 blockade reduced Th17 skewing of human T cells in vitro and abrogated the severe allograft vasculopathy and abnormal Th17 generation observed in preclinical models in which P2X7R was genetically deleted. This P2X7R intracellular region mutation thus impaired the modulatory effects of P2X7R on NLRP3 expression and function in T cells and led to NLRP3 dysregulation and Th17 skewing, delineating a high-risk group of cardiac-transplanted patients who may benefit from personalized therapy.
Francesca D’Addio, Andrea Vergani, Luciano Potena, Anna Maestroni, Vera Usuelli, Moufida Ben Nasr, Roberto Bassi, Sara Tezza, Sergio Dellepiane, Basset El Essawy, Maria Iascone, Attilio Iacovoni, Laura Borgese, Kaifeng Liu, Gary Visner, Sirano Dhe-Paganon, Domenico Corradi, Reza Abdi, Randall C. Starling, Franco Folli, Gian Vincenzo Zuccotti, Mohamed H. Sayegh, Peter S. Heeger, Anil Chandraker, Francesco Grigioni, Paolo Fiorina
T cells must migrate in order to encounter antigen-presenting cells (APCs) and to execute their varied functions in immune defense and inflammation. ATP release and autocrine signaling through purinergic receptors contribute to T cell activation at the immune synapse that T cells form with APCs. Here, we show that T cells also require ATP release and purinergic signaling for their migration to APCs. We found that the chemokine SDF-1α triggered mitochondrial ATP production, rapid bursts of ATP release, and increased migration of primary human CD4+ T cells. This process depended on pannexin-1 ATP release channels and autocrine stimulation of P2X4 receptors. SDF-1α stimulation caused localized accumulation of mitochondria with P2X4 receptors near the front of cells, resulting in a feed-forward signaling mechanism that promotes cellular Ca2+ influx and sustains mitochondrial ATP synthesis at levels needed for pseudopod protrusion, T cell polarization, and cell migration. Inhibition of P2X4 receptors blocked the activation and migration of T cells in vitro. In a mouse lung transplant model, P2X4 receptor antagonist treatment prevented the recruitment of T cells into allograft tissue and the rejection of lung transplants. Our findings suggest that P2X4 receptors are therapeutic targets for immunomodulation in transplantation and inflammatory diseases.
Carola Ledderose, Kaifeng Liu, Yutaka Kondo, Christian J. Slubowski, Thomas Dertnig, Sara Denicoló, Mona Arbab, Johannes Hubner, Kirstin Konrad, Mahtab Fakhari, James A. Lederer, Simon C. Robson, Gary A. Visner, Wolfgang G. Junger
Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1β production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction–associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.
Hsi-Min Hsiao, Ramiro Fernandez, Satona Tanaka, Wenjun Li, Jessica H. Spahn, Stephen Chiu, Mahzad Akbarpour, Daniel Ruiz-Perez, Qiang Wu, Cem Turam, Davide Scozzi, Tsuyoshi Takahashi, Hannah P. Luehmann, Varun Puri, G.R. Scott Budinger, Alexander S. Krupnick, Alexander V. Misharin, Kory J. Lavine, Yongjian Liu, Andrew E. Gelman, Ankit Bharat, Daniel Kreisel
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