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
B cells are increasingly recognised to play an important role in the ongoing control of hepatitis B virus (HBV). The development of antibodies against the viral surface antigen (HBsAg) constitutes the hallmark of resolution of acute infection and is a therapeutic goal for functional cure of chronic HBV (CHB). We characterised B cells directly ex vivo from the blood and liver of patients with CHB to investigate constraints on their antiviral potential. Unexpectedly, we found that HBsAg-specific B cells persisted in the blood and liver of many patients with CHB and were enriched for T-bet, a signature of antiviral potential in B cells. However purified, differentiated HBsAg-specific B cells from patients with CHB had defective antibody production, consistent with undetectable anti-HBs antibodies in vivo. HBsAg-specific and global B cells had an accumulation of CD21–CD27– atypical memory B cells (atMBC) with high expression of inhibitory receptors including PD-1. These atMBC demonstrated altered signalling, homing, differentiation into antibody-producing cells, survival and antiviral/pro-inflammatory cytokine production, that could be partially rescued by PD-1 blockade. Analysis of B cells within healthy and HBV-infected livers implicated the combination of this tolerogenic niche and HBV infection in driving PD-1hiatMBC and impairing B cell immunity.
Alice R. Burton, Laura J. Pallett, Laura E. McCoy, Kornelija Suveizdyte, Oliver E. Amin, Leo Swadling, Elena Alberts, Brian R. Davidson, Patrick T.F. Kennedy, Upkar S. Gill, Claudia Mauri, Paul A. Blair, Nadege Pelletier, Mala K. Maini
SUMOylation is involved in the development of several inflammatory diseases, but the physiological significance of SUMO-modulated c-Maf in autoimmune diabetes is not completely understood. Here, we report that an age-dependent attenuation of c-Maf SUMOylation in CD4+ T cells is positively correlated with the IL-21–mediated diabetogenesis in NOD mice. Using 2 strains of T cell–specific transgenic NOD mice overexpressing wild-type c-Maf (Tg-WTc) or SUMOylation site–mutated c-Maf (Tg-KRc), we demonstrated that Tg-KRc mice developed diabetes more rapidly than Tg-WTc mice in a CD4+ T cell–autonomous manner. Moreover, SUMO-defective c-Maf preferentially transactivated Il21 to promote the development of CD4+ T cells with an extrafollicular helper T cell phenotype and expand the numbers of granzyme B–producing effector/memory CD8+ T cells. Furthermore, SUMO-defective c-Maf selectively inhibited recruitment of Daxx/HDAC2 to the Il21 promoter and enhanced histone acetylation mediated by CREB-binding protein (CBP) and p300. Using pharmacological interference with CBP/p300, we illustrated that CBP30 treatment ameliorated c-Maf–mediated/IL-21–based diabetogenesis. Taken together, our results show that the SUMOylation status of c-Maf has a stronger regulatory effect on IL-21 than the level of c-Maf expression, through an epigenetic mechanism. These findings provide new insights into how SUMOylation modulates the pathogenesis of autoimmune diabetes in a T cell–restricted manner and on the basis of a single transcription factor.
Chao-Yuan Hsu, Li-Tzu Yeh, Shin-Huei Fu, Ming-Wei Chien, Yu-Wen Liu, Shi-Chuen Miaw, Deh-Ming Chang, Huey-Kang Sytwu
Autosomal dominant hyper IgE syndrome (AD-HIES), or Job’s syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF-α differentiated the responses in AD-HIES from HVs. This was associated with reduced IL-10 family signaling in blister-infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-α blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with AD-HIES suffering S. aureus infections.
Ian A. Myles, Erik D. Anderson, Noah J. Earland, Kol A. Zarember, Inka Sastalla, Kelli W. Williams, Portia Gough, Ian N. Moore, Sundar Ganesan, Cedar J. Fowler, Arian Laurence, Mary Garofalo, Douglas B. Kuhns, Mark D. Kieh, Arhum Saleem, Pamela A. Welch, Dirk A. Darnell, John I. Gallin, Alexandra F. Freeman, Steven M. Holland, Sandip K. Datta
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
Germinal centers (GCs) are major sites of clonal B cell expansion and generation of long-lived, high-affinity antibody responses to pathogens. Signaling through toll-like receptors(TLRs) on B cells promotes many aspects of GC B cell responses, including affinity-maturation, class-switching and differentiation into long-lived memory and plasma cells. A major challenge for effective vaccination is identifying strategies to specifically promote GC B cell responses. Here we have identified a mechanism of regulation of GC B cell TLR signaling, mediated by αv integrins and non-canonical autophagy. Using B cell-specific αv-knockout mice, we show that loss of αv-mediated TLR regulation increased GC B cell expansion, somatic-hypermutation, class-switching, and generation of long-lived plasma cells after immunization with virus-like particles(VLPs) or antigens associated with TLR ligand adjuvants. Furthermore, targeting αv-mediated regulation increased the magnitude and breadth of antibody responses to influenza virus vaccination. These data therefore identify a mechanism of regulation of GC B cells, which can be targeted to enhance antibody responses to vaccination.
Fiona Raso, Sara Sagadiev, Samuel Du, Emily Gage, Tanvi Arkatkar, Genita Metzler, Lynda M. Stuart, Mark T. Orr, David Rawlings, Shaun Jackson, Adam Lacy-Hulbert, Mridu Acharya
T cells play a key role in immune-mediated glomerulonephritis, but how cytotoxic T cells interact with podocytes remains unclear. To address this, we injected EGFP-specific CD8+ T cells from just EGFP death inducing (Jedi) mice into transgenic mice with podocyte-specific expression of EGFP. In healthy mice, Jedi T cells could not access EGFP+ podocytes. Conversely, when we induced nephrotoxic serum nephritis (NTSN) and injected Jedi T cells, EGFP+ podocyte transgenic mice showed enhanced proteinuria and higher blood urea levels. Morphometric analysis showed greater loss of EGFP+ podocytes, which was associated with severe crescentic and necrotizing glomerulonephritis. Notably, only glomeruli with disrupted Bowman’s capsule displayed massive CD8+ T cell infiltrates that were in direct contact with EGFP+ podocytes, causing their apoptosis. Thus, under control conditions with intact Bowman’s capsule, podocytes are not accessible to CD8+ T cells. However, breaches in Bowman’s capsule, as also noted in human crescentic glomerulonephritis, allow access of CD8+ T cells to the glomerular tuft and podocytes, resulting in their destruction. Through these mechanisms, a potentially reversible glomerulonephritis undergoes an augmentation process to a rapidly progressive glomerulonephritis, leading to end-stage kidney disease. Translating these mechanistic insights to human crescentic nephritis should direct future therapeutic interventions at blocking CD8+ T cells, especially in progressive stages of rapidly progressive glomerulonephritis.
Anqun Chen, Kyung Lee, Vivette D. D’Agati, Chengguo Wei, Jia Fu, Tian-Jun Guan, John Cijiang He, Detlef Schlondorff, Judith Agudo
Bi-allelic loss-of-function mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only one patient. We report 24 p40phox-deficient patients from 12 additional families in eight countries. These patients display eight different in-frame or out-of-frame mutations of NCF4, homozygous in 11 families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be loss-of-function, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was subnormal in the patients’ neutrophils, whereas PMA-induced DHR oxidation, which is widely used as a diagnostic test for CGD, was normal in some of the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also subnormal, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients. The patients described here suffer from hyperinflammation and peripheral infections, but they do not display any of the invasive bacterial and fungal infections seen in CGD. In conclusion, inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD
Annemarie van de Geer, Alejandro Nieto-Patlán, Douglas B. Kuhns, Anton T.J. Tool, Andrés A. Arias, Matthieu Bouaziz, Martin de Boer, José Luis Franco, Roel P. Gazendam, John L. van Hamme, Michel van Houdt, Karin van Leeuwen, Paul J.H. Verkuijlen, Timo K. van den Berg, Juan F. Alzate, Carlos A. Arango-Franco, Vritika Batura, Andrea R. Bernasconi, Barbara Boardman, Claire Booth, Siobhan O. Burns, Felipe Cabarcas, Nadine Cerf Bensussan, Fabienne Charbit-Henrion, Anniek Corveleyn, Caroline Deswarte, María Esnaola Azcoiti, Dirk Foell, John I. Gallin, Carlos Garcés, Margarida Guedes, Claas H. Hinze, Steven M. Holland, Stephen M. Hughes, Patricio Ibañez, Harry L. Malech, Isabelle Meyts, Marcela Moncada-Velez, Kunihiko Moriya, Esmeralda Neves, Matias Oleastro, Laura Perez, Vimel Rattina, Carmen Oleaga-Quintas, Neil Warner, Aleixo M. Muise, Jeanet Serafin López, Eunice Trindade, Julia Vasconselos, Severine Vermeire, Helmut Wittkowski, Austen Worth, Laurent Abel, Mary C. Dinauer, Peter D. Arkwright, Dirk Roos, Jean-Laurent Casanova, Taco W. Kuijpers, Jacinta Bustamante