The microbiome affects development and activity of the immune system, and may modulate immune therapies, but there is little direct information about this control in vivo. We studied how the microbiome affects regulation of human immune cells in humanized mice. When humanized mice were treated with a cocktail of 4 antibiotics, there was an increase in the frequency of effector T cells in the gut wall, circulating levels of IFN-γ, and appearance of anti-nuclear antibodies. Teplizumab, a non–FcR-binding anti-CD3ε antibody, no longer delayed xenograft rejection. An increase in CD8+ central memory cells and IL-10, markers of efficacy of teplizumab, were not induced. IL-10 levels were only decreased when the mice were treated with all 4 but not individual antibiotics. Antibiotic treatment affected CD11b+CD11c+ cells, which produced less IL-10 and IL-27, and showed increased expression of CD86 and activation of T cells when cocultured with T cells and teplizumab. Soluble products in the pellets appeared to be responsible for the reduced IL-27 expression in DCs. Similar changes in IL-10 induction were seen when human peripheral blood mononuclear cells were cultured with human stool samples. We conclude that changes in the microbiome may impact the efficacy of immunosuppressive medications by altering immune regulatory pathways.
Elke Gülden, Nalini K. Vudattu, Songyan Deng, Paula Preston-Hurlburt, Mark Mamula, James C. Reed, Sindhu Mohandas, Betsy C. Herold, Richard Torres, Silvio M. Vieira, Bentley Lim, Jose D. Herazo-Maya, Martin Kriegel, Andrew L. Goodman, Chris Cotsapas, Kevan C. Herold
Foxp3+ Tregs possess potent immunosuppressive activity, which is critical for maintaining immune homeostasis and self-tolerance. Defects in Treg development or function result in inadvertent immune activation and autoimmunity. Despite recent advances in Treg biology, we still do not completely understand the molecular and cellular mechanisms governing the development and suppressive function of these cells. Here, we have demonstrated an essential role of the dedicator of cytokinesis 8 (DOCK8), guanine nucleotide exchange factors required for cytoskeleton rearrangement, cell migration, and immune cell survival in controlling Treg fitness and their function. Treg-specific DOCK8 deletion led to spontaneous multiorgan inflammation in mice due to uncontrolled T cell activation and production of proinflammatory cytokines. In addition, we show that DOCK8-deficient Tregs are defective in competitive fitness and in vivo suppressive function. Furthermore, DOCK8 controls IL-2 signaling, crucial for maintenance and competitive fitness of Tregs, via a STAT5-dependent manner. Our study provides potentially novel insights into the essential function of DOCK8 in Tregs and immune regulation, and it explains the autoimmune manifestations associated with DOCK8 deficiency.
Akhilesh K. Singh, Ahmet Eken, David Hagin, Khushbu Komal, Gauri Bhise, Azima Shaji, Tanvi Arkatkar, Shaun W. Jackson, Estelle Bettelli, Troy R. Torgerson, Mohamed Oukka
Patients deficient in the guanine nucleotide exchange factor DOCK8 have decreased numbers and impaired in vitro function of Tregs and make autoantibodies, but they seldom develop autoimmunity. We show that, similarly, Dock8–/– mice have decreased numbers and impaired in vitro function of Tregs but do not develop autoimmunity. In contrast, mice with selective DOCK8 deficiency in Tregs develop lymphoproliferation, autoantibodies, and gastrointestinal inflammation, despite a normal percentage and in vitro function of Tregs, suggesting that deficient T effector cell function might protect DOCK8-deficient patients from autoimmunity. We demonstrate that DOCK8 associates with STAT5 and is important for IL-2–driven STAT5 phosphorylation in Tregs. DOCK8 localizes within the lamellar actin ring of the Treg immune synapse (IS). Dock8–/– Tregs have abnormal TCR-driven actin dynamics, decreased adhesiveness, an altered gene expression profile, an unstable IS with decreased recruitment of signaling molecules, and impaired transendocytosis of the costimulatory molecule CD86. These data suggest that DOCK8 enforces immunological tolerance by promoting IL-2 signaling, TCR-driven actin dynamics, and the IS in Tregs.
Erin Janssen, Sudha Kumari, Mira Tohme, Sumana Ullas, Victor Barrera, Jeroen M.J. Tas, Marcela Castillo-Rama, Roderick T. Bronson, Shariq M. Usmani, Darrell J. Irvine, Thorsten R. Mempel, Raif S. Geha
Subsets of mature B cell neoplasms are linked to infection with intracellular pathogens such as Epstein-Barr virus (EBV), hepatitis C virus (HCV), or Helicobacter pylori. However, the association between infection and the immunoglobulin-secreting (Ig-secreting) B proliferative disorders remains largely unresolved. We investigated whether the monoclonal IgG (mc IgG) produced by patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) targets infectious pathogens. Antigen specificity of purified mc IgG from a large patient cohort (n = 244) was determined using a multiplex infectious-antigen array (MIAA), which screens for reactivity to purified antigens or lysates from 9 pathogens. Purified mc IgG from 23.4% of patients (57 of 244) specifically recognized 1 pathogen in the MIAA. EBV was the most frequent target (15.6%), with 36 of 38 mc IgGs recognizing EBV nuclear antigen-1 (EBNA-1). MM patients with EBNA-1–specific mc IgG (14.0%) showed substantially greater bone marrow plasma cell infiltration and higher β2-microglobulin and inflammation/infection–linked cytokine levels compared with other smoldering myeloma/MM patients. Five other pathogens were the targets of mc IgG: herpes virus simplex-1 (2.9%), varicella zoster virus (1.6%), cytomegalovirus (0.8%), hepatitis C virus (1.2%), and H. pylori (1.2%). We conclude that a dysregulated immune response to infection may underlie disease onset and/or progression of MGUS and MM for subsets of patients.
Adrien Bosseboeuf, Delphine Feron, Anne Tallet, Cédric Rossi, Cathy Charlier, Laurent Garderet, Denis Caillot, Philippe Moreau, Marina Cardó-Vila, Renata Pasqualini, Wadih Arap, Alfreda Destea Nelson, Bridget S. Wilson, Hélène Perreault, Eric Piver, Pierre Weigel, François Girodon, Jean Harb, Edith Bigot-Corbel, Sylvie Hermouet
Antiphospholipid antibodies, present in one-third of lupus patients, increase the risk of thrombosis. We recently reported a key role for neutrophils — neutrophil extracellular traps (NETs), in particular — in the thrombotic events that define antiphospholipid syndrome (APS). To further elucidate the role of neutrophils in APS, we performed a comprehensive transcriptome analysis of neutrophils isolated from patients with primary APS. Moreover, APS-associated venous thrombosis was modeled by treating mice with IgG prepared from APS patients, followed by partial restriction of blood flow through the inferior vena cava. In patients, APS neutrophils demonstrated a proinflammatory signature with overexpression of genes relevant to IFN signaling, cellular defense, and intercellular adhesion. For in vivo studies, we focused on P-selectin glycoprotein ligand-1 (PSGL-1), a key adhesion molecule overexpressed in APS neutrophils. The introduction of APS IgG (as compared with control IgG) markedly potentiated thrombosis in WT mice, but not PSGL-1–KOs. PSGL-1 deficiency was also associated with reduced leukocyte vessel wall adhesion and NET formation. The thrombosis phenotype was restored in PSGL-1–deficient mice by infusion of WT neutrophils, while an anti–PSGL-1 monoclonal antibody inhibited APS IgG–mediated thrombosis in WT mice. PSGL-1 represents a potential therapeutic target in APS.
Jason S. Knight, He Meng, Patrick Coit, Srilakshmi Yalavarthi, Gautam Sule, Alex A. Gandhi, Robert C. Grenn, Levi F. Mazza, Ramadan A. Ali, Paul Renauer, Jonathan D. Wren, Paula L. Bockenstedt, Hui Wang, Daniel T. Eitzman, Amr H. Sawalha
The factors that promote the differentiation of pathogenic T cells in autoimmune diseases are poorly defined. Use of genetically modified mice has provided insight into molecules necessary for the development of autoimmunity, but the sum of the data has led to contradictory observations based on what is currently known about specific molecules in specific signaling pathways. To define the minimum signals required for development of encephalitogenic T cells that cause CNS autoimmunity, myelin-specific T cells were differentiated with various cytokine cocktails, and pathogenicity was determined by transfer into mice. IL-6+IL-23 or IL-12+IL-23 generated encephalitogenic T cells and recapitulated the essential cytokine signals provided by antigen-presenting cells, and both IL-6 and IL-12 induced IL-23 receptor expression on both mouse and human naive T cells. IL-23 signaled through both STAT3 and STAT4, and disruption in STAT4 signaling impaired CNS autoimmunity independent of IL-12. These data explain why IL-12–deficient mice develop CNS autoimmunity, while STAT4-deficient mice are resistant. CD4+ memory T cells from multiple sclerosis patients had significantly higher levels of p-STAT3/p-STAT4, and p-STAT3/p-STAT4 heterodimers were observed upon IL-23 signaling, suggesting that p-STAT3/p-STAT4 induced by IL-23 signaling orchestrate the generation of pathogenic T cells in CNS autoimmunity, regardless of Th1 or Th17 phenotype.
Priscilla W. Lee, Alan J. Smith, Yuhong Yang, Amanda J. Selhorst, Yue Liu, Michael K. Racke, Amy E. Lovett-Racke
Recent data indicate that there are different subpopulations of Th17 cells that can express a regulatory as opposed to an inflammatory gene signature. The transmembrane glycoprotein PDPN is critical in the development of multiple organs including the lymphatic system and has been described on T cells in mouse models of autoimmune Th17 inflammation. Here, we demonstrate that unlike in mice, PDPN+ T cells induced under classic Th17-polarizing conditions express transcription factors associated with Th17 cells but do not produce IL-17. Moreover, these cells express a transcriptional profile enriched for immunosuppressive and regulatory pathways and express a distinct cytokine profile compared with potentially pathogenic PDPN– Th17 cells. Ligation of PDPN by its ligand CLEC-2 ameliorates the Th17 inflammatory response. IL-17 secretion is restored with shRNA gene silencing of PDPN. Furthermore, PDPN expression is reduced via an Sgk1-mediated pathway under proinflammatory, high sodium chloride conditions. Finally, CD3+PDPN+ T cells are devoid of IL-17 in skin biopsies from patients with candidiasis, a prototypical Th17-driven skin disease. Thus, our data support the hypothesis that PDPN may serve as a marker of a nonpathogenic Th17 cell subset and may also functionally regulate pathogenic Th17 inflammation.
Alyssa N. Nylander, Gerald D. Ponath, Pierre-Paul Axisa, Mayyan Mubarak, Mary Tomayko, Vijay K. Kuchroo, David Pitt, David A. Hafler
Myasthenia gravis (MG) is a B cell–mediated autoimmune disorder of neuromuscular transmission. Pathogenic autoantibodies to muscle-specific tyrosine kinase (MuSK) can be found in patients with MG who do not have detectable antibodies to the acetylcholine receptor (AChR). MuSK MG includes immunological and clinical features that are generally distinct from AChR MG, particularly regarding responsiveness to therapy. B cell depletion has been shown to affect a decline in serum autoantibodies and to induce sustained clinical improvement in the majority of MuSK MG patients. However, the duration of this benefit may be limited, as we observed disease relapse in MuSK MG patients who had achieved rituximab-induced remission. We investigated the mechanisms of such relapses by exploring autoantibody production in the reemerging B cell compartment. Autoantibody-expressing CD27+ B cells were observed within the reconstituted repertoire during relapse but not during remission or in controls. Using two complementary approaches, which included production of 108 unique human monoclonal recombinant immunoglobulins, we demonstrated that antibody-secreting CD27hiCD38hi B cells (plasmablasts) contribute to the production of MuSK autoantibodies during relapse. The autoantibodies displayed hallmarks of antigen-driven affinity maturation. These collective findings introduce potential mechanisms for understanding both MuSK autoantibody production and disease relapse following B cell depletion.
Panos Stathopoulos, Aditya Kumar, Richard J. Nowak, Kevin C. O’Connor
Human endogenous retroviruses (HERVs), remnants of ancestral viral genomic insertions, are known to represent 8% of the human genome and are associated with several pathologies. In particular, the envelope protein of HERV-W family (HERV-W-Env) has been involved in multiple sclerosis pathogenesis. Investigations to detect HERV-W-Env in a few other autoimmune diseases were negative, except in type-1 diabetes (T1D). In patients suffering from T1D, HERV-W-Env protein was detected in 70% of sera, and its corresponding RNA was detected in 57% of peripheral blood mononuclear cells. While studies on human Langerhans islets evidenced the inhibition of insulin secretion by HERV-W-Env, this endogenous protein was found to be expressed by acinar cells in 75% of human T1D pancreata. An extensive immunohistological analysis further revealed a significant correlation between HERV-W-Env expression and macrophage infiltrates in the exocrine part of human pancreata. Such findings were corroborated by in vivo studies on transgenic mice expressing HERV-W-env gene, which displayed hyperglycemia and decreased levels of insulin, along with immune cell infiltrates in their pancreas. Altogether, these results strongly suggest an involvement of HERV-W-Env in T1D pathogenesis. They also provide potentially novel therapeutic perspectives, since unveiling a pathogenic target in T1D.
Sandrine Levet, Julie Medina, Julie Joanou, Amandine Demolder, Nelly Queruel, Kevin Réant, Matthieu Normand, Marine Seffals, Julie Dimier, Raphaële Germi, Thomas Piofczyk, Jacques Portoukalian, Jean-Louis Touraine, Hervé Perron
The role of B cells and posttranslational modifications in pathogenesis of organ-specific immune diseases is increasingly envisioned but remains poorly understood, particularly in human disorders. In celiac disease, transglutaminase 2–modified (TG2-modified; deamidated) gluten peptides drive disease-specific T cell and B cell responses, and antibodies to deamidated gluten peptides are excellent diagnostic markers. Here, we substantiate by high-throughput sequencing of IGHV genes that antibodies to a disease-specific, deamidated, and immunodominant B cell epitope of gluten (PLQPEQPFP) have biased and stereotyped usage of IGHV3-23 and IGHV3-15 gene segments with modest somatic mutations. X-ray crystal structures of 2 prototype IGHV3-15/IGKV4-1 and IGHV3-23/IGLV4-69 antibodies reveal peptide interaction mainly via germline-encoded residues. In-depth mutational analysis showed restricted selection and substitution patterns at positions involved in antigen binding. While the IGHV3-15/IGKV4-1 antibody interacts with Glu5 and Gln6, the IGHV3-23/IGLV4-69 antibody interacts with Gln3, Pro4, Pro7, and Phe8 — residues involved in substrate recognition by TG2. Hence, both antibodies, despite different interaction with the epitope, recognize signatures of TG2 processing that facilitates B cell presentation of deamidated gluten peptides to T cells, thereby providing a molecular framework for the generation of these clinically important antibodies. The study provides essential insight into the pathogenic mechanism of celiac disease.
Omri Snir, Xi Chen, Moriah Gidoni, M. Fleur du Pré, Yuguang Zhao, Øyvind Steinsbø, Knut E.A. Lundin, Gur Yaari, Ludvig M. Sollid
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