B-cell Receptor Research Articles

Design, Synthesis, and Biological Evaluation of Thieno[3,2-d]pyrimidine Derivatives as the First Bifunctional PI3Kδ Isoform Selective/Bromodomain and Extra-Terminal Inhibitors.

The concomitant inhibition of PI3Kδ and bromodomain and extra-terminal (BET) that exerts a synergistic effect on the B-cell receptor signaling pathway provides a new strategy for the treatment of aggressive diffuse large B-cell lymphoma (DLBCL). Herein, a merged pharmacophore strategy was utilized to discover a series of thieno[3,2-d]pyrimidine derivatives as the first-in-class bifunctional PI3Kδ-BET inhibitors. Through optimization, a highly potent compound (10b) was identified to possess excellent and balanced activities against PI3Kδ [inhibitory concentration (IC50) = 112 ± 8 nM] and BRD4-BD1 (IC50 = 19 ± 1 nM) and exhibited strong antiproliferative activities in DLBCL cells. Notably, this compound demonstrated good PI3Kδ selectivity over other kinases with minimal cytotoxicity in normal cells. Moreover, 10b has a good oral pharmacokinetic profile in mice and achieves outstanding antitumor activity in the SU-DHL-6 xenograft model. Taken together, these results indicate that targeting PI3Kδ and BET with a bifunctional inhibitor is a promising strategy to treat DLBCL.

Epstein–Barr virus BALF0/1 subverts the Caveolin and ERAD pathways to target B cell receptor complexes for degradation

Epstein-Barr virus (EBV) establishes persistent infection, causes infectious mononucleosis, is a major trigger for multiple sclerosis and contributes to multiple cancers. Yet, knowledge remains incomplete about how the virus remodels host B cells to support lytic replication. We previously identified that EBV lytic replication results in selective depletion of plasma membrane (PM) B cell receptor (BCR) complexes, composed of immunoglobulin and the CD79A and CD79B signaling chains. Here, we used proteomic and biochemical approaches to identify that the EBV early lytic protein BALF0/1 is responsible for EBV lytic cycle BCR degradation. Mechanistically, an immunoglobulin heavy chain (HC) cytoplasmic tail KVK motif was required for ubiquitin-mediated BCR degradation, while CD79A and CD79B were dispensable. BALF0/1 subverted caveolin-mediated endocytosis to internalize PM BCR complexes and to deliver them to the endoplasmic reticulum. BALF0/1 stimulated immunoglobulin HC cytoplasmic tail ubiquitination, which together with the ATPase valosin-containing protein/p97 drove ER-associated degradation of BCR complexes by cytoplasmic proteasomes. BALF0/1 knockout reduced the viral load of secreted EBV particles from B cells that expressed a monoclonal antibody against EBV glycoprotein 350 but not a control anti-influenza hemagglutinin antibody and increased viral particle immunoglobulin incorporation. Consistent with downmodulation of PM BCR, BALF0/1 overexpression reduced viability of a diffuse large B cell lymphoma cell line whose survival is dependent upon BCR signaling. Collectively, our results suggest that EBV BALF0/1 downmodulates immunoglobulin upon lytic reactivation to block BCR signaling and support virion release, but await the development of suitable models to test its roles in EBV reactivation in vivo.

Implications of Raftlin in different diseases: from molecular biology to diagnostic value.

Raftlin (raft-linking) protein is an essential component of the lipid raft structure and plays a crucial role in B and T cell signaling pathways. It facilitates B cell receptor (BCR) signaling by promoting calcium mobilization and tyrosine phosphorylation in the cells while colocalizing with BCR on the cell membrane. Interestingly, Raftlin is internalized in lipopolysaccharide-stimulated T cells by colocalization with Toll-like receptor 4 (TLR4), wherein it exerts a similar role as in B cells. The protein also effectuates poly(I:C) internalization into TLR3-positive endosomes in dendritic and epithelial cells through clathrin binding, thereby affecting interferon-β production. In addition, Raftlin controls the vascular endothelial cells and participates in cell growth and proliferation. Recent studies have indicated Raftlin to be a novel biomarker for the diagnosis due to its upregulated expression in malignant diseases. In this integrated study, we present the biological functions of Raftlin and its expression to provide a theoretical basis for the prevention, diagnosis, and treatment of various diseases.

Systematic evaluation of intratumoral and peripheral BCR repertoires in three cancers.

The current understanding of humoral immune response in cancer patients suggests that tumors may be infiltrated with diffuse B cells of extra-tumoral origin or may develop organized lymphoid structures, where somatic hypermutation and antigen-driven selection occur locally. These processes are believed to be significantly influenced by the tumor microenvironment through secretory factors and biased cell-cell interactions. To explore the manifestation of this influence, we used deep unbiased immunoglobulin profiling and systematically characterized the relationships between B cells in circulation, draining lymph nodes (draining LNs), and tumors in 14 patients with three human cancers. We demonstrated that draining LNs are differentially involved in the interaction with the tumor site, and that significant heterogeneity exists even between different parts of a single lymph node (LN). Next, we confirmed and elaborated upon previous observations regarding intratumoral immunoglobulin heterogeneity. We identified B cell receptor (BCR) clonotypes that were expanded in tumors relative to draining LNs and blood and observed that these tumor-expanded clonotypes were less hypermutated than non-expanded (ubiquitous) clonotypes. Furthermore, we observed a shift in the properties of complementarity-determining region 3 of the BCR heavy chain (CDR-H3) towards less mature and less specific BCR repertoire in tumor-infiltrating B-cells compared to circulating B-cells, which may indicate less stringent control for antibody-producing B cell development in tumor microenvironment (TME). In addition, we found repertoire-level evidence that B-cells may be selected according to their CDR-H3 physicochemical properties before they activate somatic hypermutation (SHM). Altogether, our work outlines a broad picture of the differences in the tumor BCR repertoire relative to non-tumor tissues and points to the unexpected features of the SHM process.

Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection.

Antibody-mediated rejection (AMR) remains a major complication after solid organ transplantation (SOT). Current treatment options are inefficient and result in drastic impairment of the general immunity. To selectively eliminate responsible alloreactive B cells characterized by anti-donor-HLA B cell receptors (BCRs), we generated Tcells overcoming rejection by antibodies (CORA-Ts) engineered with a novel chimeric receptor comprising a truncated donor-HLA molecule as antigen recognition domain. As proof-of-concept, CORA receptors based on HLA-A∗02 were developed. In co-cultures with anti-HLA-A∗02 B cell lines, CORA-Ts were specifically activated, released pro-inflammatory mediators, and exhibited strong cytotoxicity resulting in an effective reduction of anti-HLA-A∗02 antibody release. Significant reduction of growth of an anti-HLA-A∗02 B cell line could be confirmed using an invivo mouse model. Modification of the CORA receptor effectively abrogated Tcell binding, thereby avoiding Tcell sensitization. Additionally, using CRISPR-Cas9-mediated knockout of the FKBP12 gene, CORA-Ts were able to resist immunosuppressive treatment with tacrolimus, thereby allowing high efficiency in transplant patients. Our results demonstrate that CORA-Ts are able to specifically eliminate alloreactive, anti-HLA B cells, thus selectively preventing anti-HLA antibody release even under immunosuppressive conditions. This suggests CORA-Ts as potent approach to combat AMR and improve long-term graft survival in SOT patients while preserving their overall B cell immunity.

Metabolomic and transcriptomic remodeling of bone marrow myeloid cells in response to maternal obesity.

Maternal obesity puts the offspring at high risk of developing obesity and cardio-metabolic diseases in adulthood. Here, we utilized a mouse model of maternal high-fat diet (HFD)-induced obesity that recapitulates metabolic perturbations seen in humans. We show increased adiposity in the offspring of HFD-fed mothers (Off-HFD) when compared to the offspring regular diet-fed mothers (Off-RD). We have previously reported significant immune perturbations in the bone marrow of newly-weaned Off-HFD. Here, we hypothesized that lipid metabolism is altered in the bone marrow of Off-HFD vs. Off-RD. To test this hypothesis, we investigated the lipidomic profile of bone marrow cells collected from three-week-old Off-RD and Off-HFD. Diacylgycerols (DAGs), triacylglycerols (TAGs), sphingolipids and phospholipids were remarkably different between the groups, independent of fetal sex. Levels of cholesteryl esters were significantly decreased in Off-HFD, suggesting reduced delivery of cholesterol. These were accompanied by age-dependent progression of mitochondrial dysfunction in bone marrow cells. We subsequently isolated CD11b+ myeloid cells from three-week-old mice and conducted metabolomics, lipidomics, and transcriptomics analyses. The lipidomic profiles of myeloid cells were similar to bone marrow cells and included increases in DAGs and decreased TAGs. Transcriptomics revealed altered expression of genes related to immune pathways including macrophage alternative activation, B-cell receptors, and TGFβ signaling. All told, this study revealed lipidomic, metabolomic, and gene expression abnormalities in bone marrow cells broadly, and in bone marrow myeloid cells particularly, in the newly-weaned offspring of mothers with obesity, which might at least partially explain the progression of metabolic and cardiovascular diseases in their adulthood.

An engineered immunogen activates diverse HIV broadly neutralizing antibody precursors and promotes acquisition of improbable mutations.

Elicitation of HIV broadly neutralizing antibodies (bnAbs) by vaccination first requires the activation of diverse precursors, followed by successive boosts that guide these responses to enhanced breadth through the acquisition of somatic mutations. Because HIV bnAbs contain mutations in their B cell receptors (BCRs) that are rarely generated during conventional B cell maturation, HIV vaccine immunogens must robustly engage and expand B cells with BCRs that contain these improbable mutations. Here, we engineered an immunogen that activates diverse precursors of an HIV V3-glycan bnAb and promotes their acquisition of a functionally critical improbable mutation. This immunogen was validated biochemically, structurally, and in three different humanized immunoglobulin mouse models that were designed to test HIV immunogens. These results provide a blueprint for rationally designing priming immunogens that explicitly target the elicitation of antibodies with functional yet improbable mutations.

JNK1 inhibitors target distal B cell receptor signaling and overcome BTK-inhibitor resistance in CLL.

Inhibition of the proximal B cell receptor (BCR) signaling pathway by BTK inhibitors is highly effective in the treatment of CLL, but drug resistance or intolerance occurs. Here, we investigated c-Jun N-terminal protein kinase 1 (JNK1) as an alternative drug target in the distal BCR pathway. JNK1 was preferentially overexpressed and activated in poor prognostic CLL with unmutated IGHV. Proximal BCR inhibition (BTK, PI3K, or SYK inhibitors) or SYK knockdown efficiently dephosphorylated JNK1, identifying JNK1 as a critical BCR downstream kinase in CLL. JNK1 inhibition induced apoptosis in primary CLL cells, resulting in the downregulation of BCL2, MCL1, and c-JUN. JNK1 inhibition in patient-derived CLL xenografted mice and Eµ-TCL1-tg mice prevented CLL progression, reduced splenic infiltration, and restored T cell function and normal hematopoiesis. JNK1 inhibitors even remained effective in ibrutinib refractory CLL. In conclusion, our study revealed JNK1 as a promising drug target in CLL downstream of the BCR, overcoming ibrutinib resistance, blocking the protective microenvironment, and improving CLL-specific immunosuppressive mechanisms.

Extracellular vesicles from chronic lymphocytic leukemia cells promote leukemia aggressiveness by inducing the differentiation of monocytes into nurse-like cells via an RNA-dependent mechanism.

Chronic lymphocytic leukemia (CLL) cells receive several stimuli from surrounding cells, such as B-cell receptor (BCR) stimulation, and can manipulate their microenvironment via extracellular vesicle (EV) release. Here, we investigated the small RNA content (microRNA and YRNA) of CLL-EVs from leukemic cells cultured with/without BCR stimulation. We highlight an increase of miR-155-5p, miR-146a-5p, and miR-132-3p in EVs and in cells after BCR stimulation (p < 0.05, n = 25). CLL-EVs were preferentially internalized by monocytes (p = 0.0019, n = 6) and able to deliver microRNAs and the hY4 RNA. Furthermore, BCR CLL-EV induced modifications in monocytes (shape change, microRNA and gene expression, secretome) suggesting nurse-like cell (NLC) differentiation, the tumor-associated macrophages of CLL. Functionally, monocytes treated with BCR CLL-EVs protect CLL cells from spontaneous apoptosis by pro-survival cytokine production and induce their migration as well as the migration of other immune cells. We finally reported by transfection experiments that hY4 is able to induce the expression of CCL24, a key gene in M2 macrophage differentiation. In conclusion, we showed that BCR stimulation modifies the small RNA content of CLL-EVs and that the addition of leukemic EVs to monocytes leads to monocyte differentiation into NLCs establishing a protective microenvironment that supports leukemic cell survival.

Linking tumor immune infiltration to enhanced longevity in recurrence-free breast cancer.

The infiltration of tumor-infiltrating B cells and plasma cells in early-stage breast cancer has been associated with a reduced risk of distant metastasis. However, the influence of B-cell tumor infiltration on overall patient survival remains unclear. This study explored the relationship between an antitumor immune response, measured by a 14-gene B-cell/immunoglobulin (IGG) signature, and mortality risk in 9638 breast cancer patients across three datasets. Associations with tumor subtype, stage, and age were examined. IGG was characterized using spatial GeoMx profiling and single-cell RNA sequencing, and its relationship with tertiary lymphoid structures (TLSs) was evaluated. The predictive value of each of the 14 IGG genes for B-cell receptor (BCR) and T-cell receptor (TCR) clonality and longevity was also assessed, along with its association with longevity in other cancer types. High IGG signature expression was significantly associated with a 41%-47% reduction in death risk in breast cancer survivors (P < 0.001), regardless of age, tumor stage, or subtype. Similar associations were observed in other cancers, including melanoma. In breast cancer, the IGG signature was significantly linked to overall survival without relapse in patients aged 41-70 years at diagnosis. Additionally, IGG expression correlated with the presence of TLSs and higher B- and T-cell polyclonality. A specific subset of seven IGG genes strongly correlated with BCR and TCR clonality, with predictive power for identifying clonality and improved longevity, especially when combining two of these genes. This study uncovers a significant link between immune gene expression in tumors and extended longevity in breast cancer survivors, even in the absence of recurrence. The IGG signature, particularly its key gene subset, emerges as a powerful marker of sustained antitumor immunity and overall patient fitness. These findings pave the way for personalized treatment strategies that enhance both survival and long-term health outcomes.

Atypical memory B cells from natural malaria infection produced broadly neutralizing antibodies against Plasmodium vivax variants.

Expansion of atypical memory B cells (aMBCs) was demonstrated in malaria-exposed individuals. To date, the generation of P. vivax-specific aMBCs and their function in protective humoral immune responses is unknown. Here, P. vivax Duffy Binding Protein II (PvDBPII) probes were generated to detect the development and durability of specific aMBCs, and to demonstrate the capacity of these cells to produce neutralizing antibodies following natural infections. PvDBPII-specific aMBCs were elicited during malaria illness, and they persisted through the recovery phase of infections. To address biology and function of P. vivax-specific aMBCs in producing protective antibodies, a single MBC was cultured, and the secreted IgG was tested for binding and inhibition activity. The aMBC-derived clones produced antibodies with variable levels of anti-PvDBPII IgG in cultures, and some produced high antibody levels comparable to classical MBC clones. Thus, we focused our attention on the function of aMBCs in producing neutralizing antibodies. Among the aMBC clones, A1F12 and B4E11 produced broadly neutralizing antibodies against a panel of PvDBPII variants. Notably, B cell receptors (BCRs) of PvDBPII-specific aMBCs expressed unique IGHV genes, with similar usage of IGHV1-3, comparable to classical MBCs. The somatic hypermutation (SHM) rate and CDR3 length of VH and Vκ in these two MBC subsets were not significantly different. Together, our findings revealed that P. vivax infections elicited the development and persistence of P. vivax-specific aMBCs. The accumulation of aMBCs during and following infections might play an important role in producing protective antibodies against malaria.

SNX5 promotes antigen presentation in B cells by dual regulation of actin and lysosomal dynamics.

B cells rapidly adapt their endocytic pathway to promote the uptake and processing of extracellular antigens recognized through the B-cell receptor (BCR). The mechanisms coupling changes in endomembrane trafficking to the capacity of B cells to screen for antigens within lymphoid tissues remain unaddressed. We investigated the role of SNX5, a member of the sorting nexin family, which interacts with endocytic membranes to regulate vesicular trafficking and macropinocytosis. Our results show that in steady state, B cells form SNX5-rich protrusions at the plasma membrane, which dissipate upon interaction with soluble antigens, whereas B cells activated with immobilized antigens accumulate SNX5 at the immune synapse where it regulates actin-dependent spreading responses. B cells silenced for SNX5 exhibit enlarged lysosomes, which are not recruited to the synaptic membrane, decreasing their capacity to extract immobilized antigens. Overall, our findings reveal that SNX5 is critical for actin-dependent plasma membrane remodeling in B cells involved in antigen screening and immune synapse formation, as well as endolysosomal trafficking required to promote antigen extraction and presentation.

Expression Cloning of Antibodies from Single Human B Cells.

The majority of lymphomas originate from B cells at the germinal center stage. Preferential selection of B-cell clones by a limited set of antigens has been suggested to drive lymphoma development. While recent studies in B-cell chronic lymphocytic leukemia (CLL) have shown that self-reactive B-cell receptors (BCR) can generate cell-autonomous signaling and proliferation, our knowledge about the role of BCRs for the development or survival of other lymphomas remains limited. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows for unbiased characterization of the human antibody repertoire on single-cell level through the generation of recombinant monoclonal antibodies from primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells to the RT-PCR-based amplification of the expressed immunoglobulin (Ig) transcripts (IGH, IGK, and IGL) and their subsequent cloning into expression vectors for the in vitro production of recombinant monoclonal antibodies. The strategy may be used to obtain information about the clonal evolution of B-cell lymphomas by single-cell sequencing of Ig transcripts and on the antibody reactivity of human lymphoma B cells.

Stereotyped B-Cell Receptor Immunoglobulins in B-Cell Lymphomas.

Thorough examination of clonotypic B-cell receptor immunoglobulin (BcR IG) gene rearrangement sequences in patients with mature B-cell malignancies has revealed significant repertoire restrictions, leading to the identification of subsets of patients expressing highly similar, stereotyped BcR IG. This discovery strongly suggests selection by common epitopes or classes of structurally similar epitopes in the development of these tumors. Initially observed in chronic lymphocytic leukemia (CLL), where the stereotyped fraction accounts for a substantial fraction of patients, stereotyped BcR IGs have also been identified in other mature B-cell malignancies, including mantle cell lymphoma (MCL) and splenic marginal zone lymphoma (SMZL).Further comparisons across different entities have indicated that stereotyped IGs are predominantly "disease-biased," indicating distinct immune pathogenetic trajectories. Notably, accumulating evidence suggests that molecular subclassification of mature B-cell malignancies based on BcR IG stereotypy holds biological and clinical relevance. Particularly in CLL, patients belonging to the same subset due to the expression of a specific stereotyped BcR IG exhibit consistent biological backgrounds and clinical courses, especially for major and extensively studied subsets. Therefore, robust assignment to stereotyped subsets may aid in uncovering mechanisms underlying disease initiation and progression, as well as refining patient risk stratification. In this chapter, we offer an overview of recent studies on BcR IG stereotypy in mature B-cell malignancies and delineate past and present methodological approaches utilized for the identification of stereotyped BcR IG.

Enhanced Immunogenicity of Foot-and-Mouth Disease Virus-like Particles Using a Water-in-Oil-in-Water Adjuvant.

Foot-and-mouth disease (FMD) causes significant economic losses, prompting vaccination as a primary control strategy. Virus-like particles (VLPs) have emerged as promising candidates for FMD vaccines but require adjuvants to enhance their immunogenicity. In this study, we evaluated the immunogenicity of a VLP-based vaccine with a water-in-oil-in-water (W/O/W) emulsion adjuvant, named WT. The WT adjuvant was mixed with FMD VLPs to form the VLPs+WT vaccine. The size and stability of the vaccine were analyzed. BALB/c mice were immunized with the VLPs+WT vaccine, and immunological responses were assessed through antibody measurements, cytokine profiling, and gene expression analysis. In addition, splenic lymphocyte proliferation and signaling pathways were examined. The VLPs+WT vaccine exhibited a homogeneous size of 324.60 ± 2.30 nm and a viscosity of 8.76 mPa·s, indicating good stability. Immunized mice showed steady weight gain and no organ abnormalities. Compared to the VLPs group, the VLPs+WT group induced significantly higher levels of specific antibodies that persisted for 12 weeks, similar to the commercial VLPs+ISA201 vaccine. The VLPs+WT vaccine also enhanced the secretion of Th1-related (IgG2a, IFN-γ) and Th2-related (IgG1, IL-4) molecules. WT stimulated splenic lymphocyte proliferation and differentiation, primarily activating B-cell receptor signaling and phagosome pathways. It also upregulated genes associated with MHC and interferon stimulation while promoting the expression of MyD88, PI3K, AKT, p65, and p-p65 proteins. These findings suggest that WT is an effective adjuvant for FMD VLP-based vaccines, with potential for improving vaccine efficacy.

Learning the language of antibody hypervariability

Protein language models (PLMs) have demonstrated impressive success in modeling proteins. However, general-purpose "foundational" PLMs have limited performance in modeling antibodies due to the latter's hypervariable regions, which do not conform to the evolutionary conservation principles that such models rely on. In this study, we propose a transfer learning framework called Antibody Mutagenesis-Augmented Processing (AbMAP), which fine-tunes foundational models for antibody-sequence inputs by supervising on antibody structure and binding specificity examples. Our learned feature representations accurately predict mutational effects on antigen binding, paratope identification, and other key antibody properties. We experimentally validate AbMAP for antibody optimization by applying it to refine a set of antibodies that bind to a SARS-CoV-2 peptide, and obtain an 82% hit-rate and up to 22-fold increase in binding affinity. AbMAP also unlocks large-scale analyses of immune repertoires, revealing that B-cell receptor repertoires of individuals, while remarkably different in sequence, converge toward similar structural and functional coverage. Importantly, AbMAP's transfer learning approach can be readily adapted to advances in foundational PLMs. We anticipate AbMAP will accelerate the efficient design and modeling of antibodies, expedite the discovery of antibody-based therapeutics, and deepen our understanding of humoral immunity.

Recent Progress of Irreversible BTK Inhibitors

In recent years, Bruton tyrosine kinase (BTK) inhibitors that selectively inhibit B cell receptor (BCR) signaling have become a popular and effective treatment for lymphomas in the public. As the pivotal tyrosine kinase BTK regulates the BCR pathway, BTK inhibitors were developed to block the pathway by binding with BTK thereby relieving the B-cell lymphoma progression. Ibrutinib, the first-generation BTK inhibitor, can effectively inhibit BTK and alleviate various cancer diseases. However, ibrutinib's lack of selectivity has led to a series of side effects, having adverse effects on survival rates and tolerability. By altering the molecular structure, researchers have developed the next-generation irreversible BTK inhibitors aiming at increasing target specificity and decreasing off-target impacts, such as acalabrutinib, tirabrutinib, spebrutinib, branebrutinib, and zanubrutinib, who recently received approval by FDA. This article compares and analyzes the advantages and disadvantages of these irreversible BTK inhibitors, especially zanubrutinib, with ibrutinib and finally draws a conclusion.

BTK inhibition in primary central nervous system lymphoma: mechanisms, clinical efficacy, and future perspectives.

The prognosis of primary central nervous system lymphoma (PCNSL) patients is relatively poor, and there is currently no standard treatment plan. Most patients choose high-dose chemotherapy based on methotrexate. While traditional chemotherapy combined with biological therapy has achieved limited results, some patients still do not respond to treatment or cannot tolerate its toxicity and side effects. Bruton's tyrosine kinase (BTK) is a key enzyme in B-cell receptor signaling, and its activation is critical for B-cell survival and proliferation. In recent years, BTK inhibitors have shown great potential in treating lymphomas derived from various B cells because of their strong targeting ability and relatively few side effects. They may also be a reasonable treatment choice for PCNSL. This article reviews the mechanism of action, clinical research, adverse reactions, and other issues of BTK inhibitors in treating PCNSL to provide a reference for individualized treatment of patients with PCNSL.

First pilot study for newborn screening of severe T and B lymphopenias in Colombia

Congenital lymphopenias cause increased susceptibility to infections in children apparently healthy at birth. Earlier detection of these conditions would facilitate prompt treatment, prevent potentially serious disease complications and early deaths, and save healthcare resources. To perform a pilot study for neonatal screening of congenital lymphopenias by the quantification of TREC and KREC –T- and B-cell receptor excision circles– in peripheral blood samples from newborns in Medellín, Colombia. Blood samples from 1,092 newborns and six referred patients with suspected lymphopenia were collected by heel or toe-finger prick and dropped onto a filter paper. Thereafter, DNA was extracted and levels of TRECs and KRECs were measured by qPCR. The six patients with suspected lymphopenia showed undetectable or very low TREC levels. All newborns screened presented normal TREC and KREC levels. A positive correlation was found between TREC or KREC values quantified from two different filter papers. Detectable levels of the receptor excision circles decrease considerably after 24 weeks of the dried blood spot sample storage. We identified a positive association between low TREC levels and low birth weight; and a negative correlation between KREC values and prematurity. Finally, no statistical differences were found between TREC or KREC levels and delivery method. We describe the first preliminary study for the early detection of lymphopenias in Colombia. We proposed to use a cut-off value of 119 and 69 copies/μl blood of TREC and KREC, respectively for future newborn screening programs in our country.

B Cell Receptor Repertoire Analysis of the CD21lo B Cell Compartment in Healthy Individuals, Patients With Sjögren's Disease, and Patients With Radiographic Axial Spondyloarthritis.

B cells with low or absent expression of CD21 (CD21lo B cells) gained attention due to their expansion in the peripheral blood of patients with immune-mediated, rheumatic diseases. This is not only observed in typical autoimmune diseases like systemic lupus erythematosus and Sjögren's disease (SjD) but also in radiographic axial spondyloarthritis (r-axSpA), which is considered an autoinflammatory disease. To gain more insight into the origins of the heterogeneous CD21lo B-cell population, and its relation to the plasmablast (PB) compartment, we profiled the B-cell-receptor (BCR) repertoire in CD27- and CD27+ fractions of CD21lo B cells and early PBs using next-generation sequencing. Populations were sorted from peripheral blood of healthy individuals, SjD patients, and r-axSpA patients (n = 10 for each group). In healthy individuals and both patient groups, our findings indicate that CD27-CD21lo B cells, which exhibit few mutations in their BCR, may develop into CD27+CD21lo B cells and PBs, both marked by considerably more mutations. Given the known expansion of circulating CD27-CD21lo B cells in SjD and r-axSpA patients and clonal relationships with both CD27+CD21lo B cells and early PBs, these cells might actively contribute to (pathological) immune responses in rheumatic diseases with autoimmune and/or autoinflammatory characteristics.