Bebhinn Treanor

Introduction: B-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important.Methods: We identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR–Cas9. Results: We found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K–RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however, differential expression of other calcium-regulating proteins, such as ORAI1, may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this, TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly, we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. Discussion: Thus, our findings identify a role for TRPV5 in BCR signaling and B-cell activation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

Deep mining of B cell repertoires of HIV-1–infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC₅₀ value of 0.0009 µg/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 µg/mL cutoff—a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability in vivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity. Significance The high genetic diversity of HIV-1 continues to be a major barrier to the development of therapeutics for prevention and treatment. Here, we describe the design of an antibody platform that allows assembly of a highly avid, multispecific molecule that targets, simultaneously, the most conserved epitopes on the HIV-1 envelope glycoprotein. The combined multivalency and multispecificity translates into extraordinary neutralization potency and pan-neutralization of HIV-1 strains, surpassing that of the most potent anti-HIV broadly neutralizing antibody cocktails.

Despite the mechanisms of central and peripheral tolerance, the mature B cell compartment contains cells reactive for self-antigen. How these cells are poised not to respond and the mechanisms that restrain B cell responses to low-affinity endogenous antigens are not fully understood. Here, we demonstrate a critical role for the glycan-binding protein galectin-9 in setting the threshold of B cell activation and that loss of this regulatory network is sufficient to drive spontaneous autoimmunity. We further demonstrate a critical role for galectin-9 in restraining not only conventional B-2 B cells, but also innate-like B-1a cells. We show that galectin-9-deficient mice have an expanded population of B-1a cells and increased titers of B-1a-derived autoantibodies. Mechanistically, we demonstrate that galectin-9 regulates BCR and distinct TLR responses in B-1a cells, but not B-1b cells, by regulating the interaction between BCR and TLRs with the regulatory molecules CD5 and CD180, respectively. In the absence of galectin-9, B-1a cells are more readily activated and secrete increased titers of autoantibodies that facilitate autoantigen delivery to the spleen, driving autoimmune responses.

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC₅₀) values as low as 9 × 10⁻¹⁴ M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.

Despite the mechanisms of central and peripheral tolerance, the mature B cell compartment contains cells reactive for self-antigen. How these cells are poised not to respond and the mechanisms that restrain B cell responses to low affinity endogenous antigens are not fully understood. Here, we demonstrate a critical role for the glycan-binding protein galectin-9 in setting the threshold of B cell activation and that loss of this regulatory network is sufficient to drive spontaneous autoimmunity. We further demonstrate a critical role for galectin-9 in restraining not only conventional B-2 B cells, but also innate-like B-1a cells. We show that galectin-9 deficient mice have an expanded population of B1a cells and increased titers of B-1a derived autoantibodies. Mechanistically, we demonstrate that galectin-9 regulates BCR and distinct TLR responses in B-1a, but not B-1b cells, by regulating the interaction between BCR and TLRs with the regulatory molecules CD5 and CD180, respectively. In the absence of galectin-9, B-1a cells are more readily activated and secrete increased titers of autoantibodies that facilitate auto-antigen delivery to the spleen, driving autoimmune responses.

Mst1 is a serine/threonine kinase involved in cell survival, proliferation, apoptosis, and tumorigenesis. In mice, Mst1 regulates actin dynamics required for T-cell adhesion and migration, which correlate with thymic egress and entry into lymphatic tissue. The role of Mst1 in B cells and how it may control actin-dependent processes has not been well characterized. Wiskott-Aldrich syndrome protein (WASP) deficiency only moderately affects development and B-cell receptor (BCR) signaling, suggesting WASP likely associates with other molecules. We investigated whether Mst1 associates with WASP to regulate B-cell development and activation. Experimenting on Mst1/WASP double knockout (DKO) mice, we found a severe defect in the bone marrow B-cell development, and BCR signaling in the DKO mice was severely reduced. Even though WASP or Mst1 could influence the early B-cell activation, we found that the early activation events such as B-cell spreading, BCR clustering, and BCR signaling were much more impaired in the B cells from DKO mice. Furthermore, reciprocal regulation between Mst1 and WASP was observed in WASP and Mst1 KO mice, whereby the localization and function of phosphorylated WASP were affected in Mst1 KO mice. Most importantly, Mst1 inhibits the expression of WASP by decreasing the expression of WASP-interacting protein. Interestingly, we also found that WASP deficiency in patients and mice interferes with phosphorylated Mst1 localization and therefore function in B cells. Overall, our study provides a partner for WASP to regulate B-cell development and BCR signaling, as well as the reciprocal regulating molecular mechanism of one another.

Although calcium signaling and the important role of calcium release–activated calcium channels is well recognized in the context of immune cell signaling, there is a vast diversity of ion channels and transporters that regulate the entry of ions beyond calcium, including magnesium, zinc, potassium, sodium, and chloride. These ions play a critical role in numerous metabolic and cellular processes. The importance of ions in human health and disease is illustrated by the identification of primary immunodeficiencies in patients with mutations in genes encoding ion channels and transporters, as well as the immunological defects observed in individuals with nutritional ion deficiencies. Despite progress in identifying the important role of ions in immune cell development and activation, we are still in the early stages of exploring the diversity of ion channels and transporters and mechanistically understanding the role of these ions in immune cell biology. Here, we review the biology of ion signaling in B cells and the identification of critical ion channels and transporters in B‐cell development, activation, and differentiation into effector cells. Elucidating the role of ion channels and transporters in immune cell signaling is critical for expanding the repertoire of potential therapeutics for the treatment of immune disorders. Moreover, increased understanding of the role of ions in immune cell function will enhance our understanding of the potentially serious consequences of ion deficiencies in human health and disease.