Interferon regulatory factor 1 (IRF1), a transcription factor encoded within the 5q31 locus harboring systemic lupus erythematosus (SLE) associated variants, promotes inflammatory responses by T and myeloid cells. Although IFNγ-activated B cells also express IRF1, its role in B cell biology and SLE is unclear. Here, we use a mouse SLE model, single-cell multiomics, and human B cells to show that IRF1 intrinsically regulates Irf4 gene chromatin accessibility and expression in B cells to control the balance between the antibody secreting cell (ASC) lineage commitment factor, IRF4, and the B cell identity factor, IRF8. We demonstrate that IRF1, through its integration of IFNγ and TLR7 induced transcriptional programs, tips B cells toward a terminal effector inflammatory AC fate at the expense of preserving more stem-like, resting and regulatory B cells that do not elicit autoantibody-associated pathology in SLE. Thus, IRF1 serves as a central node controlling B cell-driven autoimmune disease.

We aimed to test associations of participant-reported Long COVID symptom interference with life activities with Long COVID symptoms, presence of U09.9 Long COVID diagnosis code, demographics, and clinical factors. In a subgroup, we documented coding related to Long COVID and post-exertional malaise in the electronic medical record (EMR). Using a cross-sectional analysis (n = 205) of participant data from a Long COVID survey, we tested associations with Chi-square, Fisher’s exact, or Fisher-Freeman-Halton exact statistical tests and Independent Samples T-tests. Participants were predominately female (67

Live attenuated influenza vaccination (LAIV) is the only FDA approved mucosal vaccine. Easily assayed, circulating correlates of protection after LAIV are lacking. Using fluorochrome labeled hemagglutinin (HA) antigen, we previously identified a subset of HA-specific (HA + ) IgD neg memory B cells that circulate after inactivated intramuscular vaccination (IIV), express the master transcriptional regulator, T-bet, as well as effector memory genes and predict durable antibody (Ab) responses to IIV. Here we profile the circulating HA + IgD neg memory B cell response in a cohort of immunized patients who seroconvert after LAIV to identify which, if any, circulating HA + B cells predict antibody responses after LAIV. Although we report LAIV elicits circulating T-bet + HA + IgD neg B cells that are phenotypically similar to those we described after IIV, we find no correlation between the magnitude of these cells and systemic HA-IgG responses after LAIV. Supervised and unsupervised analyses demonstrate that unlike IIV, LAIV preferentially elicits circulating HA + IgD neg B cells that co-express TBX21 and the terminal effector cell gene, Zeb2 . Consistent with their terminal differentiation status, LAIV-elicited T-bet + cells cannot be recalled as short-lived antibody-secreting cells (ASCs) after systemic or mucosal antigen re-challenge. We conclude that the transcriptional profiles and functions of HA + IgD neg B cells vary by influenza vaccine platform.

Background/Objectives: The emergence of SARS-CoV-2 variants and breakthrough infections underscores the need for next-generation vaccines capable of protecting from natural infection and/or preventing virus transmission. Intranasal vaccination offers a promising approach by eliciting local immune responses in the nasal mucosa, the primary site of infection and reservoir for transmissible virus. We evaluated two live-attenuated, respiratory syncytial virus-vectored vaccines in which the RSV F and G surface glycoproteins were replaced with a chimeric SARS-CoV-2 Spike protein from the ancestral USA/WA-1/2020 strain (MV-014-212) or the Delta variant (MV-014-212-delta). Methods: K18-hACE2 mice and LVG Syrian hamsters were vaccinated with a single intranasal dose of MV-014-212 or MV-014-212-delta. Systemic and mucosal immunity were assessed following vaccination, and protection was evaluated following Delta SARS-CoV-2 challenge. In vaccinated hamsters, morbidity, viral shedding, and lung inflammation and injury were also assessed following natural exposure to infected cagemates. Results: A single intranasal dose of either vaccine elicited systemic and mucosal immunity in K18-hACE2 mice, including serum neutralizing antibodies, Spike-specific memory B cells and plasmablasts, and Spike-specific CD8+ lung-resident memory T cells. Although MV-014-212-delta vaccination provided the best protection against the Delta variant virus challenge, both vaccines decreased viral loads in nasal discharge, lung, and brain, and reduced weight loss and mortality. In naturally acquired infection studies, vaccinated hamsters exposed to infected cagemates exhibited minimal weight loss, limited viral replication within the nasal mucosa, and attenuated lung pathology. Conclusions: Intranasal RSV-vectored vaccines can elicit broad protective respiratory immunity, suggesting that this platform could be leveraged for other respiratory pathogens.

In the twenty years since extrafollicular B cell responses were originally described, much has been learned about B cell biology. With this progress, the term "extrafollicular" has expanded beyond its initial use to describe a variety of B cell processes, resulting in ambiguity over the term. Extrafollicular responses are often not identified by location, convoluting the criteria being used to define the pathway. Here, we discuss the current understanding of B cell responses as relevant to the current uses of the term "extrafollicular." In this context, we propose a framework to classify evolving concepts in B cell biology. The use of this framework moving forward is expected to help harmonize and clarify the discussion in the field.

Interferon regulatory factor 1 (IRF1) is a transcriptional regulator of inflammation. Given the role of inflammation in autoimmunity, we hypothesized that IRF1 would influence the progression of systemic lupus erythematosus (SLE). Consistent with this, human SLE B cells expressed higher IRF1 levels and showed increased chromatin accessibility at IRF1-binding sites. To study the role of IRF1 in SLE, we analyzed lupus-prone B6.Yaa.Fcgr2b-/- (YFc) mice lacking Irf1 globally (YFc.Irf1) and only in B cells (B-YFc.Irf1). YFc.Irf1 mice had reduced ASCs and ABCs, smaller spleens, decreased serum autoantibodies, reduced glomerular IgG deposition and attenuated kidney damage. Interestingly, similar results were seen in B-YFc.Irf1 mice. Mechanistically, Irf1 tuned the response of B cells to TLR7/8 ligands. This phenotype was not limited to the YFc model. Lupus-prone B6.MRL-Faslpr/J (Lpr) mice lacking Irf1 globally (Lpr.Irf1) and in B cells (B-Lpr.Irf1) also had reduced IgG ASCs and decreased serum autoantibodies. To test whether Irf1 drives or accelerates inflammation in B cells, we analyzed aged B6 mice lacking Irf1 in all cells (B6.Irf1) and B cells (B-Irf1). Both models had decreased ASCs and ABCs and aged B6.Irf1 mice had decreased age-associated kidney pathology. These findings reveal that Irf1 modulates pathogenic B cell subsets in autoimmunity and inflammaging, suggesting IRF1 as a potential B cell target for SLE treatment. Supported by grants from NIH (R01 AI110508 & R01AI153365) Basic Autoimmunity (BA)

Donor-specific antibody responses against human leukocyte antigen (HLA) proteins mismatched between transplant donors and recipients cause allograft loss, yet the structural HLA epitopes targeted by alloreactive B cells and antibodies remain largely unresolved. We profiled the HLA-A∗01:01-specific B cell response in the transplanted kidney and blood of a recipient undergoing antibody-mediated rejection and identified immunodominant B cell and antibody responses that emerged early in the alloimmune response. These responses were focused on topographically exposed mismatched HLA residues located in the α helices along the peptide-binding groove of HLA-A∗01:01. We demonstrated that the anti-HLA-A∗01:01 B cell alloresponse converged and was maintained on this same immunodominant HLA subregion, which comprises only 20% of the HLA molecule, in a diverse group of HLA-A∗01:01-mismatched transplant recipients. Thus, the B cell and antibody alloresponses appear tightly focused on a topographically defined region on the HLA-A∗01:01 crown that is conserved across individuals expressing distinct constellations of self-HLA-A.

SLE is an autoimmune disease characterized by pathogenic autoantibodies (autoAbs) produced by terminally differentiated B lineage antibody-secreting cells (ASCs). The expanded double negative 2 (DN2) B cell subset found in SLE patients gives rise to autoAb-producing ASCs. While many B cell subsets can form ASCs, it’s unclear whether ASCs derived from specific B cell populations, isolated from either SLE or healthy donors (HD), exhibit distinct functional or pathogenic attributes. Using our novel two-step culture system, we generated ASCs in vitro from resting naïve (rNav-ASCs), resting switched memory (rSW-ASCs), and DN2 (DN2-ASCs) primary human B cell subsets isolated from HDs and SLE patients (n = 3-5). Bulk RNA-sequencing revealed differentially expressed genes and enriched pathways between ASCs generated from different B cell subsets. Notably, CXCR3, which directs cells to inflamed tissues, was upregulated in SLE-derived DN2-ASCs relative to rSW-ASCs. Moreover, we identified differences between ASCs generated from phenotypically matched subsets isolated from SLE and HD, with enrichment for inflammatory signatures within SLE rSW-ASCs. Thus, while DN2 cells are thought to be major contributors to SLE pathogenesis, the gene signature of SLE rSW-ASCs suggest rSW memory B cells may also contribute to pathogenesis. We are now performing functional assays to identify key regulators controlling the distinct attributes of ASCs derived from different B subsets in SLE patients. Funding from P01 AI125180 and the Lupus Research Alliance Basic Autoimmunity (BA)

While mRNA vaccines have been effective in combating SARS-CoV-2, the waning of vaccine-induced antibody responses and lack of vaccine-induced respiratory tract immunity contribute to ongoing infection and transmission. In this work, we compare and contrast intranasal (i.n.) and intramuscular (i.m.) administration of a SARS-CoV-2 replicon vaccine delivered by a nanostructured lipid carrier (NLC). Both i.m. and i.n. vaccines induce potent systemic serum neutralizing antibodies, bone marrow-resident immunoglobulin G-secreting cells, and splenic T cell responses. The i.n. vaccine additionally induces robust respiratory mucosal immune responses, including SARS-CoV-2-reactive lung-resident memory T cell populations. As a booster following previous i.m. vaccination, the i.n. vaccine also elicits the development of mucosal virus-specific T cells. Both the i.m.- and i.n.-administered vaccines durably protect hamsters from infection-associated morbidity upon viral challenge, significantly reducing viral loads and preventing challenged hamsters from transmitting virus to naive cagemates. This replicon-NLC vaccine’s potent systemic immunogenicity, and additional mucosal immunogenicity when delivered i.n., may be key for combating SARS-CoV-2 and other respiratory pathogens.

While human and mouse memory B cells (MBCs) can express the transcription factor T-bet, its role in regulating MBC function remains unclear. We characterized multiple transcriptionally distinct clusters of mature, somatically mutated nucleoprotein (NP)-specific MBCs in lymph nodes (LNs) and lungs of influenza-infected mice. Although none of the MBCs expressed the plasma cell (PC) lineage commitment factor Blimp1, one cluster was enriched for Tbx21+ cells. Similar to the previously described human T-bet+ effector MBC (eMBC) population, Tbx21+ mouse MBCs upregulated gene networks associated with effector metabolism, protein synthesis, and the unfolded protein response. Constitutive and inducible ablation of T-bet in murine B cells showed that T-bet expression by MBCs was required for persistence of LN and lung eMBCs with rapid in vitro and in vivo PC differentiation potential. Thus, T-bet marks NP+ eMBCs that are poised to differentiate, and it regulates maintenance of lung-resident MBCs and local PC responses following virus re-exposure.

Background/Objectives: We previously demonstrated that dendritic cell (DC) expression of CXCR5 is required for TH2 priming in mice infected with the helminth Heligmosomoides polygyrus (Hp). In this manuscript we examined how CXCR5 controls DC mediated CD4 T helper 2 cell (TH2) development. Methods: We used in vitro TH2 priming assays, RNA-seq analyses and in vivo Hp infection mouse models to identify roles for the CXCR5-expressing DCs in TH2 development. Results: We showed that migratory conventional type 2 dendritic cells (cDC2) express CXCR5 and that deletion of Cxcr5 prevents migratory DC priming of TH2 cells in vitro while overexpression of CXCR5 enhances migratory DC priming of TH2 cells in vitro. To understand how CXCR5 facilitates the TH2 priming capabilities of migratory cDC2 cells, we performed RNAseq analysis on wildtype and Cxcr5−/− DC subsets isolated from msLN of Hp-infected mice. We observed that CXCR5 expression specifically by the migratory cDC2 subset promoted a pro-proliferative transcriptional program in cDC2 cells and was required for cDC2 cell accumulation in the msLN following Hp infection. We demonstrated that CXCR5 expression specifically by cDC2 cells was necessary for upregulation of Chitinase 3-like-1 (Chi3l1), which encodes a secreted protein (Chi3l1) that regulates allergic TH2 responses. We showed that addition of recombinant Chi3l1 protein to in vitro TH2 priming cultures enhanced TH2 development and that deletion of Chi3l1 specifically in DCs resulted in fewer cDC2 cells and decreased TH2 development in vivo following Hp infection. Conclusions: CXCR5 expressed by cDC2 cells is required for induction of Chi3l1, which in turn promotes the TH2 priming capacity of these DCs. These findings provide insight into the actions of CXCR5 and Chi3l1 in helminth infection.

Systemically administered influenza vaccines provide strain-limited protection, while influenza infection of the respiratory epithelium supports development of lung resident memory B and T cells and more broadly reactive antibody responses. To test whether local antigen delivery is critical for establishing broad immunity, we directly compared respiratory tract and systemic delivery of influenza antigens to mice and hamsters using a replication-deficient adenovirus serotype 5 vector (Ad5[E1-,E2b-,E3-]). Both immunization routes elicited antibody responses in the lower respiratory tract and antigen-specific B and T cells in the draining lymph node. However, only intranasal immunization established lung-resident memory B and T cells, induced IgA responses in the upper respiratory tract directly at the site of viral entry and supported generation of IgA and IgG antibodies that bound antigenically drifted and distantly related influenza strains, including those of avian origin. Intranasal immunization accelerated viral clearance following heterologous virus challenge and was associated with limited pulmonary inflammation and fibrosis. Thus, intranasal immunization with the immunologically stealthy Ad5[E1-,E2b-,E3-] platform supports respiratory and systemic immunity to divergent influenza strains in the absence of overt lung immunopathology, suggesting that local antigen delivery may be key to development of more broadly protective "universal" flu vaccines.

Type 1 diabetes (T1D) is a chronic autoimmune disease that is caused by a combination of genetic and environmental risk factors. In this study, we sought to determine whether a known genetic risk factor, the rs1990760 single nucleotide polymorphism (SNP) (A946T) in IFIH1, resulted in a gain of function in the MDA5 protein and the effects of this mutation on the regulation of type I IFNs during infection with the diabetogenic virus coxsackievirus B3. We found that in cell lines overexpressing the risk variant IFIH1946T there was an elevated level of basal type I IFN signaling and increased basal IFN-stimulated gene expression. An investigation into the mechanism demonstrated that recombinant MDA5 with the A946T mutation had increased ATPase activity in vitro. We also assessed the effect of this SNP in primary human PBMCs from healthy donors to determine whether this SNP influenced their response to infection with coxsackievirus B3. However, we observed no significant changes in type I IFN expression or downstream induction of IFN-stimulated genes in PBMCs from donors carrying the risk allele IFIH1946T. These findings demonstrate the need for a deeper understanding of how mutations in T1D-associated genes contribute to disease onset in specific cellular contexts.

The prototypic IFN-inducible transcription factor, IRF1, not only controls inflammatory gene expression but also regulates T cell and macrophage fate specification and function. Using bone marrow chimeras (80% B6.129S2-Ighmtm1Cgn/J [µMT] + 20% B6.129S2-Irf1tm1Mak/J [Irf1-/-]), we show that IRF1 expression in B cells is required for marginal zone B (MZB) cell development and T cell-independent Ab responses. Although IFNs can induce IRF1 expression in MZB precursors, deletion of the IFN-γR (C57BL/6J [B6], B6.129S7-Ifngr1tm1Agt/J) or IFN-αR (B6[Cg]-Ifnar1tm1Agt/J) did not affect MZB cell development. Instead, BCR and TLR signals promote IRF1 expression and nuclear translocation in MZB cell precursors. In turn, IRF1 is required for Notch2-dependent gene expression in BCR- and TLR-stimulated transitional B cells and development of the MZB cell compartment. Thus, IRF1 regulates MZB-driven T cell-independent Ab responses by regulating Notch programming in MZB precursors and facilitating commitment of these cells to the MZB lineage.

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. The rules governing the immunogenicity of non-self donor HLA are poorly understood. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific B cell response in the kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We observed an immunodominant B cell antibody response focused on topographically exposed, solvent-accessible mismatched HLA residues along the peptide-binding groove - a subregion comprising only 20% of the HLA molecule. We further demonstrated that, even within a diverse cohort of transplant recipients, the B cell alloresponse consistently converges on this same immunodominant subregion on the crown of the HLA molecule. Based on these findings, we propose that B cell immunodominance in transplant rejection relies on antigenic topography, and we suggest that this link could be exploited for organ matching and therapeutics.

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.

B cells can be divided into distinct subsets based on origin, activation history and functional attributes. Most human studies, which have characterized bulk polyclonal populations of circulating B cells, suggest heterogeneity between the different B cell subsets. Indeed, we showed that memory B (Bmem) and plasma cells from the blood of the same donor exhibit distinct patterns of isotype distribution, mutational burden and have relatively limited clonal sharing. However, this heterogeneity may not extend to comparisons between the same B cell subset found within different tissues or cells that share the same reactivity. To test this, we examined influenza hemagglutinin (HA)-specific Bmem in multiple donor-matched tissues. We first generated a high-throughput bead array displaying HAs from the seasonal (H1, H3) and pandemic (H2, H5, H7, H9) flu strains and screened the serum from >80 brain-dead human tissue donors. All the donors exhibited high serum IgG reactivity against the circulating strains. We also identified serum reactivity in some donors for HAs that are not circulating in humans, suggesting that these donors likely have HA-specific IgG that are potentially broadly reactive and may have cross-reactive B cells. To directly identify B cells with cross-reactive potential, we enumerated H1 (CA09) and H2 (NETH1999) specific Bmem by flow cytometry. We show that Bmem specific for H1 or H2 are found in the spleen and blood whereas Bmem specific for both H1 and H2 are present in the mediastinal lymph node (LN), spleen and mesenteric LN. The data suggest that the repertoire of HA specific Bmem likely differ between tissues. Going forward, we will characterize the repertoire and molecular signatures of these HA-specific Bmem populations. Supported by grants from NIH (U19 AI142737,)