Type I interferons induce an epigenetically distinct memory B cell subset in chronic viral infection.

Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.

Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.

A naive CD4(+) T cell population specific for a microbial peptide:major histocompatibility complex II ligand (p:MHCII) typically consists of about 100 cells, each with a different T cell receptor (TCR). Following infection, this population produces a consistent ratio of effector cells that activate microbicidal functions of macrophages or help B cells make antibodies. We studied the mechanism that underlies this division of labor by tracking the progeny of single naive T cells. Different naive cells produced distinct ratios of macrophage and B cell helpers but yielded the characteristic ratio when averaged together. The effector cell pattern produced by a given naive cell correlated with the TCR-p:MHCII dwell time or the amount of p:MHCII. Thus, the consistent production of effector cell subsets by a polyclonal population of naive cells results from averaging the diverse behaviors of individual clones, which are instructed in part by the strength of TCR signaling.

Validation of Ligand Tetramers for the Detection of Antigen-Specific Lymphocytes.

The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.

The transcription factor KLF2 restrains CD4⁺ T follicular helper cell differentiation.

T follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4(+) T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4(+) T cells led to increased Tfh cell generation and B cell priming, whereas KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4(+) T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization and by regulation of lineage-defining transcription factors.

Cross-Reactivity with Self-Antigen Tunes the Functional Potential of Naive B Cells Specific for Foreign Antigens.

Upon Ag exposure, naive B cells expressing BCR able to bind Ag can undergo robust proliferation and differentiation that can result in the production of Ab-secreting and memory B cells. The factors determining whether an individual naive B cell will proliferate following Ag encounter remains unclear. In this study, we found that polyclonal naive murine B cell populations specific for a variety of foreign Ags express high levels of the orphan nuclear receptor Nur77, which is known to be upregulated downstream of BCR signaling as a result of cross-reactivity with self-antigens in vivo. Similarly, a fraction of naive human B cells specific for clinically-relevant Ags derived from respiratory syncytial virus and HIV-1 also exhibited an IgMLOW IgD+ phenotype, which is associated with self-antigen cross-reactivity. Functionally, naive B cells expressing moderate levels of Nur77 are most likely to proliferate in vivo following Ag injection. Together, our data indicate that BCR cross-reactivity with self-antigen is a common feature of populations of naive B cells specific for foreign Ags and a moderate level of cross-reactivity primes individual cells for optimal proliferative responses following Ag exposure.

Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells.

Listeria monocytogenes infection generates T helper 1 (Th1) effector memory cells and CC chemokine receptor 7 (CCR7)(+) cells resembling central memory cells. We tracked endogenous L. monocytogenes-specific CD4(+) T cells to determine how these memory cells are formed. Two effector cell populations were already present several days after infection. One highly expressed the T-bet transcription factor and produced Th1 memory cells in an interleukin-2 (IL-2) receptor-dependent fashion. The other resided in the T cell areas, expressed CCR7 and CXC chemokine receptor 5 (CXCR5), and like follicular helper cells depended on the Bcl6 transcription factor and inducible costimulator ligand on B cells. The CCR7(+)CXCR5(+) effector cells produced similar memory cells that generated diverse effector cell populations in a secondary response. Thus, Th1 effector memory and follicular helper-like central memory cells are produced from early effector cell populations that diverge in response to signals from the IL-2 receptor, Bcl6, and B cells.

Decrease in Numbers of Naive and Resting B Cells in HIV-Infected Kenyan Adults Leads to a Proportional Increase in Total and Plasmodium falciparum-Specific Atypical Memory B Cells.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with B cell activation and exhaustion, and hypergammaglobulinemia. How these changes influence B cell responses to coinfections such as malaria is poorly understood. To address this, we compared B cell phenotypes and Abs specific for the Plasmodium falciparum vaccine candidate apical membrane Ag-1 (AMA1) in HIV-infected and uninfected adults living in Kenya. Surprisingly, HIV-1 infection was not associated with a difference in serum AMA1-specific Ab levels. HIV-infected individuals had a higher proportion of total atypical and total activated memory B cells (MBCs). Using an AMA1 tetramer to detect AMA1-specific B cells, HIV-infected individuals were also shown to have a higher proportion of AMA1-specific atypical MBCs. However, this proportional increase resulted in large part from a loss in the number of naive and resting MBCs rather than an increase in the number of atypical and activated cells. The loss of resting MBCs and naive B cells was mirrored in a population of cells specific for an Ag to which these individuals were unlikely to have been chronically exposed. Together, the data show that changes in P. falciparum Ag-specific B cell subsets in HIV-infected individuals mirror those in the overall B cell population, and suggest that the increased proportion of atypical MBC phenotypes found in HIV-1-infected individuals results from the loss of naive and resting MBCs.

General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice.

Autoreactive B cells are associated with the development of several autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. The low frequency of these cells represents a major barrier to their analysis. Ag tetramers prepared from linear epitopes represent a promising strategy for the identification of small subsets of Ag-reactive immune cells. This is challenging given the requirement for identification and validation of linear epitopes and the complexity of autoantibody responses, including the broad spectrum of autoantibody specificities and the contribution of isotype to pathogenicity. Therefore, we tested a two-tiered peptide microarray approach, coupled with epitope mapping of known autoantigens, to identify and characterize autoepitopes using the BXD2 autoimmune mouse model. Microarray results were verified through comparison with established age-associated profiles of autoantigen specificities and autoantibody class switching in BXD2 and control (C57BL/6) mice and high-throughput ELISA and ELISPOT analyses of synthetic peptides. Tetramers were prepared from two linear peptides derived from two RNA-binding proteins (RBPs): lupus La and 70-kDa U1 small nuclear ribonucleoprotein. Flow cytometric analysis of tetramer-reactive B cell subsets revealed a significantly higher frequency and greater numbers of RBP-reactive marginal zone precursor, transitional T3, and PDL-2(+)CD80(+) memory B cells, with significantly elevated CD69 and CD86 observed in RBP(+) marginal zone precursor B cells in the spleens of BXD2 mice compared with C57BL/6 mice, suggesting a regulatory defect. This study establishes a feasible strategy for the characterization of autoantigen-specific B cell subsets in different models of autoimmunity and, potentially, in humans.

IL-4-secreting secondary T follicular helper (Tfh) cells arise from memory T cells, not persisting Tfh cells, through a B cell-dependent mechanism.

Humoral immunity requires cross-talk between T follicular helper (Tfh) cells and B cells. Nevertheless, a detailed understanding of this intercellular interaction during secondary immune responses is lacking. We examined this by focusing on the response to a soluble, unadjuvanted, pathogen-derived Ag (soluble extract of Schistosoma mansoni egg [SEA]) that induces type 2 immunity. We found that activated Tfh cells persisted for long periods within germinal centers following primary immunization. However, the magnitude of the secondary response did not appear to depend on pre-existing Tfh cells. Instead, Tfh cell populations expanded through a process that was dependent on memory T cells recruited into the reactive LN, as well as the participation of B cells. We found that, during the secondary response, IL-4 was critical for the expansion of a population of plasmablasts that correlated with increased SEA-specific IgG1 titers. Additionally, following immunization with SEA (but not with an Ag that induced type 1 immunity), IL-4 and IL-21 were coproduced by individual Tfh cells, revealing a potential mechanism through which appropriate class-switching can be coupled to plasmablast proliferation to enforce type 2 immunity. Our findings demonstrate a pivotal role for IL-4 in the interplay between T and B cells during a secondary Th2 response and have significant implications for vaccine design.

Direct visualization of endogenous Salmonella-specific B cells reveals a marked delay in clonal expansion and germinal center development.

CD4(+) T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4(+) T-cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an Ag tetramer and pull-down enrichment strategy to directly visualize OVA-specific B cells in mice, as they respond to infection with Salmonella-OVA. Surprisingly, OVA-specific B-cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B- and T-cell responses to the same coinjected Ag but this did not require the presence of iNOS. The Salmonella Pathogenicity Island 2 (SPI2) locus has been shown to be responsible for inhibition of Salmonella-specific CD4(+) T-cell responses, and an examination of SPI2-deficient bacteria demonstrated a recovery in B-cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B- and T-cell responses using SPI2-dependent mechanisms.

Heterogeneity in the differentiation and function of memory B cells.

Vaccines that induce neutralizing antibodies have led to the eradication of small pox and have severely reduced the prevalence of many other infections. However, even the most successful vaccines do not induce protective antibodies in all individuals, and can fail to induce lifelong immunity. A key to remedying these shortcomings may lie in a better understanding of long-lived memory B cells. Recent studies have revealed novel insights into the differentiation and function of these cells, and have shown that the memory B cell pool is much more heterogeneous than previously appreciated.

Macrophage scavenger receptor 1 (Msr1, SR-A) influences B cell autoimmunity by regulating soluble autoantigen concentration.

The class A macrophage scavenger receptor Msr1 (SR-A, CD204) has been reported to participate in the maintenance of immunological tolerance. We investigated the role of Msr1 in a mouse model of autoantibody-dependent arthritis. Genetic deficiency of Msr1 in K/BxN TCR transgenic mice decreased the incidence and severity of arthritis because of decreased autoantibody production. Despite normal initial activation of autoreactive CD4(+) T cells, potentially autoreactive B cells in Msr1(-/-) K/BxN mice retained a naive phenotype and did not expand. This was not due to an intrinsic B cell defect. Rather, we found that macrophages lacking Msr1 were inefficient at taking up the key autoantigen glucose-6-phosphate isomerase and that Msr1-deficient mice had elevated serum concentrations of glucose-6-phosphate isomerase. Arthritis developed normally when bone marrow from Msr1(-/-) K/BxN mice was transplanted into hosts whose macrophages did express Msr1. Thus, Msr1 can regulate the concentration of a soluble autoantigen. In this model, the absence of Msr1 led to higher levels of soluble autoantigen and protected mice from developing pathogenic autoantibodies, likely because of altered cognate interactions of autoreactive T and B cells with impaired differentiation of follicular Th cells.

Production and use of antigen tetramers to study antigen-specific B cells.

B cells generate antibodies that provide protection from infection, but also cause pathology in autoimmune and allergic conditions. Antigen-specific B cells can be detected by binding their surface antibody receptors with native antigens conjugated to fluorescent probes, a technique that has revealed substantial insight into B cell activation and function. This protocol describes the process of generating fluorescent antigen tetramer probes and delineates a process of enriching large samples based on antigen-specificity for high-resolution analyses of the antigen-specific B cell repertoire. Enrichment of tetramer-binding cells allows for detection of antigen-specific B cells as rare as 1 in 100 million cells, providing sufficient resolution to study naive B cells and IgE-expressing cells by flow cytometry. The generation of antigen tetramers involves antigen biotinylation, assessment of biotin:antigen ratio for optimal tetramer loading and polymerization around a streptavidin-fluorophore backbone. We also describe the construction of a control tetramer to exclude B cells binding to the tetramer backbone. We provide a framework to validate whether tetramer probes are detecting true antigen-specific B cells and discuss considerations for experimental design. This protocol can be performed by researchers trained in basic biomedical/immunological research techniques, using instrumentation commonly found in most laboratories. Constructing the antigen and control tetramers takes 9 h, though their specificity should be assessed before experimentation and may take weeks to months depending on the method of validation. Sample enrichment requires ~2 h but is generally time and cost neutral as fewer cells are run through the flow cytometer.

Concurrent dermoid and epidermoid cysts in an adolescent patient: a case report.

Dermoid and epidermoid cysts are benign lesions of ectodermal origin which are pathologically distinct entities, although often clinically indistinguishable. Cyst location, mobility, and appearance on MRI can help distinguish the two, however the distinction is mostly academic since both types have similar management. Co-occurrence of dermoid and epidermoid cysts together in the same patient has not been observed in the literature, however one case of an epidermoid cyst evolving into a dermoid cyst has been documented. In this case report, we identify a 16-year-old male with three separate cysts of the scalp and leg which, after histopathological analysis following surgical resection, were found to represent both dermoid and epidermoid cysts. We offer potential explanations for this rare occurrence in the absence of a genetic syndrome and highlight the importance of performing a thorough work-up of patients with multiple cysts.

Cross-protective antibodies against common endemic respiratory viruses.

Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) can cause severe disease and death in immunocompromised patients, the elderly, and those with underlying lung disease. A protective monoclonal antibody exists for RSV, but clinical use is limited to high-risk infant populations. Hence, therapeutic options for these viruses in vulnerable patient populations are currently limited. Here, we present the discovery, in vitro characterization, and in vivo efficacy testing of two cross-neutralizing monoclonal antibodies, one targeting both HPIV3 and HPIV1 and the other targeting both RSV and HMPV. The 3 × 1 antibody is capable of targeting multiple parainfluenza viruses; the MxR antibody shares features with other previously reported monoclonal antibodies that are capable of neutralizing both RSV and HMPV. We obtained structures using cryo-electron microscopy of these antibodies in complex with their antigens at 3.62 Å resolution for 3 × 1 bound to HPIV3 and at 2.24 Å for MxR bound to RSV, providing a structural basis for in vitro binding and neutralization. Together, a cocktail of 3 × 1 and MxR could have clinical utility in providing broad protection against four of the respiratory viruses that cause significant morbidity and mortality in at-risk individuals.

Establishment of isotype-switched, antigen-specific B cells in multiple mucosal tissues using non-mucosal immunization.

Although most pathogens infect the human body via mucosal surfaces, very few injectable vaccines can specifically target immune cells to these tissues where their effector functions would be most desirable. We have previously shown that certain adjuvants can program vaccine-specific helper T cells to migrate to the gut, even when the vaccine is delivered non-mucosally. It is not known whether this is true for antigen-specific B cell responses. Here we show that a single intradermal vaccination with the adjuvant double mutant heat-labile toxin (dmLT) induces a robust endogenous, vaccine-specific, isotype-switched B cell response. When the vaccine was intradermally boosted, we detected non-circulating vaccine-specific B cell responses in the lamina propria of the large intestines, Peyer's patches, and lungs. When compared to the TLR9 ligand adjuvant CpG, only dmLT was able to drive the establishment of isotype-switched resident B cells in these mucosal tissues, even when the dmLT-adjuvanted vaccine was administered non-mucosally. Further, we found that the transcription factor Batf3 was important for the full germinal center reaction, isotype switching, and Peyer's patch migration of these B cells. Collectively, these data indicate that specific adjuvants can promote mucosal homing and the establishment of activated, antigen-specific B cells in mucosal tissues, even when these adjuvants are delivered by a non-mucosal route. These findings could fundamentally change the way future vaccines are formulated and delivered.

Respiratory mucosal immunity against SARS-CoV-2 after mRNA vaccination.

SARS-CoV-2 mRNA vaccination induces robust humoral and cellular immunity in the circulation; however, it is currently unknown whether it elicits effective immune responses in the respiratory tract, particularly against variants of concern (VOCs), including Omicron. We compared the SARS-CoV-2 S-specific total and neutralizing antibody responses, and B and T cell immunity, in the bronchoalveolar lavage fluid (BAL) and blood of COVID-19-vaccinated individuals and hospitalized patients. Vaccinated individuals had significantly lower levels of neutralizing antibody against D614G, Delta (B.1.617.2), and Omicron BA.1.1 in the BAL compared with COVID-19 convalescents despite robust S-specific antibody responses in the blood. Furthermore, mRNA vaccination induced circulating S-specific B and T cell immunity, but in contrast to COVID-19 convalescents, these responses were absent in the BAL of vaccinated individuals. Using a mouse immunization model, we demonstrated that systemic mRNA vaccination alone induced weak respiratory mucosal neutralizing antibody responses, especially against SARS-CoV-2 Omicron BA.1.1 in mice; however, a combination of systemic mRNA vaccination plus mucosal adenovirus-S immunization induced strong neutralizing antibody responses not only against the ancestral virus but also the Omicron BA.1.1 variant. Together, our study supports the contention that the current COVID-19 vaccines are highly effective against severe disease development, likely through recruiting circulating B and T cell responses during reinfection, but offer limited protection against breakthrough infection, especially by the Omicron sublineage. Hence, mucosal booster vaccination is needed to establish robust sterilizing immunity in the respiratory tract against SARS-CoV-2, including infection by the Omicron sublineage and future VOCs.

Methods to Measure Antibody Neutralization of Live Human Coronavirus OC43.

The human Betacoronavirus OC43 is a common cause of respiratory viral infections in adults and children. Lung infections with OC43 are associated with mortality, especially in hematopoietic stem cell transplant recipients. Neutralizing antibodies play a major role in protection against many respiratory viral infections, but to date a live viral neutralization assay for OC43 has not been described. We isolated a human monoclonal antibody (OC2) that binds to the spike protein of OC43 and neutralizes the live virus derived from the original isolate of OC43. We used this monoclonal antibody to develop and test the performance of two readily accessible in vitro assays for measuring antibody neutralization, one utilizing cytopathic effect and another utilizing an ELISA of infected cells. We used both methods to measure the neutralizing activity of the OC2 monoclonal antibody and of human plasma. These assays could prove useful for studying humoral responses to OC43 and cross-neutralization with other medically important betacoronaviruses.

Tissue-resident CD4+ T helper cells assist the development of protective respiratory B and CD8+ T cell memory responses.

Much remains unknown about the roles of CD4+ T helper cells in shaping localized memory B cell and CD8+ T cell immunity in the mucosal tissues. Here, we report that lung T helper cells provide local assistance for the optimal development of tissue-resident memory B and CD8+ T cells after the resolution of primary influenza virus infection. We have identified a population of T cells in the lung that exhibit characteristics of both follicular T helper and TRM cells, and we have termed these cells as resident helper T (TRH) cells. Optimal TRH cell formation was dependent on transcription factors involved in T follicular helper and resident memory T cell development including BCL6 and Bhlhe40. We show that TRH cells deliver local help to CD8+ T cells through IL-21-dependent mechanisms. Our data have uncovered the presence of a tissue-resident helper T cell population in the lung that plays a critical role in promoting the development of protective B cell and CD8+ T cell responses.