Studies of human patients have shown that most anti-RBC alloantibodies are IgG1 or IgG3 subclasses, although it is unclear why transfused RBCs preferentially drive these subclasses over others. Though mouse models allow for the mechanistic exploration of class-switching, previous studies of RBC alloimmunization in mice have focused more on the total IgG response than the relative distribution, abundance, or mechanism of IgG subclass generation. Given this major gap, we compared the IgG subclass distribution generated in response to transfused RBCs relative to protein in alum vaccination, and determined the role of STAT6 in their generation.
WT mice were either immunized with Alum/HEL-OVA or transfused with HOD RBCs and levels of anti-HEL IgG subtypes were measured using end-point dilution ELISAs. To study the role of STAT6 in IgG class-switching, we first generated and validated novel STAT6 KO mice using CRISPR/cas9 gene editing. STAT6 KO mice were then transfused with HOD RBCs or immunized with Alum/HEL-OVA, and IgG subclasses were quantified by ELISA.
When compared with antibody responses to Alum/HEL-OVA, transfusion of HOD RBCs induced lower levels of IgG1, IgG2b, and IgG2c but similar levels of IgG3. Class switching to most IgG subtypes remained largely unaffected in STAT6 deficient mice in response to HOD RBC transfusion, with the one exception being IgG2b. In contrast, STAT6 deficient mice showed altered levels of all IgG subtypes following Alum vaccination.
Our results show that anti-RBC class-switching occurs via alternate mechanisms when compared with the well-studied immunogen alum vaccination.

Sustained signaling through the B cell antigen receptor (BCR) is thought to occur only when antigen(s) crosslink or disperse multiple BCR units, such as by multimeric antigens found on the surfaces of viruses or bacteria. B cell-intrinsic Toll-like receptor (TLR) signaling synergizes with the BCR to induce and shape antibody production, hallmarked by immunoglobulin (Ig) class switch recombination (CSR) of constant heavy chains from IgM/IgD to IgG, IgA or IgE isotypes, and somatic hypermutation (SHM) of variable heavy and light chains. Full B cell differentiation is essential for protective immunity, where class switched high affinity antibodies neutralize present pathogens, memory B cells are held in reserve for future encounters, and activated B cells also serve as semi-professional APCs for T cells. But the rules that fine-tune B cell differentiation remain partially understood, despite their being essential for naturally acquired immunity and for guiding vaccine development. To address this in part, we have developed a cell culture system using splenic B cells from naive mice stimulated with several biotinylated ligands and antibodies crosslinked by streptavidin reagents. In particular, biotinylated lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, and biotinylated anti-IgM were pre-assembled (multimerized) using streptavidin, or immobilized on nanoparticles coated with streptavidin, and used to active B cells in this precisely controlled, high throughput assay. Using B cell proliferation and Ig class switching as metrics for successful B cell activation, we show that the stimuli are both synergistic and dose-dependent. Crucially, the multimerized immunoconjugates are most active over a narrow concentration range. These data suggest that multimericity is an essential requirement for B cell BCR/TLRs ligands, and clarify basic rules for B cell activation. Such studies highlight the importance in determining the choice of single vs multimeric formats of antigen and PAMP agonists during vaccine design and development.

B cells are well known as key mediators of humoral immune responses via the production of antibodies. Immunoglobulin A (IgA) is the most abundantly produced antibody isotype and provides the first line of immune protection at mucosal surfaces. However, IgA has long been a divisive molecule with respect to tumor progression. IgA exerts anti- or pro-tumor effect in different tumor types. In this review, we summarize emerging evidence regarding the production and effects of IgA and IgA+ cells in the tumor microenvironment (TME). Moreover, we discuss that the TME cytokines, host diet, microbiome, and metabolites play a pivotal role in controlling the class-switch recombination (CSR) of IgA. The analysis of intratumoral Ig repertoires and determination of metabolites that influence CSR may help establish novel therapeutic targets for the treatment of cancers.

Antigens (Ags) with multivalent and repetitive structure elicit IgG production in a T-cell-independent manner. However, the mechanisms by which such T-cell-independent type-2 (TI-2) Ags induce IgG responses remain obscure. Here, we report that B-cell receptor (BCR) engagement with a TI-2 Ag but not with a T-cell-dependent (TD) Ag was able to induce the transcription of Aicda encoding activation-induced cytidine deaminase (AID) and efficient class switching to IgG3 upon costimulation with IL-1 or IFN-α in mouse B cells. TI-2 Ags strongly induced the phosphorylation of protein kinase C (PKC)δ and PKCδ mediated the Aicda transcription through the induction of BATF, the key transcriptional regulator of Aicda. In PKCδ-deficient mice, production of IgG was intact against TD Ag but abrogated against typical TI-2 Ags as well as commensal bacteria, and experimental disruption of the gut epithelial barrier resulted in fatal bacteremia. Thus, our results have revealed novel molecular requirements for class switching in the TI-2 response and highlighted its importance in homeostatic commensal-specific IgG production.
When the human body faces a potentially harmful microorganism, the immune system responds by finding and destroying the pathogen. This involves the coordination of several different parts of the immune system. B cells are a type of white blood cell that is responsible for producing antibodies: large proteins that bind to specific targets such as pathogens. B cells often need help from other immune cells known as T cells to complete antibody production. However, T cells are not required for B cells to produce antibodies against some bacteria. For example, when certain pathogenic bacteria coated with a carbohydrate called a capsule – such as pneumococcus, which causes pneumonia, or salmonella – invade our body, B cells recognize a repetitive structure of the capsule using a B-cell antigen receptor. This recognition allows B cells to produce antibodies independently of T cells. It is unclear how B cells produce antibodies in this situation or what proteins are required for this activity. To understand this process, Fukao et al. used genetically modified mice and their B cells to study how they produce antibodies independently of T cells. They found that a protein called PKCδ is critical for B cells to produce antibodies, especially of an executive type called IgG, in the T-cell-independent response. PKCδ became active when B cells were stimulated with the repetitive antigen present on the surface of bacteria like salmonella or pneumococcus. Mice that lack PKCδ were unable to produce IgG independently of T cells, leading to fatal infections when bacteria reached the tissues and blood. Understanding the mechanism behind the T cell-independent B cell response could lead to more effective antibody production, potentially paving the way for new vaccines to prevent fatal diseases caused by pathogenic bacteria.

Crosstalk between T and B cells is crucial for generating high-affinity, class-switched antibody responses. The roles of CD4+ T cells in this process have been well-characterised. In contrast, regulation of antibody responses by CD8+ T cells is significantly less defined. CD8+ T cells are principally recognised for eliciting cytotoxic responses in peripheral tissues and forming protective memory. However, recent findings have identified a novel population of effector CD8+ T cells that co-opt a differentiation program characteristic of CD4+ T follicular helper (Tfh) cells, upregulate the chemokine receptor CXCR5 and localise to B cell follicles. While it has been shown that CXCR5+CD8+ T cells mediate the removal of viral reservoirs in the context of follicular-trophic viral infections and maintain the response to chronic insults by virtue of progenitor/stem-like properties, it is not known if CXCR5+CD8+ T cells arise during acute peripheral challenges in the absence of follicular infection and whether they influence B cell responses in vivo in these settings. Using the ovalbumin-specific T cell receptor transgenic (OT-I) system in an adoptive transfer-immunisation/infection model, this study demonstrates that CXCR5+CD8+ T cells arise in response to protein immunisation and peripheral viral infection, displaying a follicular-homing phenotype, expression of cell surface molecules associated with Tfh cells and limited cytotoxic potential. Furthermore, studies assessing the B cell response in the presence of OT-I or Cxcr5-/- OT-I cells revealed that CXCR5+CD8+ T cells shape the antibody response to protein immunisation and peripheral viral infection, promoting class switching to IgG2c in responding B cells. Overall, the results highlight a novel contribution of CD8+ T cells to antibody responses, expanding the functionality of the adaptive immune system.

Positive selection of high-affinity B cells within germinal centers (GCs) drives affinity maturation of antibody responses. Here, we examined the mechanism underlying the parallel transition from immunoglobulin M (IgM) to IgG. Early GCs contained mostly unswitched IgM+ B cells; IgG+ B cells subsequently increased in frequency, dominating GC responses 14-21 days after antigen challenge. Somatic hypermutation and generation of high-affinity clones occurred with equal efficiency among IgM+ and IgG+ GC B cells, and inactivation of Ig class-switch recombination did not prevent depletion of IgM+ GC B cells. Instead, high-affinity IgG+ GC B cells outcompeted high-affinity IgM+ GC B cells via a selective advantage associated with IgG antigen receptor structure but independent of the extended cytoplasmic tail. Thus, two parallel forms of GC B-cell-positive selection, based on antigen receptor variable and constant regions, respectively, operate in tandem to ensure high-affinity IgG antibodies predominate in mature serum antibody responses.

Scavenger receptors are pattern recognition receptors that recognize both foreign and self-ligands, and initiate different mechanisms of cellular activation, often as co-receptors. The function of scavenger receptor CD36 in the immune system has mostly been studied in macrophages but it is also highly expressed by innate type B cells where its function is less explored. Here we report that CD36 is involved in macro-autophagy/autophagy in B cells, and in its absence, the humoral immune response is impaired. We found that CD36-deficient B cells exhibit a significantly reduced plasma cell formation, proliferation, mitochondrial mobilization and oxidative phosphorylation. These changes were accompanied by impaired initiation of autophagy, and we found that CD36 regulated autophagy and colocalized with autophagosome membrane protein MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). When we investigated T-cell-dependent immune responses, we found that mice with CD36 deficiency, specifically in B cells, exhibited attenuated germinal center responses, class switching, and antibody production as well as autophagosome formation. These findings establish a critical role for CD36 in B cell responses and may also contribute to our understanding of CD36-mediated autophagy in other cells as well as in B cell lymphomas that have been shown to express the receptor.Abbreviations: AICDA/AID: activation-induced cytidine deaminase; ATG5: autophagy related 5; ATP: adenosine triphosphate; BCR: B-cell receptor; CPG: unmethylated cytosine-guanosine; CQ: chloroquine; DC: dendritic cells; FOB: follicular B cells; GC: germinal center; Ig: immunoglobulin; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MZB: marginal zone B cells; NP-CGG: 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin; OCR: oxygen consumption rate; oxLDL: oxidized low-density lipoprotein; PC: plasma cells; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; SRBC: sheep red blood cells; Tfh: follicular helper T cells; TLR: toll-like receptor.

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UNASSIGNED: Chronic rhinosinusitis (CRS) describes an inflammatory condition affecting the sinonasal mucosa. As the immune system players such as immunoglobulins play prominent roles in the development of CRS, we aimed to investigate the expression of IgA subclasses and factors involved in IgA class switching in the sinonasal mucosa of CRS patients.
UNASSIGNED: Specimens were collected from the sinonasal mucosa of the healthy controls and CRS patients. Histological assessments were performed by H&E and immunohistochemistry. Real-time PCR and ELISA methods were applied to measure gene expression and protein levels extracted from tissue samples, respectively.
UNASSIGNED: We observed that total IgA and subclass-positive cells were higher in the patient groups than controls. There was a significant correlation between the number of eosinophils and total IgA and subclasses-positive cells (Pv < 0.0001). The expression of CXCL13, BAFF, AID, and germline transcripts were increased in CRSwNP patients. In contrast to IgA2 levels, IgA1 levels were significantly increased in the sinonasal tissue of CRSwNP patients (Pv < 0.01). TGF-β was significantly elevated in the sinonasal tissue of patients with CRSsNP.
UNASSIGNED: Increased protein levels of IgA subclasses and related antibody-producing cells were associated with elevated eosinophils in CRSwNP patients which may result in eosinophil pathological functions. Several therapeutic approaches might be developed to modulate the IgA production to ameliorate the inflammatory mechanisms in CRSwNP patients.

Interleukin-10 (IL-10) is a pleiotropic cytokine, which has both regulatory and stimulatory effects on different immune cell types. Different studies have reported the importance of IL-10 and Transforming growth factor-beta (TGF-β) in the regulation of B cell class switching the production of immunoglobulin A (IgA); however, the underlying mechanisms remain to be fully elucidated. The objective of this study was to investigate the TGF-β response during B stimulation of human B cells by IL-10. Pan B cells of healthy donors were negatively purified by a magnetic cell separation technique. B cells were cultured with multimeric CD40 ligand (mCD40L) and IL-10 for two and seven days. After harvesting in specific days, TGF-β receptor II and surface IgA expression was determined by flow cytometry, while IgA and TGF-β secretion was assessed by enzyme-linked immunosorbent assay. B cells endogenously expressed TGF-β receptor II and after 48 hours cultivation with mCD40L or mCD40L plus IL-10, both the expression of this receptor and the production of TGF-β were significantly increased. Notably, TGF-β levels following stimulation with mCD40L and IL-10 were higher than those produced by B cells stimulated with mCD40L alone. Furthermore, at day 7 and following IL-10 stimulation, there was a significant rise in the amount of IgA secretion by class-switched plasma cells, which was higher than stimulation with mCD40L alone. Our findings suggest that IL-10 can modulate TGF-β production and TGF-β receptor expression in mCD40-activated human B lymphocytes.