Citations

Abstract Rationale Candida albicans (CA) is ubiquitous and individuals are often immunized against it. However, skin testing reactivity with CA-antigens (CAAg) have been variable during latent tuberculosis infection (LTBI) screening. We hypothesized that the use of a CAAg in an ELISPOT assay would provide a positive control to study tuberculosis (TB)-antigen specific immune responses. Objective To test an ELISPOT with TB-antigens and control conditions, including a purified protein derivative of CA, CAAg, that differentiates subjects with LTBI from unexposed subjects. Methods An ELISPOT that detects interferon-γ from PBMCs (2×105 cells) after 2-day incubation with TB antigens, controls, including CAAg, and co-stimulatory antibodies. We studied two independent cohorts of subjects with LTBI diagnosis based on TST and IGRA results per clinical guidelines. Results One-hundred thirty-nine subjects were studied in 2010–12, including 27 patients with LTBI and 21 IGRA(−) unexposed subjects. ELISPOT with TB-antigens but also CAAg - nil (78 [21 – 182] vs. 9 [1 – 50] cfu) differentiated LTBI subjects from unexposed ones (P<0.005). A second cohort of 76 subjects were studied in 2015–17, including 14 patients with LTBI and 24 unexposed controls, which showed similar statistical results. Positive control (SEAB/antiCD3) did not differentiate these study groups. ELISPOT results with CAAg correlated with results of ELISPOT with TB antigens in all subjects (P<0.001), and in the LTBI and unexposed groups independently. Conclusion An ELISPOT assay that includes CAAg revealed immune reactivity that is prevalent in LTBI. The immune subsets that react to both CA and mycobacterial antigens are unclear, but may be relevant for both TB and candida immunity.

Abstract Background Acute respiratory exacerbations in idiopathic pulmonary fibrosis (IPF), often triggered by infection, are life threatening events characterized by rapid deterioration of lung function and associated with poor prognosis. Since microbial antigens modulate immunity by activating different pattern recognition receptors (PRR) expressed by innate immune cells (including B-cells), we sought to investigate how the activation of human B-cells through PRR stimulation by different microbial (CpG and β-glucan) and non-microbial (Mitochondrial DAMPs; MTDs) antigens resulted in the release of an inflammatory/fibrotic milieu that contributed to the pathogenesis of fibrosis in patients with IPF. Results Our results show that an inflammatory milieu is induced by stimulating circulating B-cells with both noninfectious (MTDs) and infectious antigens (CpG and β-glucan) commonly found in bacteria and fungi. Moreover, we show that the inflammatory milieu produced was specific to the inducing antigen, and that the microbial antigens (CpG and β-glucan) triggered distinct signaling pathways in B-cells. Specifically, CpG induced mTOR-activation while β-glucan was mTOR-independent and activated Src and p38. Furthermore, our results also show that B-cell aggregates are present within fibrotic areas of the lung in IPF patients and activated B-cells induce fibroblast migration, which was modified by antifibrotics (nintedanib and pirfenidone). Conclusion Our results highlight the potential contribution of activated B-cells through PRRs to the proinflammatory and profibrotic milieu seen in patients with idiopathic pulmonary fibrosis.

Abstract We hypothesize that B-lymphocytes are conducive to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive and fatal interstitial lung disease. B-lymphocytes aggregate in the lungs of IPF patients to form tertiary lymphoid follicles. How these follicles are formed and their role in the pathogenesis of IPF is not known. Hence, we sought to characterize how the activation of B-lymphocytes from IPF patients may contribute to the disease. Methods IPF patients defined by ATS guidelines were identified and recruited under local IRB #14-004714. B-lymphocytes were then isolated by negative selection. Cytokines and immunoglobulins from plasma or B-lymphocyte supernatants were measured by ELISA and signaling pathways were elucidated from whole-cell lysates by immunoblotting. Our results show that B-lymphocyte aggregates are present within fibrotic areas of the lung of IPF patients and that activated B-lymphocytes induce fibroblast migration. This effect was abrogated by nintedanib inhibition of B-lymphocyte activation. Furthermore, microbial antigen (β-glucan and CpG) activation of B-lymphocytes through pattern recognition receptors (PRRs; TLR9 and Dectin-1) contributed to the proinflammatory and profibrotic milieu seen in IPF patients. Stimulation of TLR9 and Dectin-1 resulted in activation of mTOR-dependent and -independent pathways, respectively. Rapamycin decreased TLR9 signaling, but not Dectin-1 mediated pathways. Nintedanib abrogated B-cell activation, IgM production, and the profibrotic milieu by interfering with both the mTOR-dependent and -independent pathways. In conclusion, our findings implicate activated B-lymphocytes in IPF pathogenesis highlighting the role of PRRs in this disease.

Abstract Background: Binding of the human IgM antibody B7-DC XAb to B7-DC on dendritic cells (DC) has profound immune modulatory effects. It enhances antigen presentation by DC, repolarizes T-helper (Th) 2 to Th1 cell responses, rapidly generates cytotoxic effectors and reprograms regulatory T-cells (Treg) into non-suppressive, cytokine producing autoimmune effectors. We hypothesized that co-culture of B7-DC XAb treated DC (DCXAb) with Treg results in the rapid generation of cytotoxic effectors. Methods: Bone marrow derived DC from wild type C57BL6 mice (WT-B6) were treated for 24 hours with either B7-DC XAb or a control human IgM antibody in the presence of melanoma cell lysate (B16). Subsequently, DCXAb were co-cultured with Treg (CD4+CD25+FOXP3+), Non-Treg (CD4+, CD25-), cytotoxic CD8+ T-cells isolated from WT-B6 mice and Treg from CD8 knockout mice (CD8 -/-). After re-isolation these T-cells were used in cytotoxic T-lymphocyte (CTL) assays targeting B16 and Thymic lymphoma (EL4) tumor cells. Results: Treg isolated from either WT-B6 mice or CD8 -/- mice co-cultured with B16 lysate treated DCXAb but not control antibody treated DC transformed into cytotoxic effectors targeting B16 but not EL4 tumor cells. Their level of cytotoxicity was comparable to that of CD8+ cells co-cultured with DCXAb. In contrast Non-Treg co-cultured with DCXAb did not kill in similar CTL assays. Conclusion: Co-culture of Treg with antigen treated DCXAb rapidly generates antigen specific cytotoxic effectors.

ObjectiveAutoreactive B cells are responsible for antineutrophil cytoplasmic antibody (ANCA) production in ANCA‐associated vasculitis (AAV). Rituximab (RTX) depletes circulating B cells, including autoreactive B cells. We aimed to evaluate changes and associations with relapse of the circulating autoreactive B cell pool following therapeutic B cell depletion in AAV.MethodsSequential flow cytometry was performed on 148 samples of peripheral blood mononuclear cells from 23 patients with proteinase 3 (PR3)–ANCA–positive AAV who were treated with RTX for remission induction and monitored after stopping therapy during long‐term follow‐up in a prospective clinical trial. PR3 was used as a ligand to target autoreactive PR3‐specific (PR3+) B cells. B cell recurrence was considered as the first blood sample with ≥10 B cells/μl after RTX treatment.ResultsAt B cell recurrence, PR3+ B cell frequency among B cells was higher than baseline (P < 0.01). Within both PR3+ and total B cells, frequencies of transitional and naive subsets were higher at B cell recurrence than at baseline, while memory subsets were lower (P < 0.001 for all comparisons). At B cell recurrence, frequencies of B cells and subsets did not differ between patients who experienced relapse and patients who remained in remission. In contrast, the plasmablast frequency within the PR3+ B cell pool was higher in patients who experienced relapse and associated with a shorter time to relapse. Frequencies of PR3+ plasmablasts higher than baseline were more likely to be found in patients who experienced relapse within the following 12 months compared to those in sustained remission (P < 0.05).ConclusionThe composition of the autoreactive B cell pool varies significantly following RTX treatment in AAV, and early plasmablast enrichment within the autoreactive pool is associated with future relapses.

Most gene-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are nonreplicating vectors. They deliver the gene or messenger RNA to the cell to express the spike protein but do not replicate to amplify antigen production. This study tested the utility of replication in a vaccine by comparing replication-defective adenovirus (RD-Ad) and replicating single-cycle adenovirus (SC-Ad) vaccines that express the SARS-CoV-2 spike protein. SC-Ad produced 100 times more spike protein than RD-Ad and generated significantly higher antibodies against the spike protein than RD-Ad after single immunization of Ad-permissive hamsters. SC-Ad–generated antibodies climbed over 14 weeks after single immunization and persisted for more than 10 months. When the hamsters were challenged 10.5 months after single immunization, a single intranasal or intramuscular immunization with SC-Ad-Spike reduced SARS-CoV-2 viral loads and damage in the lungs and preserved body weight better than vaccination with RD-Ad-Spike. This demonstrates the utility of harnessing replication in vaccines to amplify protection against infectious diseases.

BACKGROUND Little is known about the autoreactive B cells in antineutrophil cytoplasmic antibody–associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3–reactive (PR3 + ) B cells. METHODS Multicolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3 + B cells (total and subsets). RESULTS The frequency of PR3 + B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%–6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%–5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%–2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3 + B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3 + memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3 + B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3 + B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission ( P < 0.001). CONCLUSION This study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells. Trial registration ClinicalTrials.gov NCT00104299. Funding The Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.