Faculty Bibliography

OBJECTIVES/OBJECTIVE:The gut microbiome plays a crucial role in regulating systemic immunity and has been implicated in several chronic inflammatory diseases. Intestinal expansions of Ruminococcus gnavus (RG), a dominant gut commensal, correlate with disease flares in lupus nephritis (LN), but the underlying mechanism remains unknown. METHODS:In a Pilot cohort of patients with biopsy-proven LN, subsetted by gut microbiota community, immune status was characterised using bulk-blood RNA sequencing libraries, serum levels of representative host proteins, and levels of immunoglobulin (Ig)G antibodies to the novel lipoglycan (LG) produced by pathogenic RG strains. A Validation LN cohort was evaluated for blood transcriptomic profiles and levels of anti-LG antibodies. In murine models, mechanistic hypotheses were tested after RG gut colonisation or after intraperitoneal injection with an LG preparation, with outcomes determined by transcriptomic analyses, platelet functional readouts, and tissue histology. RESULTS:In a Pilot cohort of patients with LN, RG gut expansions were associated with high-level platelet, neutrophil, and monocyte activation. Serum levels of platelet factor 4 and release of neutrophil extracellular traps (NETs) were significantly higher in patients with high serum IgG antibody against the novel RG-specific LG, a marker of in vivo immune exposure. An LN Validation cohort confirmed these correlates and showed that anti-LG antibodies serve as a surrogate for thromboinflammatory profile in this LN-associated endotype. In mice, gut colonisation with LG-producing RG strains or a single LG injection caused megakaryocytosis and platelet activation; RG colonisation with LG-producing strains induced tubulointerstitial injury with NETosis. In vivo responses to LG toxin were Toll-like receptor 2-dependent. CONCLUSIONS:Gut expansions of the RG pathobiont may contribute to autoimmune pathogenesis through the LG toxin and cause LN flares through thromboinflammatory mechanisms in this previously unrecognised LN endotype.

Gut microbiome dysbiosis has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). However, microbiota-targeted therapeutic strategies have been lacking. Here, we report the potential of Faecalibacterium prausnitzii (strain UT1) to ameliorate gut dysbiosis and alleviate disease progression in the B6.Sle1.Yaa male mouse model of SLE. Fecal metagenomes of patients with SLE shifted carbohydrate catabolism from dietary fibers to host glycans, coinciding with depletion of F. prausnitzii. Oral administration of UT1 partially reversed lupus-associated microbiome alterations and rescued carbohydrate metabolic deficiency in lupus-prone mice. Using correlative metatranscriptomics and metabolomics, we observed restricted expression of bacterial genes related to mucin degradation, elevated pentose phosphate pathway and bile acid-modifying activities, and redirected tryptophan catabolism toward indoleacetic and indoleacrylic acids. Further host cell profiling showed that UT1 rebalanced colonic regulatory T (Treg) and T helper 17 (Th17) cell responses, suppressed systemic autoimmune activation and autoantibody production, and reduced renal pathology. Thus, our findings identify SLE-associated active microbiome signatures and provide a probiotic candidate for the treatment of lupus disease.

To understand what drives an immune response, it is important to characterize, at a molecular level, the site(s) on an immunogenic antigen that is directly contacted by a soluble antibody or B-cell antigen receptor (BCR) on the surface of a B lymphocyte. Moreover, antibody binding interactions with a microbial protein can interfere with the functional activity of a toxin (i.e., neutralization) and/or can aid in the clearance of the microbial protein from the body, further underscoring the importance of such characterization. Phage display technology is a potent tool that can be used to study any type of protein-protein interaction. In recent years, we have refined methods for the identification of the minimal binding contact sites of an antibody with an antigen. Here, we describe a workflow for optimizing antibody-mediated selection and for the identification and characterization of antigen-specific epitopes. This workflow includes (1) the generation of large libraries of random fragments of a gene of interest cloned into the validated pComb-Opti8 phagemid expression cloning vector system; (2) electroporation of these libraries into electrocompetent bacterial cells and subsequent recovery of viral particles, each of which displays the cloned gene fragment product as a fusion protein with the filamentous phage major coat protein VIII (pVIII); (3) recovery of individual phagemid clones that express the smallest functional epitopes recognized by an experimental antibody; (4) an efficient means of using high-throughput DNA sequencing to interrogate sequentially selected libraries to rapidly identify the gene subregions encoding epitopes of interest; and (5) means for the further characterization of potential antibody-epitope binding interactions.

The human immune system evolved to defend against the panoply of microbial threats. By harnessing such ability, vaccines have cumulatively saved hundreds of millions of lives. Despite such tremendous success, there have also been remarkable failures, such as the lack of a clinically proven vaccine against Staphylococcus aureus (SA), which continues to pose an urgent public health threat. In practice, it has proven challenging to identify the molecular basis for relevant epitopes for this pathogen. Here, we summarize our experience implementing an integrated approach using phage display technology for the identification of B-cell epitopes of microbial virulence factors, which we developed with a focus on SA. This approach was used to define minimal B-cell epitopes of the staphylococcal leucocidin family of pore-forming toxins (PFTs) that have been implicated in staphylococcal clinical infection. Our methodology provides proof of principle for an approach well suited for the rapid and efficient generation of modular protein-based vaccines for protection from clinical infection, which can be used to target pathogens for which no vaccine is currently available.

OBJECTIVE:The mechanisms by which the gut microbiome contributes to lupus pathogenesis remain poorly understood. The anaerobe Ruminococcus gnavus (RG) expands in patients with lupus in association with flares. The goal of this study was to determine the mechanisms by which candidate pathobiont lipoglycan-producing RG2 may contribute to autoimmunity and to identify factors promoting its expansion. METHODS:The consequences of RG colonization or depletion were evaluated in the B6.Sle1.Sle2.Sle3 triple congenic (TC) lupus model by flow cytometry and enzyme-linked immunosorbent assay. RG lysates were tested on Treg cells in vitro. Fecal microbiota transfers evaluated the contribution of the microbiome origin from lupus or control donors and dietary tryptophan. RG1 and RG2 growth and metabolome were evaluated in response to tryptophan in vitro. RESULTS:Only RG2 stably colonized TC mice, in which it induced autoantibody production and T cell activation. Depletion of anaerobes had the opposite effect, with an increased Treg frequency. RG2 induced Treg apoptosis in cocultures with dendritic cells. RG is present in TC microbiota, from which it is amplified by tryptophan. The combination of TC microbiota and high dietary tryptophan induced autoimmune activation and intestinal inflammation in healthy control mice. Finally, tryptophan enhanced RG2 growth and production of immunomodulatory metabolites. CONCLUSION/CONCLUSIONS:RG2 contributes to autoimmune activation, at least by inducing Treg apoptosis. The expansion of this pathobiont is promoted by host genetic factors and tryptophan metabolism. Thus, targeted RG2 depletion may improve disease outcomes in patients with lupus.

Mounting evidence shows that gut microbiota communities and the human immune system coexist and influence each other, and there are a number of reports of a correlation between specific changes in gut microbiota and the occurrence of autoimmune diseases. B lymphocytes play a central role in the regulation of both gut microbiota communities and in autoimmune diseases. Here, we summarize evidence of the influence of gut microbiota-B cell pathways on autoimmune diseases and how B cells regulate microorganisms, which provides mechanistic insights with relevance for identification of potential therapeutic targets and related fields.

INTRODUCTION/UNASSIGNED:Recent investigations have identified patients of African ancestry (AA) with Multiple Sclerosis (MS), who display more rapid B-cell repopulation after standard semi-annual infusions with an anti-CD20 monoclonal antibody for B cell depletion. In this study, we explored the immunologic and genetic factors, with, serum drug monitoring that may contribute to a faster rate of B-cell repletion that follows during recovery from treatment with anti-CD20 antibody. METHODS/UNASSIGNED:In AA MS patients treated with an anti-CD20 antibody that had early repopulation of peripheral blood B cells, we assessed for extrinsic factors, including the presence of anti-drug antibodies against ocrelizumab, which may contribute to early repletion. We also documented the associated serum drug levels. In addition, we examined for inheritance of intrinsic gene polymorphisms associated with B cell survival and immune function. RESULTS/UNASSIGNED:Our findings identified a subset of AA patients with early B cell repletion after anti-CD20 treatment associated with anti-drug antibodies and an absence of detectable drug. Furthermore, a separate set of AA patients with the early B cell repletion phenotype without anti-drug antibodies had significant over-representation of genetic polymorphisms that map to genes for the B cell survival factor, BAFF, to antibody-dependent cytotoxicity, and to pathways involved in inflammation, leukocyte activation and B cell differentiation. DISCUSSION/UNASSIGNED:In AA patients with MS, after anti-CD20 antibody treatment we found an unexpected high occurrence of early B cell replenishment. This was associated with the presence of anti-drug antibodies and/or specific genetic polymorphisms. Larger studies are now needed to determine whether these factors may lead to impaired therapeutic benefits of B cell targeted therapy and clinical progression, and these findings may be useful to guide future optimized personalized therapeutic strategies.

BACKGROUND/UNASSIGNED:There is a greater risk of complications from severe COVID-19 in immunocompromised patients with multiple sclerosis (pwMS) treated with certain disease-modifying therapies (DMTs), as well as a diminished vaccine response. METHODS/UNASSIGNED:In this exploratory, observational study, we recruited 28 patients with Relapsing Remitting MS (RRMS, n=24) or Secondary Progressive MS (SPMS, n=4), that were receiving treatment with either natalizumab or fumarates (diroximel or dimethyl) prior to baseline sample collection. Blood samples were collected before vaccination (baseline), between 4 weeks and 6 months post vaccination, and post booster administration. A multiplex bead immunoassay (MBI) was used to measure anti-Spike IgG, while IFNγ and IL-2 ELISpot assays were used to determine T cell activation. A 35-color spectral flow cytometry panel was used to phenotype bulk B and T cells and SARS-CoV-2-specific T cells, while dimensionality reduction was performed for further phenotypic analysis. RESULTS/UNASSIGNED:We observed a significantly increased absolute lymphocyte count (ALC) (p=0.0003) in natalizumab-treated pwMS when compared to fumarate-treated pwMS primarily due to increased circulating CD19+ B cells. Fumarate-treated pwMS exhibited a diminished Th1/Th2 ratio when compared to natalizumab-treated pwMS (p=0.0004) or healthy controls (p=0.0745), while natalizumab treatment marginally increased the Th1/Th2 ratio compared to healthy controls (p=0.1311). The observed increase in B cells in natalizumab-treated pwMS were predominantly memory B cells, and double negative (DN) B cells. However, no significant differences between the treatment groups were seen in terms of Spike IgG titers following the initial vaccination course or booster dose, nor in SARS-CoV-2-specific CD4+ responses, all of which remained robust for at least 6 months post-vaccination. The magnitude of humoral and cellular immune responses in both treatment groups were comparable to vaccinated healthy controls. Additionally, SARS-CoV-2 spike-specific CD4+ T cell phenotyping revealed a Th2 dominant response to booster dose in natalizumab-treated pwMS (p=0.0485) but not fumarate-treated pwMS. CONCLUSION/UNASSIGNED:pwMS treated with natalizumab or fumarates exhibit similarly robust and durable SARS-CoV-2 specific T cell and humoral responses following vaccination and booster dose. DMT-treated pwMS showing comparable responses to healthy individuals following initial vaccination supports the notion that treatment with these specific DMTs does not diminish strong, long-lasting immunity conferred by COVID-19 vaccination, despite the phenotypic differences modulated by each therapy.

[This corrects the article DOI: 10.3389/fimmu.2025.1590165.].

OBJECTIVES/OBJECTIVE:(1) To plot the trajectory of humoral and cellular immune responses to the primary (two-dose) COVID-19 mRNA series and the third/booster dose in B-cell-depleted multiple sclerosis (MS) patients up to 2 years post-vaccination; (2) to identify predictors of immune responses to vaccination; and (3) to assess the impact of intercurrent COVID-19 infections on SARS CoV-2-specific immunity. METHODS:Sixty ocrelizumab-treated MS patients were enrolled from NYU (New York) and University of Colorado (Anschutz) MS Centers. Samples were collected pre-vaccination, and then 4, 12, 24, and 48 weeks post-primary series, and 4, 12, 24, and 48 weeks post-booster. Binding anti-Spike antibody responses were assessed with multiplex bead-based immunoassay (MBI) and electrochemiluminescence (Elecsys®, Roche Diagnostics), and neutralizing antibody responses with live-virus immunofluorescence-based microneutralization assay. Spike-specific cellular responses were assessed with IFNγ/IL-2 ELISpot (Invitrogen) and, in a subset, by sequencing complementarity determining regions (CDR)-3 within T-cell receptors (Adaptive Biotechnologies). A linear mixed-effect model was used to compare antibody and cytokine levels across time points. Multivariate analyses identified predictors of immune responses. RESULTS:The primary vaccination induced an 11- to 208-fold increase in binding and neutralizing antibody levels and a 3- to 4-fold increase in IFNγ/IL-2 responses, followed by a modest decline in antibody but not cytokine responses. Booster dose induced a further 3- to 5-fold increase in binding antibodies and 4- to 5-fold increase in IFNγ/IL-2, which were maintained for up to 1 year. Infections had a variable impact on immunity. INTERPRETATION/CONCLUSIONS:Humoral and cellular benefits of COVID-19 vaccination in B-cell-depleted MS patients were sustained for up to 2 years when booster doses were administered.