B-cell Receptor Research Articles

Computational detection of antigen-specific B cell receptors following immunization

B cell receptors (BCRs) play a crucial role in recognizing and fighting foreign antigens. High-throughput sequencing enables in-depth sampling of the BCRs repertoire after immunization. However, only a minor fraction of BCRs actively participate in any given infection. To what extent can we accurately identify antigen-specific sequences directly from BCRs repertoires? We present a computational method grounded on sequence similarity, aimed at identifying statistically significant responsive BCRs. This method leverages well-known characteristics of affinity maturation and expected diversity. We validate its effectiveness using longitudinally sampled human immune repertoire data following influenza vaccination and SARS-CoV-2 infections. We show that different lineages converge to the same responding Complementarity Determining Region 3, demonstrating convergent selection within an individual. The outcomes of this method hold promise for application in vaccine development, personalized medicine, and antibody-derived therapeutics.

Generation of Chicken Antibody Libraries and Selection of Antigen Binders.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

The VLA-4 integrin is constitutively active in circulating chronic lymphocytic leukemia cells via BCR autonomous signaling: a novel anchor-independent mechanism exploiting soluble blood-borne ligands

In chronic lymphocytic leukemia (CLL), survival of neoplastic cells depends on microenvironmental signals at lymphoid sites where the crosstalk between the integrin VLA-4 (CD49d/CD29), expressed in ~40% of CLL, and the B-cell receptor (BCR) occurs. Here, BCR engagement inside–out activates VLA-4, thus enhancing VLA-4-mediated adhesion of CLL cells, which in turn obtain pro-survival signals from the surrounding microenvironment. We report that the BCR is also able to effectively inside–out activate the VLA-4 integrin in circulating CD49d-expressing CLL cells through an autonomous antigen-independent BCR signaling. As a consequence, circulating CLL cells exhibiting activated VLA-4 express markers of BCR pathway activation (phospho-BTK and phospho-PLC-γ2) along with higher levels of phospho-ERK and phospho-AKT indicating parallel activation of downstream pathways. Moreover, circulating CLL cells expressing activated VLA-4 bind soluble blood-borne VCAM-1 leading to increased VLA-4-dependent actin polymerization/re-organization and ERK phosphorylation. Finally, evidence is provided that ibrutinib treatment, by affecting autonomous BCR signaling, impairs the constitutive VLA-4 activation eventually decreasing soluble VCAM-1 binding and reducing downstream ERK phosphorylation by circulating CLL cells. This study describes a novel anchor-independent mechanism occurring in circulating CLL cells involving the BCR and the VLA-4 integrin, which help to unravel the peculiar biological and clinical features of CD49d+ CLL.

Holding the therapy in CLLp53: mechanisms to achieve durable responses.

Chronic lymphocytic leukemia (CLL) is a common leukemia, mainly affecting the elderly. Originating in the bone marrow, CLL involves the accumulation of B lymphocytes and progresses slowly, though 50-60% of patients will require therapy. At diagnosis, the presence of p53 protein aberrations, such as 17p deletion and TP53 mutation, arises in approximately one out of 10 patients. Even in the era of targeted therapies, these aberrations remain the most important prognostic factors. Current guidelines favor continuous BTK inhibitor therapy in patients with CLLp53, though adverse events and drug resistance may lead to discontinuation. Herein, we discuss the effects of B-cell receptor and BCL-2 inhibition, as well as the role of the immune system, in two elderly CLLp53 patients with prolonged responses to different therapies.

B cells expressing mutated IGHV1-69-encoded antigen receptors related to virus neutralization show lymphoma-like transcriptomes in patients with chronic HCV infection.

Chronic HCV infection leads to a complex interplay with adaptive immune cells that may result in B cell dyscrasias like cryoglobulinemia or lymphoma. While direct-acting antiviral therapy has decreased the incidence of severe liver damage, its effect on extrahepatic HCV manifestations such as B cell dyscrasias is still unclear. We sequenced B cell receptor (BCR) repertoires in patients with chronic HCV mono-infection and patients with HCV with a sustained virological response (SVR) after direct-acting antiviral therapy. This data set was mined for highly neutralizing HCV antibodies and compared to a diffuse large B cell lymphoma data set. The TKO model was used to test the signaling strength of selected B-BCRs in vitro. Single-cell RNA sequencing of chronic HCV and HCV SVR samples was performed to analyze the transcriptome of B cells with HCV-neutralizing antigen receptors. We identified a B cell fingerprint with high richness and somatic hypermutation in patients with chronic HCV and SVR. Convergence to specific immunoglobulin genes produced high-connectivity complementarity-determining region 3 networks. In addition, we observed that IGHV1-69 CDR1 and FR3 mutations characterizing highly neutralizing HCV antibodies corresponded to recurrent point mutations found in clonotypic BCRs of high-grade lymphomas. These BCRs did not show autonomous signaling but a lower activation threshold in an in vitro cell model for the assessment of BCR signaling strength. Single-cell RNA sequencing revealed that B cells carrying these point mutations showed a persisting oncogenic transcriptome signature with dysregulation in signaling nodes such as CARD11, MALT1, RelB, MAPK, and NFAT. We provide evidence that lymphoma-like cells derive from the anti-HCV immune response. In many patients, these cells persist for years after SVR and can be interpreted as a mechanistic basis for HCV-related B cell dyscrasias and increased lymphoma risk even beyond viral elimination.

First-in-class inhibitor of HSP110 blocks BCR activation through SYK phosphorylation in diffuse large B-cell lymphoma.

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is driven by aberrant activation of the B-cell receptor (BCR) and the TLR/MyD88 signaling pathways. The heat-shock protein HSP110 is a candidate for their regulation as it stabilizes MyD88. However, its role in overall BCR signaling remains unknown. Here, we used first-in-class HSP110 inhibitors to address this question. HSP110 inhibitors decreased the survival of several ABC-DLBCL cell lines in vitro and in vivo, and reduced the phosphorylation of BCR signaling kinases, including BTK and SYK. We identified an interaction between HSP110 and SYK and demonstrated that HSP110 promotes SYK phosphorylation. Finally, the combination of the HSP110 inhibitor with the PI3K inhibitor copanlisib decreases SYK/BTK and AKT phosphorylation synergistically, leading to suppression of tumor growth in cell line xenografts and strong reduction in patient-derived xenografts. In conclusion, by regulating the BCR/TLR signaling pathway, HSP110 inhibitors are potential drug candidates for ABC-DLBCL patients.

PLCG2 variants in cherubism

BackgroundCherubism is most commonly caused by rare heterozygous gain-of-function (GOF) missense variants in SH3BP2, which appear to signal through Phospholipase C Gamma 2 (PLCG2) to cause excessive osteoclast activity leading to expansile lesions in facial bones in childhood. GOF variants in PLCG2 lead to autoinflammatory PLCG2-associated antibody deficiency and immune dysregulation (autoinflammatory PLAID, or PLAID-GOF), characterized by variably penetrant autoinflammatory, autoimmune, infectious, and atopic manifestations. Cherubism has not been reported in PLAID to date. ObjectiveTo determine whether GOF PLCG2 variants may be associated with cherubism. MethodsClinical, laboratory, and genomic data from two patients with cherubism and other clinical symptoms observed in patients with PLCG2 variants were reviewed. Primary B-cell receptor (BCR)-induced calcium flux was assessed by flow cytometry. ResultsTwo patients with lesions consistent with cherubism but no SH3BP2 variants were found to have rare PLCG2 variants previously shown to be GOF in vitro, leading to increased BCR-induced calcium flux in one patient’s B cells. Variable humoral defects, autoinflammatory rash, and other clinical and laboratory findings consistent with PLAID were observed as well. ConclusionGOF PLCG2 variants likely represent a novel genetic driver of cherubism and should be assessed in SH3BP2-negative cases. Expansile bony lesions expand the phenotypic landscape of autoinflammatory PLAID, and bone imaging should be considered in PLAID patients.

Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein.

The early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins, including the E3/49K protein, which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45, thereby modulating activation of T and NK cells. Considering that E3/49K represents the most polymorphic viral protein among species D HAdVs, we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus, this feature is conserved regardless of the pathological associations of the respective HAdV types. It appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover, E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45, suggesting E3/49K as a potential tool to investigate CD45 specific functions. We identified B cells as new direct target of E3/49K-mediated immune modulation, representing a novel viral immunosubversive mechanism.

Synergy and antagonism in the integration of BCR and CD40 signals that control B-cell proliferation.

In response to infection or vaccination, a successful antibody response must enrich high-affinity antigen-reactive B-cells through positive selection, but eliminate auto-reactive B-cells through negative selection. B-cells receive signals from the B-cell receptor (BCR) which binds the antigen, and the CD40 receptor which is stimulated by neighboring T-cells that also recognize the antigen. How BCR and CD40 signaling are integrated quantitatively to jointly determine B-cell fate decision and proliferation remains unclear. To investigate this, we developed a differential-equations-based model of the BCR and CD40 signaling networks activating NFκB. Our model accurately recapitulates the NFκB dynamics of B-cells stimulated through their BCR and CD40 receptors, correctly predicting that costimulation induces more NFκB activity. However, when linking it to established cell fate decision models of cell survival and cell cycle control, it predicted potentiated population expansion that was not observed experimentally. We found that this discrepancy was due to a time-dependent functional antagonism exacerbated by BCR-induced caspase activity that can trigger apoptosis in founder cells, unless NFκB-induced survival gene expression protects B-cells in time. Guided by model predictions, sequential co-stimulation experiments revealed how the temporal dynamics of BCR and CD40 signaling control the fate decision between negative and positive selection of B-cell clonal expansion. Our quantitative findings highlight a complex non-monotonic integration of BCR and CD40 signals that is controlled by a balance between NFκB and cell-death pathways, and suggest a mechanism for regulating the stringency of B-cell selection during an antibody response.

B cell epitope mapping: The journey to better vaccines and therapeutic antibodies

B-cell epitope mapping is an approach that can identify and characterise specific antigen binding sites of B-cell receptors and secreted antibodies. The ability to determine the antigenic clusters of amino acids bound by B-cell clones provides unprecedented detail that will aid in developing novel and effective vaccine targets and therapeutic antibodies for various diseases. Here, we discuss conventional approaches and emerging techniques that are used to map B-cell epitopes.

Multi-compartmental diversification of neutralizing antibody lineages dissected in SARS-CoV-2 spike-immunized macaques

The continued evolution of SARS-CoV-2 underscores the need to understand qualitative aspects of the humoral immune response elicited by spike immunization. Here, we combine monoclonal antibody (mAb) isolation with deep B cell receptor (BCR) repertoire sequencing of rhesus macaques immunized with prefusion-stabilized spike glycoprotein. Longitudinal tracing of spike-sorted B cell lineages in multiple immune compartments demonstrates increasing somatic hypermutation and broad dissemination of vaccine-elicited B cells in draining and non-draining lymphoid compartments, including the bone marrow, spleen and, most notably, periaortic lymph nodes. Phylogenetic analysis of spike-specific monoclonal antibody lineages identified through deep repertoire sequencing delineates extensive intra-clonal diversification that shaped neutralizing activity. Structural analysis of the spike in complex with a broadly neutralizing mAb provides a molecular basis for the observed differences in neutralization breadth between clonally related antibodies. Our findings highlight that immunization leads to extensive intra-clonal B cell evolution where members of the same lineage can both retain the original epitope specificity and evolve to recognize additional spike variants not previously encountered.

A human autoimmune organoid model reveals IL-7 function in coeliac disease.

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

Comprehensive Analysis of Conserved B-Cell Epitopes in DENV NS1 Protein for Enhanced Rapid Diagnostic Tests Development in Indonesia

Dengue virus (DENV) constitutes a formidable public health threat within tropical and subtropical regions. The escalation of cases in Southeast Asia, notably Indonesia, is a matter of considerable concern. Timely identification of DENV infection remains imperative for efficient disease management. The non-structural protein-1 (NS1), distinguished by its heightened immunogenicity and early detectability during infection, stands as a pivotal target for diagnostic modalities. The current investigation delves into the exploration of B-cell epitopes within Indonesian DENV NS1 protein isolates, with the overarching objective of enhancing the sensitivity and specificity of early detection systems, such as the Rapid Diagnostic Test (RDT). This study has successfully delineated five B-cell epitopes that exhibit conservation across DENV serotypes within Indonesian isolates (accessions number: QBB90021.1, QBE90252.1, QBB90023.1, and UDW38833.1). These epitopes were discerned through comprehensive screening leveraging the IEDB platforms. Noteworthy variations in antigenicity, allergenicity, and toxicity profiles were observed among these identified epitopes. Molecular docking analysis substantiated a robust binding affinity between the predicted epitope and the B-cell receptor. The ensuing in silico protein-peptide docking analyses offer valuable insights into potential B-cell epitopes on the Indonesian DENV NS1 protein. The identified epitopes, particularly the SQHNYRPGY epitope characterized by its antigenic potency and non-allergenic attributes, hold promise for advancing the development of sensitive and specific RDTs tailored for DENV detection in the Indonesian context. However, it is imperative to underscore the requisite for subsequent experimental validation to affirm the efficacy of these epitopes in diagnostic applications.

JAK inhibitors attenuate hyperactivation of nonswitched memory B cells in rheumatoid arthritis patients in remission

ObjectiveTo investigate the distribution and activation of B-cell subpopulations in rheumatoid arthritis (RA) patients treated with Janus kinase inhibitors (JAKis) and to analyze their correlation with disease remission.MethodsPeripheral blood samples were collected from 23 adult healthy controls and 58 RA patients, 31 of whom were treated with JAKis and assessed during a 24-month follow-up. The number of peripheral B-cell subpopulations (including naive B cells, nonswitched memory B (NSMB) cells, switched memory B cells, and double-negative B cells), their activation, and phosphorylation of SYK and AKT upon B-cell receptor (BCR) stimulation in each population were analyzed by flow cytometry.ResultsCompared with that in healthy controls, the frequency of NSMB cells was significantly lower in new-onset untreated RA patients. However, expression of CD40, CD80, CD95, CD21low and pAKT significantly increased in these NSMB cells. Additionally, the number of NSMB cells correlated negatively with DAS28-ESR and IgG and IgA levels in these patients; expression of CD80, CD95 and CD21low on NSMB cells correlated positively with DAS28-ESR and IgG and IgA levels. After treatment with JAKis, the serum IgG concentration significantly decreased in RA patients in remission, but CD40, CD95 and pAKT levels in NSMB cells significantly decreased.ConclusionRA patients present different B-cell subpopulations, in which the frequency of NSMB cells is negatively associated with disease activity. However, treatment with JAKis can inhibit activation of NSMB cells, restore the balance of kinase phosphorylation, and facilitate disease remission in RA patients.

UBXN3B is crucial for B lymphopoiesis

The ubiquitin regulatory X (UBX) domain-containing proteins (UBXNs) are putative adaptors for ubiquitin ligases and valosin-containing protein; however, their invivo physiological functions remain poorly characterised. We recently showed that UBXN3B is essential for activating innate immunity to DNA viruses and controlling DNA/RNA virus infection. Herein, we investigate its role in adaptive immunity. We evaluated the antibody responses to multiple viruses and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza in tamoxifen-inducible global and constitutive B cell-specific Ubxn3b knockout mice; quantified various immune populations, B lineage progenitors/precursors, B cell receptor (BCR) signalling and apoptosis by flow cytometry, immunoblotting and immunofluorescence microscopy. We also performed bone marrow transfer, single-cell and bulk RNA sequencing. Both global and B cell-specific Ubxn3b knockout mice present a marked reduction in small precursor B-II (>60%), immature (>70%) and mature B (>95%) cell numbers. Transfer of wildtype bone marrow to irradiated global Ubxn3b knockouts restores normal B lymphopoiesis, while reverse transplantation does not. The mature B population shrinks rapidly with apoptosis and higher pro and activated caspase-3 protein levels were observed following induction of Ubxn3b knockout. Mechanistically, Ubxn3b deficiency leads to impaired pre-BCR signalling and cell cycle arrest. Ubxn3b knockout mice are highly vulnerable to respiratory viruses, with increased viral loads and prolonged immunopathology in the lung, and reduced production of virus-specific IgM/IgG. UBXN3B is essential for B lymphopoiesis by maintaining constitutive pre-BCR signalling and cell survival in a cell-intrinsic manner. United States National Institutes of Health grants, R01AI132526 and R21AI155820.

Lipopolysaccharide-responsive beige-like anchor is involved in regulating NF-κB activation in B cells.

Lipopolysaccharide-responsive and beige-like anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PKA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although Lrba-/- mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells. To compare and evaluate spleen-derived B cell responses to BCR crosslinking in C57BL6 Lrba-/- and Lrba+/+ mice. Spleen-derived B cells were obtained from 8 to 12-week-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinking was assessed by the F(ab')2 anti-μ chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot. Lrba-/- B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinking in the Lrba-/- mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBα, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinking in Lrba-/ - B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected. These results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter.

Effects of Alhagi maurorum Medik polysaccharide derived from different regions on the intestinal immune functions of lambs.

Introduction: Plant polysaccharide are widely studied as potential prebiotics because of their potential to protect and enhance the immunity of lambs. Methods: In this study, the polysaccharide content of Alhagi maurorum Medik from Aksu (AK) and Shanshan (SS) at different cutting periods was determined, and the functions of Alhagi maurorum Medik polysaccharide were investigated to useas an immunomodulator. Results: Our results indicated that the content of Alhagi maurorum Medik polysaccharide is the highest at the maturity stage, and the polysaccharide content of Alhagi maurorum Medik produced in Shanshan area is higher as compared to the Aksu area. The serum IgG, duodenum IgA, TNF-α, IL-4, IL-10 contents, jejunum IgA, TNF-α, IL-4, IL-17 contents, ileum IgA, IL-17 contents, duodenum villus height, crypt depth and jejunum crypt depth of lambs were significantly adjusted in the SS group as compared to CK control group and AK groups (p < 0.05). Furthemore, the sequencing results showed that SS polysaccharide promoted the release of large amounts of IgA and enhanced the immunal function of intestine by regulating the IgA production pathway and B-cell receptor signaling to activate B cells in the T-dependent pathway. Discussion: Altogether, Alhagi maurorum Medik polysaccharide from SS group holds a promising potential to be used as a valuable immunopotentiator for optimizing the immune system of intestine in lambs.

Computational estimation of clonal diversity in autoimmunity.

Diversity is the cornerstone of the adaptive immune system, crucial for its effectiveness against constantly evolving pathogens that pose threats to higher vertebrates. Accurately measuring and interpreting this diversity presents challenges for immunologists, as changes in diversity and clonotype composition can tip the balance between protective immunity and autoimmunity. In this review, we present the current methods commonly used to measure diversity from single-cell T-cell receptor and B-cell receptor sequencing. We also discuss two case studies where single-cell sequencing and diversity estimations have led to breakthroughs in autoimmune disease discovery and therapeutic innovation, and reflect upon the necessity andimportance of accurately defining and measuring lymphocyte diversity in these contexts.

Escherichia coli LTB26 mutant enhances immune responses to rotavirus antigen VP8 in a mouse model

Adjuvant is a major supplementary component of vaccines to boost adaptive immune responses. To select an efficient adjuvant from the heat-labile toxin B subunit (LTB) of E. coli, four LTB mutants (numbered LTB26, LTB34, LTB57, and LTB85) were generated by multi-amino acid random replacement. Mice have been intranasally vaccinated with human rotavirus VP8 admixed. Among the four mutants, enzyme-linked immunosorbent assay (ELISA) revealed that LTB26 had enhanced mucosal immune adjuvanticity compared to LTB, showing significantly enhanced immune responses in both serum IgG and mucosal sIgA levels. The 3D modeling analysis suggested that the enhanced immune adjuvanticity of LTB26 might be due to the change of the first LTB α-helix to a β-sheet. The molecular mechanism was studied using transcriptomic and flow cytometric (FCM) analysis. The transcriptomic data demonstrated that LTB26 enhanced immune response by enhancing B cell receptor (BCR) and major histocompatibility complex (MHC) II+-related pathways. Furthermore, LTB26 promoted Th1 and Th2-type immune responses which were confirmed by detecting IFN-γ and IL-4 expression levels. Immunohistochemical analysis demonstrated that LTB26 enhanced both Th1 and Th2 type immunity. Therefore, LTB26 was a potent mucosal immune adjuvant meeting the requirement for use in human clinics in the future.

Peripheral blood immune parameters, response, and adverse events after neoadjuvant chemotherapy plus durvalumab in early-stage triple-negative breast cancer.

We evaluated T- and B-cell receptor (TCR and BCR) repertoire diversity and 38 serum cytokines in pre- and post-treatment peripheral blood of 66 patients with triple-negative breast cancer (TNBC) who received neoadjuvant chemotherapy plus durvalumab and assessed associations with pathologic response and immune-related adverse events (irAEs) during treatment. Genomic DNA was isolated from buffy coat for TCR and BCR clonotype profiling using the Immunoseq platform and diversity was quantified with Pielou's evenness index. MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel was used to measure serum cytokine levels, which were compared between groups using moderated t-statistic with Benjamini-Hochberg correction for multiple testing. TCR and BCR diversity was high (Pielou's index > 0.75) in all samples. Baseline receptor diversities and change in diversity pre- and post-treatment were not associated with pathologic response or irAE status, except for BCR diversity that was significantly lower post-treatment in patients who developed irAE (unadjusted p = 0.0321). Five cytokines increased after treatment in patients with pathologic complete response (pCR) but decreased in patients with RD, most prominently IL-8. IFNγ, IL-7, and GM-CSF levels were higher in pre-treatment than in post-treatment samples of patients who developed irAEs but were lower in those without irAEs. Baseline peripheral blood cytokine levels may predict irAEs in patients treated with immune checkpoint inhibitors and chemotherapy, and increased post-treatment B-cell clonal expansion might mediate irAEs.