Autoimmunity
Abstract
Hypomorphic RAG1 or RAG2 mutations cause primary immunodeficiencies and can lead to autoimmunity, but the underlying mechanisms are elusive. We report here a patient carrying a c.116+2T>G homozygous splice site mutation in the first intron of RAG1, which led to aberrant splicing and greatly reduced RAG1 protein expression. B cell development was blocked at both the pro-B to pre-B transition and the pre-B to immature B cell differentiation step. The patient B cells had reduced B cell receptor repertoire diversity and decreased complementarity determining region 3 lengths. Despite B cell lymphopenia, the patient had abundant plasma cells in the BM and produced large quantities of IgM and IgG Abs, including autoantibodies. The proportion of naive B cells was reduced while the frequency of IgD–CD27– double-negative (DN) B cells, which quickly differentiated into Ab-secreting plasma cells upon stimulation, was greatly increased. Immune phenotype analysis of 52 patients with primary immunodeficiency revealed a strong association of the increased proportion of DN B and memory B cells with decreased number and proportion of naive B cells. These results suggest that the lymphopenic environment triggered naive B cell differentiation into DN B and memory B cells, leading to increased Ab production.
Authors
Qing Min, Xin Meng, Qinhua Zhou, Ying Wang, Yaxuan Li, Nannan Lai, Ermeng Xiong, Wenjie Wang, Shoya Yasuda, Meiping Yu, Hai Zhang, Jinqiao Sun, Xiaochuan Wang, Ji-Yang Wang
Abstract
BACKGROUND. Little is known about the autoreactive B cells in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3 (PR3+) B cells. METHODS. Multicolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of peripheral blood mononuclear cells from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase (MPO)-AAV, and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3+ B cells (total and subsets). RESULTS. The frequency of PR3+ B cells among circulating B cells was higher in PR3-AAV (4.77% [3.98%-6.01%]), than in MPO-AAV (3.16% [2.51%-5.22%]), and in AAV compared to HCs (1.67% [1.27%-2.16%], p<0.001 for all comparisons), implying a defective central tolerance checkpoint in patients. Only PBMC from PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving to be functionally distinct from those of MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in PR3-AAV patients. PR3+ B cells were enriched in the memory B cell compartment of PR3-AAV, and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3+ B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, p<0.05) and complete remission (p<0.001). CONCLUSIONS. This study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients in PR3-AAV, elucidating the selection process of autoreactive B cells.
Authors
Alvise Berti, Sophie Hillion, Amber M. Hummel, Young Min Son, Nedra Chriti, Tobias Peikert, Eva M. Carmona, Wayel H. Abdulahad, Peter Heeringa, Kristina M. Harris, E. William St. Clair, Paul Brunetta, Fernando Fervenza, Carol Langford, Cees Kallenberg, Peter Merkel, Paul A. Monach, Philip Seo, Robert F. Spiera, John H. Stone, Guido Grandi, Jie Sun, Jacques-Olivier Pers, Ulrich Specks, Divi Cornec
Abstract
Interleukin-33 (IL-33), a nuclear alarmin released during cell death, exerts context-specific effects on adaptive and innate immune cells eliciting potent inflammatory responses. We screened blood, skin and kidney tissues from patients with Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease driven by unabated type I interferon (IFN) production, and found increased amounts of extracellular IL-33 complexed with Neutrophil Extracellular Traps (NETs), correlating with severe, active disease. Using a combination of molecular, imaging and proteomic approaches, we show that SLE neutrophils -activated by disease immunocomplexes- release IL-33-decorated NETs that stimulate robust IFNα synthesis by plasmacytoid dendritic cells (pDCs) in an IL-33-receptor (ST2L)-dependent manner. IL33-silenced neutrophil-like cells cultured under lupus-inducing conditions generated NETs with diminished interferogenic effect. Importantly, SLE patient-derived NETs are enriched in mature bioactive isoforms of IL-33 processed by the neutrophil proteases elastase and cathepsin G. Pharmacological inhibition of these proteases neutralized IL-33-dependent IFNα production elicited by NETs. These data demonstrate a novel role for cleaved IL-33 alarmin decorating NETs in human SLE, linking neutrophil activation, type I IFN production and end-organ inflammation with skin pathology mirroring that observed in the kidneys.
Authors
Spiros Georgakis, Katerina Gkirtzimanaki, Garyfalia Papadaki, Hariklia Gakiopoulou, Elias Drakos, Maija-Leena Eloranta, Manousos Makridakis, Georgia Kontostathi, Jerome Zoidakis, Eirini Baira, Lars Rönnblom, Dimitrios T. Boumpas, Prodromos Sidiropoulos, Panayotis Verginis, George Bertsias
Abstract
The PD-1/PD-L1 pathway is a key immune checkpoint that regulates T cell activation. There is strong rationale to develop PD-1 agonists as therapeutics against autoimmunity, but progress in this area has been limited. Here, we generated T cell receptor (TCR) targeting, PD-1 agonist bispecifics called ImmTAAI molecules that mimic the ability of PD-L1 to facilitate the co-localization of PD-1 with the TCR complex at the target cell-T cell interface. PD-1 agonist ImmTAAI molecules specifically bound to target cells and were highly effective in activating the PD-1 receptor on interacting T cells to achieve immune suppression. Potent PD-1 antibody ImmTAAI molecules closely mimicked the mechanism of action of endogenously expressed PD-L1 in their localisation to the target cell-T cell interface, inhibition of proximal TCR signalling events and suppression of T cell function. At picomolar concentrations, these bispecifics suppressed cytokine production and inhibited CD8 T cell-mediated cytotoxicity in vitro. Crucially, in soluble form the PD-1 ImmTAAI molecules were inactive and hence could avoid systemic immunosuppression. This study outlines a promising new route to generate more effective, potent, tissue-targeted PD-1 agonists that can inhibit T cell function locally with the potential to treat autoimmune and chronic inflammatory diseases of high unmet need.
Authors
Adam P. Curnock, Giovanna Bossi, Jyothi Kumaran, Lindsay J. Bawden, Rita Figueiredo, Rajeevkumar Tawar, Katherine Wiseman, Emma Henderson, Sec Julie Hoong, Veronica Gonzalez, Hemza Ghadbane, David E. O. Knight, Ronan O'Dwyer, David X. Overton, Christina M. Lucato, Nicola M. G. Smith, Carlos R. Reis, Keith Page, Lorraine M. Whaley, Michelle L. McCully, Stephen Hearty, Tara M. Mahon, Peter Weber
Abstract
IgA nephropathy is caused by deposition of circulatory IgA1 in the kidney. Hypo-galactosylated IgA1 has the propensity to form poly-IgA aggregates that are prone to deposition. We purified poly-IgA from the plasma of IgA nephrology patients and showed the complex being susceptible to reducing condition, suggesting intermolecular disulfide connections between IgA units. We sought to find the cysteine residue(s) in forming intermolecular disulfide. Naturally assembled dimeric IgA, also known as secretory IgA, involves a J-chain subunit connected with two IgA1 molecules via their penultimate cysteine-471 residue on ‘tailpiece’ segment of IgA heavy chain. It is plausible that, with the absence of J-chain, the cysteine residue of mono-IgA1 might aberrantly form disulfide bond in poly-IgA formation. Mutagenesis confirmed cysteine-471 capable of promoting IgA aggregation. These discoveries prompted us to test thiol-based drugs for stabilizing cysteine. Specifically, cystine-reducing drug cysteamine used for treatment of cystinosis showed a remarkable potency in preventing IgA from self-aggregation. When administrated to rat and mouse models of IgA nephropathy, cysteamine significantly reduced glomerular IgA deposition. Collectively, our results revealed a novel molecular mechanism for aberrant formation of IgA aggregates, to which repurposed cystinosis drug cysteamine was efficacious in preventing renal IgA deposition.
Authors
Xinfang Xie, Li Gao, Pan Liu, Jicheng Lv, Wan-Hong Lu, Hong Zhang, Jing Jin
Abstract
Autoimmune disease has presented an insurmountable barrier to restoration of durable immune tolerance. Previous studies indicate that chronic therapy with metabolic inhibitors can reduce autoimmune inflammation, but it remains unknown whether acute metabolic modulation enables permanent immune tolerance to be established. In an animal model of lupus, we determined that targeting glucose metabolism with 2-deoxyglucose (2DG) and mitochondrial metabolism with metformin enables endogenous immune tolerance mechanisms to respond to tolerance induction. A 2-week course of 2DG and metformin, when combined with tolerance-inducing therapy anti-CD45RB, prevented renal deposition of autoantibodies for 6 months after initial treatment and also restored tolerance induction to allografts in lupus-prone mice. The restoration of durable immune tolerance was linked to changes in T cell surface glycosylation patterns, illustrating a role for glycoregulation in immune tolerance. These findings indicate that metabolic therapy may be applied as a powerful preconditioning to reinvigorate tolerance mechanisms in autoimmune and transplant settings that resist current immune therapies.
Authors
Christopher S. Wilson, Blair T. Stocks, Emilee M. Hoopes, Jillian P. Rhoads, Kelsey L. McNew, Amy S. Major, Daniel J. Moore
Abstract
Oligoarticular juvenile idiopathic arthritis (oligo JIA) is the most common form of chronic inflammatory arthritis in children; yet, the cause of this disease remains unknown. To understand immune responses in oligo JIA, we immunophenotyped synovial fluid T cells with flow cytometry, bulk and single-cell RNA sequencing, DNA methylation studies, and Treg suppression assays. In synovial fluid, CD4+, CD8+, and γδ T cells expressed Th1-related markers, while Th17 cells were not enriched. Th1 skewing was prominent in CD4+ T cells, including Tregs, and was associated with severe disease. Transcriptomic studies confirmed a Th1 signature in CD4+ T cells from synovial fluid. The regulatory gene expression signature was preserved in Tregs, even those exhibiting Th1 polarization. These Th1-like Tregs maintained Treg specific methylation patterns and suppressive function, supporting the stability of this Treg population in the joint. While synovial fluid CD4+ T cells displayed an overall Th1 phenotype, scRNA-seq uncovered heterogeneous effector and regulatory sub-populations, including interferon-induced Tregs, peripheral helper T cells, and cytotoxic CD4+ T cells. In conclusion, oligo JIA is characterized by Th1 polarization that encompasses Tregs but does not compromise their regulatory identity. Targeting Th1-driven inflammation and augmenting Treg function may represent important therapeutic approaches in oligo JIA.
Authors
Amélie M. Julé, Kacie J. Hoyt, Kevin Wei, Maria Gutierrez-Arcelus, Maria L. Taylor, Julie Ng, James A. Lederer, Siobhan M. Case, Margaret H. Chang, Ezra M. Cohen, Fatma Dedeoglu, Melissa M. Hazen, Jonathan S. Hausmann, Olha Halyabar, Erin Janssen, Jeffrey Lo, Mindy S. Lo, Esra Meidan, Jordan E. Roberts, Mary Beth F. Son, Robert P. Sundel, Pui Y. Lee, Talal Chatila, Peter A. Nigrovic, Lauren A. Henderson
Abstract
The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to unmask redundant functions of the NR4A family in other immune cells. Here we use a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1–/– Nr4a3–/– (DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cell-extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional, phenotypic, and transcriptional features of anergy accumulate in chimeric mice. Nevertheless, we observe upregulation of genes encoding inflammatory mediators in anergic DKO T cells, and DKO T cells exhibit enhanced capacity for IL-2 production. These studies reveal cell-intrinsic roles for the NR4A family in both central and peripheral T cell tolerance, and demonstrate that each is essential to preserve immune homeostasis.
Authors
Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman
Abstract
BACKGROUND. A previous Phase I study showed that the infusion of autologous Treg cells expanded ex-vivo into recent onset Type 1 Diabetes (T1D) patients had an excellent safety profile, however, the majority of the infused Tregs could no longer be detected in the peripheral blood three months post-infusion (NCT01210664-Treg-T1D Trial). Interleukin-2 (IL-2) has been shown to enhance human Treg cell survival and expansion at low doses in vivo. Therefore, we conducted a Phase 1 study (NCT02772679-TILT trial) combining polyclonal Treg and low-dose IL-2 and assessed the impact over time on Tregs populations and other immune cells. METHODS. T1D Patients were treated with a single infusion of autologous polyclonal Tregs, expanded ex vivo, followed by one or two 5-day courses of recombinant human low dose IL-2 (ld-IL-2) at 0.3 x 106 to 1 x 106 units/dose. Fluorescence-based flow cytometry, Cytometry by Time Of Flight (CyTOF) and 10X Genomics single cell RNAseq were used to follow the distinct immune cell populations phenotypes over time following immunotherapy. RESULTS. Multiparametric analysis revealed that the combination therapy led to an increase in the number of infused and endogenous Tregs but also resulted in a significant increase from baseline in a subset of activated NK, Mucosal-Associated Invariant T (MAIT) cells and clonal CD8+ T populations. CONCLUSIONS. These data support the hypothesis that Id-IL-2 expands exogenously administered Tregs but also can expand cytotoxic cells. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune diseases with pre-existing active immunity. TRIAL REGISTRATION. ClinicalTrials.gov NCT01210664 (Treg-T1D Trial), NCT02772679 (TILTtrial). FUNDING. Sean N. Parker Autoimmunity Research Laboratory Fund, National Center for Research Resources.
Authors
Shen Dong, Kamir J. Hiam-Galvez, Cody T. Mowery, Kevan C. Herold, Stephen E. Gitelman, Jonathan H. Esensten, Weihong Liu, Angela P. Lares, Ashley S. Leinbach, Michael Lee, Vinh Nguyen, Stanley J. Tamaki, Whitney Tamaki, Courtney M. Tamaki, Morvarid Mehdizadeh, Amy L. Putnam, Matthew H. Spitzer, C. Jimmie Ye, Qizhi Tang, Jeffrey A. Bluestone
Abstract
Immunotherapies are needed in the clinic that effectively suppress beta cell autoimmunity and reestablish long-term self-tolerance in type 1 diabetes. We previously demonstrated that nondepleting αCD4 and αCD8α antibodies establish rapid and indefinite remission in recent-onset diabetic NOD mice. Diabetes reversal by coreceptor therapy (CoRT) is induced by suppression of pathogenic effector T cells (Teff) and the selective egress of T cells from the pancreatic lymph nodes and islets that remain free of infiltration long-term. Here, we defined CoRT-induced events regulating early Teff function and pancreatic residency, and long-term tolerance. TCR-driven gene expression controlling autoreactive Teff expansion and proinflammatory activity was suppressed by CoRT, and islet T cell egress was sphingosine-1 phosphate-dependent. In both murine and human T cells, CoRT upregulated the Foxo1 transcriptional axis, which in turn was required for suppression and efficient pancreatic egress of Teff. Interestingly, long-term tolerance induced in late-preclinical NOD mice was marked by reseeding of the pancreas by a reduced CD8+ Teff pool exhibiting an exhausted phenotype. Notably, PD-1 blockade, which rescues exhausted Teff, resulted in diabetes onset in protected animals. These findings demonstrate that CoRT has distinct intrinsic effects on Teff that impact events early in induction and later in maintenance of self-tolerance.
Authors
Matthew Clark, Charles J. Kroger, Qi Ke, Rui Zhang, Karen Statum, J. Justin Milner, Aaron J. Martin, Bo Wang, Roland Tisch
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