BACKGROUND AND OBJECTIVES:Gut microbial symbionts have been shown to influence the development of autoimmunity in multiple sclerosis (MS). Emerging research points to an important relationship between the microbial-IgA interface and MS pathophysiology. IgA-secreting B cells are observed in the MS brain, and shifts in gut bacteria-IgA binding have been described in some patients with MS. However, the relationships between the gut microbiome and the host immune response, particularly regarding B-cell-depleting immunomodulation, remain underexplored. This study aimed to evaluate the composition of the gut microbiome in patients with newly diagnosed MS at baseline and after B-cell depletion, using long-read sequencing for enhanced taxonomic resolution. We further aimed to investigate the host/microbiome interface by evaluating microbe/immunoglobulin A relationships. METHODS:We collected stool samples from 43 patients with newly diagnosed, untreated MS and 42 matched healthy controls. Nineteen patients with MS initiated anti-CD20 monoclonal antibody treatment and donated additional stool samples after 6 months of treatment. We evaluated the host-microbial interface using bacterial flow cytometry and long-read 16S rRNA gene amplicon sequencing. We used Immune Coating Scores to compare the proportions of bacteria identified in the IgA-coated vs IgA-uncoated bacterial fractions. RESULTS:Patients with untreated, newly diagnosed MS showed significant reductions in IgA-bound fecal microbiota compared with controls. Using multiple linear regression models adjusted for potential confounders, we observed significant (p < 0.05) changes in the abundance and prevalence of various strain-level gut bacteria amplicon sequence variants (ASVs) within both total and IgA-coated bacterial fractions. Some changes (e.g., decreased relative abundance of a Faecalibacterium prausnitzii variant in MS) were consistent with previous reports, while others (e.g., increased relative abundance and prevalence of Monoglobus pectinyliticus in MS) were novel. Immune Coating Scores identified subsets of organisms for which normal IgA-coating patterns were disrupted at the onset of MS, as well as those (particularly Akkermansia muciniphila) whose IgA-coating became more aligned with controls after therapy. DISCUSSION:This analysis of gut microbial ASVs reveals shifts in taxonomic strains induced by immune modulation in MS.

BACKGROUND While B cell depletion is associated with attenuated antibody responses to SARS-CoV-2 mRNA vaccination, responses vary among individuals. Thus, elucidating the factors that affect immune responses after repeated vaccination is an important clinical need.METHODS We evaluated the quality and magnitude of the T cell, B cell, antibody, and cytokine responses to a third dose of BNT162b2 or mRNA-1273 mRNA vaccine in patients with B cell depletion.RESULTS In contrast with control individuals (n = 10), most patients on anti-CD20 therapy (n = 48) did not demonstrate an increase in spike-specific B cells or antibodies after a third dose of vaccine. A third vaccine elicited significantly increased frequencies of spike-specific non-naive T cells. A small subset of B cell–depleted individuals effectively produced spike-specific antibodies, and logistic regression models identified time since last anti-CD20 treatment and lower cumulative exposure to anti-CD20 mAbs as predictors of those having a serologic response. B cell–depleted patients who mounted an antibody response to 3 vaccine doses had persistent humoral immunity 6 months later.CONCLUSION These results demonstrate that serial vaccination strategies can be effective for a subset of B cell–depleted patients.FUNDING The NIH (R25 NS079193, P01 AI073748, U24 AI11867, R01 AI22220, UM 1HG009390, P01 AI039671, P50 CA121974, R01 CA227473, U01CA260507, 75N93019C00065, K24 AG042489), NIH HIPC Consortium (U19 AI089992), the National Multiple Sclerosis Society (CA 1061-A-18, RG-1802-30153), the Nancy Taylor Foundation for Chronic Diseases, Erase MS, the Robert Leet and Clara Guthrie Patterson Trust, and the Claude D. Pepper Older Americans Independence Center at Yale (P30 AG21342).

Supplementary Figure S1, related to Experimental Procedures: Molecular characterization of primary BTICs. Supplementary Figure S2, related to Supplementary Figure 1: BTICs recover their proliferative and invasive capacities in vitro. Supplementary Figure S3, related to Supplementary Figure 3: BTICs express G6PC and its inhibition decreases cell migration, invasion and proliferation. Supplementary Figure S4, related to Supplementary Figure 3: G6PC knockdowns exhibit decreased cell invasion and migration. Supplementary Figure S5, related to Supplementary Figure 4: Pharmacological inhibition of G6PC induces an increase in the expression of the astrocytic marker GFAP in the recovery group. Supplementary Figure S6, related to Supplementary Figure 5: G6PC knockdown activates glycogen synthase (GYS1) and inhibits glycogen phosphorylase (PYGL). Supplementary Figure S7, related to Supplementary Figure 6: Invasion and proliferation capacity of BTICs in vivo.

T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4+ T cell subsets associated with plasma -blast expansion and clinical outcome. Peripheral helper T cells (Tph cells; denoted as PD-1highCXCR5-CD4+ T cells) are significantly increased, as are plasmablasts. Tph cells exhibit "B cell help"signatures and induce plasmablast differentiation in vitro. Interestingly, expanded plasmablasts show increased CXCR3 expres-sion, which is positively correlated with higher frequency of activated Tph cells and better clinical outcome. Mechanistically, Tph cells help B cell differentiation and produce more interferon g (IFNg), which induces CXCR3 expression on plasmablasts. These results elucidate a role for Tph cells in regulating protective B cell response during acute viral infection.

Vitiligo is a common autoimmune skin disease that affects 1-2% of the population. Disease is characterized by CD8+ effector T cells that target melanocytes, the pigment producing cells. This targeted destruction leads to patchy depigmentation, which can result in social stigma and emotional distress. Individuals with vitiligo exhibit a polyclonal expansion of T cells that target melanocyte proteins, such as MART1, TYROSINASE, or PMEL. The functional role of diverse polyclonal responses in driving disease is largely unknown. We sought to determine whether vitiligo is driven by a single dominant clone or rather a collaboration of clones. To do this, we recruited a total of 21 active untreated vitiligo patients and coupled single cell gene expression data with paired alpha/beta T cell receptor (TCR) to mark clonotypic expression on autoreactive CD8+ T cells in vitiligo lesions as well as PBMCs. We assessed their autoreactive status using dextramer labeling. We found that vitiligo lesions have multiple expanded CD8+ T cell clones within lesions and observed an overlap in clonal landscape across separate lesions as well as non-lesional skin. The CD8+ T cell population split into two populations based on gene transcription, highly activated and less activated. Expanded clones, defined as clonal populations with the greatest number of cells, predominately cluster in the highly activated population, expressing increased levels of IFNG and GZMB compared to clones that were not expanded in both lesional and non-lesional skin. These observations suggest that vitiligo is driven by the collaboration of multiple expanded clones that represent T cells with increased cytotoxic function. Further investigation about the diversity in phenotype of the different clones within lesions and between separate lesions on the same patient will help to determine whether different clones have specified roles during their collaborative attack on melanocytes.

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100A hi /HLA-DR lo classical monocytes and activated LAG-3 hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8 + clones, unmutated IGHG + B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19.

B cell depletion in patients with relapsing-remitting multiple sclerosis (RRMS) markedly prevents new MRI-detected lesions and disease activity, suggesting the hypothesis that altered B cell function leads to the activation of T cells driving disease pathogenesis. Here, we performed comprehensive analyses of CD40 ligand- (CD40L-) and IL-21-stimulated memory B cells from patients with MS and healthy age-matched controls, modeling the help of follicular helper T cells (Tfh cells), and found a differential gene expression signature in multiple B cell pathways. Most striking was the impaired TIGIT expression on MS-derived B cells mediated by dysregulation of the transcription factor TCF4. Activated circulating Tfh cells (cTfh cells) expressed CD155, the ligand of TIGIT, and TIGIT on B cells revealed their capacity to suppress the proliferation of IL-17-producing cTfh cells via the TIGIT/CD155 axis. Finally, CCR6+ cTfh cells were significantly increased in patients with MS, and their frequency was inversely correlated with that of TIGIT+ B cells. Together, these data suggest that the dysregulation of negative feedback loops between TIGIT+ memory B cells and cTfh cells in MS drives the activated immune system in this disease.

To characterize taxonomic and functional shifts in the gut microbiota of multiple sclerosis (MS) patients induced by B-cell depletion.

Cutaneous T-cell lymphoma (CTCL) is a life-debilitating malignancy of lymphocytes homing to the skin. Although CTCL is thought to arise from a combination of genetic, epigenetic, and environmental factors, specific triggers are unclear. The skin is colonized by a unique microbiota and is heavily influenced by its interactions. We hypothesized that adaptive immune responses to skin commensals lead to clonal T-cell proliferation and transformation in the appropriate genetic background. We therefore collected lesional and nonlesional skin microbiota from patients with CTCL to study T cell interactions using skin T cell explants and peripheral, skin-homing CD4+ T cells. By various methods, we identified Bacillus safensis in CTCL lesions, a rare human commensal in healthy skin, and showed that it can induce malignant T cell activation and cytokine secretion. Taken together, our data suggest microbial triggers in the skin microbiota of patients with CTCL as potential instigators of tumorigenesis.

SummaryA dysregulated immune response against coronavirus-2 (SARS-CoV-2) plays a critical role in the outcome of patients with coronavirus disease 2019 (COVID-19). A significant increase in circulating plasmablasts is characteristic of COVID-19 though the underlying mechanisms and its prognostic implications are not known. Here, we demonstrate that in the acute phase of COVID-19, activated PD-1highCXCR5−CD4+ T cells, peripheral helper T cells, (Tph) are significantly increased and promote inflammatory tissue-homing plasmablasts in patients with stable but not severe COVID-19. Analysis of scRNA-seq data revealed that plasmablasts in stable patients express higher levels of tissue-homing receptors including CXCR3. The increased Tph cells exhibited “B cell help” signatures similar to that of circulating T follicular helper (cTfh) cells and promoted B cell differentiation in vitro. Compared with cTfh cells, Tph cells produced more IFNγ, inducing tissue-homing chemokine receptors on plasmablasts. Finally, expansion of activated Tph cells was correlated with the frequency of CXCR3+ plasmablasts in the acute phase of patients with stable disease. Our results demonstrate a novel role for Tph cells in acute viral immunity by inducing ectopic, antibody secreting plasmablasts.

Background Diets deficient in fibers are prevalent in modern societies and implicated in gut microbial dysbiosis contributing to the pathogenesis of chronic inflammatory disorders.1 A particular type of dietary fiber, resistant starch (RS) type 2, was shown to ameliorate disease in murine models of systemic lupus erythematosus (SLE).2 This effect was mediated by improvement of the gut barrier and growth inhibition of a translocating Lactobacillus strain. This Lactobacillus sp. was shown to drive lupus-related pathology in mice via the type I interferon pathway, and its genus was also enriched in a subset of SLE patients. Whether diet enriched in RS have similar effects in patients is unknown. We aimed to understand how dietary RS content influences gut microbial community structures in SLE and SLE-related antiphospholipid syndrome (APS) patients with well-defined microbiomes.3,4 Methods Stools and dietary information were collected from 12 SLE (n=28), 15 APS (n=44) patients and 20 control subjects (n=48) for up to 3 visits (0, 4 and 8 weeks) as previously described (3,4). Microbiota composition was defined by 16S rRNA V4 region sequencing. The FDA reference list was used to calculate the RS content. Patients' diets were classified as low RS content if less than 2.5 g per day, medium RS if 2.5 to 15 g, and high RS above 15 g. Results Lactobacillus spp. were significantly enriched in SLE patients (p=0.002) compared to non-disease controls. APS patients showed a similar trend (p=0.06), but SLE patients displayed higher relative abundance compared to APS (p=0.011). No significant association was observed between low-to-medium RS content and Lactobacillus. High RS content was not achieved in routine diets of SLE and APS patients in these cohorts. However, medium RS was associated with an outgrowth of Bifidobacterium in SLE patients (p=0.016). Also, medium RS correlated in APS patients with a reduction of cardiolipin-synthesizing bacteria from the Coriobacteriaceae family (p=0.011) including Collinsella (p=0.009) and Slackia genera (p=0.033), previously linked to APS.5,6 Conclusions The content of RS in patients` regular diets has a distinct impact on the gut microbiota composition depending on the autoimmune disorder. Medium levels of dietary RS were associated in SLE with increased Bifidobacterium, short-chain fatty acid producing bacteria known to promote immune homeostasis, and with decreased cardiolipin-producing commensals in APS. It remains to be tested in an interventional trial if high RS content corrects the outgrowth of Lactobacillus in these patients, but moderate levels of RS may provide already beneficial effects on other taxa potentially involved in the pathogenesis of these disorders. References Thorburn et al. 2014, Immunity 19, 833-842. Zegarra-Ruiz et al. 2019, Cell Host Microbe 25, 113-127. Greiling et al. 2018, Science Transl Med 10, 1–15. Ruff et al. 2019, Cell Host Microbe 26, 1–14. Aguiar et al. 2016, Arthritis Rheum 68 (suppl 10). Bellochi et al. 2019, J Clin Med 8, 1291, 1-15. Acknowledgments The work was supported by grants from the National Institutes of Health (NIH) (R01AI118855, T32AI07019), Arthritis National Research Foundation, Arthritis Foundation, Lupus Research Alliance, and Maren Foundation. Trial Registrations ClinicalTrials.gov Identifiers: NCT02394964 (The Human Microbiome in Immune-Mediated Diseases) and NCT01787305 (Pilot Study of Gut Commensals in Antiphospholipid Syndrome).

Immunomodulatory medications are a mainstay of treatment for autoimmune diseases and malignancies. In addition to their direct effects on immune cells, these medications also impact the gut microbiota. Drug-induced shifts in commensal microbes can lead to indirect but important changes in the immune response. We performed a comprehensive literature search focusing on immunotherapy/microbe interactions. Immunotherapies were categorized into 5 subtypes based on their mechanisms of action: cell trafficking inhibitors, immune checkpoint inhibitors, immunomodulators, antiproliferative drugs, and inflammatory cytokine inhibitors. Although no consistent relationships were observed between types of immunotherapy and microbiota, most immunotherapies were associated with shifts in specific colonizing bacterial taxa. The relationships between colonizing microbes and drug efficacy were not well-studied for autoimmune diseases. In contrast, the efficacy of immune checkpoint inhibitors for cancer was tied to the baseline composition of the gut microbiota. There was a paucity of high-quality data; existing data were generated using heterogeneous sampling and analytic techniques, and most studies involved small numbers of participants. Further work is needed to elucidate the extent and clinical significance of immunotherapy effects on the human microbiome.

Host–microbiota interactions are fundamental for the development of the immune system. Drastic changes in modern environments and lifestyles have led to an imbalance of this evolutionarily ancient process, coinciding with a steep rise in immune-mediated diseases such as autoimmune, allergic and chronic inflammatory disorders. There is an urgent need to better understand these diseases in the context of mucosal and skin microbiota. This Review discusses the mechanisms of how the microbiota contributes to the predisposition, initiation and perpetuation of immune-mediated diseases in the context of a genetically prone host. It is timely owing to the wealth of new studies that recently contributed to this field, ranging from metagenomic studies in humans and mechanistic studies of host–microorganism interactions in gnotobiotic models and in vitro systems, to molecular mechanisms with broader implications across immune-mediated diseases. We focus on the general principles, such as breaches in immune tolerance and barriers, leading to the promotion of immune-mediated diseases by gut, oral and skin microbiota. Lastly, the therapeutic avenues that either target the microbiota, the barrier surfaces or the host immune system to restore tolerance and homeostasis will be explored.

Given the immense antigenic load present in the microbiome, we hypothesized that microbiota mimotopes can be a persistent trigger in human autoimmunity via cross-reactivity. Using antiphospholipid syndrome (APS) as a model, we demonstrate cross-reactivity between non-orthologous mimotopes expressed by a common human gut commensal, Roseburia intestinalis (R. int), and T and B cell autoepitopes in the APS autoantigen β2-glycoprotein I (β2GPI). Autoantigen-reactive CD4+ memory T cell clones and an APS-derived, pathogenic monoclonal antibody cross-reacted with R. int mimotopes. Core-sequence-dependent anti-R. int mimotope IgG titers were significantly elevated in APS patients and correlated with anti-β2GPI IgG autoantibodies. R. int immunization of mice induced β2GPI-specific lymphocytes and autoantibodies. Oral gavage of susceptible mice with R. int induced anti-human β2GPI autoantibodies and autoimmune pathologies. Together, these data support a role for non-orthologous commensal-host cross-reactivity in the development and persistence of autoimmunity in APS, which may apply more broadly to human autoimmune disease.

Multiple sclerosis (MS) is a genetically mediated autoimmune disease characterized by inflammation in the central nervous system (CNS). Disease onset is thought to occur when autoreactive T cells orchestrate a cascade of events in the CNS resulting in white and grey matter inflammation and axonal degeneration. It is unclear what triggers the activation of CNS-reactive T cells and their polarization into inflammatory subsets. Mounting evidence from animal and human studies supports the hypothesis that the gut microbiome affects MS pathogenesis. We investigated the association between the gut microbiome and inflammatory T cell subsets in relapsing-remitting MS patients and healthy controls. Gut microbiome composition was characterized by sequencing the V4 region of the 16S rRNA gene from fecal DNA, and inflammatory T cell subsets were characterized by flow cytometry. We identified an altered gut microbiome in MS patients, including decreased abundance of Coprococcus, Clostridium, and an unidentified Ruminococcaceae genus. Among circulating immune cells, patients had increased expression of CXCR3 in both CD4 and CD8 T cells, and both CD4+CXCR3+ and CD8+CXCR3+ populations expressing the gut-homing α4β7 integrin receptor were increased. Finally, we show that alpha diversity inversely correlated with a CXCR3+ Th1 phenotype in MS. These findings indicate the presence of an aberrant gut-immune axis in patients with MS.

Pathogenic autoantibodies in neuromyelitis optica require antigen array assembly and a specific epitope to initiate robust complement activation.