Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19.

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.

Tyrosine kinase 2 modulates splenic B cells through type I IFN and TLR7 signaling.

Tyrosine kinase 2 (TYK2) is involved in type I interferon (IFN-I) signaling through IFN receptor 1 (IFNAR1). This signaling pathway is crucial in the early antiviral response and remains incompletely understood on B cells. Therefore, to understand the role of TYK2 in B cells, we studied these cells under homeostatic conditions and following in vitro activation using Tyk2-deficient (Tyk2-/-) mice. Splenic B cell subpopulations were altered in Tyk2-/- compared to wild type (WT) mice. Marginal zone (MZ) cells were decreased and aged B cells (ABC) were increased, whereas follicular (FO) cells remained unchanged. Likewise, there was an imbalance in transitional B cells in juvenile Tyk2-/- mice. RNA sequencing analysis of adult MZ and FO cells isolated from Tyk2-/- and WT mice in homeostasis revealed altered expression of IFN-I and Toll-like receptor 7 (TLR7) signaling pathway genes. Flow cytometry assays corroborated a lower expression of TLR7 in MZ B cells from Tyk2-/- mice. Splenic B cell cultures showed reduced proliferation and differentiation responses after activation with TLR7 ligands in Tyk2-/- compared to WT mice, with a similar response to lipopolysaccharide (LPS) or anti-CD40 + IL-4. IgM, IgG, IL-10 and IL-6 secretion was also decreased in Tyk2-/- B cell cultures. This reduced response of the TLR7 pathway in Tyk2-/- mice was partially restored by IFNα addition. In conclusion, there is a crosstalk between TYK2 and TLR7 mediated by an IFN-I feedback loop, which contributes to the establishment of MZ B cells and to B cell proliferation and differentiation.

Aberrant alteration of peripheral B lymphocyte subsets in hepatocellular carcinoma patients.

Although B lymphocytes are widely known to participate in the immune response, the conclusive roles of B lymphocyte subsets in the antitumor immune response have not yet been determined. Single-cell data from GEO datasets were first analyzed, and then a B cell flow cytometry panel was used to analyze the peripheral blood of 89 HCC patients and 33 healthy controls recruited to participate in our research. Patients with HCC had a higher frequency of B10 cells and a lower percentage of MZB cells than healthy controls. And the changes in B cell subsets might occur at an early stage. Moreover, the frequency of B10 cells decreased after surgery. Positively correlated with B10 cells, the elevated IL-10 level in HCC serum may be a new biomarker in HCC identification. For the first time, our results suggest that altered B cell subsets are associated with the development and prognosis of HCC. Increased B10 cell percentage and IL-10 in HCC patients suggest they might augment the development of liver tumors. Hence, B cell subsets and related cytokines may have predictive value in HCC patients and could be potential targets for immunotherapy in HCC.

Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

Genetic loss of NFAT2 (NFATc1) impairs B cell development of B1 and B2 B cells.

NFAT2 activity was shown to be of critical importance in B cell receptor signaling, development and proliferation; however its role in B cell development in the periphery is still not completely understood. We confirmed that NFAT2 deletion leads to impaired B1 B cell development, supported by our finding of limited B1 progenitors in the bone marrow and spleen of NFAT2 deficient mice. Moreover, we show for the first time that loss of NFAT2 increases immature B cells in particular transitional T2 and T3 as well as mature follicular B cells while marginal zone B cells are decreased. We further demonstrate that NFAT2 regulates the expression of B220, CD23, CD38, IgM/IgD and ZAP70 in murine B cells. In vivo analyses revealed decreased proliferation and increased apoptosis of NFAT2 deficient B cells. In summary, this study provides an extensive analysis of the role of NFAT2 in peripheral B lymphocyte development.

Germinal center formation, immunoglobulin production and hindlimb nociceptive sensitization after tibia fracture.

Emerging evidence suggests that Complex Regional Pain Syndrome (CRPS) is in part a post-traumatic autoimmune disease mediated by an adaptive immune response after limb injuries. We previously observed in a murine tibial fracture model of CRPS that pain-related behaviors were dependent upon adaptive immune mechanisms including the neuropeptide-dependent production of IgM for 5 months after injury. However, the time course of induction of this immune response and the demonstration of germinal center formation in lymphoid organs has not been evaluated. Using the murine fracture model, we employed behavioral tests of nociceptive sensitization and limb dysfunction, serum passive transfer techniques, western blot analysis of IgM accumulation, fluorescence-activated cell sorting (FACS) of lymphoid tissues and immunohistochemistry to follow the temporal activation of the adaptive immune response over the first 3 weeks after fracture. We observed that: 1) IgM protein levels in the skin of the fractured mice were elevated at 3 weeks post fracture, but not at earlier time points, 2) serum from fracture mice at 3 weeks, but not 1 and 2 weeks post fracture, had pro-nociceptive effects when passively transferred to fractured muMT mice lacking B cells, 3) fracture induced popliteal lymphadenopathy occurred ipsilateral to fracture beginning at 1 week and peaking at 3 weeks post fracture, 4) a germinal center reaction was detected by FACS analysis in the popliteal lymph nodes from injured limbs by 3 weeks post fracture but not in other lymphoid tissues, 5) germinal center formation was characterized by the induction of T follicular helper cells (Tfh) and germinal center B cells in the popliteal lymph nodes of the injured but not contralateral limbs, and 6) fracture mice treated with the Tfh signaling inhibitor FK506 had impaired germinal center reactions, reduced IgM levels, reduced nociceptive sensitization, and no pronociceptive serum effects after administration to fractured muMT mice. Collectively these data demonstrate that tibia fracture induces an adaptive autoimmune response characterized by popliteal lymph node germinal center formation and Tfh cell dependent B cell activation, resulting in nociceptive sensitization within 3 weeks.

Canonical NF-κB signaling is uniquely required for the long-term persistence of functional mature B cells.

Although canonical NF-κB signaling is crucial to generate a normal mature B-cell compartment, its role in the persistence of resting mature B cells is controversial. To resolve this conflict, we ablated NF-κB essential modulator (NEMO) and IκB kinase 2 (IKK2), two essential mediators of the canonical pathway, either early on in B-cell development or specifically in mature B cells. Early ablation severely inhibited the generation of all mature B-cell subsets, but follicular B-cell numbers could be largely rescued by ectopic expression of B-cell lymphoma 2 (Bcl2), despite a persisting block at the transitional stage. Marginal zone (MZ) B and B1 cells were not rescued, indicating a possible role of canonical NF-κB signals beyond the control of cell survival in these subsets. When canonical NF-κB signaling was ablated specifically in mature B cells, the differentiation and/or persistence of MZ B cells was still abrogated, but follicular B-cell numbers were only mildly affected. However, the mutant cells exhibited increased turnover as well as functional deficiencies upon activation, suggesting that canonical NF-κB signals contribute to their long-term persistence and functional fitness.

α-Galactosylceramide stimulates splenic lymphocyte proliferation in vitro and increases antibody production in vivo in late neonatal-age mice.

The neonatal stage is characterized by weak responses to various infections and vaccines, thus the development of efficient formulas to improve vaccine effectiveness is of high priority. The glycolipid alpha galactosylceramide (αGalCer) is known as a potent immune modulator due mainly to natural killer (NK) T cell activation. Using a mouse tetanus toxoid (TT) immunization model, we observed that neonatal mice given αGalCer at the time of primary immunization on postnatal day (pnd) 17 had a significantly higher TT-specific immunoglobulin (Ig)M response as well as a memory IgG response, while αGalCer given on pnd 7 resulted in only marginal boosting. Consistently, immunostaining of the spleen sections from αGalCer-treated pnd 17 immunized neonates showed a higher number of Ki67(+) cells in the splenic germinal centre area, suggesting a stronger response after immunization. In-vitro kinetic studies revealed that spleen cells from newborn to pnd 7 neonates did not respond to αGalCer stimulation, whereas cell proliferation was increased markedly by αGalCer after pnd 7, and became dramatic around neonatal pnd 17-18, which was accompanied by increased B, T and NK T cell populations in the spleen. In addition, in pnd 17 spleen cells, αGalCer significantly stimulated the production of NK T cytokines, interleukin (IL)-4 and interferon (IFN)-γ, and promoted the proliferation of CD23(+) B cells, a subset of B cells enriched in germinal centres. These data suggest that αGalCer is an effective immune stimulus in the late neonatal stage, and thus may be useful in translational studies to test as a potential adjuvant to achieve a more efficient response to immunization.

Current understanding of the immune potential of B-cell subsets in malarial pathogenesis.

In the past several decades, our understanding of how B cells are generated and what function they perform has continued to advance. It is widely accepted that B-cell subsets play a critical role in mediating immune response. Surprisingly, human and murine malarial infections cause major alterations in the composition of B-cell subsets in both the spleen and periphery. Multiple B-cell subsets are well characterized in murine models following primary and secondary infection, although in human malarial infection, these subsets are not well defined. Furthermore, a rare known function of B cells includes the potential role of regulating the activities of other cells in the body as regulatory cells. Plasmodium infection strongly alters the frequency of these regulatory B cells indicating the immunoregulatory function of B cells in malarial. It is important to note that these subsets, taken together, form the cellular basis of humoral immune responses, allowing protection against a wide array of Plasmodium antigens to be achieved. However, it remains a challenge and an important area of investigation to understand how these B-cell subsets work together to provide protection against Plasmodium infection.

Overview of human B-cell development and antibody deficiencies.

B cells are a key component of the humoral (antibody-mediated) immune response which is responsible for defense against a variety of pathogens. Here we provide an overview of the current understanding of B cell development and function and briefly describe inborn errors of immunity associated with B cell development defects which can manifest as immune deficiency, malignancy, autoimmunity, or allergy. The knowledge and application of B cell biology are essential for laboratory evaluation and clinical assessment of these B cell disorders.

Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation.

Integrin-mediated interactions between hematopoietic cells and their microenvironment are important for the development and function of immune cells. Here, the role of the integrin adaptor Kindlin-3 in B cell homeostasis is studied. Comparing the individual steps of B cell development in B cell-specific Kindlin-3 or alpha4 integrin knockout mice, we found in both conditions a phenotype of reduced late immature, mature, and recirculating B cells in the bone marrow. In the spleen, constitutive B cell-specific Kindlin-3 knockout caused a loss of marginal zone B cells and an unexpected expansion of follicular B cells. Alpha4 integrin deficiency did not induce this phenotype. In Kindlin-3 knockout B cells VLA-4 as well as LFA-1-mediated adhesion was abrogated, and short-term homing of these cells in vivo was redirected to the spleen. Upon inducible Kindlin-3 knockout, marginal zone B cells were lost due to defective retention within 2 weeks, while follicular B cell numbers were unaltered. Kindlin-3 deficient follicular B cells displayed higher IgD, CD40, CD44, CXCR5, and EBI2 levels, and elevated PI3K signaling upon CXCR5 stimulation. They also showed transcriptional signatures of spontaneous follicular B cell activation. This activation manifested in scattered germinal centers in situ, early plasmablasts differentiation, and signs of IgG class switch.

Short-chain fatty acids regulate B cells differentiation via the FFA2 receptor to alleviate rheumatoid arthritis.

BACKGROUND AND PURPOSE: Short-chain fatty acids (SCFAs) are metabolites from gut microbes involved in the host's inflammatory response and immunity. The aim of this study was to investigate the role of SCFAs in rheumatoid arthritis (RA) and possible mechanisms. EXPERIMENTAL APPROACH: Gut microbiota diversity in mice was analysed by 16S rDNA sequencing. SCFAs levels were analysed by gas chromatography mass spectrometry. T and B cells were analysed by flow cytometry. Bone damage was analysed by micro-CT and X-ray. Histopathological status was analysed by HE staining. Proteins in tissues were analysed by immunohistochemistry and PCR. Mice with CD19+ B cells deficient in FFA2 receptors were used to explore the molecular mechanisms involved. KEY RESULTS: Levels of acetate, propionate, butyrate, and valerate were decreased in RA patients, and the first three correlated positively with the frequency of Bregs but not Tregs in peripheral blood. Administration of the three SCFAs prior to the onset of collagen-induced arthritis in mice improved arthritic symptoms, increased the Bregs frequency, and decreased transitional B and follicular B cell frequency. However, the preceding phenomena could not be observed in mice with CD19+ B cells deficient in FFA2 receptors. The effects of the three SCFAs in RA were dependent on FFA2 receptors but were independent of the other five B cell receptors (FFA3 receptor, HCA2 receptor, PPARγ, Olfr-78, and AhR). CONCLUSIONS AND IMPLICATIONS: SCFAs regulate B cells differentiation via FFA2 receptors to alleviate RA. This provides new insights into the treatment of RA from an immunological and microbiological perspective.

Homeostatic apoptosis prevents competition-induced atrophy in follicular B cells.

While the intrinsic apoptosis pathway is thought to play a central role in shaping the B cell lineage, its precise role in mature B cell homeostasis remains elusive. Using mice in which mature B cells are unable to undergo apoptotic cell death, we show that apoptosis constrains follicular B (FoB) cell lifespan but plays no role in marginal zone B (MZB) cell homeostasis. In these mice, FoB cells accumulate abnormally. This intensifies intercellular competition for BAFF, resulting in a contraction of the MZB cell compartment, and reducing the growth, trafficking, and fitness of FoB cells. Diminished BAFF signaling dampens the non-canonical NF-κB pathway, undermining FoB cell growth despite the concurrent triggering of a protective p53 response. Thus, MZB and FoB cells exhibit a differential requirement for the intrinsic apoptosis pathway. Homeostatic apoptosis constrains the size of the FoB cell compartment, thereby preventing competition-induced FoB cell atrophy.

Infection of B Cell Follicle-Resident Cells by Friend Retrovirus Occurs during Acute Infection and Is Maintained during Viral Persistence.

B cell follicles of the spleen and lymph nodes are immune privileged sites and serve as sanctuaries for infected CD4+ cells in HIV infection. It is assumed that CD8+ T cell responses promote the establishment of the reservoir, as B cell follicles do not permit CD8+ T cell entry. Here we analyzed the infected cell population in the Friend retrovirus (FV) infection and investigated whether FV can similarly infect follicular cells. For analysis of FV-infected cells, we constructed a recombinant FV encoding the bright fluorescent protein mWasabi and performed flow cytometry with cells isolated from spleens, lymph nodes and bone marrow of FV-mWasabi-infected mice. Using t-stochastic neighbor embedding for data exploration, we demonstrate how the target cell population changes during the course of infection. While FV was widely distributed in erythrocytes, myeloid cells, B cells, and CD4+ T cells in the acute phase of infection, the bulk viral load in the late phase was carried by macrophages and follicular B and CD4+ T cells, suggesting that FV persists in cells that are protected from CD8+ T cell killing. Importantly, seeding into follicular cells was equally observed in CD8+ T cell-depleted mice and in highly FV-susceptible mice that mount a very weak immune response, demonstrating that infection of follicular cells is not driven by immune pressure. Our data demonstrate that infection of cells in the B cell follicle is a characteristic of the FV infection, making this murine retrovirus an even more valuable model for development of retrovirus immunotherapy approaches.IMPORTANCE Human immunodeficiency virus is notorious for its ability to avoid clearance by therapeutic interventions, which is partly attributed to the establishment of reservoirs in latently infected cells and cells that reside in immunologically privileged B cell follicles. In the work presented here, we show that cells of the B cell follicle are equally infected by a simple mouse gammaretrovirus. Using fluorescently labeled Friend retrovirus, we found that B cells and T cells in the B cell follicle, while not carrying the bulk of the virus load, were indeed infected by Friend virus in the early acute phase of the infection and persisted in the chronic infection. Our results suggest that infection of follicular cells may be a shared property of lymphotropic viruses and propose the FV infection of mice as a useful model to study strategies for follicular reservoir elimination.

Marginal Zone B Cells Induce Alloantibody Formation Following RBC Transfusion.

Red blood cell (RBC) alloimmunization represents a significant immunological challenge for some patients. While a variety of immune constituents likely contribute to the initiation and orchestration of alloantibodies to RBC antigens, identification of key immune factors that initiate alloantibody formation may aid in the development of a therapeutic modality to minimize or prevent this process. To define the immune factors that may be important in driving alloimmunization to an RBC antigen, we determined the specific immune compartment and distinct cells that may initially engage transfused RBCs and facilitate subsequent alloimmunization. Our findings demonstrate that the splenic compartment is essential for formation of anti-KEL antibodies following KEL RBC transfusion. Within the spleen, transfused KEL RBCs are found within the marginal sinus, where they appear to specifically co-localize with marginal zone (MZ) B cells. Consistent with this, removal of MZ B cells completely prevented alloantibody formation following KEL RBC transfusion. While MZ B cells can mediate a variety of key downstream immune pathways, depletion of follicular B cells or CD4 T cells failed to similarly impact the anti-KEL antibody response, suggesting that MZ B cells may play a key role in the development of anti-KEL IgM and IgG following KEL RBC transfusion. These findings highlight a key contributor to KEL RBC-induced antibody formation, wherein MZ B cells facilitate antibody formation following RBC transfusion.

Interleukin-10 production and T cell-suppressive capacity in B cell subsets from atherosclerotic apoE -/- mice.

The evidence regarding the role of regulatory B cells (Breg) in atherosclerosis are scarce, and there are contradictory data about their atheroprotective properties. Due to the demonstrated protective function of Breg in different inflammatory diseases mainly through interleukin-10 (IL-10) production, the knowledge of their participation in atherosclerosis immunopathology would be very valuable. To further study which B cell subsets participate in IL-10 production and their regulatory role, splenocytes from apolipoprotein-E-deficient mice were evaluated by ex vivo and in vitro cultures. Atherosclerotic mice had increased frequency of IL-10+ B cells, which presented high CD1d, CD19, and IgM, but variable CD5, CD21, and CD23 expression. IL-10+ B cells were not enriched in B cell subsets previously reported as Breg. Increased frequency of IL-10+ B cells with transitional 1-like (T1-like) and follicular (FO) and reduced CD5+ and marginal zone (MZ) phenotypes were observed ex vivo. Increased frequency of IL-10+ B cells with T1-like and MZ, and decreased IL-10+ FO and T2 phenotypes were also observed in vitro. To determine regulatory capacity of B cells in the atherosclerotic model, each subset were co-cultured with CD4+CD25- T cells. CD5+, FO, MZ, and T1-like cells from atherosclerotic mice exhibited regulation in an IL-10-dependent manner. However, only FO cells decreased both frequency of interferon gamma (IFN-γ)+ and tumor necrosis factor alpha (TNF-α)+ and proliferation of T cells. Finally, splenocytes showed increased frequency of IFN-γ+ and TNF-α+ cells only when FO-depleted B cells were evaluated. These results suggest that mainly FO B cells can modulate in some level the inflammatory responses observed in atherosclerosis.

Cell extrinsic alterations in splenic B cell maturation in Flt3-ligand knockout mice.

B lymphopoiesis in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of immature B cells is reduced in Flt3-ligand (FL-/-) mice leading to deficiencies in splenic B cells. Here, we sought to understand the cellular basis of the spleen B cell deficiency in FL-/- mice. Significant reductions in transitional (TS) and follicular (FO) B cells were found in FL-/- mice, and increased frequencies, but not absolute numbers, of marginal zone (MZ) B cells. BAFF-R expression on splenic B cells and serum levels of B cell activating factor (BAFF) was comparable to wildtype (WT) mice. Mixed BM chimeras revealed that the reductions in TS and FO B cells were cell extrinsic. FL administration into FL-/- mice restored the deficiency in TS B cells and normalized the MZ compartment. Ki67 analysis revealed a significant decrease in the proliferative capacity of TS B cells in FL-/- mice. A Bcl2 transgene did not rescue TS cells in FL-/- mice, uncoupling FL-deficiency to Bcl2-dependent survival pathways. Upregulation of CD1d expression and adoptive transfer experiments suggested MZ skewing in FL-/- mice. These findings support an integral role for Flt3 signaling in peripheral B cell maturation.

B cell autophagy mediates TLR7-dependent autoimmunity and inflammation.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5(f/f)). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.

Reversible expression of CD138 on mature follicular B cells is downregulated by IL-4.

CD138, known as a marker of plasma cells, was reported to be expressed to an intermediate level in the murine bone marrow precursor B cells. Here an intermediate level of CD138 expression was also noted in a subpopulation of splenic follicular B cells, which were distinguishable from CD138(high) plasma cells, whereas the majority of transitional or marginal zone B cells did not express CD138. These CD138(int) B cells were IgM(low)IgD(high) mature B cells, located within follicular B cell zone, and expressed a lower level of CD21 than CD138(-) follicular B cells. During in vitro culture of splenic cells, the proportion of CD138(int) B cells increased, which was noticeably reversed by the addition of IL-4 to the culture. The experiments with sorted CD138(int) cells showed that IL-4-mediated regulation of the CD138 expression was B cell-intrinsic and independent of in vitro B cell death. Our results demonstrate that mouse CD138(int) B cells characterize a subpopulation of IgM(low)IgD(high) mature follicular B cells. The CD138 expression on follicular B cells may represent a reversible status, reflecting a dynamic state probably influenced by IL-4.