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Background Involvement of B cells in the pathogenesis of rheumatoid arthritis (RA) is supported by the presence of disease-specific autoantibodies and the efficacy of treatment directed against B cells. B cells that express low levels of or lack the B cell receptor (BCR) co-receptor CD21, CD21 −/low B cells, have been linked to autoimmune diseases, including RA. In this study, we characterized the CD21 + and CD21 −/low B cell subsets in newly diagnosed, early RA (eRA) patients and investigated whether any of the B cell subsets were associated with autoantibody status, disease activity and/or joint destruction. Methods Seventy-six eRA patients and 28 age- and sex-matched healthy donors were recruited. Multiple clinical parameters were assessed, including disease activity and radiographic joint destruction. B cell subsets were analysed in peripheral blood (PB) and synovial fluid (SF) using flow cytometry. Results Compared to healthy donors, the eRA patients displayed an elevated frequency of naïve CD21 + B cells in PB. Amongst memory B cells, eRA patients had lower frequencies of the CD21 + CD27 + subsets and CD21 −/low CD27 + IgD + subset. The only B cell subset found to associate with clinical factors was the CD21 −/low double-negative (DN, CD27 − IgD − ) cell population, linked with the joint space narrowing score, i.e. cartilage destruction. Moreover, in SF from patients with established RA, the CD21 −/low DN B cells were expanded and these cells expressed receptor activator of the nuclear factor κB ligand (RANKL). Conclusions Cartilage destruction in eRA patients was associated with an expanded proportion of CD21 −/low DN B cells in PB. The subset was also expanded in SF from established RA patients and expressed RANKL. Taken together, our results suggest a role for CD21 −/low DN in RA pathogenesis.

SARS-CoV-2 infection represents a global health problem that has affected millions of people. The fine host immune response and its association with the disease course have not yet been fully elucidated. Consequently, we analyze circulating B cell subsets and their possible relationship with COVID-19 features and severity. Using a multiparametric flow cytometric approach, we determined B cell subsets frequencies from 52 COVID-19 patients, grouped them by hierarchical cluster analysis, and correlated their values with clinical data. The frequency of CD19 B cells is increased in severe COVID-19 compared to mild cases. Specific subset frequencies such as transitional B cell subsets increase in mild/moderate cases but decrease with the severity of the disease. Memory B compartment decreased in severe and critical cases, and antibody-secreting cells are increased according to the severity of the disease. Other non-typical subsets such as double-negative B cells also showed significant changes according to disease severity. Globally, these differences allow us to identify severity-associated patient clusters with specific altered subsets. Finally, respiratory parameters, biomarkers of inflammation, and clinical scores exhibited correlations with some of these subpopulations. The severity of COVID-19 is accompanied by changes in the B cell subpopulations, either immature or terminally differentiated. Furthermore, the existing relationship of B cell subset frequencies with clinical and laboratory parameters suggest that these lymphocytes could serve as potential biomarkers and even active participants in the adaptive antiviral response mounted against SARS-CoV-2.

B cells are fundamental players in the pathophysiology of autoimmune diseases of the central nervous system, such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). A deeper understanding of disease-specific B cell functions has led to the differentiation of both diseases and the development of different treatment strategies. While NMOSD is strongly associated with pathogenic anti-AQP4 IgG antibodies and proinflammatory cytokine pathways, no valid autoantibodies have been identified in MS yet, apart from certain antigen targets that require further evaluation. Although both diseases can be effectively treated with B cell depleting therapies, there are distinct differences in the peripheral B cell subsets that influence CNS inflammation. An increased peripheral blood double negative B cells (DN B cells) and plasmablast populations has been demonstrated in NMOSD, but not consistently in MS patients. Furthermore, DN B cells are also elevated in rheumatic diseases and other autoimmune entities such as myasthenia gravis and Guillain-Barré syndrome, providing indirect evidence for a possible involvement of DN B cells in other autoantibody-mediated diseases. In MS, the peripheral memory B cell pool is affected by many treatments, providing indirect evidence for the involvement of memory B cells in MS pathophysiology. Moreover, it must be considered that an important effector function of B cells in MS may be the presentation of antigens to peripheral immune cells, including T cells, since B cells have been shown to be able to recirculate in the periphery after encountering CNS antigens. In conclusion, there are clear differences in the composition of B cell populations in MS and NMOSD and treatment strategies differ, with the exception of broad B cell depletion. This review provides a detailed overview of the role of different B cell subsets in MS and NMOSD and their implications for treatment options. Specifically targeting DN B cells and plasmablasts in NMOSD as opposed to memory B cells in MS may result in more precise B cell therapies for both diseases.

ObjectivesThe role of B cells in COVID‐19, beyond the production of specific antibodies against SARS-CoV-2, is still not well understood. Here, we describe the novel landscape of circulating double-negative (DN) CD27− IgD− B cells in COVID‐19 patients, representing a group of atypical and neglected subpopulations of this cell lineage.MethodsUsing multiparametric flow cytometry, we determined DN B cell subset amounts from 91 COVID-19 patients, correlated those with cytokines, clinical and laboratory parameters, and segregated them by principal components analysis.ResultsWe detected significant increments in the DN2 and DN3 B cell subsets, while we found a relevant decrease in the DN1 B cell subpopulation, according to disease severity and patient outcomes. These DN cell numbers also appeared to correlate with pro- or anti-inflammatory signatures, respectively, and contributed to the segregation of the patients into disease severity groups.ConclusionThis study provides insights into DN B cell subsets’ potential role in immune responses against SARS‐CoV‐2, particularly linked to the severity of COVID‐19.

Disease-associated, high-affinity pathological autoantibody production is a well-described consequence of immune dysregulation affecting B cells in systemic sclerosis (SSc), including the distribution of B-cell subsets. We have previously shown that the increased relative frequency of CD19+CD27+IgD− switched memory B cells is associated with the severe form of SSc. This study sought to analyze memory B cell subsets using an extended range of markers for further subdivision based on CD19, IgD, CD27, CD38 and CD95 phenotype, to define relationship between the alterations of memory B cell subsets and the clinical features of SSc. Peripheral blood samples were obtained from 21 SSc patients, including 14 diffuse (dcSSc) and 7 limited (lcSSc) cutaneous SSc patients, with disease duration of 2.7 ( ± 1.6) years. After purification of CD19+ B cells, multiparametric flow cytometry was performed and the frequencies of CD19+IgD−CD27−CD38+ double negative (DN) 1, CD19+IgDloCD27+CD38+ unswitched, CD19+IgD−CD27+CD38+CD95− resting switched and CD19+IgD−CD27+CD38−CD95+ activated switched memory (ASM) B cells were determined, and correlated with clinical features of SSc. The dcSSc patients had a higher frequency of ASM B cells (p = 0.028) compared to lcSSc patients. The percentage of ASM B cells was elevated in anti-Scl-70 (anti-topoisomerase I) antibody positive patients compared to negative patients (p = 0.016). Additionally, the frequency of ASM B cells was also increased in patients with pulmonary fibrosis (p = 0.003) suggesting that patients with severe form of SSc have higher ASM B cell ratios. Furthermore, the ratio of DN1 B cells was decreased (p = 0.029), while the level of anti-citrate synthase IgG natural autoantibody was elevated (p = 0.028) in patients with active disease. Our observations on the increase of ASM B cells in dcSSc and in patients with pulmonary fibrosis may point to the association of this alteration with the severe form of the disease. Functionally the correlation of ASM B cells as effector memory-plasma cell precursors with anti-topoisomerase I antibody positivity could reflect their contribution to pathological autoantibody production, whereas the decrease of memory precursor DN B cells and the increase of anti-citrate synthase IgG autoantibody may have potential significance in the assessment of disease activity.

The dialogue between T and B cells can be regulated by different mechanisms, such as co-inhibitory receptors, which therefore play a crucial role in preventing autoimmune diseases such as systemic lupus erythematosus (SLE). B and T lymphocyte attenuator (BTLA) is a co-inhibitory receptor expressed on many myeloid and lymphoid cells. Although peripheral B cells express a very high amount of BTLA, previous works in the context of autoimmunity mainly focused on T cells, and whether BTLA expression on B cells plays a role in the lupus pathogenesis is still unclear. In the present study, we examine the expression of BTLA, as well as its ligand HVEM (Herpesvirus Entry Mediator), on various B cell subsets in lupus patients compared to healthy controls (HCs). We evidenced the existence of double-negative (DN; IgD−CD27−) memory B cells expressing very low levels of BTLA, which are enhanced in active lupus patients. An in-depth analysis revealed that these BTLAlow DN cells mainly correspond to the newly reported DN3 B cell subset, originally described in the context of SARS-CoV2 infection. These cells display an activated and antibody-secreting cell phenotype, and we propose that their low BTLA expression may favor their expansion and rapid differentiation into plasmablasts in lupus patients.

Recently, evidence has supported a significant role for immune and oxidative-mediated damage underlying the pathogenesis of different types of retinal diseases, including retinitis pigmentosa (RP). Our study aimed to evaluate the presence of immune cells and mediators in patients with RP using flow cytometric analysis of peripheral blood (PB) and aqueous humor (AH) samples. We recruited 12 patients with RP and nine controls undergoing cataract surgery. Flow cytometric analysis of PB and AH samples provided a membrane staining that targeted surface molecules (CD14, CD16, CD19, CD3, CD4, CD8, and CD161) identifying monocytes, natural killer (NK) cells, B cells, T cells, and T subpopulations, respectively. Moreover, lymphocytes were polyclonally stimulated to evaluate cytokine (CK) production at single-cell level. The circulating immune cell distribution was comparable between patients with RP and controls. Conversely, in the AH of controls we could detect no cells, while in the RP AH samples we found infiltrating leukocytes, consisting of T (CD3+), B (CD19+), NK (CD16+CD3-) cells, and monocytes (CD14+). In patients with RP, the frequency of most infiltrating immune cell populations was similar between the AH and PB. However, among T cell subpopulations, the frequency of CD3+CD4+ T cells was significantly lower in the RP AH compared to RP PB, whereas CD3+CD4-CD8- double-negative (DN) T cells were significantly higher in the RP AH compared to RP PB. Cytokine production analysis revealed a trend toward an increased frequency of CD3+CD8-CD161+IFN-ɣ-producing cells and a decreased frequency of CD3+CD8+IL-4-producing cells in the RP AH compared to RP PB. The detection of immune cells, particularly DN T cells, and a Th1-skewed phenotype in RP AH suggests immune-mediated and inflammatory mechanisms in the disease.

In thyroid cancer, the tumor immune microenvironment (TIME) plays a crucial role in cancer development, progression and response to treatment. Like many other cancers, thyroid cancer creates a complex network of interactions with immune cells directly (cell-to-cell) and via humoral mediators (i.e., cytokines). This dynamic microenvironment undergoes constant modification, which can lead to changes in the immunophenotype that might explain cancer progression, dedifferentiation and resistance to treatment. According to the cancer immunoediting hypothesis, cancerous tumors can shape their immune microenvironment to create an immunosuppressive milieu that allows them to evade classic immune surveillance. One mechanism by which this occurs is through the reprogramming of immune cells, often shifting their phenotypes from cytotoxic to regulatory. Recent research has shed light on cellular components and molecular interactions within the thyroid cancer TIME. Immune cells such as Tumor-Associated Lymphocytes (TALs), myeloid-derived suppressor cells (MDSCs), Tumor-Associated Macrophages (TAMs) and Double-Negative (DN) T cells seem to play key roles in shaping the immune response to thyroid cancer. Additionally, cytokines, chemokines and other signaling molecules contribute to the communication and regulation of immune cells within that microenvironment. By studying these interactions, researchers aim to uncover not just potential therapeutic targets but also biomarkers of thyroid cancer that could provide clues on severity and progression. Based on that knowledge, strategies such as the use of immune checkpoint inhibitors, antigen-specific targeted immunotherapies, and immunomodulatory agents are being explored to enhance the anti-tumor immune response and overcome cancer immunosuppressive mechanisms. In this review, we analyze the available literature and provide our own experience to unravel the complexity of the thyroid immune microenvironment. Continued research in this area holds promise for improving outcomes through the identification of immune markers of severity/progression of thyroid cancer and the development of innovative immunotherapeutic approaches.

Pediatric gout is a condition that differs from traditional adult gout and has attracted significant attention. This study aims to explore the molecular mechanisms underlying pediatric gout. We analyzed peripheral blood samples from pediatric gout patients and healthy controls using single-cell RNA sequencing (scRNA-seq). Statistical tests were employed to analyze the data and identify significant differences between the groups. Our findings revealed that CD14+ monocytes and DN T cells play crucial roles in pediatric gout. CD14+ monocytes are essential for recognizing and phagocytosing monosodium urate (MSU) crystals, triggering inflammation. DN T cells may be involved in the adaptive immune response within gouty joints. These cells not only contribute to the inflammatory response but also interact with other immune cells, amplifying the inflammatory cascade. Comparative analysis with adult gout studies highlighted both similarities and differences in cellular and molecular mechanisms between children and adults. The CD14+ monocytes may be interact with other immune cells through the TNF-α/NF-κB signaling pathway. Targeting this pathway may offer therapeutic potential for managing pediatric gout. The results provide a foundation for new diagnostic markers and therapeutic targets for pediatric gout. They also pave the way for future research and the development of targeted therapies that can effectively manage and potentially prevent the debilitating effects of gout in children. Understanding the unique molecular mechanisms in pediatric gout could influence treatment strategies and improve patient outcomes.

Double-negative T (DNT) cells are a rare and unconventional T-lymphocyte subpopulation lacking both CD4 and CD8 markers. Their immunopathological roles and clinical relevance have yet to be elucidated. Beyond autoimmune lymphoproliferative syndrome (ALPS), these cells may also play a role in rheumatic disorders, including systemic lupus erythematosus (SLE); indeed, these two diseases share several autoimmune manifestations (including nephritis). Moreover, one of the main experimental murine models used to investigate lupus, namely the MRL/lpr mouse, is characterized by an expansion of DNT cells, which can support the production of pathogenic autoantibodies and/or modulate the immune response in this context. However, lupus murine models are not completely consistent with their human SLE counterpart, of course. In this mini review, we summarize and analyze the most relevant clinical studies investigating the DNT cell population in SLE patients. Overall, based on the present literature review and analysis, DNT cell homeostasis seems to be altered in patients with SLE. Indeed, most of the available clinical studies (which include both adults and children) reported an increased DNT cell percentage in SLE patients, especially during the active phases, even though no clear correlation with disease activity and/or inflammatory parameters has been clearly established. Well-designed, standardized, and longitudinal clinical studies focused on DNT cell population are needed, in order to further elucidate the actual contribution of these cells in SLE pathogenesis and their interactions with other immune cells (also implicated and/or altered in SLE, such as basophils), and clarify whether their expansion and/or immunophenotypic aspects may have any immunopathological relevance (and, then, represent potential disease markers and, in perspective, even therapeutic targets) or are just an unspecific epiphenomenon of autoimmunity.