ScRNA-seq of CD79A+ B cells n=5 early-stage LUAD lesions and normal adjacent tissues (NAT)

Abstract Introduction Most if not all the IgM secreted early after Influenza virus infection by B-1 and B-2 cells is derived from antibody-secreting cells (ASCs) developing in rapidly forming extrafollicular (EF) B cell responses. We previously demonstrated that the lack of the early-derived IgM significantly reduces the later-induced IgG response to influenza A virus (IAV) infection. The mechanisms underlying this effect on IgG remain to be fully explored. Here we hypothesize that EF-derived IgM affects antigen retention and stimulation of germinal center (GC) B cells, promoting stronger selection for memory and plasma cell differentiation in GCs. Methods Flow cytometry, ELISPOT, ELISA. Results To investigate the impact of reduced EF response output on GC functionality, we infected secreted IgM knockout (s-IgM KO) mice with IAV and analyzed the draining mediastinal lymph nodes (medLN) for alterations in GC B cell frequencies at day 14 post-infection. Our initial studies demonstrate significant reductions in both frequency and total numbers of GC B cells in s-IgM KO mice compared to wild-type (WT) controls. Moreover, we found that s-IgM KO mice harbored significantly fewer CD80+,CD73+, Influenza hemagglutinin (HA)-specific memory B cells at 30 days post infection (p.i) and decreased HA+ plasma cells in the bone marrow compared to WT mice at 45 days p.i. Antigen-specific serum and bone marrow antibody levels were assessed by ELISA and ELISPOT, and female s-IgMKO mice showed a reduction in IgG levels compared to WT after 45 days p.i. Conclusion Collectively, this data suggests that early EF-derived IgM responses are critical to support maximal GC outputs. Understanding these mechanisms may provide valuable insights for optimizing vaccine strategies against IAV infections. Funding Source N/A Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Lyme disease, an emerging tickborne disease caused by Borrelia burgdorferi (Bb), can result in myriad symptoms of unknown causes in humans. Here, we show that Bb rapidly disrupts the gastrointestinal immune barrier in infected mice, inducing a "leaky gut" syndrome, characterized by increased gut permeability, systemic endotoxemia, elevated LPS binding protein and intestinal fatty acid binding protein, and altered blood leukocyte profiles. Patients with acute Lyme disease showed similar blood changes that correlated with disease symptoms and liver function measurements and largely resolved after antibiotic treatment. Mechanistically, Bb infection triggered mild early inflammation in the small intestine and suppressed humoral immunity to the microbiota, resulting in reduced IgA coating of fecal microbes and decreased antimicrobial serum IgG, despite the increased gut permeability. These defects preceded shifts in later observed gut bacterial composition and short-chain fatty acid production. Together, our findings identify mucosal barrier dysfunction and impaired antimicrobial humoral immunity as potential drivers of Bb-induced disease.

Anti-human antibodies for flow cytometry

Introduction Advanced age is associated with increased morbidity and mortality following acute viral infections, including SARS-CoV-2. Despite this, most preclinical models rely on young animals and fail to account for age-related immune remodeling. How advanced aging alters antiviral immune responses and contributes to immune-mediated pathology remains incompletely understood.Methods Young (2-6 months), aged (15-18 months), and advanced aged (20-29 months) mice were infected with murine cytomegalovirus (MCMV) or influenza virus. Survival, viral burden, cytokine production, immune cell phenotypes, and tissue pathology were assessed using flow cytometry, histology, serum cytokine analysis, and RNA sequencing. Mouse findings were compared with publicly available transcriptomic datasets from SARS-CoV-2-infected human cohorts across age groups.Results Advanced aged mice exhibited markedly increased mortality and organ pathology following viral infection despite maintaining viral loads comparable to younger mice. These outcomes were associated with heightened systemic and tissue inflammatory cytokine production, reduced antigen-specific T cell responses, and increased frequencies of NK cells and non-antigen-specific bystander T cell activation. Coagulopathy with thrombolytic clot formation was observed exclusively in advanced aged mice. Transcriptomic analysis revealed enrichment of inflammatory and coagulation pathways in influenza-infected advanced aged mice, paralleling findings in elderly humans with SARS-CoV-2 infection, who also displayed reduced expression of T cell-associated genes.Discussion These findings demonstrate that advanced age profoundly alters antiviral immune responses, shifting immunity away from effective antigen-specific T cell responses toward inflammatory and innate pathways that contribute to immune-mediated pathology. The results highlight the importance of modeling advanced aging in preclinical studies and suggest that age-dependent immune imbalance may underlie increased inflammation, coagulopathy, and mortality during viral infection in both mice and humans.

CD11c+ B cells are present in close proximity to CD4+ T cells within tumor

Tumor-infiltrating B lymphocytes (TIL-B) are increasingly recognized as favorable prognostic markers in multiple cancer types, and the mechanisms underlying this are being actively investigated. In this study of TIL-Bs, we identified CD79A as a reliable quantifier of B lymphocytes and evaluated transcriptomic data for 15 distinct tumors using 8,720 samples of treatment naïve patients from The Cancer Genome Atlas and normal tissues from Gene Tissue Expression. B-lymphocyte infiltration correlated with survival for some but not all tumors. In lung adenocarcinoma (LUAD), CD79A levels were strongly predictive of overall survival, whereas CD8A transcripts were not, indicating that leukocytic infiltration per se does not explain the B cell's impact. Single-cell RNA sequencing and flow cytometry identified increased relative numbers of CD11c+ B cells in patients with treatment-naïve LUAD compared with normal tissue and blood. In LUAD, CD11c+ TIL-Bs were localized near CD4+ T cells, and in vitro stimulation with anti-IgG with/without CD40 agonist resulted in expansion and rapid differentiation. Stimulation also induced IL12, IL21, and TNFα secretion, which are cytokines known to enhance antitumor immunity. Overall, the data indicate that CD11c+ TIL-Bs are a potential target for anticancer therapeutic approaches and/or a potential prognostic biomarker for cancer prognosis.

Protective immunity is induced by both, wild-type measles virus (WT MeV) and live-attenuated (LA) measles virus vaccine (LAMV), affording the opportunity to compare common and selective immunological underpinnings of immune protection. Here, peripheral blood mononuclear cells (PBMCs) were collected from rhesus macaques at several timepoints after infection with either virus for single-cell RNA/VDJ sequence analysis. Data revealed acute immune activation at 10/11 days post infection (dpi) with WT MeV, with increased frequencies of CD8 T cells, monocytes and NK cells, and B and T cell expansion and activation, most notably IgA+ B cells and cytotoxic CD8 T cells. Two interferon-stimulated genes (ISGs), IFI6 and IFI27, were upregulated selectively across PBMCs, with strongest ISGylation and JAK-STAT activation in monocytes. Furthermore, WT MeV-infected PBMCs were enriched among IgA+ B cells and Th17 CD4 T cells, expressing the highest levels of ISGs. However, lymphocyte depletion, with absolute or relative reduction in B, CD4, and CD8 T cells, preferential ISG expression in activated/memory but not naive B and T cells, as well as dampened acute NK cell activation suggested acute immune suppression. Subsequent immune reconstitution was implicated by increased frequencies of IgM+ memory B cell subtypes with lower IGHV somatic hypermutation (SHM) rates at 42/43/56 dpi. LAMV elicited distinct humoral and cellular responses, with greater induction of IgG+ than IgA+ B cells and differential cytotoxic CD8 T cells encoding distinct TRBV/TRAV genes, compared to WT MeV. LAMV infection induced no signs of immune suppression. Together, these data provide insights into distinct features of WT MeV and LAMV-induced immunity.

Identification of CD11c on enriched TIL B cell cultures

Abstract Introduction Borrelia burgdorferi (Bb), the causative agent of Lyme disease, causes persistent infections in their reservoir hosts, including mice. B cell responses to Bb can control disease but cannot clear infection. Prior work in the lab identified a rapid collapse of infection-induced germinal centers (GC) and impaired long-term humoral immunity to Bb and unrelated vaccine antigens. IL-10, a potent immunoregulatory cytokine, is strongly induced during Bb infection, limiting Bb tissue clearance. However, the mechanisms by which IL10 acts to limit effective immunity remains unknown. Here we investigated whether blockade of IL10 can improve adaptive immunity to Bb. Methods Groups of C57BL/6 mice were treated with anti—IL-10 receptor antibody (αIL-10R) or control rat IgG prior to infection with Bb. Some mice were immunized in addition with influenza virions in alum. Serum antibody responses were measured by ELISA. Flow cytometric analysis assessed GC B and T follicular helper (Tfh) cell populations, and PCR for Bb flaB on various tissues quantified bacterial loads. Results On day 30 after infection, αIL-10R—treated mice had increased total and Bb-specific serum IgG, as well as enhanced responses to influenza vaccination. Flow cytometry demonstrated increased GC B and Tfh populations, and qPCR revealed reduced Bb loads compared to control-treated mice. Using IL-10—GFP reporter mice, we identified CD4+ T and B cells as predominant sources of IL-10. Conclusion Ongoing studies using CD4-Cre and mb1-Cre IL-10flx/flx mice will define the impact of each IL10 cell source on humoral immune responses to Bb and on GC maintenance. These results demonstrate the critical role of Bb-induced IL10 in suppression of effective adaptive immunity to Bb, identifying IL-10 as a potential therapeutic target for improving immunity and Bb clearance in Lyme disease patients. Funding Source NIH/NIAID R01 AI157007 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Abstract Introduction B-1 cells are a fetal/neonatal-derived B cell population located in small frequencies in the spleen. They are also the predominant B cell subset in the body cavities of common laboratory mice. B-1 cells differ from conventional (B-2) cells in that they maintain their numbers throughout life not by bone marrow replenishment but rather by “self-renewal”, a poorly understood process in which B-1 cells divide following unknown stimuli. Using cell population data obtained in an unbiased phenotypic screen, conducted by flow cytometry of 56 cell populations in 640 mouse lines each carrying a single gene deletion, we identified ppp2r5a as a candidate gene required for the selective control of B-1 cell maintenance and/or generation. Ppp2r5a encodes a regulatory subunit of protein phosphatase PP2A; a phosphase known to participate in cell activation, proliferation and cell death through its function in the WNT/Beta-catenin pathway. Here we tested the hypothesis that PP2A regulation is necessary for B-1 cell self-renewal. Methods Flow cytometry, in vitro cell culture, ELISA assy. Results Ppp2r5a-/- mice showed significantly lower frequencies of B-1 cells and B-1 derived plasma cells (PC) in spleen and BM, respectively, but not the body cavities. Consistent with B-1 cell’s role as producers of natural antibodies, mice had lower concentrations of serum IgM, IgG and IgG3 compared to wild type controls. Compared to B-1 cells from wild type mice, Ppp2r5a-/- B-1 cells showed increased proliferation in response to TLR4 agonist stimulation, but also increased cell death, as assessed by flow cytometric staining for Caspase 3 and 7. In addition, Ppp2r5a-/- B-1 cells showed dysregulated expression of mitochondria and Beta-catenin. Conclusion Together, these data implicate the WNT/ Beta-catenin pathway as an important regulator of B-1 cell dynamics. Funding Source NIHR21AI172208 (NB) and NIH UM1 OD023221 (KCKL) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Cytokine analysis reference ranges and reagent list

CD11c+ tumor B cells exhibit an activated and antigen presenting phenotype

The rapid onset of innate immune defenses is critical for early control of viral replication in an infected host, yet it can also lead to irreversible tissue damage, especially in the respiratory tract. Intricate regulatory mechanisms must exist that modulate inflammation, while controlling the infection. Here, B cells expressing choline acetyl transferase (ChAT), an enzyme required for production of the metabolite and neurotransmitter acetylcholine (ACh) are identified as such regulators of the immediate early response to influenza A virus. Lung tissue ChAT + B cells are shown to interact with a7 nicotinic Ach receptor-expressing lung interstitial macrophages in mice within 24h of infection to control their production of TNFa, shifting the balance towards reduced inflammation at the cost of enhanced viral replication. Thus, innate-stimulated B cells are key participants of an immediate-early regulatory cascade that controls lung tissue damage after viral infection.

Extrafollicular (EF) B cell responses are essential for protective immunity, but the signals that direct naïve B cells toward an EF fate remain unclear. Our previous work showed that deleting both adaptor proteins, MyD88 and TRIF, reduced EF cell numbers post-influenza infection. In separate studies, we show that TLR3 activation appears to be upstream of TRIF and TLR7 activation upstream of MyD88 signaling, but what role each of these adaptor proteins play in EF formation remains undefined. To address this, we performed flow cytometric analysis of CTV-labeled naïve B cells stimulated with TLR3, TLR7, or combined TLR3 + 7 agonists over a 96-hour period. Stimulation through TLR7 (CL097) promoted robust proliferation without plasma-cell differentiation, while TLR3 stimulation (POLY(I:C)) induced activation, as measured by increased cell surface expression of co-stimulatory receptors. Notably, TLR3 + 7 co-stimulation synergistically enhanced both proliferation and plasma-cell differentiation. qPCR analysis of key plasma-cell differentiation factors, including Pax5, Bach2, BLIMP1 and IRF4, all of which were strongly upregulated by combined TLR3 + 7 stimulation. To further dissect the transcriptional targets downstream of each TLR signal alone or in combination, we are using bulk RNA-Seq analysis with the goal to delineate the signaling pathways that explain the synergistic effects of these two distinct TLR stimuli on the kinetics of plasma cell differentiation in vitro. Funding sources: R01AI148652 and R01AI184867 Immune Response Regulation: Molecular Mechanisms (IRM)

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.

Lyme disease, the most widespread tick-borne disease in North America, is caused by the bacterium Borrelia burgdorferi (Bb). Approximately 10-15% of infections result in neuroborreliosis, common symptoms of which include headaches, facial palsy, and long-term cognitive impairment. Previous studies of Bb dissemination focus on assessing Bb transmigration at static time points rather than analyzing the complex dynamic process of extravasation. Furthermore, current in vitro models lack crucial physiological factors such as flow, demonstrating a need for more robust models for studying Bb dissemination to understand its dynamics and mechanisms. Here, a 3D tissue-engineered microvessel model is used and fluorescently-labeled Bb is perfused to model vascular dissemination in non-tissue-specific (iEC) and brain-specific (iBMEC) microvessels while acquiring time-lapse images in real time. In iECs, extravasation involves two steps: adhesion to the endothelium and transmigration into the extracellular matrix, which can be modulated through glycocalyx degradation or inflammation. In contrast, Bb extravasation in iBMECs is an extremely rare event regardless of glycocalyx degradation or inflammation. In addition, circulating Bb do not induce endothelial activation in iECs or iBMECs, but induces barrier dysfunction in iECs. These findings provide a further understanding of Bb vascular dissemination.

Initial description of extrafollicular B cell responses (EF) identified them as short-lived clusters of rapidly proliferating B cells and plasmablasts in splenic bridging channels and red pulp as well as lymph node medullary cord areas. Their physical location guided the nomenclature: outside, or at the edges of B cell follicles and near T cell zones of secondary lymphoid organs, thus distinct from the follicular situated germinal centers (GCs). Because EFs are often induced transiently and to both T-dependent and T-independent antigens, and because they generate IgM and class-switched antibodies often with no or little signs of somatic hypermutations, they were thought to be less impactful than GC-derived antibodies. However, highly protective antibodies are generated by these EFs rapidly after acute infections and their induction often correlates with pathogen clearance, while in autoimmunity EF-derived antibodies have been implicated as pathogenic drivers of disease. Moreover, subsets of memory B cells, including some CD11c+ "atypical" B cells/ABCs are generated independently of GC. Their diverse appearance and impact have initiated an ongoing debate about whether all "non-GC responses" are necessarily EF responses. The current debate is reflected also in the articles compiled for this issue of Immunological Reviews considering EF responses. Here, I briefly summarize the steps leading to B cell activation and EF and GC formation, providing context for the contributed reviews that span a breadth of topics from descriptions of non-GC responses in jawed non-mammalian vertebrates as possible orthologues of mammalian EF to the molecular and metabolic requirements and CD4 T cell helper quality of EF, tissue-specific B cell responses, and discussions on the origins and classifications of "atypical" memory B cells in mice and man.