The transcription factor Hhex cooperates with the corepressor Tle3 to promote memory B cell development.

Memory B cells (MBCs) are essential for long-lived humoral immunity. However, the transcription factors involved in MBC differentiation are poorly defined. Here, using single-cell RNA sequencing analysis, we identified a population of germinal center (GC) B cells in the process of differentiating into MBCs. Using an inducible CRISPR-Cas9 screening approach, we identified the hematopoietically expressed homeobox protein Hhex as a transcription factor regulating MBC differentiation. The corepressor Tle3 was also identified in the screen and was found to interact with Hhex to promote MBC development. Bcl-6 directly repressed Hhex in GC B cells. Reciprocally, Hhex-deficient MBCs exhibited increased Bcl6 expression and reduced expression of the Bcl-6 target gene Bcl2. Overexpression of Bcl-2 was able to rescue MBC differentiation in Hhex-deficient cells. We also identified Ski as an Hhex-induced transcription factor involved in MBC differentiation. These findings establish an important role for Hhex-Tle3 in regulating the transcriptional circuitry governing MBC differentiation.

Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers.

Tbet+CD11c+ B cells arise during type 1 pathogen challenge, aging, and autoimmunity in mice and humans. Here, we examined the developmental requirements of this B cell subset. In acute infection, T follicular helper (Tfh) cells, but not Th1 cells, drove Tbet+CD11c+ B cell generation through proximal delivery of help. Tbet+CD11c+ B cells developed prior to germinal center (GC) formation, exhibiting phenotypic and transcriptional profiles distinct from GC B cells. Fate tracking revealed that most Tbet+CD11c+ B cells developed independently of GC entry and cell-intrinsic Bcl6 expression. Tbet+CD11c+ and GC B cells exhibited minimal repertoire overlap, indicating distinct developmental pathways. As the infection resolved, Tbet+CD11c+ B cells localized to the marginal zone where splenic retention depended on integrins LFA-1 and VLA-4, forming a competitive memory subset that contributed to antibody production and secondary GC seeding upon rechallenge. Therefore, Tbet+CD11c+ B cells comprise a GC-independent memory subset capable of rapid and robust recall responses.

Follicular dendritic cells restrict interleukin-4 availability in germinal centers and foster memory B cell generation.

B cells within germinal centers (GCs) enter cycles of antibody affinity maturation or exit the GC as memory cells or plasma cells. Here, we examined the contribution of interleukin (IL)-4 on B cell fate decisions in the GC. Single-cell RNA-sequencing identified a subset of light zone GC B cells expressing high IL-4 receptor-a (IL4Ra) and CD23 and lacking a Myc-associated signature. These cells could differentiate into pre-memory cells. B cell-specific deletion of IL4Ra or STAT6 favored the pre-memory cell trajectory, and provision of exogenous IL-4 in a wild-type context reduced pre-memory cell frequencies. IL-4 acted during antigen-specific interactions but also influenced bystander cells. Deletion of IL4Ra from follicular dendritic cells (FDCs) increased the availability of IL-4 in the GC, impaired the selection of affinity-matured B cells, and reduced memory cell generation. We propose that GC FDCs establish a niche that limits bystander IL-4 activity, focusing IL-4 action on B cells undergoing selection and enhancing memory cell differentiation.

Production of IL-10 by CD4(+) regulatory T cells during the resolution of infection promotes the maturation of memory CD8(+) T cells.

Memory CD8(+) T cells are critical for host defense upon reexposure to intracellular pathogens. We found that interleukin 10 (IL-10) derived from CD4(+) regulatory T cells (Treg cells) was necessary for the maturation of memory CD8(+) T cells following acute infection with lymphocytic choriomeningitis virus (LCMV). Treg cell-derived IL-10 was most important during the resolution phase, calming inflammation and the activation state of dendritic cells. Adoptive transfer of IL-10-sufficient Treg cells during the resolution phase 'restored' the maturation of memory CD8(+) T cells in IL-10-deficient mice. Our data indicate that Treg cell-derived IL-10 is needed to insulate CD8(+) T cells from inflammatory signals, and reveal that the resolution phase of infection is a critical period that influences the quality and function of developing memory CD8(+) T cells.

A central role for Notch in effector CD8(+) T cell differentiation.

Activated CD8(+) T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We found that the signaling receptor Notch controls this 'choice'. Notch promoted the differentiation of immediately protective TECs and was correspondingly required for the clearance of acute infection with influenza virus. Notch activated a major portion of the TEC-specific gene-expression program and suppressed the MPC-specific program. Expression of Notch was induced on naive CD8(+) T cells by inflammatory mediators and interleukin 2 (IL-2) via pathways dependent on the metabolic checkpoint kinase mTOR and the transcription factor T-bet. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of an infection.

The Interleukin-2-mTORc1 Kinase Axis Defines the Signaling, Differentiation, and Metabolism of T Helper 1 and Follicular B Helper T Cells.

The differentiation of CD4(+) helper T cell subsets with diverse effector functions is accompanied by changes in metabolism required to meet their bioenergetic demands. We find that follicular B helper T (Tfh) cells exhibited less proliferation, glycolysis, and mitochondrial respiration, accompanied by reduced mTOR kinase activity compared to T helper 1 (Th1) cells in response to acute viral infection. IL-2-mediated activation of the Akt kinase and mTORc1 signaling was both necessary and sufficient to shift differentiation away from Tfh cells, instead promoting that of Th1 cells. These findings were not the result of generalized signaling attenuation in Tfh cells, because they retained the ability to flux calcium and activate NFAT-transcription-factor-dependent cytokine production. These data identify the interleukin-2 (IL-2)-mTORc1 axis as a critical orchestrator of the reciprocal balance between Tfh and Th1 cell fates and their respective metabolic activities after acute viral infection.

Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus.

Innate lymphoid cells (ILCs), a heterogeneous cell population, are critical in orchestrating immunity and inflammation in the intestine, but whether ILCs influence immune responses or tissue homeostasis at other mucosal sites remains poorly characterized. Here we identify a population of lung-resident ILCs in mice and humans that expressed the alloantigen Thy-1 (CD90), interleukin 2 (IL-2) receptor a-chain (CD25), IL-7 receptor a-chain (CD127) and the IL-33 receptor subunit T1-ST2. Notably, mouse ILCs accumulated in the lung after infection with influenza virus, and depletion of ILCs resulted in loss of airway epithelial integrity, diminished lung function and impaired airway remodeling. These defects were restored by administration of the lung ILC product amphiregulin. Collectively, our results demonstrate a critical role for lung ILCs in restoring airway epithelial integrity and tissue homeostasis after infection with influenza virus.

Transcription factor STAT3 and type I interferons are corepressive insulators for differentiation of follicular helper and T helper 1 cells.

Follicular helper T (Tfh) cells are required for the establishment of T-dependent B cell memory and high affinity antibody-secreting cells. We have revealed herein opposing roles for signal transducer and activator of transcription 3 (STAT3) and type I interferon (IFN) signaling in the differentiation of Tfh cells following viral infection. STAT3-deficient CD4(+) T cells had a profound defect in Tfh cell differentiation, accompanied by decreased germinal center (GC) B cells and antigen-specific antibody production during acute infection with lymphocytic choriomeningitis virus. STAT3-deficient Tfh cells had strikingly increased expression of a number of IFN-inducible genes, in addition to enhanced T-bet synthesis, thus adopting a T helper 1 (Th1) cell-like effector phenotype. Conversely, IFN-αß receptor blockade restored Tfh and GC B cell phenotypes in mice containing STAT3-deficient CD4(+) T cells. These data suggest mutually repressive roles for STAT3 and type I IFN signaling pathways in the differentiation of Tfh cells following viral infection.

CD4+ T cell help guides formation of CD103+ lung-resident memory CD8+ T cells during influenza viral infection.

Tissue-resident memory T (Trm) cells provide enhanced protection against infection at mucosal sites. Here we found that CD4(+) T cells are important for the formation of functional lung-resident CD8(+) T cells after influenza virus infection. In the absence of CD4(+) T cells, CD8(+) T cells displayed reduced expression of CD103 (Itgae), were mislocalized away from airway epithelia, and demonstrated an impaired ability to recruit CD8(+) T cells to the lung airways upon heterosubtypic challenge. CD4(+) T cell-derived interferon-γ was necessary for generating lung-resident CD103(+) CD8(+) Trm cells. Furthermore, expression of the transcription factor T-bet was increased in "unhelped" lung Trm cells, and a reduction in T-bet rescued CD103 expression in the absence of CD4(+) T cell help. Thus, CD4(+) T cell-dependent signals are important to limit expression of T-bet and allow for the development of CD103(+) CD8(+) Trm cells in the lung airways following respiratory infection.

CD301b⁺ dermal dendritic cells drive T helper 2 cell-mediated immunity.

Unlike other types of T helper (Th) responses, whether the development of Th2 cells requires instruction from particular subset of dendritic cells (DCs) remains unclear. By using an in vivo depletion approach, we have shown that DCs expressing CD301b were required for the generation of Th2 cells after subcutaneous immunization with ovalbumin (OVA) along with papain or alum. CD301b⁺ DCs are distinct from epidermal or CD207⁺ dermal DCs (DDCs) and were responsible for transporting antigen injected subcutaneously with Th2-type adjuvants. Transient depletion of CD301b⁺ DCs resulted in less effective accumulation and decreased expression of CD69 by polyclonal CD4⁺ T cells in the lymph node. Moreover, despite intact cell division and interferon-γ production, CD301b⁺ DC depletion led to blunted interleukin-4 production by OVA-specific OT-II transgenic CD4⁺ T cells and significantly impaired Th2 cell development upon infection with Nippostrongylus brasiliensis. These results reveal CD301b⁺ DDCs as the key mediators of Th2 immunity.

Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity.

Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential "universal" vaccine.

Defective CD8 T cell responses in aged mice are due to quantitative and qualitative changes in virus-specific precursors.

Aging is associated with suboptimal CD8 T cell responses to viral infections. It is not clear whether these poor responses are due to environmental influences or quantitative and qualitative changes in the pool of responding CD8 T cells. Our studies demonstrated several deleterious age-related changes in the pool of Ag-specific CD8 T cells that respond to infection. The majority of CD8 T cells from uninfected aged mice was CD44(Hi) and had increased expression of inhibitory receptors including PD1, LAG3, 2B4, and CD160. These aged CD44(Hi) CD8 T cells were transcriptionally similar to exhausted CD8 T cells found during chronic infections. In addition, the number of virus-specific precursors in aged mice prior to infection was decreased up to 10-fold, and many of these Ag-specific precursors had high expression of CD44 and PD1. Finally, TCR transgenic studies demonstrated that the CD44(Hi) Ag-specific CD8 T cells from unimmunized aged and young mice were qualitatively inferior compared with CD44(Lo) CD8 T cells from aged or young donors. Thus, a decrease in precursor frequency as well as qualitative changes of CD8 T cells during aging are directly related to impaired immunity.

In Vivo CRISPR/Cas9-Mediated Gene Ablation in Murine B Cells.

CRISPR-Cas9 genome editing is a powerful tool for assessing the functional role of candidate genes. In vitro CRISPR/Cas9 screens have been used to rapidly assess the role of thousands of genes in the differentiation and function of immune populations. However, the physiological relevance of a gene is often dependent on signals received in the tissue microenvironment, such as exposure to growth factors, chemokines, cytokines, and cell contact-dependent signals, which may not be recapitulated in an in vitro setting. Additionally, in vitro approaches are not sufficient to induce the differentiation of all cell populations limiting the cell types that can be screened. This has posed a major barrier to understanding the genes regulating the differentiation of germinal center B cells. Here, we describe an approach to perform an in vivo Crispr-Cas9 screen to specifically ablate genes in activated B cells. Using this approach, we have been able to reveal novel transcriptional regulators of germinal center B cell differentiation following viral infection.

Polarization of the memory B cell response.

Memory B cells are long-lived cells that are induced following infection or vaccination. Upon antigen re-encounter, memory B cells rapidly differentiate into antibody-secreting or germinal center B cells. While memory B cells are an important component of long-term protective immunity following vaccination, they also contribute to the progression of diseases such as autoimmunity and allergy. Numerous subsets of memory B cells have been identified in mice and humans that possess important phenotypic and functional differences. Here, we review the transcriptional circuitry governing memory B cell differentiation and function. We then summarize emerging evidence that the inflammatory environment in which memory B cells develop has an important role in shaping their phenotype and examine the pathways regulating the development of memory B cells during a type 1-skewed and type-2 skewed immune response.

Functional heterogeneity in the memory B-cell response.

Most vaccines induce robust antibody and memory B-cell (MBC) responses that are capable of mediating protective immunity. However, antibody titers wane following vaccination necessitating the administration of booster vaccines to maintain a protective antibody titer. MBCs are stably maintained following vaccination and can rapidly give rise to antibody-secreting cells or undergo further affinity maturation upon antigen re-encounter. Repeated antigen encounter results in the development of MBCs that encode antibodies capable of mediating broadly protective immunity against viruses such as SARS-CoV-2 and influenza. Here, we summarize emerging evidence that MBCs are a heterogeneous population composed of transcriptionally and phenotypically distinct subsets that have discrete roles in mediating protective immunity upon antigen re-encounter and examine the implications of these findings for the development of vaccines capable of eliciting broadly protective immunity.

CD62L expression marks a functionally distinct subset of memory B cells.

The memory B cell response consists of phenotypically distinct subsets that differ in their ability to respond upon antigen re-encounter. However, the pathways regulating the development and function of memory B cell subsets are poorly understood. Here, we show that CD62L and CD44 are progressively expressed on mouse memory B cells and identify transcriptionally and functionally distinct memory B cell subsets. Bcl6 is important in regulating memory B cell subset differentiation with overexpression of Bcl6 resulting in impaired CD62L+ memory B cell development. Bcl6 regulates memory B cell subset development through control of a network of genes, including Bcl2 and Zeb2. Overexpression of Zeb2 impairs the development of CD62L+ memory B cells. Importantly, CD62L is also differentially expressed on human memory B cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and identifies phenotypically distinct populations. Together, these data indicate that CD62L expression marks functionally distinct memory B cell subsets.

Cell-intrinsic defects in the proliferative response of antiviral memory CD8 T cells in aged mice upon secondary infection.

Although previous studies have demonstrated delayed viral clearance and blunted effector T cell responses in aged mice during infection, memory CD8 T cells and especially secondary responses have received less attention. In this study, we show that modest differences in the number of memory CD8 T cells formed in aged versus young animals were associated with altered memory CD8 T cell differentiation. Aged immune mice had increased morbidity and mortality upon secondary viral challenge, suggesting changes in T cell immunity. Indeed, virus-specific memory CD8 T cells from aged mice showed substantially reduced proliferative expansion upon secondary infection using multiple challenge models. In addition, this defect in recall capacity of aged memory CD8 T cells was cell-intrinsic and persisted upon adoptive transfer into young mice. Thus, the poor proliferative potential of memory T cells and altered memory CD8 T cell differentiation could underlie age-related defects in antiviral immunity.

Development and function of tissue-resident memory B cells.

Barrier tissues are the primary site of infection for pathogens likely to cause future pandemics. Tissue-resident lymphocytes can rapidly detect pathogens upon infection of barrier tissues and are critical in preventing viral spread. However, most vaccines fail to induce tissue-resident lymphocytes and are instead reliant on circulating antibodies to mediate protective immunity. Circulating antibody titers wane over time following vaccination leaving individuals susceptible to breakthrough infections by variant viral strains that evade antibody neutralization. Memory B cells were recently found to establish tissue residence following infection of barrier tissues. Here, we summarize emerging evidence for the importance of tissue-resident memory B cells in the establishment of protective immunity against viral and bacterial challenge. We also discuss the role of tissue-resident memory B cells in regulating the progression of non-infectious diseases. Finally, we examine new approaches to develop vaccines capable of eliciting barrier immunity.

The germinal centre B cell response to SARS-CoV-2.

The germinal centre (GC) response is critical for the generation of affinity-matured plasma cells and memory B cells capable of mediating long-term protective immunity. Understanding whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination elicits a GC response has profound implications for the capacity of responding B cells to contribute to protection against infection. However, direct assessment of the GC response in humans remains a major challenge. Here we summarize emerging evidence for the importance of the GC response in the establishment of durable and broad immunity against SARS-CoV-2 and discuss new approaches to modulate the GC response to better protect against newly emerging SARS-CoV-2 variants. We also discuss new findings showing that the GC B cell response persists in the draining lymph nodes for at least 6 months in some individuals following vaccination with SARS-CoV-2 mRNA-based vaccines.

SARS-CoV-2 booster vaccination rescues attenuated IgG1 memory B cell response in primary antibody deficiency patients.

Background: Although SARS-CoV-2 vaccines have proven effective in eliciting a protective immune response in healthy individuals, their ability to induce a durable immune response in immunocompromised individuals remains poorly understood. Primary antibody deficiency (PAD) syndromes are among the most common primary immunodeficiency disorders in adults and are characterized by hypogammaglobulinemia and impaired ability to mount robust antibody responses following infection or vaccination. Methods: Here, we present an analysis of both the B and T cell response in a prospective cohort of 30 individuals with PAD up to 150 days following initial COVID-19 vaccination and 150 days post mRNA booster vaccination. Results: After the primary vaccination series, many of the individuals with PAD syndromes mounted SARS-CoV-2 specific memory B and CD4+ T cell responses that overall were comparable to healthy individuals. Nonetheless, individuals with PAD syndromes had reduced IgG1+ and CD11c+ memory B cell responses following the primary vaccination series, with the defect in IgG1 class-switching rescued following mRNA booster doses. Boosting also elicited an increase in the SARS-CoV-2-specific B and T cell response and the development of Omicron-specific memory B cells in COVID-19-naïve PAD patients. Individuals that lacked detectable B cell responses following primary vaccination did not benefit from booster vaccination. Conclusion: Together, these data indicate that SARS-CoV-2 vaccines elicit memory B and T cells in most PAD patients and highlights the importance of booster vaccination in immunodeficient individuals.