The sphingosine 1-phosphate receptor S1P2 maintains the homeostasis of germinal center B cells and promotes niche confinement.

Mice deficient in sphingosine 1-phosphate receptor type 2 (S1P(2)) develop diffuse large B cell lymphoma. However, the role of S1P(2) in normal germinal center (GC) physiology is unknown. Here we show that S1P(2)-deficient GC B cells outgrew their wild-type counterparts in chronically established GCs. We found that antagonism of the kinase Akt mediated by S1P(2) and its downstream mediators Gα(12), Gα(13) and p115RhoGEF regulated cell viability and was required for growth control in chronically proliferating GCs. Moreover, S1P(2) inhibited GC B cell responses to follicular chemoattractants and helped confine cells to the GC. In addition, S1P(2) overexpression promoted the centering of activated B cells in the follicle. We suggest that by inhibiting Akt activation and migration, S1P(2) helps restrict GC B cell survival and localization to an S1P-low niche at the follicle center.

Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B-cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short-lived plasma cells, long-lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock-out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre- and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long-lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage.

An Overview of l-Amino Acid Oxidase Functions from Bacteria to Mammals: Focus on the Immunoregulatory Phenylalanine Oxidase IL4I1.

l-amino acid oxidases are flavin adenine dinucleotide-dependent enzymes present in all major kingdom of life, from bacteria to mammals. They participate in defense mechanisms by limiting the growth of most bacteria and parasites. A few mammalian LAAOs have been described, of which the enzyme "interleukin-4 induced gene 1" (IL4I1) is the best characterized. IL4I1 mainly oxidizes l-phenylalanine. It is a secreted enzyme physiologically produced by antigen presenting cells of the myeloid and B cell lineages and T helper type (Th) 17 cells. Important roles of IL4I1 in the fine control of the adaptive immune response in mice and humans have emerged during the last few years. Indeed, IL4I1 inhibits T cell proliferation and cytokine production and facilitates naïve CD4⁺ T-cell differentiation into regulatory T cells in vitro by limiting the capacity of T lymphocytes to respond to clonal receptor stimulation. It may also play a role in controlling the germinal center reaction for antibody production and limiting Th1 and Th17 responses. IL4I1 is expressed in tumor-associated macrophages of most human cancers and in some tumor cell types. Such expression, associated with its capacity to facilitate tumor growth by inhibiting the anti-tumor T-cell response, makes IL4I1 a new potential druggable target in the field of immunomodulation in cancer.

A role for host activation-induced cytidine deaminase in innate immune defense against KSHV.

Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens.

Analysis of mice deficient in both REV1 catalytic activity and POLH reveals an unexpected role for POLH in the generation of C to G and G to C transversions during Ig gene hypermutation.

Multiple DNA polymerases are involved in the generation of somatic mutations during Ig gene hypermutation. Mice expressing a catalytically inactive REV1 (REV1AA) exhibit reduction of both C to G and G to C transversions and moderate decrease of A/T mutations, whereas DNA polymerase η (POLH) deficiency causes greatly reduced A/T mutations. To investigate whether REV1 and POLH interact genetically and functionally during Ig gene hypermutation, we established REV1AA Polh(-/-) mice and analyzed Ig gene hypermutation in the germinal center (GC) B cells. REV1AA Polh(-/-) mice were born at the expected ratio and developed normally with no apparent gross abnormalities. B-cell development, maturation, Ig gene class switch and the GC B-cell expansion were not affected in these mice. REV1AA Polh(-/-) B cells also exhibited relatively normal sensitivity to etoposide and ionizing radiation. Analysis of somatic mutations in the J(H)4 intronic region revealed that REV1AA Polh(-/-) mice had a further decrease of overall mutation frequency compared with REV1AA or Polh(-/-) mice, indicating that the double deficiency additively affected the generation of mutations. Remarkably, REV1AA Polh(-/-) mice had nearly absent C to G and G to C transversions, suggesting that POLH is essential for the generation of residual C to G and G to C transversions observed in REV1AA mice. These results reveal genetic interactions between REV1 catalytic activity and POLH and identify an alternative pathway, mediated by non-catalytic REV1 and POLH, in the generation of C to G and G to C transversions.

A disintegrin and metalloproteinase 10 regulates antibody production and maintenance of lymphoid architecture.

A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.

Low protease activity in B cell follicles promotes retention of intact antigens after immunization.

The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited.

Phosphoinositide 3-kinase activity in T cells regulates the magnitude of the germinal center reaction.

The generation of high-affinity Abs is essential for immunity and requires collaboration between B and T cells within germinal centers (GCs). By using novel mouse models with a conditional deletion of the p110δ catalytic subunit of the PI3K pathway, we established that p110δ is required in T cells, but not in B cells, for the GC reaction. We found the formation of T follicular helper (T(FH)) cells to be critically dependent on p110δ in T cells. Furthermore, by deleting phosphatase and tensin homolog deleted on chromosome 10, which opposes p110δ in activated T cells, we found a positive correlation between increased numbers of T(FH) cells and GC B cells. These results are consistent with the hypothesis that T cell help is the limiting factor in the GC reaction. P110δ was not required for the expression of B cell lymphoma 6, the downregulation of CCR7, or T cell entry into primary follicles. Instead, p110δ was the critical catalytic subunit for ICOS downstream signaling and the production of key T(FH) cytokines and effector molecules. Our findings support a model in which the magnitude of the GC reaction is controlled by the activity of the PI3K pathway in T(FH) cells.

Evolution of ectopic lymphoid neogenesis and in situ autoantibody production in autoimmune nonobese diabetic mice: cellular and molecular characterization of tertiary lymphoid structures in pancreatic islets.

A pivotal role for tertiary lymphoid structures (TLSs) in promoting Ag-specific humoral responses during chronic inflammation is emerging in several autoimmune conditions, including rheumatoid arthritis, Sjogren's syndrome, and autoimmune thyroiditis. However, there is limited evidence on the cellular and molecular mechanisms underlying TLS formation and their contribution to autoimmunity in the pancreas during autoimmune insulitis. In this study, we performed a detailed and comprehensive assessment of the evolution of TLSs during autoimmune insulitis in 126 female NOD mice from 4 to 38 wk of age. We demonstrated that during progression from peri- to intrainsulitis in early diabetic mice, T and B cell infiltration follows a highly regulated process with the formation of lymphoid aggregates characterized by T/B cell segregation, follicular dendritic cell networks, and differentiation of germinal center B cells. This process is preceded by local upregulation of lymphotoxins alpha/beta and lymphoid chemokines CXCL13 and CCL19, and is associated with infiltration of B220(+)/IgD(+)/CD23(+)/CD21(-) follicular B cells expressing CXCR5. Despite a similar incidence of insulitis, late diabetic mice displayed a significantly reduced incidence of fully organized TLSs and reduced levels of lymphotoxins/lymphoid chemokines. Upon development, TLSs were fully functional in supporting in situ autoreactive B cell differentiation, as demonstrated by the expression of activation-induced cytidine deaminase, the enzyme required for Ig affinity maturation and class switching, and the presence of CD138(+) plasma cells displaying anti-insulin reactivity. Overall, our work provides direct evidence that TLSs are of critical relevance in promoting autoimmunity and chronic inflammation during autoimmune insulitis and diabetes in NOD mice.

Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting.

Subjects with X-linked hyper-IgM syndrome (X-HIgM) have a markedly reduced frequency of CD27(+) memory B cells, and their Ig genes have a low level of somatic hypermutation (SHM). To analyze the nature of SHM in X-HIgM, we sequenced 209 nonproductive and 926 productive Ig heavy chain genes. In nonproductive rearrangements that were not subjected to selection, as well as productive rearrangements, most of the mutations were within targeted RGYW, WRCY, WA, or TW motifs (R = purine, Y = pyrimidine, and W = A or T). However, there was significantly decreased targeting of the hypermutable G in RGYW motifs. Moreover, the ratio of transitions to transversions was markedly increased compared with normal. Microarray analysis documented that specific genes involved in SHM, including activation-induced cytidine deaminase (AICDA) and uracil-DNA glycosylase (UNG2), were up-regulated in normal germinal center (GC) B cells, but not induced by CD40 ligation. Similar results were obtained from light chain rearrangements. These results indicate that in the absence of CD40-CD154 interactions, there is a marked reduction in SHM and, specifically, mutations of AICDA-targeted G residues in RGYW motifs along with a decrease in transversions normally related to UNG2 activity.

Activation-induced cytidine deaminase expression and activity in the absence of germinal centers: insights into hyper-IgM syndrome.

Somatic hypermutation normally occurs as a consequence of the expression of activation-induced cytidine deaminase (AID) by Ag-activated, mature B cells during T cell-dependent germinal center responses. Nonetheless, despite their inability to express CD154 and initiate GC responses, patients with type 1 hyper-IgM syndrome (HIGM1) support populations of IgM(+)IgD(+)CD27(+) B cells that express mutated Ig genes. The origin of these mutated B cells is unknown; the IgM(+)IgD(+)CD27(+) cells do not express AID and appear to acquire mutations independent of stringent selection by Ag. Here, we demonstrate that immature/transitional 1 B cells from the bone marrow of CD154-deficient mice express AID and acquire Ig mutations that lack the hallmarks of antigenic selection via BCR signaling. Comparable levels of AID expression was found in developmentally immature B cells recovered from murine fetal liver and from human immature/transitional 1 B cells recovered from umbilical cord blood. AID expression in human fetal liver was also robust, approaching that of human tonsil tissue and the human germinal center B cell line, Ramos. These observations led us to conclude that AID expression in developing human B cells is the origin of the mutated IgM(+)IgD(+)CD27(+) B cells present in HIGM1 patients, and we propose that both mice and humans share a latent, AID-dependent pathway for the preimmune diversification of B lymphocytes that is more prominent in chicken, sheep, and rabbits.

SMS2 deficiency impairs PKCδ-regulated B cell tolerance in the germinal center.

B cell tolerance prevents autoimmunity by deleting or deactivating autoreactive B cells that otherwise may cause autoantibody-driven disorders, including systemic lupus erythematosus (lupus). Lupus is characterized by immunoglobulin Gs carrying a double-stranded (ds)-DNA autospecificity derived mainly from somatic hypermutation in the germinal center (GC), pointing to a checkpoint breach of GC B cell tolerance that leads to lupus. However, tolerance mechanisms in the GC remain poorly understood. Here, we show that upregulated sphingomyelin synthase 2 (SMS2) in anti-dsDNA GC B cells induces apoptosis by directly activating protein kinase C δ (PKCδ)'s pro-apoptotic activity. This tolerance mechanism prevents lupus autoimmunity in C57/BL6 mice and can be stimulated pharmacologically to inhibit lupus pathogenesis in lupus-prone NZBWF1 mice. Patients with lupus consistently have substantially reduced SMS2 expression in B cells and to an even greater extent in autoimmune-prone, age-associated B cells, suggesting that patients with lupus have insufficient SMS2-regulated B cell tolerance.

Downregulated expression of SHP-1 in Burkitt lymphomas and germinal center B lymphocytes.

We wish to identify developmental changes in germinal center B cells that may contribute to their rapid growth. SHP-1 is an SH2 domain-containing phosphotyrosine phosphatase that negatively regulates activation of B cells and other cells of hematopoietic lineages. We have found that in all 13 EBV-negative and 11 EBV-positive Burkitt lymphomas with a nonlymphoblastoid phenotype, the mean concentration of SHP-1 was reduced to 5% of that of normal B and T cells. The possibility that this diminished expression of SHP-1 was related to the germinal center phenotype of Burkitt lymphomas was supported by the low to absent immunofluorescent staining for SHP-1 in germinal centers, and by the inverse relationship between the concentration of SHP-1 and the expression of the germinal center marker CD38 on purified tonsillar B cells. In CD38-high B cells, SHP-1 concentration was 20% of that of mantle zone B cells from the same donor. This reduction in SHP-1 is comparable to that of cells from motheaten viable mev/mev mice in which there is dysregulated, spontaneous signaling by cytokine and antigen receptors. Therefore, germinal center B cells may have a developmentally regulated, low threshold for cellular activation.

Polymerase chain reaction selects a novel disintegrin proteinase from CD40-activated germinal center dendritic cells.

To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction-based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

Increased expression of activation-induced cytidine deaminase is associated with anti-CCP and rheumatoid factor in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID(+) germinal centres were observed in six of the 12 RA synovial samples, and AID(+) cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID(+) cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (P < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10(+)IgM(+)CD20(+) B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (P < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (P

Btk regulates localization, in vivo activation, and class switching of anti-DNA B cells.

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk-/- mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk-/- mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk-/- mice. A transgene expressing a low level of Btk in B cells (Btk(lo)) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk(lo) B cells. anti-DNA IgG was not observed in 56R.Btk(lo) mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.

Cdk9/Cyclin T1 complex: a key player during the activation/differentiation process of normal lymphoid B cells.

Cdk9/Cyclin T1 complex is very important in controlling specific differentiative pathways of several cell types. Limited data are available regarding the expression of Cdk9/Cyclin T1 in hematopoietic and lymphoid tissues. Cdk9/Cyclin T1 expression seems to be related to particular stages of lymphoid differentiation/activation. In this study, we observed that the expression level of Cdk9/Cyclin T1 in vivo increases in memory B cells compared to naïve B cells, and in activated B cells, compared to non-activated ones. The expression level of the Cdk9/Cyclin T1 complex does not increase in cells induced to differentiate in vitro. In addition, we showed that Cdk9 interacts with E12 and E47, specifically activated during Germinal Center (GC) reaction. Taken together this data suggests an active role for the Cdk9/Cyclin T1 complex during lymphoid differentiation through germinal center reaction.

Methyltransferase Nsd2 Ensures Germinal Center Selection by Promoting Adhesive Interactions between B Cells and Follicular Dendritic Cells.

Antibody affinity maturation, which is an antigen-based selection process for B cells, occurs in germinal centers (GCs). GCB cells must efficiently recognize, acquire, and present antigens from follicular dendritic cells (FDCs) to receive positive selection signals from T helper cells. Previous studies showed that GCB cells undergo adhesive interactions with FDCs, but the regulatory mechanisms underlying the cell adhesions and their functional relevance remain unclear. Here, we identified H3K36me2 methyltransferase Nsd2 as a critical regulator of GCB cell-FDC adhesion. Nsd2 deletion modestly reduced GC responses but strongly impaired B cell affinity maturation. Mechanistically, Nsd2 directly regulated expression of multiple actin polymerization-related genes in GCB cells. Nsd2 loss reduced B cell adhesion to FDC-expressed adhesion molecules, thus affecting both B cell receptor (BCR) signaling and antigen acquisition. Overall, Nsd2 coordinates GCB positive selection by enhancing both BCR signaling and T cell help.

Methods to Study the Role of Cdc42, Rac1, and Rac2 in B-Cell Cytoskeletal Responses.

B-cell migration and adhesion are critical to form a germinal center response, the site for B-cell production of high-affinity antibodies. Here, we describe two assays that can be used to examine B-cell cytoskeletal responses needed during the germinal center response: B-cell spreading and homotypic adhesion. Spreading of B cells is dependent on Cdc42, while Rac1 and Rac2 are necessary for homotypic adhesion. These in vitro assays can be used to examine functional responses of B cells mediated by the cell cytoskeleton, for example when comparing B cells from different gene knockout animals.