Germinal centers (GCs) are specialized sites in lymphoid tissues where antigen-specific B cells undergo proliferation, affinity maturation, and differentiation into high-affinity memory B cells and long-lived plasma cells following immunization or infection. The specific signals and mechanisms that are involved in GC B cell initiation, maintenance, and differentiation are not completely understood. Here, we investigated the expression and function of brain acid-soluble protein 1 (BASP1). Previously, no specific role for BASP1 in adaptive immunity has been established. BASP1 expression is induced in early GC B cells and increases over time as the GC progresses. We demonstrate, using short hairpin RNA knockdown and conditional deletion in mice, that BASP1 functions to maintain GC responses by promoting GC B cell survival and reducing memory B cells, and increasing plasma cell output. Finally, BASP1 induces a unique transcriptional program in GC B cells, enriched for interferon-γ- and interleukin-12-responsive genes. Thus, BASP1 regulates the survival and transcriptome of GC B cells as well as alters the nature of the memory B cell subset and plasma cell generation.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1β as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

Immunological memory provided by memory B cells, memory T cells and long-lived (memory) plasma cells yields long-lasting, effective cellular and humoral immune protection to previously encountered pathogens and vaccines. Memory B cells provide a fast and potent anamnestic antibody response following a reencounter with antigen, thereby providing an extra arm of immunity to pathogens that are not cleared by pre-existing antibodies. In addition, they qualify as potent antigen-presenting and immunoregulatory cells in secondary immune reactions. We are only beginning to understand the heterogeneity of memory B cells and their compartmentalisation. Plasma cells persist, potentially for a lifetime, in specialised niches in the tissues, especially in bone marrow and gut, but also in inflamed tissue. The molecular mechanisms governing the long-term persistence of memory B and plasma cells, in the apparent absence of antigen, are only now beginning to be unravelled. While long-lived plasma cells provide essential protection against pathogens, their longevity and resistance to irradiation, immunosuppression and therapies targeting B cells can be problematic for the treatment of chronic antibody-mediated diseases.

Memory B cells (MBCs) are phenotypically and functionally diverse, but their developmental origins remain undefined. Murine MBCs can be divided into subsets by expression of CD80 and PD-L2. Upon re-immunization, CD80/PD-L2 double-negative (DN) MBCs spawn germinal center B cells (GCBCs), whereas CD80/PD-L2 double-positive (DP) MBCs generate plasmablasts but not GCBCs. Using multiple approaches, including generation of an inducible GCBC-lineage reporter mouse, we demonstrate in a T cell-dependent response that DN cells formed independently of the germinal center (GC), whereas DP cells exhibited either extrafollicular (DP EX ) or GCBC (DP GC ) origins. Chromatin and transcriptional profiling revealed similarity of DN cells with an early memory precursor. Reciprocally, GCBC-derived DP cells shared distinct genomic features with GCBCs, while DP EX cells had hybrid features. Upon restimulation, DP EX cells were more prone to divide, while DP GC cells differentiated toward IgG1 + plasmablasts. Thus, MBC functional diversity is generated through distinct developmental histories, which imprint characteristic epigenetic patterns onto their progeny, thereby programming them for divergent functional responses.

Both B cell receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBCs) to synergistically induce c-MYC and phosphorylated S6 ribosomal protein (p-S6), markers of positive selection. How interleukin-21 (IL-21), a key T follicular helper (T FH )–derived cytokine, affects GCBCs is unclear. Like BCR and CD40 signals, IL-21 receptor (IL-21R) plus CD40 signals also synergize to induce c-MYC and p-S6 in GCBCs. However, IL-21R plus CD40 stimulation differentially affects GCBC fate compared with BCR plus CD40 ligation—engaging unique molecular mechanisms—as revealed by bulk RNA sequencing (RNA-seq), single-cell RNA-seq, and flow cytometry of GCBCs in vitro and in vivo. Whereas both signal pairs induced BLIMP1 in some GCBCs, only the IL-21R/CD40 combination induced IRF4 hi /CD138 + cells, indicative of plasma cell differentiation, along with CCR6 + /CD38 + memory B cell precursors. These findings reveal a second positive selection pathway in GCBCs, document rewired IL-21R signaling in GCBCs, and link specific T FH - and Ag-derived signals to GCBC differentiation.

Additional COVID-19 vaccines that are safe, easy to manufacture, and immunogenic are needed for global vaccine equity. Here, we developed a recombinant type 5 adenovirus vector encoding for the SARS-CoV-2-S1 subunit antigen and nucleocapsid as a fusion protein (Ad5.SARS-CoV-2-S1N) delivered to BALB/c mice through multiple vaccine administration routes. A single subcutaneous (S.C.) immunization with Ad5.SARS-CoV-2-S1N induced a similar humoral response, along with a significantly higher S1-specific cellular response, as a recombinant type 5 adenovirus vector encoding for S1 alone (Ad5.SARS-CoV-2-S1). Immunogenicity was improved by homologous prime boost strategies, using either S.C. or intranasal (I.N.) delivery of Ad5.SARS-CoV-2-S1N, and further improved through heterologous prime boost, with traditional intramuscular (I.M.) injection, using subunit recombinant S1 protein. Priming with low dose (1×10 10 v.p.) of Ad5.SARS-CoV-2-S1N and boosting with either wildtype recombinant rS1 or B.1.351 recombinant rS1 induced a robust neutralizing response, that was sustained against immune evasive Beta and Gamma SARS-CoV-2 variants, along with a long-lived plasma cell response in the bone marrow 29 weeks post vaccination. This novel Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity in mice and supports the further development of COVID-19 based vaccines incorporating the nucleoprotein as a target antigen.

Optimal vaccines are needed for sustained suppression of SARS-CoV-2 and other novel coronaviruses. Here, we developed a recombinant type 5 adenovirus vector encoding the gene for the SARS-CoV-2 S1 subunit antigen (Ad5.SARS-CoV-2-S1) for COVID-19 immunization and evaluated its immunogenicity in mice. A single immunization with Ad5.SARS-CoV-2-S1 via S.C. injection or I.N delivery induced robust antibody and cellular immune responses. Vaccination elicited significant S1-specific IgG, IgG1, and IgG2a endpoint titers as early as 2 weeks, and the induced antibodies were long lasting. I.N. and S.C. administration of Ad5.SARS-CoV-2-S1 produced S1-specific GC B cells in cervical and axillary LNs, respectively. Moreover, I.N. and S.C. immunization evoked significantly greater antigen-specific T-cell responses compared to unimmunized control groups with indications that S.C. injection was more effective than I.N. delivery in eliciting cellular immune responses. Mice vaccinated by either route demonstrated significantly increased virus-specific neutralization antibodies on weeks 8 and 12 compared to control groups, as well as BM antibody forming cells (AFC), indicative of long-term immunity. Thus, this Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity following delivery to mice by S.C. and I.N. routes of administration, supporting the further development of Ad-based vaccines against COVID-19 and other infectious diseases for sustainable global immunization programs.

Memory B cells (MBCs) protect the body from recurring infections. MBCs differ from their naive counterparts (NBCs) in many ways, but functional and surface marker differences are poorly characterized. In addition, although mice are the prevalent model for human immunology, information is limited concerning the nature of homology in B cell compartments. To address this, we undertook an unbiased, large-scale screening of both human and mouse MBCs for their differential expression of surface markers. By correlating the expression of such markers with extensive panels of known markers in high-dimensional flow cytometry, we comprehensively identified numerous surface proteins that are differentially expressed between MBCs and NBCs. The combination of these markers allows for the identification of MBCs in humans and mice and provides insight into their functional differences. These results will greatly enhance understanding of humoral immunity and can be used to improve immune monitoring.

Clinical definitions of asthma fail to capture the heterogeneity of immune dysfunction in severe, treatment-refractory disease. Applying mass cytometry and machine learning to bronchoalveolar lavage (BAL) cells, we find that corticosteroid-resistant asthma patients cluster largely into two groups: one enriched in interleukin (IL)-4(+) innate immune cells and another dominated by interferon (IFN)-gamma(+) T cells, including tissue-resident memory cells. In contrast, BAL cells of a healthier population are enriched in IL-10(+) macrophages. To better understand cellular mediators of severe asthma, we developed the Immune Cell Linkage through Exploratory Matrices (ICLite) algorithm to perform deconvolution of bulk RNA sequencing of mixed-cell populations. Signatures of mitosis and IL-7 signaling in CD206(-)FceRI(+)CD127(+)IL-4(+) innate cells in one patient group, contrasting with adaptive immune response in T cells in the other, are preserved across technologies. Transcriptional signatures uncovered by ICLite identify T-cell-high and T-cell-poor severe asthma patients in an independent cohort, suggesting broad applicability of our findings.

Germinal center B cells (GCBCs) are critical for generating long-lived humoral immunity. How GCBCs meet the energetic challenge of rapid proliferation is poorly understood. Dividing lymphocytes typically rely on aerobic glycolysis over oxidative phosphorylation for energy. Here we report that GCBCs are exceptional among proliferating B and T cells, as they actively oxidize fatty acids (FAs) and conduct minimal glycolysis. In vitro, GCBCs had a very low glycolytic extracellular acidification rate but consumed oxygen in response to FAs. [13C6]-glucose feeding revealed that GCBCs generate significantly less phosphorylated glucose and little lactate. Further, GCBCs did not metabolize glucose into tricarboxylic acid (TCA) cycle intermediates. Conversely, [13C16]-palmitic acid labeling demonstrated that GCBCs generate most of their acetyl-CoA and acetylcarnitine from FAs. FA oxidation was functionally important, as drug-mediated and genetic dampening of FA oxidation resulted in a selective reduction of GCBCs. Hence, GCBCs appear to uncouple rapid proliferation from aerobic glycolysis.

Long non-coding RNAs are a unique class of molecules involved in an exceptional variety of cellular processes, including transcriptional, translational, and epigenetic regulation. Here, we report the initial characterization of a lncRNA expressed specifically in the germinal center--organized sites of B cell proliferation, somatic hyper-mutation, and cellular differentiation that develop in response to antigenic challenge. The precise signals dictating how and when differentiated cells, such as memory B cells and long-lived plasma cells, exit the GC reaction remain incompletely understood. Thus, additional levels of molecular regulation are likely at work in the coordination of these diverse processes. We identify GCLnc1--a novel, nuclear-localized lncRNA--as a regulator of the GC reaction. As GCLnc1 is located adjacent to the murine Bcl6 locus, this lncRNA has the potential to modulate expression of key transcription factors controlling B cell identity and/or differentiation. Interestingly, forced over-expression of GCLnc1 in B cells led to upregulation of TFs associated with plasma cell identity, such as IRF4 and Blimp-1. When adoptively transferred in vivo, over-expression of GCLnc1 led to transduced B cells predominantly adopting a non-GC phenotype at the typical peak GC response, in addition to enhancing expression of IRF4 and Blimp-1. Genetic deletion of GCLnc1 led to reduced frequency of GC B cells, decreased Bcl6 expression, and dysregulated light zone/dark zone distribution in response to NP-KLH immunization. Collectively, we suggest GCLnc1 plays a key role in the integration of signaling events during the GC reaction, and may modulate the processes that drive GC exit.

Although human B cells have been extensively studied, most reports have used peripheral blood as a source. Here, we used a unique tissue resource derived from healthy organ donors to deeply characterize human B-cell compartments across multiple tissues and donors. These datasets revealed that B cells in the blood are not in homeostasis with compartments in other tissues. We found striking donor-to-donor variability in the frequencies and isotype of CD27+ memory B cells (MBCs). A comprehensive antibody-based screen revealed markers of MBC and allowed identification of novel MBC subsets with distinct functions defined according to surface expression of CD69 and CD45RB. We defined a tissue-resident MBC phenotype that was predominant in the gut but absent in blood. RNA-sequencing of MBC subsets from multiple tissues revealed a tissue-resident MBC gene signature as well as gut- and spleen-specific signatures. Overall, these studies provide novel insights into the nature and function of human B-cell compartments across multiple tissues.

The B cell response to Ehrlichia muris is dominated by plasmablasts (PBs), with few-if any-germinal centers (GCs), yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs) that express the transcription factor T-bet and harbor V-region mutations. Because Ehrlichia prominently infects the liver, we investigated the nature of liver B cell response and that of the spleen. B cells within infected livers proliferated and underwent somatic hypermutation (SHM). Vh-region sequencing revealed trafficking of clones between the spleen and liver and often subsequent local clonal expansion and intraparenchymal localization of T-bet+ MBCs. T-bet+ MBCs expressed MBC subset markers CD80 and PD-L2. Many T-bet+ MBCs lacked CD11b or CD11c expression but had marginal zone (MZ) B cell phenotypes and colonized the splenic MZ, revealing T-bet+ MBC plasticity. Hence, liver and spleen are generative sites of B cell responses, and they include V-region mutation and result in liver MBC localization.

SummaryGerminal centers (GC) are sites of rapid B‐cell proliferation in response to certain types of immunization. They arise in about 1 week and can persist for several months. In GCs, B cells differentiate in a unique way and begin to undergo somatic mutation of the Ig V regions at a high rate. GC B cells (GCBC) thus undergo clonal diversification that can affect the affinity of the newly mutant B‐cell receptor (BCR) for its driving antigen. Through processes that are still poorly understood, GCBC with higher affinity are selectively expanded while those with mutations that inactivate the BCR are lost. In addition, at various times during the extended GC reaction, some GCBC undergo differentiation into either long‐lived memory B cells (MBC) or plasma cells. The cellular and molecular signals that govern these fate decisions are not well‐understood, but are an active area of research in multiple laboratories. In this review, we cover both the history of this field and focus on recent work that has helped to elucidate the signals and molecules, such as key transcription factors, that coordinate both positive selection as well as differentiation of GCBC.

B cell immune responses initiate after recognition of foreign or self antigen, often but not always in the context of an innate immune stimulus (eg, due to infection) or adjuvant (as in a vaccination). This process typically begins in B cell zones including the follicular areas of secondary lymphoid tissues and the marginal zone of the B cell. Recognition of Ag by BCR initially leads to B cell activation that triggers chemokine receptor modulation and relocation to T zone/red pulp border areas, locations at which activated B cells could encounter cognate activated T cells.1, 2 Canonically, the response continues at this site with T-B interactions leading to mutual activation, proliferation, and differentiation. Within a few days, a portion of dividing B cells differentiates into short-lived plasmablasts that secrete antibody while also continuing to divide and interact with T cells. Also around this time, a fraction of both dividing B cells and T cells reenter nearby follicles and continue to interact and differentiate toward germinal center (GC) phenotypes, ultimately generating the GC response comprised of GC B cells (GCBC) and T follicular helper cells (TFH), both of which express the signature transcription factor (TF) BCL6.3, 4 Meanwhile, within a few days, the initial response at the T-B border decays and the short-lived plasmablasts die off. Within the GC, T-B interactions continue while AID-driven somatic hypermutation of IgV regions is fully induced,5-7 resulting in nearly every daughter cell having at least one sequence difference from the parent. This diversity forms the substrate for Ag-driven selection, which may be mediated by a combination of signals from both BCR and T cell help.8-11 The latter reads out the ability of GCBC to effectively present Ag that is captured through the BCR. Via cycles of mutation, selection, and division, clones with increased affinity—as well as increased intraclonal diversity—evolve and expand within the GC.12, 13 A small fraction of GCBC differentiates into long-lived progeny: memory B cells (MBC) and long-lived plasma cells. Interestingly, recent work shows that MBC are formed during the early part of the response, including before the GC even forms, while long-lived plasma cells form only at the latter part of the GC reaction, when GCBC have more mutations and overall higher affinity.14 Notably, the GC reaction also spins off some short-lived PCs earlier in the process.2 This canonical pathway has overshadowed in terms of scientific attention several alternative immune response pathways that have also been long known but less well studied15; in fact, these pathways may be much more commonly engaged in pathogen and vaccine responses than is typically thought to be the case. These responses are loosely termed "extrafollicular," since they represent the persistence of the initial response that had started outside of the follicle. In cases of certain infections, for example Salmonella, these EF responses can last for weeks in the absence of a GC response.16, 17 T cells can and do play a role in promoting these responses, often differentiating into a BCL6-expressing T "extrafollicular" helper cell type.18, 19 EF responses to certain types of Ags can proceed in the absence of T cells, so-called T-independent responses.20, 21 Also in the category of "alternative" pathways would be included those that induce so-called "age-related B cells" (ABCs), also termed T-bet positive B cells.22 The biological significance of these alternative pathways is less well understood. What are the conditions that preferentially induce alternative B cell responses? Do they induce memory, undergo affinity maturation, and lead to generation of long-lived plasma cells? These are all the active and reemerging areas of research. A major concept in putting together this volume of reviews was to include coverage of these types of response alongside updated reviews of processes and cells related to the classical GC pathway itself. Hence the title of this volume: Germinal Center and Extrafollicular B Cell Responses. This volume has assembled reviews from many contributors to our understanding of these modes of B cell response. They can be grouped loosely together based on specific subtopics as well as with respect to some global themes. The figure (Figure 1) represents how the reviews relate to these aspects of the B cell immune response and its malignant transformation. The stages or processes on which each review focuses are indicated on the cartoon, with some reviews appearing in multiple places. This "visual abstract" will allow the reader interested in a particular aspect to direct oneself to the relevant reviews. Below we further detail some of these topics and global themes. With apologies to co-authors who are fully credited in the reviews themselves, for clarity here we will refer to the senior authors of each article as the singular writer. Several reviews focus on aspects of the mechanics of the GC itself, albeit at different levels. Shulman, Qi, and Haberman all focus on B cell:T cell interactions in the GC. Shulman covers the key role that SLAM family members, plexins, and ultimately integrins play in shaping adhesive interactions and at the same time signaling to cells and thereby affecting other key signals such as CD40:CD40L. Qi and Haberman focus on the details of the reciprocal B-T interaction in the GC and steps leading up to the fully mature GC. Shlomchik and Shulman discuss signaling events within GC B cells that underpin positive selection; Shlomchik emphasizes how intracellular signals from both BCR and CD40 are reprogrammed in GCBC compared to naïve B cells in order to promote positive selection. Qi emphasizes reciprocal B-T interactions that lead to T cell-mediated positive selection and affinity maturation of GCBC. In a complementary way, Kurosaki discusses signals and TF networks that regulate the differentiation of GCBC into plasma cell precursors that beget LLPC. Related to this aspect, Good-Jacobson takes a "inside to out" perspective on how B cells intrinsically reprogram themselves by alterations in epigenetic DNA and chromatin marking, that in turn regulate GC, plasma, and memory B cell function and differentiation. Her review integrates work from her lab and others to both describe progressive changes in expression of key enzymes that modulate epigenetic marks as well as control B cell responses to immunization. The review is a comprehensive tour through multiple key genetic experiments that have explored the effects of genetic deletion or manipulation of these DNA and chromatin modifying systems. Multiple reviews focus on the T cell component of the GC. Qi discusses signals from B cells to TFH that either elicit direct T cell help or serve to maintain the TFH phenotype. Craft reviews the developmental processes that lead to TFH differentiation, as well as the heterogeneity among the group of T cells that express the signature TF BCL6 and are specialized to interact with responding B cells. His review discusses both cytokine signals and surface molecule expression that are key to each of these stages, linking these signals and molecules to expression of key TFs that in turn coordinate TFH differentiation. Baumjohann takes a unique perspective on TFH, using the filter of microRNA expression and function to outline how these control TFH formation, differentiation, and function. He reveals how these microRNAs interact with and regulate more well-known TF and signaling networks inherent to TFH. Finally, in this domain, Graca reviews a unique and still-mysterious subset of TFH. Graca and colleagues were among three groups that simultaneously reported the existence of BCL6-expressing TFH that also expressed FoxP3, the quintessential TF associated with regulatory T cells23-25; these TFH were designated T follicular regulatory T cells, or TFR. Overall, a predominance of evidence (but not all) suggests that TFR negatively regulate humoral immunity; nonetheless, the origins and functions of TFR remain controversial. In this volume, Graca presents a comprehensive overview of this subject, including both human and mouse and covering TFR-like cells in the blood and tissues. As alluded to, not all B cell responses take the GC path. Indeed, the literature indicates that many scenarios lead to EF or similar responses. These include Salmonella infection, Ehrlichia infection, malarial infection, multiple "T-independent" immune responses including responses to encapsulated bacteria, and lupus autoimmunity in some animal models and a significant subset of human patients. The responding B cell type for such responses has sometimes been identified as B-1 or marginal zone, though follicular B cells can also contribute to such responses. Allman reviews and provides an interesting perspective on a spectrum of such responses, proposing that—counter to most dogma—T cell-independent EF responses can and often do generate long-lived humoral immunity including LLPC and MBC. Sanz focuses on the B cell response in patients with humans, arguing cogently for a major contribution by the EF pathway. Immune responses involving T-bet expressing B cells seem in a category of their own, yet they seem to emanate from potentially different sources and result in different outputs, depending on the context. They are often associated with the same types of responses in which the EF pathway predominates, such as Ehrlichia infection or SLE. However, other types of infection, for example influenza, that elicit GC responses can also elicit T-bet expressing responding cells and longer-living progeny. In his contribution to this volume, Cancro reviews and provides a synthesis of this rapidly evolving area of B cell biology. Sanz adds the perspective of human lupus with respect to the origins and function of T-bet positive cells in that context. Plasma cells and plasmablasts (collectively termed "antibody-forming cells," AFC) are terminally differentiated cells that are generated at various points by all of the B cell response pathways. Due to either their scarcity or in the case of plasmablasts, their ephemeral nature, insights into the origins and heterogeneity of this lineage are limited compared to the importance of these cells as the definitive source of all antibody. Yet, progress is being made in terms of understanding TF networks that generate them (as discussed by both Kurosaki and Bhattacharya). In particular, Bhattacharya reviews the metabolic underpinning of plasma cell longevity, and relates this to reported metabolic states of other differentiated cells in the B lineage, including GCBC and MBC. Sanz and Cancro focus more on short-lived AFC, especially those that express T-bet. Allman again covers the spectrum of short- and long-lived AFC. Effective B cell responses carry with them a significant liability for malignant transformation. Indeed, the vast majority of lymphomas are of B cell lineage and most are thought to derive from GCBC or post-GCBC. Aside from the intense proliferation and clonal expansion associated with GCBC responses, which in itself is a risk factor for transformation, AID is strongly expressed in GCBC, mediating class switch and somatic hypermutation of IgV regions. The off-target effects of AID, as well as aberrant class switching both contribute to meaningful genome instability that can be directly traced as causative of many types of B cell lymphoma.26, 27 The mechanisms by which GC-derived lymphomas arise, and how that relates both to lymphoma heterogeneity and subtypes as well as to normal GCBC biology, are the subjects of four reviews in this volume (Amati, Casola, Melnick, and Pasqualucci). Amati and Casola focus on Myc-driven lymphomas. Amati focuses on the mechanisms by which Myc overexpression induces transformation in lymphoma. The context is with respect to various second genetic hits, especially BCL2 overexpression, that synthetically lead to lymphoma. These insights in turn have implications for treatment strategies, as Amati explains. Casola delves into the role of BCR signals in conferring fitness to Myc-driven lymphomas in both animal models and human disease. He covers the very interesting observation that BCR signals confer advantage such that lower fitness of BCR-negative lymphomas is only revealed in the face of competitor BCR-positive cells in vitro and in vivo. There are important signaling and clinical implications and correlations to this, which are covered as well as the review concludes. Melnick and Pasqualucci, on the other hand, each review a number of genetic alterations that are commonly seen in non-Hodgkin's lymphoma, covering monumental efforts by their and multiple other labs to piece together how these mutations lead to transformation. These mutations are often in genes encoding epigenetic modifiers, including those discussed by Good-Jacobson; they are expected to have pervasive effects in altering GCBC proliferation and differentiation, thus promoting transformation. Among the additional mechanisms considered by both reviews are: TF reprogramming and networks (including MYC), BCR and innate immune signals, TNFR-family signals, evasion of immune surveillance, metabolic reprogramming, blocking apoptosis (eg, BCL2), and blocking differentiation. The dissection of these various mutations and how they function in combination has profound implications for lymphoma classification, targeted therapy development and accordingly, personalized medicine. In addition to the perspective of cellular differentiation as just discussed, it is interesting to view the collection of reviews in this volume in terms of processes that they discuss (Table 1). Signaling is a major subject in reviews by Shlomchik, Shulman, Haberman in normal GCBC and by Casola, Melnick, and Pasqualucci in GCBC-derived lymphomas. TF networks are described in multiple reviews. They are covered in normal B lineage cells by Kurosaki, Melnick, Pasqualucci, Good-Jacobson, and Cancro and in malignant cells by Amati, Melnick, and Pasqualucci. Both Craft and Graca discuss how TF networks control TFH/TFR differentiation and function. Baumjohann, Good-Jacobson, Melnick, and Pasqualucci in turn focus on the related topic of microRNA and epigenetic control of cell processes. Metabolic reprogramming is another theme found across reviews and cell types. This is the central topic of Bhattacharya with emphasis on plasma cells. Amati, Casola, Melnick, and Pasqualucci all touch on this topic in GCBC-derived lymphomas, particularly as MYC has pervasive metabolic effects. Craft, in parallel, describes how metabolism can control TFH differentiation. B-T interactions, naturally, are a subject in multiple reviews, most notably: Craft, Graca, Haberman, Kurosaki, Shulman, and Qi. All of these describe the bidirectional orchestration of differentiation that occurs into and leading up to the GC reaction. Lastly, several reviews relate their basic topics to autoimmune disease. These include contributions by Sanz, Baumjohann, Cancro, Graca, and Craft that discuss immunoregulation and how that can go wrong leading to self-reactivity and disease. Taken together the reviews in this volume thus represent an up to date and in-depth cross-section of the many related topics around B cell immune responses. They are multi-layered, like the responses themselves, and overlap in terms of cellular processes and themes that should be both informative and thought-provoking. It is hoped that this short introduction can serve as a guide and set of perspectives by which the reader can approach these reviews. At a larger scale, the insights reviewed in this volume have implications for understanding basic biology, disease, immune responses, malignancy, and therapy. The authors thank Rebecca Elsner and Danny Wikenheiser of the Shlomchik lab for their useful comments. Supported by NIH grants (1R01 AI105018-05 and R01 AI043603-18) to MJS. The authors declare no conflict of interest.

B cell antigen receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBCs) to optimize selection for high-affinity B cells. In GCBC, BCR signals are constrained, but the mechanisms are not well understood. Here we describe a GC-specific, AKT-kinase-driven negative feedback loop that attenuates BCR signaling. Mass spectrometry revealed that AKT target activity was altered in GCBCs compared with naive B cells. Retargeting was linked to differential AKT T308 and S473 phosphorylation, in turn controlled by GC-specific upregulation of phosphoinositide-dependent protein kinase PDK1 and the phosphatase PTEN. In GCBCs, AKT preferentially targeted CSK, SHP-1 and HPK1, which are negative regulators of BCR signaling. We found that phosphorylation enhances enzymatic activity of these proteins, creating a negative feedback loop that dampens upstream BCR signaling. AKT inhibition relieved this negative feedback and enhanced activation of BCR-proximal kinase LYN, as well as downstream BCR signaling molecules in GCBCs.

We have recently shown (Luo et al., Immunity, in press) that, compared to naïve B cells (NBC), both B cell receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBC). BCR signaling in GCBC induces only transient activation of Syk, leading to partial activation of the PI3K-AKT pathway, generating p-Foxo1 but not p-S6. There is minimal BCR-directed activation of other downstream pathways such as NF-κB. Conversely for CD40 ligation, only NF-κB but not PI3K is activated in GCBC. Initially we found that Lyn, the most upstream kinase in the BCR cascade, has more inhibitory vs activating phosphorylation in GCBC. To understand how this occurs, and why AKT targets only selected substrates in GCBC, we used an AKT-substrate-specific mAb to immunoprecipitate AKT targets in GCBC. We analyzed these by mass spectrometry to identify potential GCBC-specific AKT targets. This analysis revealed that AKT itself was retargeted in GCBC compared to NBC. In GCBC, AKT appeared to preferentially target negative regulators, including CSK, a kinase of the inhibitory Tyr of Lyn. In vitro kinase and functional studies confirmed that AKT could phosphorylate CSK. We then demonstrated that this phosphorylation results in markedly increased CSK enzymatic activity to phosphorylate Lyn. Consistent with this, we found Lyn activity in GC B cells is inhibited by CSK catalyzed phosphorylation. Critically, inhibiting AKT substantially enhanced activation vs inhibitory Tyr phosphorylation of Lyn, and enhanced Syk phosphorylation and downstream signals upon BCR stimulation in GCBC. Taken together, our findings identify and document a unique AKT negative feedback loop that dampens proximal BCR signaling in GC B cells.

Positive selection of germinal center (GC) B cells is driven by B cell receptor (BCR) affinity and requires help from follicular T helper cells. The transcription factors c-Myc and Foxo1 are critical for GC B cell selection and survival. However, how different affinity-related signaling events control these transcription factors in a manner that links to selection is unknown. Here we showed that GC B cells reprogram CD40 and BCR signaling to transduce via NF-κB and Foxo1, respectively, whereas naive B cells propagate both signals downstream of either receptor. Although either BCR or CD40 ligation induced c-Myc in naive B cells, both signals were required to highly induce c-Myc, a critical mediator of GC B cell survival and cell cycle reentry. Thus, GC B cells rewire their signaling to enhance selection stringency via a requirement for both antigen receptor- and T cell-mediated signals to induce mediators of positive selection.