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Macrophages are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL). Macrophage estimation by immunohistochemistry shows varying prognostic significance across studies in DLBCL, and does not provide a comprehensive analysis of macrophage subtypes. Here, using digital spatial profiling with whole transcriptome analysis of CD68+ cells, we characterize macrophages in distinct spatial niches of reactive lymphoid tissues (RLTs) and DLBCL. We reveal transcriptomic differences between macrophages within RLTs (light zone /dark zone, germinal center/ interfollicular), and between disease states (RLTs/ DLBCL), which we then use to generate six spatially-derived macrophage signatures (MacroSigs). We proceed to interrogate these MacroSigs in macrophage and DLBCL single-cell RNA-sequencing datasets, and in gene-expression data from multiple DLBCL cohorts. We show that specific MacroSigs are associated with cell-of-origin subtypes and overall survival in DLBCL. This study provides a spatially-resolved whole-transcriptome atlas of macrophages in reactive and malignant lymphoid tissues, showing biological and clinical significance.

Traditional bolus vaccines typically require multiple doses, which complicates the vaccination process and may cause missed shots, leading to sub-optimal immunity and reduced vaccine effectiveness. Herein, a gel-based long-acting vaccine system with self-adjuvant properties based on laponite was constructed to simplify vaccination procedures and improve vaccine effectiveness. Firstly, the gel system could recruit multiple types of immune cells to form immune niches. Secondly, it could achieve sustained delivery of antigens to lymph nodes by active transport and passive drainage. Then, the gel system triggered the formation of a large number of germinal centers, which elicited enhanced and durable humoral immune responses, as well as strong cellular immune responses. As a result, it eventually showed good prophylactic and therapeutic effects in a variety of tumor models including melanoma, colorectal cancer and peritoneal metastasis models. By further combining the immunoadjuvant CpG ODN and cytokine IL-12, the effect of the gel-vaccine could be further enhanced. In a murine peritoneal metastasis model of colorectal carcinoma, a single administration of the gel-vaccine resulted in complete tumor eradication in 8/9 mice. In summary, this study developed an immunologically active gel-vaccine system. And as a robust and versatile vaccine platform, by loading different antigens and adjuvants, this gel-vaccine system is expected to realize its better therapeutic potential. [Display omitted] • The laponite-based gel system recruited multiple types of immune cells to form immune niches. • It achieved sustained delivery of antigens to lymph nodes by active transport and passive drainage. • It triggered the formation of a large number of germinal centers, which elicited enhanced and durable immune responses. • The laponite-based gel delivery system could simplify vaccination procedures and improve vaccine effectiveness. [ABSTRACT FROM AUTHOR]

Germinal center (GC) B cells with functional B cell receptors (BCRs) transition from the dark zone (DZ) to the light zone (LZ) through BCR tonic-like signals (checkpoint 1). Antigen-induced BCR crosslinking provides a survival signal and primes LZ GC B cells to receive synergistic CD4+ follicular helper T (Tfh) cell help signals (checkpoint 2). A strong and sustained BCR signal induces reactive oxygen species (ROS)-mediated apoptosis, but this can be counteracted by the Tfh help signal (checkpoint 3). The balance between BCR and Tfh cell help signals ensures the clonal expansion that occurs in the DZ. Positive selection of germinal center B cells depends on the affinity of their antigen receptors. Emerging evidence shows that optimal B cell receptor (BCR) signaling, as well as a balance between BCR and T cell help signals, is necessary for antibody affinity maturation in the generation of antibody diversity. When mature B cells are activated by antigens, the selection of these activated B cells takes place particularly during T cell-dependent immune responses in which an improved antibody repertoire is generated through somatic hypermutation in germinal centers (GCs). In this process the importance of antigen presentation by GC B cells, and subsequent T follicular helper (Tfh) cell help in positive selection of GC B cells, has been well appreciated. By contrast, the role of B cell receptor (BCR) signaling per se remains unclear. Strong experimental support for the involvement of BCR signaling in GC B cell selection has now been provided. Interestingly, these studies suggest that several checkpoints operating through the BCR ensure affinity maturation. [ABSTRACT FROM AUTHOR]

Follicular helper CD4hi T cells (TFH) are a major cellular pool for the maintenance of the HIV reservoir. Therefore, the delineation of the follicular (F)/germinal center (GC) immune landscape will significantly advance our understanding of HIV pathogenesis. We have applied multiplex confocal imaging, in combination with the relevant computational tools, to investigate F/GC in situ immune dynamics in viremic (vir-HIV), antiretroviral-treated (cART HIV) People Living With HIV (PLWH) and compare them to reactive, non-infected controls. Lymph nodes (LNs) from viremic and cART PLWH could be further grouped based on their TFH cell densities in high-TFH and low-TFH subgroups. These subgroups were also characterized by different in situ distributions of PD1hi TFH cells. Furthermore, a significant accumulation of follicular FOXP3hiCD4hi T cells, which were characterized by a low scattering in situ distribution profile and strongly correlated with the cell density of CD8hi T cells, was found in the cART-HIV low-TFH group. An inverse correlation between plasma viral load and LN GrzBhiCD8hi T and CD16hiCD15lo cells was found. Our data reveal the complex GC immune landscaping in HIV infection and suggest that follicular FOXP3hiCD4hi T cells could be negative regulators of TFH cell prevalence in cART-HIV. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusions Recently, it has been questioned whether vaccination of patients with inflammatory (auto)immune diseases under anti‐tumor necrosis factor (TNF) treatment leads to impaired vaccine‐induced immune responses and protection against breakthrough infections. However, the effects of TNF blockade on short‐ and long‐term immune responses after repeated vaccination remain unclear. Vaccination studies have shown that initial short‐term IgG antibodies (Abs) carry highly galactosylated and sialylated Fc glycans, whilst long‐term IgG Abs have low levels of galactosylation and sialylation and are most likely generated by long‐lived plasma cells (PCs) derived primarily from the germinal center (GC) response. Thus, IgG Fc glycosylation patterns may be applicable to distinguish short‐ and long‐term vaccine responses after repeated vaccination under the influence of anti‐TNF treatment.We used COVID‐19 vaccination as a model to investigate vaccine‐induced IgG subclass levels and Fc glycosylation patterns, B cell subsets, and effector functions of short‐ and long‐term Ab responses after up to three vaccinations in patients on anti‐TNF or other immunosuppressive treatments and in healthy individuals. Using TriNetX, a global healthcare database, we determined the risk of SARS‐CoV‐2 breakthrough infections in vaccinated patients treated with anti‐TNF or other immunosuppressive drugs.Anti‐TNF treatment reduced the long‐term abundance of all anti‐S IgG subclasses with low levels of galactosylation and sialylation. Re‐activation of potential memory B cells initially generated highly galactosylated and sialylated IgG antibodies, which were progressively reduced after each booster dose in anti‐TNF‐treated patients, especially in the elderly. The reduced short‐ and long‐term IgG (1) levels in anti‐TNF‐treated patients correlated with diminished functional activity and an increased risk for the development of COVID‐19.The data suggest that anti‐TNF treatment reduces both GC‐dependent long‐lived PCs and GC‐dependent memory B cell‐derived short‐lived PCs, hence both the long‐ and short‐term IgG subclass responses, respectively, after repeated vaccination. We propose that anti‐TNF therapy, especially in the elderly, reduces the benefit of booster vaccination. [ABSTRACT FROM AUTHOR]

Tertiary lymphoid structures (TLS) represent the ectopic aggregations of immune cells arising during chronic inflammation or tumor progression. In cancer, TLS are often associated with beneficial clinical outcomes in patients undergoing immunotherapy, underscoring their prognostic and predictive significance. Mature TLS, characterized by germinal centers and areas of T-cell and B-cell aggregation, are considered primary locations for activating and maintaining both humoral and cellular anti-tumor immune effects. Despite their recognized importance, the mechanisms driving the formation of mature TLS in cancer and their influence on the immune response within tumors remain insufficiently understood. Therefore, this review aims to comprehensively explore the structural composition, development mechanisms, maturity impact factors, immunological function, and innovative therapeutic strategies of mature TLS within the tumor microenvironment. The research summarized herein offers novel insights and considerations for therapeutic approaches to promote TLS generation and maturation in patients with cancer, representing a promising avenue for future cancer therapies. [ABSTRACT FROM AUTHOR]

Heterologous prime-boost has broken the protective immune response bottleneck of the COVID-19 vaccines. however, the underlying mechanisms have not been fully elucidated. Here, we investigated antibody responses and explored the response of germinal center (GC) to priming with inactivated vaccines and boosting with heterologous adenoviral-vectored vaccines or homologous inactivated vaccines in mice. Antibody responses were dramatically enhanced by both boosting regimens. Heterologous immunization induced more robust GC activation, characterized by increased Tfh cell populations and enhanced helper function. Additionally, increased B-cell activation and antibody production were observed in a heterologous regimen. Libra-seq was used to compare the differences of S1-, S2- and NTD-specific B cells between homologous and heterologous vaccination, respectively. S2- specific CD19+ B cells presented increased somatic hypermutations (SHMs), which were mainly enriched in plasma cells. Moreover, a heterologous booster dose promoted the clonal expansion of B cells specific to S2 and NTD regions. In conclusion, the functional role of Tfh and B cells following SARS-CoV-2 heterologous vaccination may be important for modulating antibody responses. These findings provide new insights for the development of SARS-CoV- 2 vaccines that induce more robust antibody response. [ABSTRACT FROM AUTHOR]

In this article for the Highlight of 2023 series, we discuss recent advances in the fundamental biology of the germinal center response. These discoveries provide important insights as to how the germinal center contributes to protection against infection, and also highlights opportunities for future vaccine development. [ABSTRACT FROM AUTHOR]

We propose a stochastic framework to describe the evolution of the B-cell repertoire during germinal center (GC) reactions. Our model is formulated as a multitype age-dependent branching process with time-varying immigration. The immigration process captures the mechanism by which founder B cells initiate clones by gradually seeding GC over time, while the branching process describes the temporal evolution of the composition of these clones. The model assigns a type to each cell to represent attributes of interest. Examples of attributes include the binding affinity class of the B cells, their clonal family, or the nucleotide sequence of the heavy and light chains of their receptors. The process is generally non-Markovian. We present its properties, including as t → ∞ when the process is supercritical, the most relevant case to study expansion of GC B cells. We introduce temporal alpha and beta diversity indices for multitype branching processes. We focus on the dynamics of clonal dominance, highlighting its non-stationarity, and the accumulation of somatic hypermutations in the context of sequential immunization. We evaluate the impact of the ongoing seeding of GC by founder B cells on the dynamics of the B-cell repertoire, and quantify the effect of precursor frequency and antigen availability on the timing of GC entry. An application of the model illustrates how it may help with interpretation of BCR sequencing data. [ABSTRACT FROM AUTHOR]

Abstract Background The role of the scavenger receptor CD36 in cell metabolism and the immune response has been investigated mainly in macrophages, dendritic cells, and T cells. However, its involvement in B cells has not been comprehensively examined. Methods To investigate the function of CD36 in B cells, we exposed Cd36 fl/fl MB1 cre mice, which lack CD36 specifically in B cells, to apoptotic cells to trigger an autoimmune response. To validate the proteins that interact with CD36 in primary B cells, we conducted mass spectrometry analysis following anti-CD36 immunoprecipitation. Immunofluorescence and co-immunoprecipitation were used to confirm the protein interactions. Results The data revealed that mice lacking CD36 in B cells exhibited a reduction in germinal center B cells and anti-DNA antibodies in vivo. Mass spectrometry analysis identified 30 potential candidates that potentially interact with CD36. Furthermore, the interaction between CD36 and the inhibitory Fc receptor FcγRIIb was first discovered by mass spectrometry and confirmed through immunofluorescence and co-immunoprecipitation techniques. Finally, deletion of FcγRIIb in mice led to decreased expression of CD36 in marginal zone B cells, germinal center B cells, and plasma cells. Conclusions Our data indicate that CD36 in B cells is a critical regulator of autoimmunity. The interaction of CD36-FcγRIIb has the potential to serve as a therapeutic target for the treatment of autoimmune disorders.

The opioid epidemic continues to be a major public health issue that includes millions of people who inject drugs (PWID). PWID have increased incidence of serious infections, including HIV as well as metabolic and inflammatory sequelae. We sought to discern the extent of systemic alterations in humoral immunity associated with injection drug use, including alterations in the plasma proteome and its regulation of B cell responsiveness. Comprehensive plasma proteomics analysis of HIV negative/hepatitis C negative individuals with a history of recent injection heroin use was performed using mass spectrometry and ELISA. The effects of plasma from PWID and healthy controls on the in vitro proliferation and transcriptional profile of B cell responses to stimulation were determined by flow cytometry and RNA-Seq. The plasma proteome of PWID was distinct from healthy control individuals, with numerous immune-related analytes significantly altered in PWID, including complement (C3, C5, C9), immunoglobulin (IgD, IgM, kappa light chain), and other inflammatory mediators (CXCL4, LPS binding protein, C-reactive protein). The plasma of PWID suppressed the in vitro proliferation of B cells. Transcriptome analysis indicated that PWID plasma treatment increased B cell receptor and CD40 signaling and shifted B cell differentiation from plasma cell-like toward germinal center B cell-like transcriptional profiles. These results indicate that the systemic inflammatory milieu is substantially altered in PWID and may impact their B cell responses. [ABSTRACT FROM AUTHOR]

The germinal center response or reaction (GCR) is a hallmark event of adaptive humoral immunity. Unfolding in the B cell follicles of the secondary lymphoid organs, a GC culminates in the production of high-affinity antibody-secreting plasma cells along with memory B cells. By interacting with follicular dendritic cells (FDC) and T follicular helper (Tfh) cells, GC B cells exhibit complex spatiotemporal dynamics. Driving the B cell dynamics are the intracellular signal transduction and gene regulatory network that responds to cell surface signaling molecules, cytokines, and chemokines. As our knowledge of the GC continues to expand in depth and in scope, mathematical modeling has become an important tool to help disentangle the intricacy of the GCR and inform novel mechanistic and clinical insights. While the GC has been modeled at different granularities, a multiscale spatial simulation framework - integrating molecular, cellular, and tissue-level responses - is still rare. Here, we report our recent progress toward this end with a hybrid stochastic GC framework developed on the Cellular Potts Model-based CompuCell3D platform. Tellurium is used to simulate the B cell intracellular molecular network comprising NF-kB, FOXO1, MYC, AP4, CXCR4, and BLIMP1 that responds to B cell receptor (BCR) and CD40-mediated signaling. The molecular outputs of the network drive the spatiotemporal behaviors of B cells, including cyclic migration between the dark zone (DZ) and light zone (LZ) via chemotaxis; clonal proliferative bursts, somatic hypermutation, and DNA damage-induced apoptosis in the DZ; and positive selection, apoptosis via a death timer, and emergence of plasma cells in the LZ. Our simulations are able to recapitulate key molecular, cellular, and morphological GC events, including B cell population growth, affinity maturation, and clonal dominance. This novel modeling framework provides an opensource, customizable, and multiscale virtual GC simulation platform that enables qualitative and quantitative in silico investigations of a range of mechanistic and applied research questions on the adaptive humoral immune response in the future. [ABSTRACT FROM AUTHOR]

Weakened germinal center responses by the aged immune system result in diminished immunity against pathogens and reduced efficacy of vaccines. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have decreased expansion of antigen‐specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with accumulated Tfh cells, even in naïve mice. Despite increased numbers, aged Tfh had reduced expression of master transcription factor BCL6 and increased expression of the ectonucleotidase CD39. In vitro activation revealed that proliferative capacity was maintained in aged CD4+ T cells, but not the costimulatory molecule CD40L. When activated in vitro by aged antigen‐presenting cells, young CD4+ naïve T cells generated reduced numbers of activated cells with upregulated CD40L. To determine the contribution of cell‐extrinsic influences on antigen‐specific Tfh induction, young, antigen‐specific B and CD4+ T cells were adoptively transferred into aged hosts prior to peptide immunization. Transferred cells had reduced expansion and differentiation into germinal center B cell and Tfh and reduced antigen‐specific antibody titers when compared to young hosts. Young CD4+ T cells transferred aged hosts differentiated into Tfh cells with reduced PD‐1 and BCL6 expression, and increased CD39 expression, though they maintained their mitochondrial capacity. These results highlight the role of the lymphoid microenvironment in modulating CD4+ T cell differentiation, which contributes to impaired establishment and maintenance of germinal centers. [ABSTRACT FROM AUTHOR]

The development of psoriasis is mainly driven by the dysregulation of T cells within the skin, marking a primary involvement of these cells in the pathogenesis. Although B cells are integral components of the immune system, their role in the initiation and progression of psoriasis is not as pivotal as that of T cells. The paradox of B cell suggests that, while it is crucial for adaptive immunity, B cells may contribute to the exacerbation of psoriasis. Numerous ideas proposed that there are potential relationships between psoriasis and B cells especially within germinal centers (GCs). Recent research projected that B cells might be triggered by autoantigens which then induced molecular mimicry to alter B cells activity within GC and generate autoantibodies and pro‐inflammatory cytokines, form ectopic GC, and dysregulate the proliferation of keratinocytes. Hence, in this review, we gathered potential evidence indicating the participation of B cells in psoriasis within the context of GC, aiming to enhance our comprehension and advance treatment strategies for psoriasis thus inviting many new researchers to investigate this issue. [ABSTRACT FROM AUTHOR]

Background: The role of the scavenger receptor CD36 in cell metabolism and the immune response has been investigated mainly in macrophages, dendritic cells, and T cells. However, its involvement in B cells has not been comprehensively examined. Methods: To investigate the function of CD36 in B cells, we exposed Cd36fl/flMB1cre mice, which lack CD36 specifically in B cells, to apoptotic cells to trigger an autoimmune response. To validate the proteins that interact with CD36 in primary B cells, we conducted mass spectrometry analysis following anti-CD36 immunoprecipitation. Immunofluorescence and co-immunoprecipitation were used to confirm the protein interactions. Results: The data revealed that mice lacking CD36 in B cells exhibited a reduction in germinal center B cells and anti-DNA antibodies in vivo. Mass spectrometry analysis identified 30 potential candidates that potentially interact with CD36. Furthermore, the interaction between CD36 and the inhibitory Fc receptor FcγRIIb was first discovered by mass spectrometry and confirmed through immunofluorescence and co-immunoprecipitation techniques. Finally, deletion of FcγRIIb in mice led to decreased expression of CD36 in marginal zone B cells, germinal center B cells, and plasma cells. Conclusions: Our data indicate that CD36 in B cells is a critical regulator of autoimmunity. The interaction of CD36-FcγRIIb has the potential to serve as a therapeutic target for the treatment of autoimmune disorders. [ABSTRACT FROM AUTHOR]

Germinal center (GC) responses are essential for establishing protective, longlasting immunity through the differentiation of GC B cells (BGC) and plasma cells (BPC), along with the generation of antigen-specific antibodies. Among the various pathways influencing immune responses, the STING (Stimulator of Interferon Genes) pathway has emerged as significant, especially in innate immunity, and extends its influence to adaptive responses. In this study, we examined how the STING ligand cGAMP can modulate these key elements of the adaptive immune response, particularly in enhancing GC reactions and the differentiation of BGC, BPC, and follicular helper T cells (TFH). Employing in vivo models, we evaluated various antigens and the administration of cGAMP in Alum adjuvant, investigating the differentiation of BGC, BPC, and TFH cells, along with the production of antigen-specific antibodies. cGAMP enhances the differentiation of BGC and BPC, leading to increased antigen-specific antibody production. This effect is shown to be type I Interferon-dependent, with a substantial reduction in BPC frequency upon interferon (IFN)-b blockade. Additionally, cGAMP's influence on TFH differentiation varies over time, which may be critical for refining vaccine strategies. The findings elucidate a complex, antigen-specific influence of cGAMP on T and B cell responses, providing insights that could optimize vaccine efficacy. [ABSTRACT FROM AUTHOR]

Humanized immune system (HIS) mice have been developed and used as a small surrogate model to study human immune function under normal or disease conditions. Although variations are found between models, most HIS mice show robust human T cell responses. However, there has been unsuccessful in constructing HIS mice that produce high-affinity human antibodies, primarily due to defects in terminal B cell differentiation, antibody affinity maturation, and development of primary follicles and germinal centers. In this review, we elaborate on the current knowledge about and previous attempts to improve human B cell development in HIS mice, and propose a potential strategy for constructing HIS mice with improved humoral immunity by transplantation of human follicular dendritic cells (FDCs) to facilitate the development of secondary follicles. [ABSTRACT FROM AUTHOR]

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Extrafollicular plasmablast responses (EFRs) are considered to generate antibodies of low affinity that offer little protection from infections. Paradoxically, high avidity antigen-B cell receptor engagement is thought to be the main driver of B cell differentiation, whether in EFRs or slower-developing germinal centers (GCs). Here we show that influenza infection rapidly induces EFRs, generating protective antibodies via Toll-like receptor (TLR)-mediated mechanisms that are both B cell intrinsic and extrinsic. B cell-intrinsic TLR signals support antigen-stimulated B cell survival, clonal expansion, and the differentiation of B cells via induction of IRF4, the master regulator of B cell differentiation, through activation of NF-kB c-Rel. Provision of sustained TLR4 stimulation after immunization shifts the fate of virus-specific B cells towards EFRs instead of GCs, prompting rapid antibody production and improving their protective capacity over antigen/alum administration alone. Thus, inflammatory signals act as B cell fate-determinants for the rapid generation of protective antiviral extrafollicular responses.

OBJECTIVE: To determine the Frequency of Germinal Center B (GCB) type Diffuse Large B Cell Lymphoma (DLBCL) in Karachi, Pakistan, and to screen the DLBCL patients for EZH2 mutation. METHODOLOGY: A cross-sectional study was conducted on DLBCL patients, and IHC analysis, PCR, and sequencing of the EZH2 gene were used. This study was conducted from August 2016 to July 2020. Surgical specimens received in the histopathology section of DDRRL centrally and from different collection units throughout Sindh having been diagnosed with DLBCL were collected. Three markers (CD10, BCL6, and MUM1) were used for subtyping. DNA was manually extracted from the FFPE Blocks of DLBCL cases using phenol: chloroform: isopropanol alcohol. Polymerase chain reaction (PCR) was performed, and samples were sent from Dow University to Korea (Macrogen) for sequencing. The amplified product was 189bp. Primers were used for EZH2 to flank the region containing the tyrosine domain on exon 16 and exon 18. RESULTS: Out of 116 cases, 60.3% and 39.7% were male and female, respectively. The mean age was 49.73±16.38 years. The mean survival duration of cases diagnosed with GCB was 8.21±7.39 months. Polymorphism of EZH2 mutation (Tyr 701 Asn) was found to be positive in GCB-DLBCL for three samples (16.6%). CONCLUSION: In the current study, Sanger sequencing was used to detect any mutation in the coding region of EZH2, and a unique mutation was reported (Tyr701Asn).

Background: B cells are essential for providing humoral protection against acute influenza A virus (IAV) infection. FcγRIIB, a regulator of antibody (Ab) production, influences immune responses during pathogen infections, but its specific impact on humoral protection and B cell-mediated responses against IAV remains unclear. Methods: To investigate FcγRIIB's role in host defense and B cell function during acute IAV infection, we generated mice with systemic FcγRIIB deficiency, functional impairment, and B cell-specific FcγRIIB deletion. We infected these mice with PR8 (H1N1) or Hkx31 (H3N2) IAVs and evaluated body weight preservation, survival rates, Ab production, viral neutralization, Ab affinity maturation, and germinal center B cell development. Results: Mice lacking FcγRIIB or with impaired function showed improved protection, preserved body weight, and increased survival rates during IAV infection. Notably, mice with haploinsufficient FcγRIIB function displayed protective effects. Selective deficiency of FcγRIIB in B cells led to enhanced Ab production, resulting in elevated IAV-specific Abs in the serum with superior viral neutralizing potency. However, the impact on the affinity maturation index of virus-specific Abs was modest. Accordingly, FcγRIIB-deficient B cells maintained normal germinal center B cell development during IAV infection, whereas wild-type mice exhibited delayed differentiation. Conclusion: Our research underscores the pivotal role of FcγRIIB in host defense and B cell-mediated immunity during acute IAV infection. Additionally, our discoveries hold implications for antiviral treatments, particularly during the initial stages of IAV infection, aimed at enhancing the host's humoral immune response.

Introduction: The microphthalmia transcription factor Mitf has been shown to regulate B cell activation and tolerance. However, the underlying B cell-specific mechanisms responsible, and those that distinguish Mitf from closely related Mitf/TFE (MiT) transcription factors Tfe3, Tfeb, and Tfec, remain obscure. Methods: Two complementary mouse models of Mitf and MiT deficiency were used: the Mitfmi-vga9/mi-vga9 systemic loss-of-function mutation, and B-cell specific MiT family inactivation via transgenic expression of a trans-dominant negative (TDN) protein (TDN-B). These models were employed to identify MiT family candidate target genes and pathways. Results: Both models displayed spontaneous splenomegaly coincident with elevated plasma cell numbers, autoantibody titers, and proteinuria. These abnormalities appeared dependent on T helper cells, but independent of other non-B cell intrinsic effects of systemic Mitf inactivation. MiT inactivation in B cells augmented aspects of lupus-like autoimmune disease on the C57BL/6-Faslpr/lpr background. In both models, RNAseq of ex vivo resting B cells showed transcriptional upregulation of genes that control cell cycle, germinal center responses, and plasma cell differentiation. Among the genes strongly upregulated in both models were Socs6, Isp53 (Baiap1), S1pR2, and IgG2b/c. Mitf null B cells, but not TDN-B cells, showed evidence of type I interferon dysregulation. Discussion: These studies clarify Mitf's role as 1) a key regulator of a B cell intrinsic germinal center program that influences self-tolerance through novel target genes, and 2) a regulator of systemic inflammatory processes that can impact the B cell microenvironment. This distinction of Mitf's function from that of related MiT transcription factors advances our understanding of B cell regulation and autoimmunity. [ABSTRACT FROM AUTHOR]

Modern vaccine development is having a golden period, with a variety of novel subunit technologies being introduced into clinical development in recent years. This opens the opportunity to find the best platform to use for novel vaccine antigen candidates through head-to-head comparative studies. Seldom appreciated is, however, the fact that these different technologies often do not have the same optimal antigen dose ratio, prime-boost regime and peak timepoint for measuring immunity. Instead, the preclinical studies that make the basis for platform selection use standard protocols not optimized for individual vaccines and fail to make selection on an informed basis. Here, I discuss the opportunities we have to optimize vaccine platform technologies through a better understanding of vaccine priming kinetics, the optimal antigen dose and sampling time and location. [ABSTRACT FROM AUTHOR]

The germinal center response or reaction (GCR) is a hallmark event of adaptive humoral immunity. Unfolding in the B cell follicles of the secondary lymphoid organs, a GC culminates in the production of high-affinity antibody-secreting plasma cells along with memory B cells. By interacting with follicular dendritic cells (FDC) and T follicular helper (Tfh) cells, GC B cells exhibit complex spatiotemporal dynamics. Driving the B cell dynamics are the intracellular signal transduction and gene regulatory network that responds to cell surface signaling molecules, cytokines, and chemokines. As our knowledge of the GC continues to expand in depth and in scope, mathematical modeling has become an important tool to help disentangle the intricacy of the GCR and inform novel mechanistic and clinical insights. While the GC has been modeled at different granularities, a multiscale spatial simulation framework – integrating molecular, cellular, and tissue-level responses – is still rare. Here, we report our recent progress toward this end with a hybrid stochastic GC framework developed on the Cellular Potts Model-based CompuCell3D platform. Tellurium is used to simulate the B cell intracellular molecular network comprising NF-κB, FOXO1, MYC, AP4, CXCR4, and BLIMP1 that responds to B cell receptor (BCR) and CD40-mediated signaling. The molecular outputs of the network drive the spatiotemporal behaviors of B cells, including cyclic migration between the dark zone (DZ) and light zone (LZ) via chemotaxis; clonal proliferative bursts, somatic hypermutation, and DNA damage-induced apoptosis in the DZ; and positive selection, apoptosis via a death timer, and emergence of plasma cells in the LZ. Our simulations are able to recapitulate key molecular, cellular, and morphological GC events, including B cell population growth, affinity maturation, and clonal dominance. This novel modeling framework provides an open-source, customizable, and multiscale virtual GC simulation platform that enables qualitative and quantitative in silico investigations of a range of mechanistic and applied research questions on the adaptive humoral immune response in the future.

Follicular helper CD4hi T cells (TFH) are a major cellular pool for the maintenance of the HIV reservoir. Therefore, the delineation of the follicular (F)/germinal center (GC) immune landscape will significantly advance our understanding of HIV pathogenesis. We have applied multiplex confocal imaging, in combination with the relevant computational tools, to investigate F/GC in situ immune dynamics in viremic (vir-HIV), antiretroviral-treated (cART HIV) People Living With HIV (PLWH) and compare them to reactive, non-infected controls. Lymph nodes (LNs) from viremic and cART PLWH could be further grouped based on their TFH cell densities in high-TFH and low-TFH subgroups. These subgroups were also characterized by different in situ distributions of PD1hi TFH cells. Furthermore, a significant accumulation of follicular FOXP3hiCD4hi T cells, which were characterized by a low scattering in situ distribution profile and strongly correlated with the cell density of CD8hi T cells, was found in the cART-HIV low-TFH group. An inverse correlation between plasma viral load and LN GrzBhiCD8hi T and CD16hiCD15lo cells was found. Our data reveal the complex GC immune landscaping in HIV infection and suggest that follicular FOXP3hiCD4hi T cells could be negative regulators of TFH cell prevalence in cART-HIV.

A hallmark of T cell dependent (TD) humoral immune responses is the generation of long–lived memory B cells. The generation of these cells occurs primarily in the germinal center (GC) reaction, where antigen–activated B cells undergo affinity maturation as a major consequence of the combined processes of proliferation, somatic hypermutation of their immunoglobulin V (IgV) region genes, and selection for improved affinity of their B–cell antigen receptors. As many B cells also undergo class–switching to IgG or IgA in these TD responses, there was traditionally a focus on class–switched memory B cells in both murine and human studies on memory B cells. However, it has become clear that there is also a large subset of IgM–expressing memory B cells, which have important phenotypic and functional similarities but also differences to class–switched memory B cells. There is an ongoing discussion about the origin of distinct subsets of human IgM+ B cells with somatically mutated IgV genes. We argue here that the vast majority of human IgM–expressing B cells with somatically mutated IgV genes in adults is indeed derived from GC reactions, even though a generation of some mostly lowly mutated IgM+ B cells from other differentiation pathways, mainly in early life, may exist. [ABSTRACT FROM AUTHOR]

The gammaherpesviruses, including human Epstein-Barr virus, human Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4), are ubiquitous pathogens that directly contribute to the genesis of a wide variety of malignancies, including B cell lymphomas. In vivo, these viruses infect naïve B cells and then usurp B cell signaling pathways to drive infected cells, independent of antigen stimulation, through germinal center (GC) reactions and thereby establish lifelong latency in the memory B cell compartment. However, the specific molecular determinants by which these viruses establish chronic latent infection in B cells in vivo remain largely unknown. The E3 ubiquitin ligase Cullin 4b (Cul4b) is well recognized as a central player in regulating cell proliferation, DNA damage repair, and gene transcription by catalyzing the ubiquitination and degradation of numerous cellular proteins. Here, we determined whether B cell-intrinsic expression of Cul4b is required for chronic gammaherpesvirus infection in vivo. Through MHV68 infection of mice with B cell-specific Cul4b deficiency, we found that loss of Cul4b expression in B cells severely impeded the establishment of latency at peripheral sites. In particular, a lack of Cul4b expression in B cells significantly attenuated the expansion of virus-infected GC B cells, suggesting that gammaherpesviruses may manipulate a previously unknown function of Cul4b in GC B cell biology. Cumulatively, these findings demonstrate that Cul4b promotes chronic gammaherpesvirus infection in vivo. [ABSTRACT FROM AUTHOR]

B-cell lymphomas are a group of heterogeneous neoplasms resulting from the clonal expansion of mature B cells arrested at various stages of differentiation. Specifically, two lymphoma subtypes arise from germinal centers (GCs), namely follicular lymphoma (FL) and GC B-cell diffuse large B-cell lymphoma (GCBDLBCL). In addition to recent advances in describing the genetic landscape of FL and GCB-DLBCL, tumor microenvironment (TME) has progressively emerged as a central determinant of early lymphomagenesis, subclonal evolution, and late progression/transformation. The lymphoma-supportive niche integrates a dynamic and coordinated network of immune and stromal cells defining microarchitecture and mechanical constraints and regulating tumor cell migration, survival, proliferation, and immune escape. Several questions are still unsolved regarding the interplay between lymphoma B cells and their TME, including the mechanisms supporting these bidirectional interactions, the impact of the kinetic and spatial heterogeneity of the tumor niche on B-cell heterogeneity, and how individual genetic alterations can trigger both B-cell intrinsic and B-cell extrinsic signals driving the reprogramming of non-malignant cells. Finally, it is not clear whether these interactions might promote resistance to treatment or, conversely, offer valuable therapeutic opportunities. A major challenge in addressing these questions is the lack of relevant models integrating tumor cells with specific genetic hits, non-malignant cells with adequate functional properties and organization, extracellular matrix, and biomechanical forces. We propose here an overview of the 3D in vitro models, xenograft approaches, and genetically-engineered mouse models recently developed to study GC B-cell lymphomas with a specific focus on the pros and cons of each strategy in understanding B-cell lymphomagenesis and evaluating new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Germinal centers (GCs) are the sites of secondary antibody diversification and underlie the mechanism of action of many vaccination strategies. Activation-induced deaminase (AID) triggers secondary antibody diversification through the introduction of somatic changes in immunoglobulin genes which lead to the generation of antibodies of higher affinity and more specialized effector functions. However, AID can also target other genomic regions, giving rise to mutations and chromosome translocations with oncogenic potential. Many human lymphomas originate from mature B cells that have undergone the GC reaction, such as the diffuse large B cell lymphoma, the follicular lymphoma and Burkitt lymphoma, and carry chromosome translocations. Mature B cell lymphomagenesis has been modeled in the mouse by the genetic introduction of chromosome translocations. Here, we present an in-depth characterization of one such model, λ-MYC mice. We found that young pre-tumor stage mice had a prominent block in early B cell differentiation that resulted in the generation of very aggressive tumors lacking surface B cell receptor (BCR) expression, indicating that a large fraction of tumors in λ-MYC mice arise from B cell precursors rather than from mature B cells. Further, we assessed the contribution of AID to B cell lymphomagenesis in λ-MYC mice by using a genetic tracer of historical AID expression. Only a fraction of tumors contained cells of GC origin as defined by AID expression. AID-experienced tumors associated with longer survival and resembled mature B cell lymphomas. Thus, AID expression defines Burkitt lymphomagenesis in λ-MYC mice. [ABSTRACT FROM AUTHOR]

Several oral bacteria, including Prevotella melaninogenica (Pm), have aquaporin (AQP) proteins homologous to human AQP5, a major water channel protein targeted in Sjogren's syndrome. This study aimed to understand the antigenic characteristics that induce autoantibodies against an AQP5 "E" epitope (AQP5E) in a mouse model using C57BL/6 mice. Immunization with a PmE‐L peptide derived from Pm AQP, which contains amino acid mismatches both at the B‐ and T‐cell epitopes, efficiently induced anti‐AQP5E autoantibodies accompanied by increased germinal center (GC) B and follicular helper T cells in the draining lymph nodes. However, PmE, a peptide lacking a T‐cell epitope, and AQP5E‐L, an AQP5‐derived self‐peptide, hardly induced either anti‐AQP5E autoantibodies or GC responses. Surprisingly, OTII‐AQP5E, a peptide that replaced the self T‐cell epitope of AQP5E‐L with an ovalbumin‐derived foreign T‐cell epitope, was not any better than AQP5E‐L in the induction of anti‐AQP5E autoantibodies and GC response, despite the substantial expansion of CD4+ T cells and production of anti‐OTII‐AQP5E antibodies. The complex of biotinylated PmE‐L peptide and highly immunogenic streptavidin (SA) induced a strong extrafollicular B‐cell response skewed toward the expansion of SA‐specific B cells. However, the expansion of AQP5E‐specific GC B cells was limited, resulting in the inefficient induction of anti‐AQP5E autoantibodies. Collectively, our results have demonstrated that anti‐AQP5E autoantibody production is only allowed when foreign B‐ and T‐cell epitopes drive a strong GC response of AQP5E‐specific B cells for affinity maturation. This study helps explain why cross‐reactive anti‐AQP5 autoantibodies are not produced during the immune response to Pm in most healthy people. [ABSTRACT FROM AUTHOR]

Toll‐like receptor 7 (TLR7) triggers antiviral immune responses through its capacity to recognize single‐stranded RNA. TLR7 loss‐of‐function mutants are associated with life‐threatening pneumonia in severe COVID‐19 patients. Whereas TLR7‐driven innate induction of type I IFN appears central to control SARS‐CoV2 virus spreading during the first days of infection, the impact of TLR7‐deficiency on adaptive B‐cell immunity is less clear. In the present study, we examined the role of TLR7 in the adaptive B cells response to various pathogen‐like antigens (PLAs). We used inactivated SARS‐CoV2 and a PLA‐based COVID‐19 vaccine candidate designed to mimic SARS‐CoV2 with encapsulated bacterial ssRNA as TLR7 ligands and conjugated with the RBD of the SARS‐CoV2 Spike protein. Upon repeated immunization with inactivated SARS‐CoV2 or PLA COVID‐19 vaccine, we show that Tlr7‐deficiency abolished the germinal center (GC)‐dependent production of RBD‐specific class‐switched IgG2b and IgG2c, and neutralizing antibodies to SARS‐CoV2. We also provide evidence for a non‐redundant role for B‐cell‐intrinsic TLR7 in the promotion of RBD‐specific IgG2b/IgG2c and memory B cells. Together, these data demonstrate that the GC reaction and class‐switch recombination to the Myd88‐dependent IgG2b/IgG2c in response to SARS‐CoV2 or PLAs is strictly dependent on cell‐intrinsic activation of TLR7 in B cells. [ABSTRACT FROM AUTHOR]

IntroductionThe microphthalmia transcription factor Mitf has been shown to regulate B cell activation and tolerance. However, the underlying B cell-specific mechanisms responsible, and those that distinguish Mitf from closely related Mitf/TFE (MiT) transcription factors Tfe3, Tfeb, and Tfec, remain obscure.MethodsTwo complementary mouse models of Mitf and MiT deficiency were used: the Mitfmi-vga9/mi-vga9 systemic loss-of-function mutation, and B-cell specific MiT family inactivation via transgenic expression of a trans-dominant negative (TDN) protein (TDN-B). These models were employed to identify MiT family candidate target genes and pathways.ResultsBoth models displayed spontaneous splenomegaly coincident with elevated plasma cell numbers, autoantibody titers, and proteinuria. These abnormalities appeared dependent on T helper cells, but independent of other non-B cell intrinsic effects of systemic Mitf inactivation. MiT inactivation in B cells augmented aspects of lupus-like autoimmune disease on the C57BL/6-Faslpr/lpr background. In both models, RNAseq of ex vivo resting B cells showed transcriptional upregulation of genes that control cell cycle, germinal center responses, and plasma cell differentiation. Among the genes strongly upregulated in both models were Socs6, Isp53 (Baiap1), S1pR2, and IgG2b/c. Mitf null B cells, but not TDN-B cells, showed evidence of type I interferon dysregulation.DiscussionThese studies clarify Mitf’s role as 1) a key regulator of a B cell intrinsic germinal center program that influences self-tolerance through novel target genes, and 2) a regulator of systemic inflammatory processes that can impact the B cell microenvironment. This distinction of Mitf's function from that of related MiT transcription factors advances our understanding of B cell regulation and autoimmunity.

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous human virus that promotes B-cell activation and maturation through expression of latency proteins and non-coding RNAs. In this study, we provide evidence that EBV mimics the molecular phenotype of germinal center (GC) B cells. EBV infection of primary human B cells promotes their rapid proliferation and GC dark zone (DZ)-like gene expression profile. Following this transient hyperproliferative period, the activation of NF-κB target genes, including Bcl2a1 (BFL-1), simulates the transition from the DZ to the T-cell supported light zone (LZ). We previously characterized the regulatory landscape of EBV+ B cells at the Bcl2a1 locus defining a key role for the viral EBV nuclear antigen (EBNA) 3A protein in promoting three-dimensional chromatin architecture correlated with BFL-1 expression. Here, we define the global chromatin accessibility of tonsillar B cells and find that naïve and memory B cells have highly similar accessibility profiles that differ substantially from those of DZ and LZ B cells. Notably, multiple regions within the Bcl2a1 locus are significantly more accessible in DZ and LZ versus naïve and memory subsets. However, we found that BFL-1 upregulation from DZ to LZ correlates with a significant increase in three-dimensional (3-D) chromatin association between accessible upstream enhancer regions and the BFL-1 transcriptional start site. These elements were critical for BFL-1 expression in lymphoblastoid cell lines (LCLs). Moreover, increased BFL-1 expression in LCLs protected against extrinsic apoptosis. Collectively, these results suggest a conserved mechanism underlying BFL-1 upregulation that promotes survival of both LZ and EBV+ immortalized B cells.IMPORTANCEEpstein-Barr virus has evolved with its human host leading to an intimate relationship where infection of antibody-producing B cells mimics the process by which these cells normally recognize foreign antigens and become activated. Virtually everyone in the world is infected by adulthood and controls this virus pushing it into life-long latency. However, immune-suppressed individuals are at high risk for EBV+ cancers. Here, we isolated B cells from tonsils and compare the underlying molecular genetic differences between these cells and those infected with EBV. We find similar regulatory mechanism for expression of an important cellular protein that enables B cells to survive in lymphoid tissue. These findings link an underlying relationship at the molecular level between EBV-infected B cells in vitro with normally activated B cells in vivo. Our studies also characterize the role of a key viral control mechanism for B cell survival involved in long-term infection.

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

This Brief Review delves into B cell responses in the context of allergy. The primary contribution of B cells to allergy is the production of IgE, the Ab isotype that triggers immediate hypersensitivity reactions through the release of mediators from mast cells and basophils. B cells may also have protective roles in allergy, such as through the production of IgG or as regulatory B cells. In this review, I focus on the basic principles of B cell differentiation and discuss features relevant to allergic immune responses. In particular, I discuss: (1) class-switch recombination; (2) plasma cell differentiation; (3) germinal centers and affinity maturation; and (4) memory B cells and recall responses, with an emphasis on IgE, IgG1, and IgG4. I also consider how B cells may contribute to allergic responses independent of Ab production-for example, by serving as APCs.

Antibody-mediated adaptive immunity must provide effective long-term protection with minimal adverse effects, against rapidly mutating pathogens, in a human population with diverse ages, genetics, and immune histories. In order to grasp and leverage the complexities of the antibody response, we advocate for a mechanistic understanding of the multiscale germinal center (GC) reaction - the process by which precursor B-cells evolve high-affinity antigen-specific antibodies, forming an effector repertoire of plasma and memory cells for decades-long protection. The regulatory dynamics of B-cells within the GC are complex, and unfold across multiple interacting spatial and temporal scales. At the organism scale, over weeks to years, the antibody sequence repertoire formed by various B-cell clonal lineages modulates antibody quantity and quality over time. At the tissue and cellular scale, over hours to weeks, B-cells undergo selection via spatially distributed interactions with local stroma, antigen, and helper T-cells. At the molecular scale, over seconds to days, intracellular signaling, transcriptional, and epigenetic networks modulate B-cell fates and shape their clonal lineages. We summarize our current understanding within each of these scales, and identify missing links in connecting them. We suggest that quantitative multi-scale mathematical models of B-cell and GC reaction dynamics provide predictive frameworks that can apply basic immunological knowledge to practical challenges such as rational vaccine design.

The role of CD4 T regulatory cells is well established in peripheral tolerance and the pathogenesis of the murine model and human autoimmune diseases. CD4 T regulatory cells (CD4 Tregs) have been investigated in common variable immunodeficiency (CVID). Recently, additional members have been added to the club of regulatory lymphocytes. These include CD8 T regulatory (CD8 Tregs), B regulatory (Bregs), and T follicular helper regulatory (TFR) cells. There are accumulating data to suggest their roles in both human and experimental models of autoimmune disease. Their phenotypic characterization and mechanisms of immunoregulation are evolving. Patients with CVID may present or are associated with an increased frequency of autoimmunity and autoimmune diseases. In this review, we have primarily focused on the characteristics of CD4 Tregs and new players of the regulatory club and their changes in patients with CVID in relation to autoimmunity and emphasized the complexity of interplay among various regulatory lymphocytes. We suggest future careful investigations of phenotypic and functional regulatory lymphocytes in a large cohort of phenotypic and genotypically defined CVID patients to define their role in the pathogenesis of CVID and autoimmunity associated with CVID.

Introduction: Diffuse Large B-cell Lymphoma (DLBCL) is the most common Non-Hodgkin’s Lymphoma (NHL). Using Gene Expression Profiling (GEP) DLBCL has been subtyped into two groups of prognostic importance, Germinal Center B-cell (GCB) like and activated B-cell like. GCB DLBCL has a better survival and can be identified using Hans algorithm with three immunohistochemical markers Cluster Differentiation (CD10), B-cell lymphoma 6 (BCL6) and Multiple Myeloma Oncogene-1 (MUM1). Aim: To analyse DLBCL using Hans algorithm as both GCB lymphoma and non GCB lymphoma have different treatment and prognosis. Materials and Methods: This was a cross-sectional study conducted in the Department of Pathology, Government Medical College, Kozhikode, Kerala, India, from January 2019 to December 2020. A total of 97 DLBCL cases received in the Department of Pathology, from January 2016 to December 2019 were included in the study were subtyped using Hans algorithm. CD10, BCL6 and MUM1 were considered positive, if more than 30% of the tumour cells showed staining by the respective antibodies. The relation between DLBCL subtypes and the age, gender, symptoms, site of initial involvement, organomegaly, Ann Arbor stage, treatment response and overall survival. Findings in the patients were analysed using Chi-square test. Statistical Package for Social Sciences (SPSS) software version 18.0. Overall survival was estimated using Kaplan-Meier method. Results: The median age of study population was 60 years (age range: 31-85 years) and there were 55 (56.7%) males and 42 (43.3%) females. Out of the 97 DLBCL cases 47 (48.5%) were GCB and 50 (51.5%) were non GCB subtype. Statistical analysis was done only in 88 patients (excluded nine recurrent lymphoma patients, which may have a different outcome). There was significant association (p-value=0.003) between stage and subtypes as majority of the non GCB cases presented in an advanced stage. The rate of complete remission with Rituximab Cyclophosphamid Hydroxydaunorubicin Oncovin Prednisone (RCHOP) chemotherapy was higher in GCB (58.75%) compared to non GCB (15.25%) subtypes (p-value=0.001). Overall survival rate of GCB was 74.4% and non GCB was 31% with a p-value of 0.001. There was no statistically significant relation between DLBCL subtypes and other clinicopathological factors. Conclusion: In the present study, the patients within the GCB subtype had better treatment response and overall survival rate compared to non GCB subtype. Non germinal center subtype presented in advanced stage and had a worse prognosis. Therefore, it is essential to subtype DLBCL in all cases to identify non GCB subtype, which may need additional treatment after RCHOP chemotherapy.

The germinal center (GC) reaction is a coordinated and dynamic ensemble of cells and processes that mediate the maturation and selection of high-affinity GC B cells (GCBs) from lower-affinity precursors and ultimately results in plasma cell and memory cell fates that exit the GC. It is of great interest to identify intrinsic and extrinsic factors that control the selection process. The transcription factor IRF4, induced upon BCR and CD40 signaling, is essential for the acquisition of plasma cell and GCB cell fates. We hypothesized that beyond this early requirement, IRF4 continuously operates at later phases of the B cell response. We show that IRF4 is expressed in GCBs at levels greater than seen in resting cells and plays a role in efficient selection of high-affinity GCBs. Halving Irf4 gene copy number in an Ag-specific murine B cell model, we found that Ag presentation, isotype switching, GC formation and zonation, somatic hypermutation rates, and proliferation were comparable with cells with a full Irf4 allelic complement. In contrast, Irf4 haploinsufficient GCBs exhibited impaired generation of high-affinity cells. Mechanistically, we demonstrate suboptimal Blimp-1 regulation among high-affinity Irf4 haploinsufficient GCBs. Furthermore, in cotransfer settings, we observed a marked disadvantage of Irf4 haploinsufficient cells for GC entry, evidential of ineffective recruitment of T cell help. We propose that, analogous to its role in early GC entry, IRF4 continues to function in the late phase of the Ab response to promote productive T follicular helper cell interactions and to activate optimal Blimp-1 expression during GC selection and affinity maturation.

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type–specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology (IL21, IL21R, IL4R, and BCL6) and transcription factors regulating B cell differentiation (POU2AF1 and HHEX). Together, these analyses provide a powerful new cell type–resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

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.

Follicular CXCR5+ PD-1+ CD8 T cells (CD8 Tfc) arise in multiple models of systemic autoimmunity yet their functional contribution to disease remains in debate. Here we define the follicular localization and functional interactions of CD8 Tfc with B cells during autoimmune disease. The absence of functional T regulatory cells in autoimmunity allows for CD8 Tfc development that then expands with lymphoproliferation. CD8 Tfc are identifiable within the lymph nodes and spleen during systemic autoimmunity, but not during tissue-restricted autoimmune disease. Autoimmune CD8 Tfc cells are polyfunctional, producing helper cytokines IL-21, IL-4, and IFNγ while maintaining cytolytic proteins CD107a, granzyme B, and TNF. During autoimmune disease, IL-2-KO CD8 T cells infiltrate the B cell follicle and germinal center, including the dark zone, and in vitro induce activation-induced cytidine deaminase in naïve B cells via IL-4 secretion. CD8 Tfc represent a unique CD8 T cell population with a diverse effector cytokine repertoire that can contribute to pathogenic autoimmune B cell response.

B cell clones compete for entry into and dominance within germinal centers (GCs), where the highest-affinity B cell receptors (BCRs) are selected. However, diverse and low-affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesize that a negative feedback loop may operate within B cells to preferentially restrain high-affinity clones from monopolizing the early GC niche. Here, we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We show that NUR77 expression scales with antigen stimulation and restrains B cell expansion. Although NUR77 is dispensable for regulating GC size when GCs are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. We propose that this is important to preserve early clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

Background Arterial/aortic tertiary lymphoid organs (ATLOs), characterized by germinal centers, control local arterial immune responses. T follicular helper cells (Tfh), resident in germinal centers, regulate immunoglobulin production and germinal center development. They consist of Tfh1, Tfh2, and Tfh17 subsets. T follicular regulatory (Tfr) cells possess suppressive functions as regulatory T cells and migrate into germinal centers. Immunoglobulin G4 (IgG4)–related diseases manifest in vascular lesions as frequently formed inflammatory aneurysms (IgG4‐related abdominal aortic aneurysm [IgG4‐AAAs]). IgG4‐AAAs contain several ATLOs. Methods and Results We performed whole‐slide immunohistochemical image analysis in surgical specimens of IgG4‐AAAs (n=21), non–IgG4‐related inflammatory AAAs (n=17), atherosclerotic AAAs (n=10), and Takayasu arteritis (n=5). IgG4‐AAA was characterized by numerous, large, irregular‐shaped ATLOs, and higher numbers of Tfr and Tfh2 cells than Tfh1 cells were present compared with others. The morphologic abnormalities (in number, area, and form) of ATLOs in IgG4‐AAAs and the increased number of Tfr cells are closely related to the activity of IgG4‐related diseases. All T‐cell subsets were more enriched within ATLOs than outside ATLOs. In particular, an increase in Tfr cells in IgG4‐AAAs was associated with ATLO formation. Increased Tfh17 cells were found in Takayasu arteritis, and atherosclerotic AAA and non–IgG4‐related inflammatory AAAs were characterized by increased Tfh1 cells. Conclusions In the classification of vascular lesions, considering the imbalance in T‐cell subsets, IgG4‐AAA should be positioned as adventitial vasculitis with predominant Tfr and Tfh2 cells, accompanied by the abnormal appearance of ATLOs.

A hallmark of T cell dependent (TD) humoral immune responses is the generation of long–lived memory B cells. The generation of these cells occurs primarily in the germinal center (GC) reaction, where antigen–activated B cells undergo affinity maturation as a major consequence of the combined processes of proliferation, somatic hypermutation of their immunoglobulin V (IgV) region genes, and selection for improved affinity of their B–cell antigen receptors. As many B cells also undergo class–switching to IgG or IgA in these TD responses, there was traditionally a focus on class–switched memory B cells in both murine and human studies on memory B cells. However, it has become clear that there is also a large subset of IgM–expressing memory B cells, which have important phenotypic and functional similarities but also differences to class–switched memory B cells. There is an ongoing discussion about the origin of distinct subsets of human IgM+ B cells with somatically mutated IgV genes. We argue here that the vast majority of human IgM–expressing B cells with somatically mutated IgV genes in adults is indeed derived from GC reactions, even though a generation of some mostly lowly mutated IgM+ B cells from other differentiation pathways, mainly in early life, may exist.

B-cell lymphomas are a group of heterogeneous neoplasms resulting from the clonal expansion of mature B cells arrested at various stages of differentiation. Specifically, two lymphoma subtypes arise from germinal centers (GCs), namely follicular lymphoma (FL) and GC B-cell diffuse large B-cell lymphoma (GCB-DLBCL). In addition to recent advances in describing the genetic landscape of FL and GCB-DLBCL, tumor microenvironment (TME) has progressively emerged as a central determinant of early lymphomagenesis, subclonal evolution, and late progression/transformation. The lymphoma-supportive niche integrates a dynamic and coordinated network of immune and stromal cells defining microarchitecture and mechanical constraints and regulating tumor cell migration, survival, proliferation, and immune escape. Several questions are still unsolved regarding the interplay between lymphoma B cells and their TME, including the mechanisms supporting these bidirectional interactions, the impact of the kinetic and spatial heterogeneity of the tumor niche on B-cell heterogeneity, and how individual genetic alterations can trigger both B-cell intrinsic and B-cell extrinsic signals driving the reprogramming of non-malignant cells. Finally, it is not clear whether these interactions might promote resistance to treatment or, conversely, offer valuable therapeutic opportunities. A major challenge in addressing these questions is the lack of relevant models integrating tumor cells with specific genetic hits, non-malignant cells with adequate functional properties and organization, extracellular matrix, and biomechanical forces. We propose here an overview of the 3D in vitro models, xenograft approaches, and genetically-engineered mouse models recently developed to study GC B-cell lymphomas with a specific focus on the pros and cons of each strategy in understanding B-cell lymphomagenesis and evaluating new therapeutic strategies.

Secondary immunoglobulin diversification by somatic hypermutation and class switch recombination in B cells is instrumental for an adequate adaptive humoral immune response. These genetic events may, however, also introduce aberrations into other cellular genes and thereby cause B cell malignancies. While the basic mechanism of somatic hypermutation and class switch recombination is now well understood, their regulation and in particular the mechanism of their specific targeting to immunoglobulin genes is still rather mysterious. In this review, we summarize the current knowledge on the mechanism and regulation of secondary immunoglobulin diversification and discuss known mechanisms of physiological targeting to immunoglobulin genes and mistargeting to other cellular genes. We summarize open questions in the field and provide an outlook on future research. [ABSTRACT FROM AUTHOR]