Regulated selection of germinal-center cells into the memory B cell compartment.

Despite the importance of memory B cells in protection from reinfection, how such memory cells are selected and generated during germinal-center (GC) reactions remains unclear. We found here that light-zone (LZ) GC B cells with B cell antigen receptors (BCRs) of lower affinity were prone to enter the memory B cell pool. Mechanistically, cells in this memory-prone fraction had higher expression of the transcriptional repressor Bach2 than that of their counterparts with BCRs of higher affinity. Haploinsufficiency of Bach2 resulted in reduced generation of memory B cells, independently of suppression of the gene encoding the transcription factor Blimp-1. Bach2 expression in GC cells was inversely correlated with the strength of help provided by T cells. Thus, we propose an instructive model in which weak help from T cells maintains relatively high expression of Bach2, which predisposes GC cells to enter the memory pool.

Influenza vaccination strategies targeting the hemagglutinin stem region.

Influenza is one of the best examples of highly mutable viruses that are able to escape immune surveillance. Indeed, in response to influenza seasonal infection or vaccination, the majority of the induced antibodies are strain-specific. Current vaccine against the seasonal strains with the strategy of surveillance-prediction-vaccine does not cover an unmet virus strain leading to pandemic. Recently, antibodies targeting conserved epitopes on the hemagglutinin (HA) protein have been identified, albeit rarely, and they often showed broad protection. These antibody discoveries have brought the feasibility to develop a universal vaccine. Most of these antibodies bind the HA stem domain and accumulate in the memory B cell compartment. Broadly reactive stem-biased memory responses were induced by infection with antigenically divergent influenza strains and were able to eradicate these viruses, together indicating the importance of generating memory B cells expressing high-quality anti-stem antibodies. Here, we emphasize recent progress in our understanding of how such memory B cells can be generated and discuss how these advances may be relevant to the quest for a universal influenza vaccine.

Proenkephalin+ regulatory T cells expanded by ultraviolet B exposure maintain skin homeostasis with a healing function.

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological self-tolerance and suppress various immune responses. Here we report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in "wound healing involved in inflammatory responses" and the "neuropeptide signaling pathway," as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK+ UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis.

Repurposing the psoriasis drug Oxarol to an ointment adjuvant for the influenza vaccine.

Aluminum precipitates have long been used as adjuvants for human vaccines, but there is a clear need for safer and more effective adjuvants. Here we report in a mouse model that the psoriasis drug Oxarol ointment is a highly effective vaccine adjuvant. By applying Oxarol ointment onto skin, humoral responses and germinal center (GC) reactions were augmented, and the treated mice were protected from death caused by influenza virus infection. Keratinocyte-specific vitamin D3 receptor (Vdr) gene expression was required for these responses through induction of the thymic stromal lymphopoietin (Tslp) gene. Experiments involving administration of recombinant TSLP or, conversely, anti-TSLP antibody demonstrated that TSLP plays a key role in the GC reactions. Furthermore, cell-type-specific Tslpr gene deletion or diphtheria toxin-mediated deletion of specific cell types revealed that CD11c+ cells excluding Langerhans cells were responsible for the Oxarol-mediated GC reactions. These results indicate that active vitamin D3 is able to enhance the humoral response via Tslp induction in the skin and serves as a new vaccine adjuvant.

IgG Fc sialylation is regulated during the germinal center reaction following immunization with different adjuvants.

BACKGROUND: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects. OBJECTIVE: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants. METHODS: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns. RESULTS: Different adjuvants induce distinct IgG+ GC B-cell responses with specific transcriptomes and expression levels of the α2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3- follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-in-oil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-γ+ TFH1 cells, IL-6/IL-23-dependent IL-17A+ TFH17 cells, and high ratios of TFH cells to Foxp3+ follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor. CONCLUSION: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC.

Requirement for memory B-cell activation in protection from heterologous influenza virus reinfection.

While two memory compartments, memory B cells and long-lived plasma cells, are thought to contribute to the successful establishment of memory recall responses, the unique roles of each cellular compartment are still unclear. Herein, by tracing influenza anti-hemagglutinin (HA)-specific antibodies in mice, we demonstrate that pre-existing antibodies secreted by long-lived plasma cells are essential for protection from reinfection with the same influenza virus, whereas protection from secondary infection with an antigenically distinct influenza virus requires memory B-cell activation. These activated memory B cells were largely specific for the conserved HA stem region, and generated sufficient levels of antibodies for protection from heterologous reinfection. Given that the anti-stem plasmablasts derived from the memory B cells were higher affinity than those from naive B cells, our results suggest that maturation of anti-stem memory B cells during primary influenza infection and their subsequent activation are required for protection from reinfection by mutant viruses.

FcγRIIb on B Cells and Myeloid Cells Modulates B Cell Activation and Autoantibody Responses via Different but Synergistic Pathways in Lupus-Prone Yaa Mice.

C57BL/6 (B6).FcγRIIb-/- Yaa mice spontaneously develop lethal lupus nephritis. To define the cell type-specific role of FcγRIIb in Yaa-associated lupus, we established B cell- (CD19Cre Yaa), myeloid cell- (C/EBPαCre Yaa), and dendritic cell- (DC) (CD11cCre Yaa) specific FcγRIIb-deficient B6.Yaa mouse strains. CD19Cre Yaa mice developed milder lupus than B6.FcγRIIb-/- Yaa mice, indicating that FcγRIIb deficiency on B cells is not sufficient for the development of severe disease. Surprisingly, C/EBPαCre Yaa mice also showed autoantibody production and mild lupus similar to that in CD19Cre Yaa mice, whereas CD11cCre Yaa mice stayed disease free. These observations indicate that FcγRIIb deficiency in B cells and myeloid cells, but not DCs, contributes to the severe disease in B6.FcγRIIb-/- Yaa mice. Flow cytometric analysis showed that the frequency of peripheral Gr-1- but not Gr-1+ monocyte was increased in B6.FcγRIIb-/- Yaa and C/EBPαCre Yaa but not CD19Cre Yaa mice, suggesting a link between FcγRIIb deficiency on myeloid cells and the high frequency of Gr-1- monocytes. RNA sequencing revealed that compared with Gr-1+ monocytes, Gr-1- monocytes expressed higher levels of the B cell-stimulating cytokines BSF-3, IL-10, and IL-1ß, the DC markers CD11c, CD83, and Adamdec1, and the antiapoptotic factors Bcl2 and Bcl6. In conclusion, in Yaa-associated lupus nephritis, FcγRIIb on B cells and myeloid cells modulates B cell activation via different but synergistic pathways. Gr-1- monocytes are the most likely candidate myeloid cells involved.

Tolerogenic immunoreceptor ILT3/LILRB4 paradoxically marks pathogenic auto-antibody-producing plasmablasts and plasma cells in non-treated SLE.

Plasmablasts and plasma cells (PBs and PCs) producing pathogenic auto-antibodies in patients with systemic autoimmune diseases could be a better target for specific therapies for the disease than general immunosuppression or pan- or activated B-cell targeting. Our previous study indicated that leukocyte immunoglobulin-like receptor (LILR) B4 (B4, also known as ILT3/LIR-5/CD85k), a tolerogenic receptor in antigen-presenting cells, is ectopically expressed on the PB/PC surface in healthy individuals. Here, we show that the enlarged population size of PBs/PCs with augmented B4 expression is characteristic in non-treated systemic lupus erythematosus (SLE). Paradoxically, the transcription frequency of the anti-double-strand DNA immunoglobulin-coding VH sequence in the B4+ population of non-treated SLE was significantly higher than that in B4- cells. B4+ and B4- PBs/PCs were suggested to be developmentally equivalent based on the simultaneous generation of these populations upon activation of memory B cells in vitro B4 expression was found to be induced efficiently by IL-2, while IFN-α effectively induced B4+ PBs/PCs in vitro Utilizing the elevated B4 will support opening a new avenue for identifying the mechanism for generation of, and additional molecular markers for, pathogenic cells.

Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge.

The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.

The chromatin regulator DMAP1 modulates activity of the nuclear factor B (NF-B) transcription factor Relish in the Drosophila innate immune response.

The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-B family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-B protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling.

Diminished neutralizing activity against the XBB1.5 strain in 55.9% of individuals post 6 months COVID-19 mRNA booster vaccination: insights from a pseudovirus assay on 1,353 participants in the Fukushima vaccination community survey, Japan.

This study investigates the neutralizing activity against the XBB1.5 variant and the ancestral strain in a population post-bivalent vaccination using a pseudo virus assay validated with authentic virus assay. While bivalent booster vaccination and past infections enhanced neutralization against the XBB 1.5 strain, individuals with comorbidities showed reduced responses. The study suggests the need for continuous vaccine updates to address emerging SARS-CoV-2 variants and highlights the importance of monitoring real-world immune responses.

Potent neutralizing broad-spectrum antibody against SARS-CoV-2 generated from dual-antigen-specific B cells from convalescents.

Several antibody therapeutics have been developed against SARS-CoV-2; however, they have attenuated neutralizing ability against variants. In this study, we generated multiple broadly neutralizing antibodies from B cells of convalescents, by using two types of receptor-binding domains, Wuhan strain and the Gamma variant as bait. From 172 antibodies generated, six antibodies neutralized all strains prior to the Omicron variant, and the five antibodies were able to neutralize some of the Omicron sub-strains. Structural analysis showed that these antibodies have a variety of characteristic binding modes, such as ACE2 mimicry. We subjected a representative antibody to the hamster infection model after introduction of the N297A modification, and observed a dose-dependent reduction of the lung viral titer, even at a dose of 2 mg/kg. These results demonstrated that our antibodies have certain antiviral activity as therapeutics, and highlighted the importance of initial cell-screening strategy for the efficient development of therapeutic antibodies.

Light chain editing generates polyreactive antibodies in chronic graft-versus-host reaction.

The chronic graft-versus-host (cGvH) reaction is a model of induced lupus caused by alloreactive CD4(+) T cells from a Bm-12 mouse in a C57BL/6 recipient. We used this cGvH reaction in C57BL/6 anti-DNA H chain transgenic mice, 56R/B6, to understand the structure, specificity, and origin of the induced autoantibodies (auto-Abs). We found anti-DNA Abs that reacted to several different antigens, such as phosphatidylserine, myelin basic protein, thyroglobulin, histone, insulin, cytochrome C, and beta-galactosidase. This polyreactivity was found for Abs from B cells that expressed the 56R H chain transgene with "editor" L chains that did not completely veto autoreactivity. We suggest that such incomplete editing results in polyreactivity and that incompletely edited polyreactive B cells influence the subsequent expression of pathogenic auto-Abs in disease. We also found B cells that coexpress kappa and lambda L chain. These B cells contributed to the autoimmune response and are possibly in the marginal zone of the spleen.

TLR9/MyD88 signaling is required for class switching to pathogenic IgG2a and 2b autoantibodies in SLE.

Loss of tolerance in systemic lupus erythematosus (SLE) leads to the generation of autoantibodies, which accumulate in end-organs where they induce disease. Here we show that immunoglobulin (Ig)G2a and 2b autoantibodies are the pathogenic isotypes by recruiting FcgammaRIV expressing macrophages. Class switching, but not development, of IgM anti-self B cells to these pathogenic subclasses requires the innate immune receptor Toll-like receptor (TLR)9 and MyD88 signaling. In their absence, switching of autoreactive B cells to the IgG2a and 2b subclasses is blocked, resulting in reduced pathology and mortality. In contrast, switching of anti-self B cells to IgG1 is not perturbed and generation of nonautoreactive IgG2a and 2b antibodies is not impaired in TLR9-deficient mice. Thus, the TLR9 pathway is a potential target for therapeutic intervention in SLE.

Nanoplasmonic multiplex biosensing for COVID-19 vaccines.

The ongoing emergence of severe acute respiratory syndrome caused by the new coronavirus (SARS-CoV-2) variants requires swift actions in identifying specific antigens and optimizing vaccine development to maximize the humoral response of the patient. Measuring the specificity and the amount of antibody produced by the host immune system with high throughput and accuracy is critical to develop timely diagnostics and therapeutic strategies. Motivated by finding an easy-to-use and cost-effective alternative to existing serological methodologies for multiplex analysis, we develop a proof-of-concept multiplex nanoplasmonic biosensor to capture the humoral response in serums against multiple antigens. Nanoplasmonic sensing relies on the wavelength shift of the localized surface plasmon resonance (LSPR) peak of gold nanostructures upon binding interactions between the antibodies and the immobilized antigens. Here the antigens are first immobilized on different sensing areas by using a mono-biotinylation system based on the high affinity interaction between biotin and streptavidin. We then validate the multiplex platform by detecting the presence of 3 monoclonal antibodies against 3 antigens (2 different hemagglutinins (HAs) from influenza viruses, and the SARS-CoV-2 Spike RBD (receptor binding domain)). We also measure the humoral response in murine sera collected before and after its immunization with the SARS-CoV-2 Spike protein, in good agreement with the results obtained by the ELISA assay. Our nanoplasmonic assays have successfully demonstrated multiple serum antibody profiling, which can be further integrated with microfluidics as an effective high throughput screening platform in future studies for the ongoing SARS-CoV-2 vaccine development.

Potent SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from patients with COVID-19-convalescent, and identified antibodies that exhibited the comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced. Our antibodies showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

Rap1 Is Essential for B-Cell Locomotion, Germinal Center Formation and Normal B-1a Cell Population.

Integrin regulation by Rap1 is indispensable for lymphocyte recirculation. In mice having B-cell-specific Rap1a/b double knockouts (DKO), the number of B cells in lymph nodes decreased to approximately 4% of that of control mice, and B cells were present in the spleen and blood. Upon the immunization with NP-CGG, DKO mice demonstrated the defective GC formation in the spleen, and the reduced NP-specific antibody production. In vitro, Rap1 deficiency impaired the movement of activated B cells along the gradients of chemoattractants known to be critical for their localization in the follicles. Furthermore, B-1a cells were almost completely absent in the peritoneal cavity, spleen and blood of adult DKO mice, and the number of B-cell progenitor/precursor (B-p) were reduced in neonatal and fetal livers. However, DKO B-ps normally proliferated, and differentiated into IgM+ cells in the presence of IL-7. CXCL12-dependent migration of B-ps on the VCAM-1 was severely impaired by Rap1 deficiency. Immunostaining study of fetal livers revealed defects in the co-localization of DKO B-ps and IL-7-producing stromal cells. This study proposes that the profound effects of Rap1-deficiency on humoral responses and B-1a cell generation may be due to or in part caused by impairments of the chemoattractant-dependent positioning and the contact with stromal cells.

Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal.

A still unanswered question is what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells; conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development. We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate.

Influenza virus infection expands the breadth of antibody responses through IL-4 signalling in B cells.

Influenza viruses are a major public health problem. Vaccines are the best available countermeasure to induce effective immunity against infection with seasonal influenza viruses; however, the breadth of antibody responses in infection versus vaccination is quite different. Here, we show that nasal infection controls two sequential processes to induce neutralizing IgG antibodies recognizing the hemagglutinin (HA) of heterotypic strains. The first is viral replication in the lung, which facilitates exposure of shared epitopes that are otherwise hidden from the immune system. The second process is the germinal center (GC) response, in particular, IL-4 derived from follicular helper T cells has an essential role in the expansion of rare GC-B cells recognizing the shared epitopes. Therefore, the combination of exposure of the shared epitopes and efficient proliferation of GC-B cells is critical for generating broadly-protective antibodies. These observations provide insight into mechanisms promoting broad protection from virus infection.