Allostimulation leads to emergence of a human B cell population with increased expression of HLA class I antigen presentation-associated molecules and the immunoglobulin receptor FcRL5.

In the extensive literature characterizing lymphocyte contributions to transplant-related pathologies including allograft rejection and graft-versus-host disease, T cell-focused investigation has outpaced investigation of B cells. Most B cell-related reports describe regulatory and antibody-producing functions, with less focus on the potential role of antigen-presenting capacity. Using in vitro human mixed lymphocyte reactions (MLRs) to model allostimulation, we analyzed responder B cells using transcriptional analysis, flow cytometry, and microscopy. We observed emergence of an activated responder B cell subpopulation phenotypically similar to that described in individuals with graft-versus-host disease or allograft rejection. This population had markedly increased expression of FcRL5 (Fc receptor like 5) and molecules associated with human leukocyte antigen class I antigen presentation. Consistent with this phenotype, these cells demonstrated increased internalization of irradiated cell debris and dextran macromolecules. The proportion of this subpopulation within MLR responders also correlated with emergence of activated, cytotoxic CD8+ T cells. B cells of similar profile were quite infrequent in unstimulated blood from healthy individuals but readily identifiable in disaggregated human splenocytes and increased in both cases upon allostimulation. Further characterization of the emergence and function of this subpopulation could potentially contribute to identification of novel biomarkers and targeted therapeutics relevant to curbing transplant-related pathology.

Regulation of B-cell function and expression of CD11c, T-bet, and FcRL5 in response to different activation signals.

CD11c, FcRL5, or T-bet are commonly expressed by B cells expanding during inflammation, where they can make up >30% of mature B cells. However, the association between the proteins and differentiation and function in the host response remains largely unclear. We have assessed the co-expression of CD11c, T-bet, and FcRL5 in an in vitro B-cell culture system to determine how stimulation via the BCR, toll-like receptor 9 (TLR9), and different cytokines influence CD11c, T-bet, and FcRL5 expression. We observed different expression dynamics for all markers, but a largely overlapping regulation of CD11c and FcRL5 in response to BCR and TLR9 activation, while T-bet was strongly dependent on IFN-γ signaling. Investigating plasma cell differentiation and APC functions, there was no association between marker expression and antibody secretion or T-cell help. Rather the functions were associated with TLR9-signalling and B-cell-derived IL-6 production, respectively. These results suggest that the expression of CD11c, FcRL5, and T-bet and plasma cell differentiation and improved APC functions occur in parallel and are regulated by similar activation signals, but they are not interdependent.

Upregulated Fcrl5 disrupts B cell anergy and causes autoimmune disease.

B cell anergy plays a critical role in maintaining self-tolerance by inhibiting autoreactive B cell activation to prevent autoimmune diseases. Here, we demonstrated that Fc receptor-like 5 (Fcrl5) upregulation contributes to autoimmune disease pathogenesis by disrupting B cell anergy. Fcrl5-a gene whose homologs are associated with human autoimmune diseases-is highly expressed in age/autoimmunity-associated B cells (ABCs), an autoreactive B cell subset. By generating B cell-specific Fcrl5 transgenic mice, we demonstrated that Fcrl5 overexpression in B cells caused systemic autoimmunity with age. Additionally, Fcrl5 upregulation in B cells exacerbated the systemic lupus erythematosus-like disease model. Furthermore, an increase in Fcrl5 expression broke B cell anergy and facilitated toll-like receptor signaling. Thus, Fcrl5 is a potential regulator of B cell-mediated autoimmunity by regulating B cell anergy. This study provides important insights into the role of Fcrl5 in breaking B cell anergy and its effect on the pathogenesis of autoimmune diseases.

Mass cytometry identifies characteristic immune cell subsets in bronchoalveolar lavage fluid from interstitial lung diseases.

Immune cells have been implicated in interstitial lung diseases (ILDs), although their phenotypes and effector mechanisms remain poorly understood. To better understand these cells, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF), connective-tissue disease (CTD)-related ILD, and sarcoidosis, using two panels including 64 markers. Among myeloid cells, we observed the expansion of CD14+ CD36hi CD84hiCCR2- monocyte populations in IPF. These CD14+ CD36hi CD84hi CCR2- subsets were also increased in ILDs with a progressive phenotype, particularly in a case of acute exacerbation (AEx) of IPF. Analysis of B cells revealed the presence of cells at various stages of differentiation in BALF, with a higher percentage of IgG memory B cells in CTD-ILDs and a trend toward more FCRL5+ B cells. These FCRL5+ B cells were also present in the patient with AEx-IPF and sarcoidosis with advanced lung lesions. Among T cells, we found increased levels of IL-2R+ TIGIT+ LAG3+ CD4+ T cells in IPF, increased levels of CXCR3+ CD226+ CD4+ T cells in sarcoidosis, and increased levels of PD1+ TIGIT+ CD57+ CD8+ T cells in CTD-ILDs. Together, these findings underscore the diverse immunopathogenesis of ILDs.

Erratum: Upregulated Fcrl5 disrupts B cell anergy and causes autoimmune disease.

[This corrects the article DOI: 10.3389/fimmu.2023.1276014.].

Alternative B Cell Differentiation During Infection and Inflammation.

Long-term protective immunity to infectious disease depends on cell-mediated and humoral immune responses. Induction of a strong humoral response relies on efficient B cell activation and differentiation to long-lived plasma cells and memory B cells. For many viral or bacterial infections, a single encounter is sufficient to induce such responses. In malaria, the induction of long-term immunity can take years of pathogen exposure to develop, if it occurs at all. This repeated pathogen exposure and suboptimal immune response coincide with the expansion of a subset of B cells, often termed atypical memory B cells. This subset is present at low levels in healthy individuals as well but it is observed to expand in an inflammatory context during acute and chronic infection, autoimmune diseases or certain immunodeficiencies. Therefore, it has been proposed that this subset is exhausted, dysfunctional, or potentially autoreactive, but its actual role has remained elusive. Recent reports have provided new information regarding both heterogeneity and expansion of these cells, in addition to indications on their potential role during normal immune responses to infection or vaccination. These new insights encourage us to rethink how and why they are generated and better understand their role in our complex immune system. In this review, we will focus on recent advances in our understanding of these enigmatic cells and highlight the remaining gaps that need to be filled.

Humoral immunity in hepatitis B virus infection: Rehabilitating the B in HBV.

Insights into the immunopathogenesis of chronic HBV infections are fundamental in the quest for novel treatment approaches aimed at a functional cure. While much is known about the ineffective HBV-specific T-cell responses that characterise persistent HBV replication, B cells have been left largely understudied. However, an important role for humoral immunity during the natural history of HBV infections, as well as after functional cure, has been inadvertently revealed by the occurrence of HBV flares following B cell-depleting treatments. Herein, we review our current understanding of the role of the humoral immune response in chronic HBV, both at the level of HBV-specific antibody production and at the phenotypic and broader functional level of B cells. The recent development of fluorescently labelled HBV proteins has given us unprecedented insights into the phenotype and function of HBsAg- and HBcAg-specific B cells. This should fuel novel research into the mechanisms behind dysfunctional HBsAg-specific and fluctuating, possibly pathogenic, HBcAg-specific B-cell responses in chronic HBV. Finally, novel immunomodulatory treatments that partly target B cells are currently in clinical development, but a detailed assessment of their impact on HBV-specific B-cell responses is lacking. We plead for a rehabilitation of B-cell studies related to both the natural history of HBV and treatment development programmes.

A broad and systematic approach to identify B cell malignancy-targeting TCRs for multi-antigen-based T cell therapy.

CAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of multi-antigen-targeting approaches. We propose that a T cell receptor (TCR)-based strategy would increase the number of potential antigenic targets, as peptides from both intracellular and extracellular proteins can be recognized. Here, we aimed to isolate a broad range of promising TCRs targeting multiple antigens for treatment of B cell malignancies. As a first step, 28 target genes for B cell malignancies were selected based on gene expression profiles. Twenty target peptides presented in human leukocyte antigen (HLA)-A∗01:01, -A∗24:02, -B∗08:01, or -B∗35:01 were identified from the immunopeptidome of B cell malignancies and used to form peptide-HLA (pHLA)-tetramers for T cell isolation. Target-peptide-specific CD8 T cells were isolated from HLA-mismatched healthy donors and subjected to a stringent stepwise selection procedure to ensure potency and eliminate cross-reactivity. In total, five T cell clones specific for FCRL5 in HLA-A∗01:01, VPREB3 in HLA-A∗24:02, and BOB1 in HLA-B∗35:01 recognized B cell malignancies. For all three specificities, TCR gene transfer into CD8 T cells resulted in cytokine production and efficient killing of multiple B cell malignancies. In conclusion, using this systematic approach we successfully identified three promising TCRs for T cell therapy against B cell malignancies.

Variants of Novel Immunomodulatory Fc Receptor Like 5 Gene Are Associated With Multiple Sclerosis Susceptibility in the Polish Population.

Fc receptors have been shown to play a role in several autoimmune diseases. We aimed to test, for the first time, whether some of the single nucleotide variants in the FCRL5 gene were associated with multiple sclerosis (MS) susceptibility and clinical manifestations in the Polish population. The case-control study included 94 individuals with MS and 160 healthy subjects. We genotyped two single nucleotide variants of the FCRL5 gene: rs2012199 and rs6679793. The age of onset, disease duration, and clinical condition of the MS subjects were analyzed. For statistical analysis, we used the chi-squared test confirmed with Fisher's exact test. We observed the significant differences in the distribution of investigated FCRL5 genotypes between MS subjects and healthy controls. The CC and CT genotypes, as well as the C allele of rs2012199, were significantly more common in the MS subjects, as were genotypes AA and AG, and allele A of rs6679793. We noted that decreased MS susceptibility was associated with the T allele rs2012199 (OR = 0.37, p = 0.0002) and G allele rs6679793 (OR = 0.6, p = 0.02). Our results support the role of the FCRL5 locus in MS predisposition and extend the evidence of its influence on autoimmunity.

Potential functions of atypical memory B cells in Plasmodium-exposed individuals.

Naturally acquired iummunity against clinical malaria is slow to develop, taking years of repeated exposure to parasites to acquire sufficiently broad and potent antibody responses. Increasing evidence suggests that Plasmodium infection and the resulting immune stimulation contribute to changes in the B cell compartment. In particular, accumulation of atypical memory B cells (atMBCs) is common in Plasmodium-exposed individuals. Similarities to B cell subsets present in other acute and chronic disease settings have provided insight into the development and potential function of these cells; however, their contribution to protection against malaria is still poorly understood. Here, we discuss recent findings that have increased our understanding of atMBCs and outline outstanding questions related to their function and development in the protective immune response to malaria.

FCRL5+ Memory B Cells Exhibit Robust Recall Responses.

FCRL5+ atypical memory B cells (atMBCs) expand in many chronic human infections, including recurrent malaria, but studies have drawn opposing conclusions about their function. Here, in mice infected with Plasmodium chabaudi, we demonstrate expansion of an antigen-specific FCRL5+ population that is distinct from previously described FCRL5+ innate-like murine subsets. Comparative analyses reveal overlapping phenotypic and transcriptomic signatures between FCRL5+ B cells from Plasmodium-infected mice and atMBCs from Plasmodium-exposed humans. In infected mice, FCRL5 is expressed on the majority of antigen-specific germinal-center-derived memory B cells (MBCs). Upon challenge, FCRL5+ MBCs rapidly give rise to antibody-producing cells expressing additional atypical markers, demonstrating functionality in vivo. Moreover, atypical markers are expressed on antigen-specific MBCs generated by immunization in both mice and humans, indicating that the atypical phenotype is not restricted to chronic settings. This study resolves conflicting interpretations of atMBC function and suggests FCRL5+ B cells as an attractive target for vaccine strategies.

Genetic risk of FCRL3 and FCRL5 polymorphisms in children with asthma and allergic rhinitis in a Chinese Han population.

OBJECTIVES: Asthma and allergic rhinitis (AR) frequently occur as comorbid diseases of the upper airways. Single-nucleotide polymorphisms (SNPs) in the FCRL3 and FCRL5 genes have recently been shown to be associated with various immune-related disorders. This study evaluated the association of FCRL3 and FCRL5 polymorphisms with asthma and allergic rhinitis (AR) in a Han Chinese population. METHODS: Seven single nucleotide polymorphisms (SNPs) of the FCRL3 and FCRL5 were genotyped in 300 asthmatic children, and 206 healthy unrelated individuals using PCR-restriction fragment length polymorphism (PCR-RFLP) assay. Genotyping was validated by direct sequencing. RESULTS: Our results showed that the frequencies of the rs6692977 CT genotype and T allele within FCRL5 were significantly higher in asthma with comorbid AR compared to healthy controls (Bonferroni-corrected p (Pc) = 3.75 × 10-6; Pc = 0.006, respectively), whereas these of the CC genotype and C allele were significantly lower (Pc = 4.15 × 10-5; Pc = 0.006, respectively). The frequencies of the rs7528684 A allele (Pc = 1.80 × 10-3) and the rs10489678 G allele (Pc = 0.04) within FCRL3 were higher in asthma with comorbid AR than in controls. However, no differences in the tested genetic polymorphisms were detected between asthma and healthy individuals. CONCLUSION: This study identified novel SNPs in FCRL3 and FCRL5 significantly associated with the risk for asthma with comorbid AR in the Chinese population. The genetic variants may play role in the development of the asthma phenotype in children with asthma.

Plasmodium-specific atypical memory B cells are short-lived activated B cells.

A subset of atypical memory B cells accumulates in malaria and several infections, autoimmune disorders and aging in both humans and mice. It has been suggested these cells are exhausted long-lived memory B cells, and their accumulation may contribute to poor acquisition of long-lasting immunity to certain chronic infections, such as malaria and HIV. Here, we generated an immunoglobulin heavy chain knock-in mouse with a BCR that recognizes MSP1 of the rodent malaria parasite, Plasmodium chabaudi. In combination with a mosquito-initiated P. chabaudi infection, we show that Plasmodium-specific atypical memory B cells are short-lived and disappear upon natural resolution of chronic infection. These cells show features of activation, proliferation, DNA replication, and plasmablasts. Our data demonstrate that Plasmodium-specific atypical memory B cells are not a subset of long-lived memory B cells, but rather short-lived activated cells, and part of a physiologic ongoing B-cell response.

High-throughput characterization of the functional impact of IgG Fc glycan aberrancy in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) encompasses all forms of chronic idiopathic arthritis that arise before age 16. Previous studies have found JIA to be associated with lower Fc galactosylation of circulating IgG, but the overall spectrum of glycan changes and the net impact on IgG function are unknown. Using ultra performance liquid chromatography (UPLC), we compared IgG glycosylation in 54 subjects with recent-onset untreated JIA with 98 healthy pediatric controls, paired to biophysical profiling of affinity for 20 IgG receptors using a high-throughput multiplexed microsphere assay. Patients with JIA exhibited an increase in hypogalactosylated and hyposialylated IgG glycans, but no change in fucosylation or bisection, together with alteration in the spectrum of IgG ligand binding. Supervised machine learning demonstrated a robust capacity to discriminate JIA subjects from controls using either glycosylation or binding data. The binding signature was driven predominantly by enhanced affinity for Fc receptor like protein 5 (FcRL5), a noncanonical Fc receptor expressed on B cells. Affinity for FcRL5 correlated inversely with galactosylation and sialylation, a relationship confirmed through enzymatic manipulation. These results demonstrate the capacity of combined structural and biophysical IgG phenotyping to define the overall functional impact of IgG glycan changes and implicate FcRL5 as a potential cellular sensor of IgG glycosylation.

Emerging antibody-drug conjugates for treating lymphoid malignancies.

INTRODUCTION: Antibody-drug conjugates (ADC) are monoclonal antibodies (Mabs) attached to biologically active drugs through specialized chemical linkers. They deliver and release cytotoxic agents at the tumor site, reducing the likelihood of systemic exposure and therefore toxicity. These agents should improve the potency of chemotherapy by increasing the accumulation of cytotoxic the drug within or near the neoplastic cells with reduced systemic effects. Areas covered: A literature review was conducted of the MEDLINE database PubMed for articles in English examining Mabs, B-cell receptor pathway inhibitors and immunomodulating drugs. Publications from 2000 through April 2017 were scrutinized. Conference proceedings from the previous five years of the American Society of Hematology, European Hematology Association, American Society of Clinical Oncology, and ACR/ARHP Annual Scientific Meetings were searched manually. Additional relevant publications were obtained by reviewing the references from the chosen articles. Expert opinion: Newer ADCs show promise as treatment for several hematologic malignancies, especially lymphoma, multiple myeloma, and leukemia. However, definitive data from ongoing and future clinical trials will aid in better defining the status of these agents in the treatment of these diseases.

Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5/CD3 and Is a Requirement for Myeloma Cell Killing.

The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

Hepatitis C viraemia reversibly maintains subset of antigen-specific T-bet+ tissue-like memory B cells.

BACKGROUND: Chronic antigen exposure and/or ageing increases the frequency of T-box expressed in T cells (T-bet)-expressing B-lymphocytes in mice. The frequency and significance of B-cell T-bet expression during chronic hepatitis C (HCV) infection in human subjects has never been described. METHODS: Healthy controls, cirrhotic and noncirrhotic HCV-infected patients, and non-HCV patients with cirrhosis were recruited. Peripheral blood mononuclear cells were phenotyped for expression of T-bet and related markers by flow cytometry. In a subset of patients who underwent antiviral therapy and were cured of HCV infection (sustained virological response), the dynamics of T-bet expression in B cells was monitored. After cure, convalescent B cells were tested for T-bet expression after re-exposure to infected plasma or recombinant HCV proteins. RESULTS: Forty-nine patients including 11 healthy donors, 30 hepatitis C-infected individuals (nine with liver cancer, 13 with cirrhosis, eight without cirrhosis) and eight patients with cirrhosis due to non-HCV-related cause were recruited. We found that B cells in patients with chronic HCV exhibited increased frequency of T-bet+ B cells relative to noninfected individuals (median 11.5% v. 2.2%, P<.0001) but that there were no significant differences between noncirrhotic, cirrhotic and cancer-bearing infected individuals. T-Bet+ B cells expressed higher levels of CD95, CXCR3, CD11c, CD267 and FcRL5 compared to T-bet- B cells and predominantly exhibit a tissue-like memory CD27- CD21- phenotype independent of HCV infection. T-bet+ B cells in HCV-infected patients were more frequently class-switched IgD- IgG+ (40.4% vs. 26.4%, P=.012). Resolution of HCV infection with direct-acting antiviral (DAA) therapy leads to a marked reduction in the frequency of T-bet+ B cells (median 14.1% pretreatment v. 6.7% end of treatment v. 6.1% SVR12, P≤.01). Re-exposure of convalescent (cured) B cells to viremic plasma and recombinant HCV E2 protein led to re-expression of T-bet. CONCLUSION: Chronic antigenemia in chronic HCV infection induces and maintains an antigen-specific T-bet+ B cell. These B cells share markers with tissue-like memory B cells. Antigen-driven T-bet expression may be a critical suppressor of B-cell activation in chronic HCV infection.

Study of the FCRL5 gene single nucleotide p olymorphism in patients with ankylosing spondylitis / 中华风湿病学杂志

ObjectiveTo investigate the association between the FCRL5 gene (rs6427384 and rs12036228) single nucleotide polymorphism(SNP) and patients with ankylosing spondylitis (AS). Methods SNP of FCRL5 gene (rs6427384 and rs12036228) was analyzed in 169 patients with AS and 184 healthy controls by ligase detection reaction based on high temperature(LDR)-PCR. The distribution of FCRL5 genotypes and allele frequencies were detected between the two groups. Chi-square test, one-way ANOVA were used for statistical analysis. ResultsSignificant differences were found in the distribution of allele frequencies of FCRL5 gene between AS patients and health controls(P＜0.05). The C allele frequencies of FCRL5 gene at positions of rs6427384 and rs12036228 were 17.3％, 92.3％ and 25.0％, 87.2％ respectively in the AS group and the control group. And the T allele frequencies of rs6427384 and rs12036228 were 82.7％, 7.7％ and 75.0％, 12.8％ in the AS group and the control group. The percentages of CC, CT and TT genotype(rs6427384) were 3.7％, 27.2％, and 69.1％ in the AS group, which were significantly different from those of the control group(3.9％, 42.2％ and 53.9％ ) (x2=8.7637, P=0.0125 ). Staging of sacroiliitis in X ray were significantly different during AS patients whose genotype represented as CC, CT and TT (rs6427384)(x2=34.159, P=0.0001 ). Incidences of the initial symptoms (low back pain or inflammation of periphery joint)in the AS group were obviously differed among patients with different genotypes (rs6427384) (x2=7.254, P=0.027), so did the mean duration of morning stiffness (F=4.159, P=0.018) and the average scores of BASDAI (F=4.461, P=0.014). Incidences of the initial symptoms in the AS group were also conspicuously different between the AS patients with different genotypes (rs 12036228 ) (x2=6.640, P=0.036 ). ConclusionOur study suggests that the SNP(rs6427384 and rs12036228) of FCRL5 may be a susceptibility factor for AS in Anhui Han population and the genotype may influence the clinical phenotype of AS.