Autoreactive Cells Research Articles

Daratumumab As a Treatment for Adult Immune Thrombocytopenia: A Phase II Study with Safety Run-in (the DART Study)

Daratumumab As a Treatment for Adult Immune Thrombocytopenia: A Phase II Study with Safety Run-in (the DART Study)

Nucleic Acids as Novel Therapeutic Modalities to Address Multiple Sclerosis Onset and Progression.

The issue of treating Multiple Sclerosis (MS) begins with disease-modifying treatments (DMTs) which may cause lymphopenia, dyspnea, and many other adverse effects. Consequently, further identification and evaluation of alternative treatments are crucial to monitoring their long-term outcomes and hopefully, moving toward personalized approaches that can be translated into clinical treatments. In this article, we focused on the novel therapeutic modalities that alter the interaction between the cellular constituents contributing to MS onset and progression. Furthermore, the studies that have been performed to evaluate and optimize drugs' efficacy, and particularly, to show their limitations and strengths are also presented. The preclinical trials of novel approaches for multiple sclerosis treatment provide promising prospects to cure the disease with pinpoint precision. Considering the fact that not a single treatment could be effective enough to cover all aspects of MS treatment, additional researches and therapies need to be developed in the future. Since the pathophysiology of MS resembles a jigsaw puzzle, researchers need to put a host of pieces together to create a promising window towards MS treatment. Thus, a combination therapy encompassing all these modules is highly likely to succeed in dealing with the disease. The use of different therapeutic approaches to re-induce self-tolerance in autoreactive cells contributing to MS pathogenesis is presented. A Combination therapy using these tools may help to deal with the clinical disabilities and symptoms of the disease in the future.

Virus Infection Is an Instigator of Intestinal Dysbiosis Leading to Type 1 Diabetes.

In addition to genetic predisposition, environmental determinants contribute to a complex etiology leading to onset of type 1 diabetes (T1D). Multiple studies have established the gut as an important site for immune modulation that can directly impact development of autoreactive cell populations against pancreatic self-antigens. Significant efforts have been made to unravel how changes in the microbiome function as a contributor to autoimmune responses and can serve as a biomarker for diabetes development. Large-scale longitudinal studies reveal that common environmental exposures precede diabetes pathology. Virus infections, particularly those associated with the gut, have been prominently identified as risk factors for T1D development. Evidence suggests recent-onset T1D patients experience pre-existing subclinical enteropathy and dysbiosis leading up to development of diabetes. The start of these dysbiotic events coincide with detection of virus infections. Thus viral infection may be a contributing driver for microbiome dysbiosis and disruption of intestinal homeostasis prior to T1D onset. Ultimately, understanding the cross-talk between viral infection, the microbiome, and the immune system is key for the development of preventative measures against T1D.

Reduced Follicular Regulatory T Cells in Spleen and Pancreatic Lymph Nodes of Patients With Type 1 Diabetes.

In the attempt to understand the origin of autoantibody (AAb) production in patients with and at risk for type 1 diabetes (T1D), multiple studies have analyzed and reported alterations in T follicular helper (Tfh) cells in presymptomatic AAb+ subjects and patients with T1D. Yet, whether the regulatory counterpart of Tfh cells, represented by T follicular regulatory (Tfr) cells, is similarly altered is still unclear. To address this question, we performed analyses in peripheral blood, spleen, and pancreatic lymph nodes (PLN) of organ donor subjects with T1D. Blood analyses were also performed in living AAb- and AAb+ subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed among T1D, AAb-, and AAb+ adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared with nondiabetic control subjects. Furthermore, adoptive transfer of Tfr cells delayed disease development in a mouse model of T1D, a finding that could indicate that Tfr cells play an important role in peripheral tolerance and regulation of autoreactive Tfh cells. Together, our findings provide evidence of Tfr cell alterations within disease-relevant tissues in patients with T1D, suggesting a role for Tfr cells in defective humoral tolerance and disease pathogenesis.

Astroglial and Microglial Purinergic P2X7 Receptor as a Major Contributor to Neuroinflammation during the Course of Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system that leads to the progressive disability of patients. A characteristic feature of the disease is the presence of focal demyelinating lesions accompanied by an inflammatory reaction. Interactions between autoreactive immune cells and glia cells are considered as a central mechanism underlying the pathology of MS. A glia-mediated inflammatory reaction followed by overproduction of free radicals and generation of glutamate-induced excitotoxicity promotes oligodendrocyte injury, contributing to demyelination and subsequent neurodegeneration. Activation of purinergic signaling, in particular P2X7 receptor-mediated signaling, in astrocytes and microglia is an important causative factor in these pathological processes. This review discusses the role of astroglial and microglial cells, and in particular glial P2X7 receptors, in inducing MS-related neuroinflammatory events, highlighting the importance of P2X7R-mediated molecular pathways in MS pathology and identifying these receptors as a potential therapeutic target.

AIRE-overexpressing BMDCs suppress TFH cells through ICOSL to prevent and attenuate autoimmune diabetes in NOD mice

The strong genetic association between autoimmune regulator (AIRE) and autoimmune diseases indicates its critical role in immune tolerance. AIRE deficiency is thought to promote the development of follicular helper T (TFH) cells, which are considered to be essential in B cell proliferation. Excessive TFH cell generation is a key step towards the development of autoimmune diseases, including type 1 diabetes. However, the potential mechanism by which AIRE contributes to the generation and function of the TFH cell population has remained elusive. We show that AIRE reduced TFH cell generation by inhibiting the expression of inducible costimulatory ligand (ICOSL), interleukin (IL)-6 and IL-27 in dendritic cells (DCs). To understand the precise impact of AIRE-overexpressing bone marrow-derived DCs (AIRE-BMDCs) on type 1 diabetes progression and the associated molecular mechanisms, we transferred AIRE-BMDCs to recipient NOD mice and found that transplantation of AIRE-BMDCs can prevent or delay the onset of diabetes, attenuate diabetes after the establishment of overt hyperglycaemia, and lead to the inhibition of autoreactive pathological TFH cells and germinal centre (GC) B cells. To further determine the potential mechanism underlying this TFH cell depletion, BMDCs were cotransferred with recombinant mouse ICOSL (ICOSLG protein). We demonstrated that NOD mice were more susceptible to diabetes when they received AIRE-BMDCs and ICOSLG than when they received only mock-vehicle BMDCs (GFP-BMDCs). In addition, we did not observe the reversal of diabetes in any mice subjected to this cotransfer system. A single cycle of ICOSLG treatment temporarily promoted TFH cell proliferation and GC development. Our results reveal a mechanistic role of AIRE-BMDCs in the initiation of TFH cell differentiation, and the AIRE-mediated decrease in ICOSL expression in BMDCs plays a critical role. The effect of decreased ICOSL expression in type 1 diabetes will guide the design and evaluation of parallel studies in patients.

Autologous Hematopoietic Stem Cell Transplantation (AHSCT) in Advanced, Treatment Refractory Multiple Sclerosis (MS) — a Phase II Clinical Trial

Aim: Ablation of auto-reactive immune cells followed by autologous hematopoietic stem cell transplantation (AHSCT) in the treatment of multiple sclerosis (MS) has been explored for the previous two decades. Morbidity and mortality rates originally limited referral for AHSCT in auto-immune disease but a reduction in treatment toxicity has facilitated increased clinical trials in the field. Here we present the outcomes of patients withadvanced, treatment refractory MS undergoing AHSCT in a Phase II study at our institute. We evaluate the safety and efficacy of AHSCT with intermediate-intensity immunosuppression in patients with highly aggressive MS.Methods: A single-centre, phase II clinical trial of AHSCT for patients with active relapsing remitting (RRMS) and secondary progressive MS (SPMS) commenced at St Vincent's Hospital in December 2010. Patients eligible for AHSCT had an Expanded Disability Status Scale (EDSS) of 2.0-7, had failed two prior disease-modifying therapies and displayed evidence of ongoing disease activity manifesting as clinical relapses and/or new MRI lesions in the year prior to AHSCT. CD34+ mobilisation with cyclophosphamide and G-CSF was performed, followed by conditioning with a BEAM (carmustine, etoposide, cytarabine, melphalan) + ATG (antithymocyte globulin) chemotherapeutic regimen. Patients underwent clinical assessment and peripheral blood collection for tissue banking at 3, 6, 12 months and yearly to 5 years. Gadolinium enhanced MRI was performed at 6, 12 months and yearly to 5 years. The primary outcomes were event-free survival and overall survival. Event free survival (EFS) was defined as no clinical or radiological relapses and no evidence of disability progression (by EDSS). Polychromic multi-parameter flow cytometry was performed for evaluation of post-transplant immune reconstitution.Results: Forty patients (26 with RRMS and 14 with SPMS) completed AHSCT, with a median follow-up of 24 months (range 3 to 63 months). Median EDSS prior to AHSCT was 6, with 17/40 having an EDSS >6, indicating a significant burden of neurologic disability. Patients were heavily pre-treated with immunosuppressive drugs (DMTs). Fifty-six percent of patients failed >4 disease modifying therapies prior to AHSCT with 2/3 having failed high efficacy treatment with Natalizumab (alpha-4 integrin inhibitor).Progression-free survival, clinical relapse-free survival (RFS), and MRI activity-free survival for the cohort were 80%, 90% and 95% respectively. Event free survival in the RRMS cohort was 90%. Sustained improvement in neurologic function by EDSS was seen in 15/40 (37.5%) of patients; 11 (42.3%) RRMS patients and 4 (28.6%) SPMS patients. MS quality of life score (MSQOL) - physical improved by 14.4 points (p=0.004) and MSQOL - mental improved by 10.3 points (p=0.08). Treatment related mortality was 0%. Adverse effects due to AHSCT were consistent with expected toxicities. One patient required admission to the intensive care unit in the post-transplant period due to serum sickness. There were no significant late neurologic adverse effects noted and no recorded cases of progressive multifocal leukoencephalopathy noted.Conclusion: The relapse-free survival (RFS) in our treatment refractory MS cohort undergoing AHSCT is significantly higher compared to RFS rates in MS patients treated with high-efficacy immunosuppressive therapies. Patients undergoing AHSCT at our institution had comparable rates of EFS to other recent prospective, single centre trials of AHSCT in spite of having higher baseline EDSS, greater evidence of pre-AHSCT disease activity, more heavily pre-treated (Burt et al. 2015, Atkins et al 2016) and use of a less aggressive conditioning regimen (Atkins et al. 2016). In summary, results of this phase II study suggest that AHSCT is effective in inducing sustained remission in the majority of aggressive RRMS and SPMS patients who failed multiple DMTs. DisclosuresNo relevant conflicts of interest to declare.

Current state of antigen-specific immunotherapy for type 1 diabetes.

Update on antigen-specific immunotherapy (ASIT) in type 1 diabetes (T1D) with focus on deoxyribonucleic acid (DNA)-induced immunization and the current obstacles to further research and clinical realization. In T1D, immune system imbalances together with malfunctioning islet-specific processes cause autoreactive immune cells to destroy beta cells in the islets. ASIT may restore self-tolerance; however, the approach has yet to fully meet its promise and may require co-administration of antigen (preproinsulin) and suitable immune response modifiers. A self-tolerant immune system may be regained using ASIT where T effector cells are repressed and/or T regulatory cells are induced. Administration of exogenous antigens has been safe in T1D. Conversely, adequate and lasting beta cell preservation has yet to be tested in sufficiently large clinical trials in suitable patients and may require targeting of multiple parts of the immunopathophysiology using combination therapies. DNA-based induction of native antigen expression to ensure important posttranscriptional modifications and presentation to the immune system together with tolerance-enhancing immune response modifiers (i.e., cytokines) may be more efficacious than exogenous antigens given alone. Progress is limited mainly by the scarcity of validated biomarkers to track the effects of ASIT in T1D.

Modulation of Intestinal ILC3 for the Treatment of Type 1 Diabetes.

Gut-associated lymphoid tissue (GALT) is crucial for the maintenance of the intestinal homeostasis, but it is also the potential site of the activation of autoreactive cells and initiation/propagation of autoimmune diseases in the gut and in the distant organs. Type 3 innate lymphoid cells (ILC3) residing in the GALT integrate signals from food ingredients and gut microbiota metabolites in order to control local immunoreactivity. Notably, ILC3 secrete IL-17 and GM-CSF that activate immune cells in combating potentially pathogenic microorganisms. ILC3 also produce IL-22 that potentiates the strength and integrity of epithelial tight junctions, production of mucus and antimicrobial peptides thus enabling the proper function of the intestinal barrier. The newly discovered function of small intestine ILC3 is the secretion of IL-2 and the promotion of regulatory T cell (Treg) generation and function. Since the intestinal barrier dysfunction, together with the reduction in small intestine ILC3 and Treg numbers are associated with the pathogenesis of type 1 diabetes (T1D), the focus of this article is intestinal ILC3 modulation for the therapy of T1D. Of particular interest is free fatty acids receptor 2 (FFAR2), predominantly expressed on intestinal ILC3, that can be stimulated by available selective synthetic agonists. Thus, we propose that FFAR2-based interventions by boosting ILC3 beneficial functions may attenuate autoimmune response against pancreatic β cells during T1D. Also, it is our opinion that treatments based on ILC3 stimulation by functional foods can be used as prophylaxis in individuals that are genetically predisposed to develop T1D.

1142-P: Update on BCG Clinical Trial Programs in Patients with Advanced Type 1 Diabetes

The bacillus Calmette-Guerin (BCG) vaccine was first introduced a century ago for tuberculosis prevention and is today being tested in clinical trials for treatment of diverse forms of autoimmunity, including type 1 diabetes (T1D). Long-term follow up of a Phase I study of the BCG vaccine in longstanding T1D revealed potential disease-modulating effects after repeated BCG vaccination, including long-term reductions of HbA1c, death of autoreactive cells, modest restoration of insulin secretion and induction of beneficial regulatory T cells (Tregs). Here we provide an update on the BCG clinical trial program current underway at Massachusetts General Hospital (MGH): enrollment numbers, study timelines and interim outcomes for key T1D biomarkers. To date, 236 patients have been enrolled in the randomized and open-label MGH trials and 143 patients have received at least two doses of BCG, 34 of whom are being followed in open-label, unblinded trials. Enrollment of 150 participants in a pediatric study is planned for 2021. Data from ongoing analysis of the open-label BCG trials shows lowered insulin needs and decreases in HbA1c as compared to reference populations. Patients are also being monitored by RNAseq, epigenetics, Treg cell numbers and Treg signature gene expression to better define the mechanism of beneficial effects. We show a gradual and stable 3-year time course of demethylation of signature Treg genes known to be associated with Treg potency, including Foxp3, which was observed to be over-methylated at baseline in T1D and, over 3 years after BCG vaccination, demethylated to a level close to controls without T1D. The clinical time course of BCG-related improvements (i.e., improvement in HbA1C) appears to correlate with the time course of Treg induction through epigenetic modifications. Disclosure W. Kuhtreiber: None. H. Takahashi: None. R. Keefe: None. K. Nelson: None. N. Ng: None. J. Braley: None. H. Zheng: None. D. L. Faustman: None.

Fatal Immune Response Associated with Anti-PD1: Pathological Insight

The recent introduction of immune checkpoint inhibitor therapy with anti- PD-1 agents has shown encouraging responses in gastrointestinal oncology. However, it is also known to possibly result in uncontrolled T cell autoreactivity, leading to a broad range of immune related adverse events. We present a patient who developed sequential immune-related hematologic and hepatic complications following pembrolizumab therapy for metastatic colon cancer. In particular, we describe in detail the histopathologic and immunohistochemical findings in the liver and bone marrow, adding to the spectrum of morphological changes which may be observed in association with PD-1 inhibitor therapy. The report also highlights the occurrence of these adverse reactions even after discontinuation of immunotherapy, eventually resulting in the death of the patient.

CD47 Deficiency Ameliorates Ocular Autoimmune Inflammation.

Autoimmune uveitis is a sight-threatening ocular inflammatory condition in which the retina and uveal tissues become a target of autoreactive immune cells. The CD47 is a ubiquitously expressed transmembrane protein which plays multiple roles in fundamental cellular functions including phagocytosis, proliferation, and adhesion. Signal regulatory protein alpha (SIRPα), one of the CD47 ligands, is predominantly expressed in myeloid lineage cells such as dendritic cells (DCs) or macrophages, and CD47-SIRPα signaling pathway is implicated in the development of autoimmune diseases. Our current study demonstrates how CD47 depletion is effective in the prevention of experimental autoimmune uveitis (EAU), an animal model of human autoimmune uveitis, in animals deficient of CD47 (CD47-/-). Systemic suppression of SIRPα+ DCs in animals deficient in CD47 resulted in the inability of autoreactive CD4+ T cells to develop, which is crucial to induction of EAU. Of interest, retinal microglia, the resident immune cell of the retina, express SIRPα, however these cells were not operative in EAU suppression in response to CD47 depletion. These results identify CD47 as a significant regulator in the development of SIRPα+ DCs that is vital to disease induction in EAU.

Neutralization of IL-6 inhibits formation of autoreactive TH17 cells but does not prevent loss of renal function in experimental autoimmune glomerulonephritis

In anti-glomerular basement membrane glomerulonephritis (anti-GBM GN), antibodies and T cells directed against the Goodpasture antigen, the non-collagenous domain of the α3-chain of type IV collagen (α3(IV)NC1), provoke renal inflammation resulting in rapidly progressing crescentic GN. Interleukin 6 (IL-6) is a pleiotropic cytokine with both pro- and anti-inflammatory activities, and IL-6 blockade is successfully used for treatment of diseases associated with acute and chronic inflammation. However, the role of IL-6 in anti-GBM GN is unclear. Here, we use the mouse model of experimental autoimmune glomerulonephritis (EAG) to study the role of IL-6 in anti-GBM GN. DBA/1J mice were immunized with α3(IV)NC1 and developed fatal crescentic GN. Treatment of mice with neutralizing anti-IL-6 antibodies impaired the generation of α3(VI)NC1-specific TH1 and TH17 cells. However, despite lasting reduction of the TH17 cell response, antibody treatment did not prevent crescentic GN. Antibody treatment was also ineffective in a therapeutic setting with pre-existing autoantibodies and T cells. In conclusion, our results indicate that although the blockade of IL-6 impairs the development of autoimmunity against α3(VI)NC1, this treatment does not ameliorate crescentic GN both in a preemptive and a therapeutic approach.

Neurologic Manifestations of COVID-19 in Children: Emerging Pathophysiologic Insights.

The COVID-19 pandemic has affected mortality and morbidity across all ages, including children. Neurologic manifestations of coronavirus disease 2019, ranging from headaches to cerebrovascular stroke, may involve the CNS or peripheral nervous system. Neurologic involvement is also noted in the multisystem inflammatory syndrome in children, a pediatric condition that occurs weeks after infection with severe acute respiratory syndrome coronavirus 2, the virus that causes COVID-19. Knowledge about mechanisms of neurologic disease and immunologic response to severe acute respiratory syndrome coronavirus 2 is scarce but rapidly growing. Here, we concisely review experimental and clinical data relevant to pathophysiologic hypotheses regarding neurologic manifestations of coronavirus disease 2019. We highlight important knowledge gaps in differences between adults and children in their response to coronavirus disease 2019.

Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.

STING Agonist Mitigates Experimental Autoimmune Encephalomyelitis by Stimulating Type I IFN-Dependent and -Independent Immune-Regulatory Pathways.

The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of IFN genes (STING) pathway induces a powerful type I IFN (IFN-I) response and is a prime candidate for augmenting immunity in cancer immunotherapy and vaccines. IFN-I also has immune-regulatory functions manifested in several autoimmune diseases and is a first-line therapy for relapsing-remitting multiple sclerosis. However, it is only moderately effective and can induce adverse effects and neutralizing Abs in recipients. Targeting cGAMP in autoimmunity is unexplored and represents a challenge because of the intracellular location of its receptor, STING. We used microparticle (MP)-encapsulated cGAMP to increase cellular delivery, achieve dose sparing, and reduce potential toxicity. In the C57BL/6 experimental allergic encephalomyelitis (EAE) model, cGAMP encapsulated in MPs (cGAMP MPs) administered therapeutically protected mice from EAE in a STING-dependent fashion, whereas soluble cGAMP was ineffective. Protection was also observed in a relapsing-remitting model. Importantly, cGAMP MPs protected against EAE at the peak of disease and were more effective than rIFN-β. Mechanistically, cGAMP MPs showed both IFN-I-dependent and -independent immunosuppressive effects. Furthermore, it induced the immunosuppressive cytokine IL-27 without requiring IFN-I. This augmented IL-10 expression through activated ERK and CREB. IL-27 and subsequent IL-10 were the most important cytokines to mitigate autoreactivity. Critically, cGAMP MPs promoted IFN-I as well as the immunoregulatory cytokines IL-27 and IL-10 in PBMCs from relapsing-remitting multiple sclerosis patients. Collectively, this study reveals a previously unappreciated immune-regulatory effect of cGAMP that can be harnessed to restrain T cell autoreactivity.

Pathogenic ILC1 and NKp46+ ILC3 Are Defined by Core and Tissue-Specific Transcriptional Signatures in Autoimmune Neuroinflammation

Abstract During multiple sclerosis, myelin-specific CD4+T cells invade the central nervous system (CNS) where they initiate a chronic inflammatory disease, leading to demyelination and neurodegeneration. Therefore, it is crucial to identify immunological factors allowing disease-inducing T cells to enter the CNS. Our previous study revealed a novel function of T-bet-expressing NKp46+ innate lymphocytes (ILC) as CNS gatekeepers, which control functional stability and access of autoreactive Th17 cells to the CNS. However, the identity of NKp46+ ILC subset(s) that regulate neuroinflammation and their mechanism of action are still poorly characterized. T-bet-expressing NKp46+ ILC encompass three subsets – conventional natural killer (NK) cells, ILC1 and NKp46+ ILC3. Here, we demonstrate that the pathogenic role of T-bet-expressing NKp46+ ILC maps to ILC1 and NKp46+ ILC3 but not NK cells. Single cell RNA-Seq analysis revealed that meninges-infiltrating NK cells exhibit undifferentiated phenotype devoid of expression of effector molecules and reminiscent of blood NK cells, explaining the functional redundancy of NK cells in the disease process. By contrast, NKp46+ ILC1/3, which directly interact with myelin-specific CD4+ T cells, were defined by the core set of 13 genes, which was not shared by splenic or meningeal NK cells. A subset of the ILC-specific genes was highly expressed by meningeal, but not splenic, ILCs, suggesting their importance in disease pathology. Thus, our transcriptional analysis identified modules of gene expression that coordinate functional polarization of ILCs in meningeal microenvironment and provides a blueprint for the discovery of ILC-specific targeted therapy for the treatment of CNS autoimmune disorder.

ICOS is critical for T follicular regulatory cell differentiation

Abstract The Inducible Costimulator (ICOS) is a T cell costimulatory receptor critical for humoral immunity. ICOS-deficient patients suffer from recurrent infections due to lack of protective antibodies. However, some patients also display signs of antibody-mediated autoimmunity. These findings may reflect a dual role of ICOS: facilitating the differentiation and function of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. While Tfh cells are known to provide help to B cells to produce high affinity antibodies, the main role of Tfr cells seems to be preventing autoantibody generation. Using Foxp3-cre-mediated ICOS knockout (ICOS FC) mice, we show that T regulatory (Treg)-specific ICOS deletion drastically reduces the number of Tfr cells without altering Treg cell numbers. Single cell RNA sequencing further revealed shifts in transitory Tfr precursor populations in immunized ICOS FC mice. Importantly, we observed a lowered ratio of antigen-specific germinal center B (GCB) cells and increased anti-nuclear antibodies in ICOS FC mice, suggesting a rise in autoreactive GCB cells. We also noted variations in isotype composition of total and virus-specific antibodies in infected ICOS FC mice. Mechanistically, our data suggests that ICOS could promote the Treg-to-Tfr transition by regulating CXCR5 expression. Thus, our study demonstrates that ICOS is critical for Tfr cell generation and supports the role of Tfr cells in preventing generation of autoantibodies during germinal center reactions.

Specific traits of the B cell response to self-antigens revealed by single cell transcriptomics

Abstract In an autoimmune environment, B cells spontaneously participate and are selected in germinal centers (GC). In previous work, we investigated epitope spreading in a mixed bone marrow chimera model that combines B cells from lupus-like mice with those from wild type (WT) B6 mice (Degn et al Cell 2017). In the current study, we identify novel biomarkers of autoimmune B cells at the population and single cell level. Cohorts of autoimmune and WT immunized chimeric mice were prepared. To track WT-derived B cells, we used the tamoxifen inducible AID-CreERT2-EYFP mice. WT chimeras were immunized and boosted. Splenic B cells were sorted based EYFP+ signal and processed for droplet-based gene expression and BCR repertoire single cell RNAseq. To detect specific traits of autoreactive B cells relative to immunized, unsupervised cluster analysis, differential gene expression and pseudotime analysis were used. Results show higher antibody repertoire diversity in the autoimmune relative to immune B cells, but striking similarity for transcriptomic usage. However, distinct gene expression was identified for the autoreactive B cells such as CXCL10 usage by GC B cells and SLPI expression by autoreactive plasma cells. Importantly, we were able to identify DN2- and DN4-like memory B cells in both conditions. DN2 cells, presumed to derive directly from naïve B cells, have been recently pointed as a source of pathogenic extrafollicular antibody secreting cells in humans. Overall, results reveal the variety of characteristics that B cells, in their many states, adopt in the response to self and should open the way to new approaches to control their pathogenicity in autoimmune disease.

The Ubiquitin Ligase Itch Regulates Germinal Center Selection

Abstract More than 23 million Americans suffer from autoimmune diseases driven by somatically mutated autoantibodies. High levels of somatic hypermutation of immunoglobulin genes occurs in germinal center (GC) B cells. Some of these mutations render autoreactivity, but cells bearing autoreactive receptors are eliminated due to strict selection of GC B cells by mechanisms that are still poorly defined. Inappropriate positive selection signals can rescue autoreactive cells from elimination and drive autoimmune disease. The E3 ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop debilitating, multi-faceted, potentially fatal, autoimmune disease; yet how Itch regulates GC B cell fate or function is not well understood. By studying Itch deficient mice, we have recently shown that Itch directly limits B cell activity to shape antibody responses. While Itch-deficient mice displayed normal numbers of pre-immune B cell populations, they showed elevated numbers of GC B cells. Mixed bone marrow chimeras revealed that Itch acts within B cells to skew the proportions of light zone and dark zone GC B cells. Proteomic profiling of GC B cells uncovered that Itch deficient cells exhibit hallmarks of positive selection. These results support a novel role for Itch in limiting positive selection of GC B cells to restrict GC size. In this role, Itch may shape the GC-derived antibody repertoire to prevent autoimmunity.