LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells.

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

Elevated unphosphorylated STAT1 and IRF9 in T and B cells of primary sjögren's syndrome: Novel biomarkers for disease activity and subsets.

OBJECTIVES: Autoreactive B cells and interferon (IFN) signature are hallmarks of primary sjögren's syndrome (pSS), but how IFN signaling pathways influence autoantibody production and clinical manifestations remain unclear. More detailed studies hold promise for improved diagnostic methodologies and personalized treatment. METHODS: We analyzed peripheral blood T and B cell subsets from 34 pSS patients and 38 healthy donors (HDs) at baseline and upon stimulation regarding their expression levels of type I and II IFN signaling molecules (STAT1/2, IRF1, IRF9). Additionally, we investigated how the levels of these molecules correlated with serological and clinical characteristics and performed ROC analysis. RESULTS: Patients showed elevated IFN pathway molecules, including STAT1, STAT2 and IRF9 among most T and B cell subsets. We found a reduced ratio of phosphorylated STAT1 and STAT2 in patients in comparison to HDs, although B cells from patients were highly responsive by increased phosphorylation upon IFN stimulation. Correlation matrices showed further interrelations between STAT1, IRF1 and IRF9 in pSS. Levels of STAT1 and IRF9 in T and B cells correlated with the IFN type I marker Siglec-1 (CD169) on monocytes. High levels of STAT1 and IRF9 within pSS B cells were significantly associated with hypergammaglobulinemia as well as anti-SSA/anti-SSB autoantibodies. Elevated STAT1 levels were found in patients with extraglandular disease and could serve as a biomarker for this subgroup (p < 0.01). Notably, IRF9 levels in T and B cells correlated with EULAR Sjögren's syndrome disease activity index (ESSDAI). CONCLUSION: Here, we provide evidence that in active pSS patients, enhanced IFN signaling incl. unphosphorylated STAT1 and STAT2 with IRFs entertain chronic T and B cell activation. Furthermore, increased STAT1 levels candidate as biomarker of extraglandular disease, while IRF9 levels can serve as biomarker for disease activity.

Altered increase in STAT1 expression and phosphorylation in severe COVID-19.

The interferon pathway, a key antiviral defense mechanism, is being considered as a therapeutic target in COVID-19. Both, substitution of interferon and JAK/STAT inhibition to limit cytokine storms have been proposed. However, little is known about possible abnormalities in STAT signaling in immune cells during SARS-CoV-2 infection. We investigated downstream targets of interferon signaling, including STAT1, STAT2, pSTAT1 and 2, and IRF1, 7 and 9 by flow cytometry in 30 patients with COVID-19, 17 with mild, and 13 with severe infection. We report upregulation of STAT1 and IRF9 in mild and severe COVID-19 cases, which correlated with the IFN-signature assessed by Siglec-1 (CD169) expression on peripheral monocytes. Interestingly, Siglec-1 and STAT1 in CD14+ monocytes and plasmablasts showed lower expression among severe cases compared to mild cases. Contrary to the baseline STAT1 expression, the phosphorylation of STAT1 was enhanced in severe COVID-19 cases, indicating a dysbalanced JAK/STAT signaling that fails to induce transcription of interferon stimulated response elements (ISRE). This abnormality persisted after IFN-α and IFN-γ stimulation of PBMCs from patients with severe COVID-19. Data suggest impaired STAT1 transcriptional upregulation among severely infected patients may represent a potential predictive biomarker and would allow stratification of patients for certain interferon-pathway targeted treatments.

B cells in systemic lupus erythematosus.

PURPOSE OF REVIEW: New insight into altered B cell distribution including newly identified subsets and abnormalities in systemic lupus erythematosus (SLE) as well as their role in immune protection are summarized in this review. RECENT FINDINGS: SLE carries characteristic B cell abnormalities, which offer new insights into B cell differentiation and their disturbances including discoveries of pathogenic B cell subsets and intrinsic B cell abnormalities. A recent study in SLE found that antigen-experienced B cell subsets lacking expression of CD27 and IgD defined by their lack of CXCR5 and CD19low expression are expanded in SLE and represent plasmablasts likely escaping proper selection. In terms of therapeutic targeting with broader coverage than rituximab, second-generation anti-CD20, anti-CD38 and CD19-CART treatment experiences have advanced our understanding recently. However, the key role of qualitative and quantitative B cell requirements in connection with T cells became apparent during SARS-Cov2 infection and vaccination, especially in patients with gradual B cell impairments by rituximab, mycophenolate mofetil and cyclophosphamide. SUMMARY: Identification and characterization relevant B cell subsets together with altered regulatory mechanisms in SLE facilitates new approaches in targeting pathogenic B cells but require consideration of preservation of protection.

B and T Cell Responses after a Third Dose of SARS-CoV-2 Vaccine in Kidney Transplant Recipients.

BACKGROUND: Accumulating evidence sugges ts solid organ transplant recipients, as opposed to the general population, show strongly impaired responsiveness toward standard SARS-CoV-2 mRNA-based vaccination, demanding alternative strategies for protectio n o f this vulnerable group. METHODS: In line with recent recommendations, a third dose of either heterologous ChAdOx1 (AstraZeneca) or homologous BNT162b2 (BioNTech) was administered to 25 kidney transplant recipients (KTR) without humoral response after two doses of BNT162b2, followed by analysis of serological responses and vaccine-specific B- and T-cell immunity. RESULTS: Nine out of 25 (36%) KTR under standard immunosuppressive treatment seroconverted until day 27 after the third vaccination, whereas one patient developed severe COVID-19 infection immediately after vaccination. Cellular analysis 7 days after the third dose showed significantly elevated frequencies of viral spike-protein receptor-binding domain-specific B cells in humor al responders as compared with nonresponders. Likewise, portions of spike-reactive CD4 + T helper cells were significantly elevated in patients who were seroconverting. Furthermore, overall frequencies of IL-2 + , IL-4 + , and polyfunctional CD4 + T cells significantly increased after the third dose, whereas memory/effector differentiation remained unaffected. CONCLUSIONS: Our data suggest a fraction of transplant recipients benefit from triple vaccination, where seroconversion is associated with quantitative and qualitative changes of cellular immunity. At the same time, the study highlights that modified vaccination approaches for immunosuppressed patients remain an urgent medical need. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/JASN/2021_11_23_briggsgriffin112321.mp3.

Elevated STAT1 expression but not phosphorylation in lupus B cells correlates with disease activity and increased plasmablast susceptibility.

OBJECTIVES: SLE is characterized by two pathogenic key signatures, type I IFN and B-cell abnormalities. How these signatures are interrelated is not known. Type I-II IFN trigger activation of Janus kinase (JAK) - signal transducer and activator of transcription (STAT). JAK-STAT inhibition is an attractive therapeutic possibility for SLE. We assess STAT1 and STAT3 expression and phosphorylation at baseline and after IFN type I and II stimulation in B-cell subpopulations of SLE patients compared with other autoimmune diseases and healthy controls (HD) and related it to disease activity. METHODS: Expression of STAT1, pSTAT1, STAT3 and pSTAT3 in B and T cells of 21 HD, 10 rheumatoid arthritis (RA), seven primary Sjögren's (pSS) and 22 SLE patients was analysed by flow cytometry. STAT1 and STAT3 expression and phosphorylation in PBMCs (peripheral blood mononuclear cells) of SLE patients and HD after IFNα and IFNγ incubation were further investigated. RESULTS: SLE patients showed substantially higher STAT1 but not pSTAT1 in B- and T-cell subsets. Increased STAT1 expression in B-cell subsets correlated significantly with SLEDAI and Siglec-1 on monocytes, a type I IFN marker. STAT1 activation in plasmablasts was IFNα dependent while monocytes exhibited dependence on IFNγ. CONCLUSION: Enhanced expression of STAT1 by B-cell candidates as a key node of two immunopathogenic signatures (type I IFN and B-cells) related to important immunopathogenic pathways and lupus activity. We show that STAT1 is activated upon IFNα exposure in SLE plasmablasts. Thus, Jak inhibitors, targeting JAK-STAT pathways, hold a promise to block STAT1 expression and control plasmablast induction in SLE.

Cytokine-producing B cells: a translational view on their roles in human and mouse autoimmune diseases.

B-cell depletion therapy has beneficial effects in autoimmune diseases. This is only partly explained by an elimination of autoantibodies. How does B-cell depletion improve disease? Here, we review preclinical studies showing that B cells can propagate autoimmune disorders through cytokine production. We also highlight clinical observations indicating the relevance of these B-cell functions in human autoimmunity. Abnormalities in B-cell cytokine production have been observed in rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and systemic lupus erythematosus. In the first two diseases, B-cell depletion erases these abnormalities, and improves disease progression, suggesting a causative role for defective B-cell cytokine expression in disease pathogenesis. However, in the last two disorders, the pathogenic role of B cells and the effect of B-cell depletion on cytokine-producing B cells remain to be clarified. A better characterization of cytokine-expressing human B-cell subsets, and their modulation by B cell-targeted therapies might help understanding both the successes and failures of current B cell-targeted approaches. This may even lead to the development of novel strategies to deplete or amplify selectively pathogenic or protective subsets, respectively, which might be more effective than global depletion of the B-cell compartment.

Postactivated B cells in systemic lupus erythematosus: update on translational aspects and therapeutic considerations.

PURPOSE OF REVIEW: This review summarizes recent insights and current understanding of the role of postactivated B cells in SLE and related pathogenic and potential therapeutic implications. RECENT FINDING: B cells are considered key players in SLE and experience from various B-cell-targeted therapies underlines their clinical relevance. In the last years, new insights have been obtained on B-cell abnormalities within the complex pathophysiology of SLE. These insights involve a revised understanding of BCR signaling, that has been reported to be hyperresponsive in the past, but newer studies suggest a postactivation functiotype in terms of reduced BCR and TLR signaling. Despite comprehensive efforts to delineate B-cell abnormalities on assessing large-scale genomic, epigenomic and proteomic data, understanding functional impairments of cellular interactions and subcellular functions remains crucial. A recently identified enhanced protein tyrosine phosphatase (PTP) activity was found in relation to diminished BCR responses in SLE. This finding together with reduced cytokine production upon TLR9 activation appears to mark postactivated lupus B cells. Other studies identified increased PTP activity in line with a gain-of-function mutation of phosphatase PTPN22, one of the strongest SLE risk alleles. Improved understanding of these B cell abnormalities in SLE holds promise to gain further insights in mechanisms of autoimmunity and pave the way for selective therapies targeting key principles of chronic autoimmunity. SUMMARY: SLE B cells (similar as previously described for lupus T cells) are characterized by a postactivation (exhausted) functiotype mandating consideration for innovative therapies.

Recent thymic emigrants are the preferential precursors of regulatory T cells differentiated in the periphery.

Most Forkhead box P3(+) (Foxp3(+)) CD4 regulatory T cell (Treg) precursors are newly formed thymocytes that acquire Foxp3 expression on antigen encounter in the thymus. Differentiation of Treg, however, can also occur in the periphery. What limits this second layer of self- and nonself-reactive Treg production in physiological conditions remains to be understood. In this work, we tested the hypothesis that, similarly to thymic Treg, the precursors of peripheral Treg are immature T cells. We show that CD4(+)CD8(-)Foxp3(-) thymocytes and recent thymic emigrants (RTEs), contrarily to peripheral naïve mature cells, efficiently differentiate into Treg on transfer into lymphopenic mice. By varying donor and recipient mice and conducting ex vivo assays, we document that the preferential conversion of newly formed T cells does not require intrathymic preactivation, is cell-intrinsic, and correlates with low and high sensitivity to natural inhibitors and inducers of Foxp3 expression, such as IL-6, T-cell receptor triggering, and TGF-ß. Finally, ex vivo analysis of human thymocytes and peripheral blood T cells revealed that human RTE and newly developed T cells share an increased potential to acquire a FOXP3(bright)CD25(high) Treg phenotype. Our findings indicating that RTEs are the precursors of Tregs differentiated in the periphery should guide the design of Treg-based therapies.

Naturally secreted immunoglobulins limit B1 and MZ B-cell numbers through a microbiota-independent mechanism.

B-cell numbers and immunoglobulin (Ig) titers can increase several logs during immune responses. In contrast to this plasticity and despite constant renewal, B-cell numbers are stable in the absence of immunization. We assessed the role of serum Igs in maintaining specific B-cell subset homeostasis at steady state. Using mice genetically deficient in secreted IgM only (secretory µ chain-deficient), in switched Igs and hypermutated IgM (activation-induced cytidine deaminase-deficient), or fully agammaglobulemic (AID(-/-)µS(-/-)), we dissected the contribution of different Ig classes to 4 phenotypes associated with loss of serum Igs: 1) increased splenic B-cell numbers, mostly of the B1 and marginal zone (MZ) B-cell subtypes; 2) enlarged germinal centers (GCs) in spleen and mesenteric lymph nodes; 3) enrichment in IRF4(+)CD138(-) plasmablast-like cells; and 4) overexpression of IgM in several cell subsets. Complementation experiments based on either mixed bone marrow reconstitution of chimeras or Ig infusion, and analysis of mice raised in germ-free conditions reveal a negative feedback mechanism in which MZ and B1 cell numbers are under the control of naturally secreted Igs as the result of an intrinsic property of the immune system, whereas GC development is under indirect control of secreted Igs that limit bacterial species triggering GC reactions.

CD8 T cells induce T-bet-dependent migration toward CXCR3 ligands by differentiated B cells produced during responses to alum-protein vaccines.

Antibody-forming cells (AFCs) expressing the chemokine receptor CXCR3 are recruited to sites of inflammation where they help clear pathogens but may participate in autoimmune diseases. Here we identify a mechanism that induces CXCR3 expression by AFC and germinal center (GC) B cells. This happens when CD8 T cells are recruited into CD4 T cell-dependent B-cell responses. Ovalbumin-specific CD4 T cells (OTII) were transferred alone or with ovalbumin-specific CD8 T cells (OTI) and the response to subcutaneous alum-precipitated ovalbumin was followed in the draining lymph nodes. OTII cells alone induce T helper 2-associated class switching to IgG1, but few AFC or GC B cells express CXCR3. By contrast, OTI-derived IFN-γ induces most responding GC B cells and AFCs to express high levels of CXCR3, and diverse switching to IgG2a, IgG2b, with some IgG1. Up-regulation of CXCR3 by GC B cells and AFCs and their migration toward its ligand CXCL10 are shown to depend on B cells' intrinsic T-bet, a transcription factor downstream of the IFN-γR signaling. This model clarifies how precursors of long-lived AFCs and memory B cells acquire CXCR3 that causes their migration to inflammatory foci.

Differential response of IgM and IgG memory B cell populations to CD40L: insights of T cell - memory B cell interactions.

Memory B cells (mBCs) are characterized by their long-term stability, fast reactivation, and capability to rapidly differentiate into antibody-secreting cells (ASCs). However, the role of T cells in the differentiation of mBCs, in contrast to naive B cells, remains to be delineated. We study the role of T cells in mBC responses, using CD40L stimulation and autologous T-B co-cultures. Our results showed that increased CD40L levels led to a selective increased proliferation of IgM+ mBC, which did not class-switched, resulting in higher frequencies of IgM+ ASCs and a lower frequency of IgG+ ASCs. The IgG+/IgA+ mBCs were unaffected. We further compared the transcription of immune-related genes in IgM+ and IgG+ pre-plasmablasts cultured at high (500 ng/mL) and low (50 ng/mL) CD40L levels. In response to increased CD40L levels, both populations exhibited a core response to genes related to activation (TRAF1, AKT3, CD69, and CD80). However, they differed in genes related to cytokine/chemokine/homing interactions (CCL3/4/17, LTA, NKX2-3, BCL2 and IL21R) and cell-cell interactions (HLADR, CD40, and ICOSL), which were largely confined to IgG+ cells. Our findings revealed that in co-cultures with a high T-ratio, the response was similar to that found in cultures with high CD40L levels. These results suggest that IgG+ mBCs have a greater capacity for proliferation and T cell interaction, and weaker migration capabilities, leading to a preference for an IgG response over IgM in the short term. This adaptable response could fine-tune the memory repertoire with different functions of IgG versus IgM mBCs.

Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow.

Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.

Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo.

Regulatory T (T reg) cells exert powerful down-modulatory effects on immune responses, but it is not known how they act in vivo. Using intravital two-photon laser scanning microscopy we determined that, in the absence of T reg cells, the locomotion of autoantigen-specific T cells inside lymph nodes is decreased, and the contacts between T cells and antigen-loaded dendritic cells (DCs) are of longer duration. Thus, T reg cells can exert an early effect on immune responses by attenuating the establishment of stable contacts during priming of naive T cells by DCs.

Cutting edge: Intrathymic differentiation of adaptive Foxp3+ regulatory T cells upon peripheral proinflammatory immunization.

Thymocytes differentiate into CD4(+) Foxp3(+) regulatory T cells (T(R)) upon interaction between their TCR and peptide-MHC II complexes locally expressed in the thymus. Conversion of naive CD4(+) T cells into T(R) can additionally take place in the periphery under noninflammatory conditions of Ag encounter. In this study, making use of TCR transgenic models naturally devoid of Foxp3(+) cells, we report de novo generation of T(R) upon a single footpad injection of Ag mixed with a classic proinflammatory adjuvant. Abrupt T(R) differentiation upon immunization occurred intrathymically and was essential for robust tolerance induction in a mouse model of spontaneous encephalomyelitis. This phenomenon could be attributed to a specific feature of thymocytes, which, in contrast to mature peripheral CD4(+) T cells, were insensitive to the inhibitory effects of IL-6 on the induction of Foxp3 expression. Our findings uncover a pathway for T(R) generation with major implications for immunity and tolerance induction.

B- and Plasma Cell Subsets in Autoimmune Diseases: Translational Perspectives.

B lymphocytes play a central role in immunity owing to their unique antibody-producing capacity that provides protection against certain infections and during vaccination. In autoimmune diseases, B cells can gain pathogenic relevance through autoantibody production, antigen presentation, and proinflammatory cytokine secretion. Recent data indicate that B and plasma cells can function as regulators through the production of immunoregulatory cytokines and/or employing checkpoint molecules. In this study, we review the key findings that define subsets of B and plasma cells with pathogenic and protective functions in autoimmunity. In addition to harsh B-cell depletion, we discuss the strategies that have the potential to reinstall the balance of pathogenic and protective B cells with the potential of more specific and personalized therapies.

Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators.

Human B lymphocytes are attractive targets for immunotherapies in autoantibody-mediated diseases. Gene editing technologies could provide a powerful tool to determine gene regulatory networks regulating B cell differentiation into plasma cells, and identify novel therapeutic targets for prevention and treatment of autoimmune disorders. Here, we describe a new approach that uses CRISPR-Cas9 technology to target genes in primary human B cells in vitro for identifying plasma cell regulators. We found that sgRNA and Cas9 components can be efficiently delivered into primary human B cells through RD114-pseudotyped retroviral vectors. Using this system, we achieved approximately 80% of gene knockout efficiency. We disrupted expression of a triad of transcription factors, IRF4, PRDM1, and XBP1, and showed that human B cell survival and plasma cell differentiation are severely impaired. Specifically, that IRF4, PRDM1, and XBP1 were expressed at different stages during plasma cell differentiation, IRF4, PRDM1, and XBP1-targeted B cells failed to progress to the pre-plasmablast, plasma cell state, and plasma cell survival, respectively. Our method opens a new avenue to study gene functions in primary human B cells and identify novel plasma cell regulators for therapeutic applications.

CD39 and CD326 Are Bona Fide Markers of Murine and Human Plasma Cells and Identify a Bone Marrow Specific Plasma Cell Subpopulation in Lupus.

Antibody-secreting cells (ASCs) contribute to immunity through production of antibodies and cytokines. Identification of specific markers of ASC would allow selective targeting of these cells in several disease contexts. Here, we performed an unbiased, large-scale protein screening, and identified twelve new molecules that are specifically expressed by murine ASCs. Expression of these markers, particularly CD39, CD81, CD130, and CD326, is stable and offers an improved resolution for ASC identification. We accessed their expression in germ-free conditions and in T cell deficient mice, showing that at least in part their expression is controlled by microbial- and T cell-derived signals. Further analysis of lupus mice revealed the presence of a subpopulation of LAG-3- plasma cells, co-expressing high amounts of CD39 and CD326 in the bone marrow. This population was IgM+ and correlated with IgM anti-dsDNA autoantibodies in sera. Importantly, we found that CD39, CD81, CD130, and CD326 are also expressed by human peripheral blood and bone marrow ASCs. Our data provide innovative insights into ASC biology and function in mice and human, and identify an intriguing BM specific CD39++CD326++ ASC subpopulation in autoimmunity.

B Cell Characteristics at Baseline Predict Vaccination Response in RTX Treated Patients.

Background: Vaccination is considered as most efficient strategy in controlling SARS-CoV-2 pandemic spread. Nevertheless, patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) are at increased risk to fail humoral and cellular responses upon vaccination. The ability to predict vaccination responses is essential to guide adequate safety and optimal protection in these patients. Methods: B- and T- cell data before vaccination were evaluated for characteristics predicting vaccine responses in altogether 15 patients with autoimmune inflammatory rheumatic diseases receiving RTX. Eleven patients with rheumatoid arthritis (RA) on other therapies, 11 kidney transplant recipients (KTR) on regular immunosuppression and 15 healthy controls (HC) served as controls. A multidimensional analysis of B cell subsets via UMAP algorithm and a correlation matrix were performed in order to identify predictive markers of response in patients under RTX therapy. Results: Significant differences regarding absolute B cell counts and specific subset distribution pattern between the groups were identified at baseline. In this context, the majority of B cells from vaccination responders of the RTX group (RTX IgG+) were naïve and transitional B cells, whereas vaccination non-responders (RTX IgG-) carried preferentially plasmablasts and double negative (CD27-IgD-) B cells. Moreover, there was a positive correlation between neutralizing antibodies and B cells expressing HLA-DR and CXCR5 as well as an inverse correlation with CD95 expression and CD21low expression by B cells among vaccination responders. Summary: Substantial repopulation of the naïve B cell compartment after RTX therapy appeared to be essential for an adequate vaccination response, which seem to require the additional capability of antigen presentation and germinal center formation. Moreover, expression of exhaustion markers represent negative predictors of vaccination responses.