YY1 Is Required for Germinal Center B Cell Development.

YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction

Therapeutic vaccination for chronic hepatitis B in the Trimera mouse model

Therapeutic vaccination for chronic hepatitis B in the Trimera mouse modelrnRaja Vuyyuru and Wulf O. BöcherrnHepatitis B is a liver disease caused by Hepatitis B virus (HBV). It ranges in severity from a mild illness, lasting a few weeks (acute), to a serious long-term (chronic) illness that can lead either to liver disease or liver cancer. Acute infection is self limiting in most adults, resulting in clearance of virus from blood and liver and the development of lasting immunity. However 5% of acutely infected patients do not resolve primary HBV infection, leading to chronic infection with persistent viral replication in the liver. The strength of the initial antiviral immune response elicited to Hepatitis B determines the subsequent clinical outcome. A strong and broad T cell response leads to spontaneous resolution. Conversely, a weak T cell response favours viral persistence and establishment of chronic disease. While treatments using interferon-alpha or nucleos(t)ide analogues can reduce disease progression, they rarely lead to complete recovery. The lack of a suitable small animal model hampered efforts to understand the mechanisms responsible for immune failure in these chronic patients.rnIn current study we used Trimera mice to study the efficacy of potential vaccine candidates using HBV loaded dendritic cells in HBV chronic infection in vivo. The Trimera mouse model is based on Balb/c mice implanted with SCID mouse bone marrow and human peripheral blood mononuclear cells (PBMC) from HBV patients, and thus contains the immune system of the donor including their HBV associated T cell defect.rnIn our present study, strong HBV specific CD4+ and CD8+ T cell responses were enhanced by therapeutic vaccination in chronic HBV patients. These T cell responses occurred independently of either the course of the disease or the strength of their underlying HBV specific T cell failure. These findings indicate that the Trimera mouse model represents a novel experimental tool for evaluating potential anti-HBV immunotherapeutic agents. This in vivo data indicated that both the HBV specific CD4+ cell and CD8+ responses were elicited in the periphery. These HBV specific T cells proliferated and secreted cytokines upon restimulation in Trimera mice. The observation that these HBV specific T cells are not detectable directly ex vivo indicates that they must be immune tolerant or present at a very low frequency in situ. HBV specific T cell responses were suppressed in Trimera mice under viremic conditions, suggesting that viral factors might be directly involved in tolerizing or silencing antiviral T cell responses. Thus, combination of an effective vaccine with antiviral treatment to reduce viremia might be a more effective therapeutic strategy for the future. Such approaches should be tested in Trimera mice generated in HBV or HBs expressing transgenic mice before conducting clinical trials.r

The characteristics of Borrelia hermsii infection in human hematopoeitic stem cell-engrafted mice mirror those of human relapsing fever

Rodents are natural reservoirs for a variety of species of Borrelia that cause relapsing fevers in humans. The murine model of this disease recapitulates many of the clinical manifestations of the human disease and has revealed that T cell-independent antibody responses are required to resolve the bacteremic episodes. However, it is not clear whether such protective humoral responses are mounted in humans

Interleukin-7-dependent B lymphocytes are required for the anti-pneumococcal polysaccharide response and protective immunity to Streptococcus pneumoniae

Unlike human adults or adult mice, young children or young mice respond poorly to pneumococcal polysaccharides (PPS). In mice, B1b lymphocytes are the major responders to a variety of bacterial polysaccharides including PPS. Despite having B1b cells, young mice are severely impaired in responding to PPS, suggesting that B cells in the young are distinct from those in adults. Since B lymphopoeisis early in life is largely Interleukin-7 (IL-7)-independent, while in adults it is IL-7-dependent, we hypothesize that B cells developed in the presence of IL-7 are required for generating anti-PPS antibody responses. In support of this, we found that despite having B1b cells, young wildtype and adult mice deficient either in IL-7 or IL-7Rα are severely impaired in responding to Pneumovax®23 vaccine, and do not survive pneumococcal challenge. Furthermore, we found that transgenic expression of IL-7 promotes the anti-PPS response in young and confers protective immunity to young mice. To translate these findings to human infants we have utilized neonatal NOD/SCID/gcnull mice engrafted with human umbilical cord blood CD34+ hematopoietic stem cells to create a Human Immune System mouse (HISmouse) model. We have found that these HISmice generate several B cell subsets including B1 (CD19+CD20+CD27+CD43+CD70-CD69-) and the majority of them exhibit an immature phenotype. Moreover, just as young children, HISmice responded poorly to PPS. IL-7 is produced mainly by non-hematopoietic stromal cells, and unlike the human IL-7, the murine IL-7 is poor stimulator of human B lymphocyte development. Although our data indicate that IL-7-dependent B cells are crucial for generating anti-polysaccharide response, we also found that enforced expression of a polysaccharide (a1,3, dextran)-specific B cell antigen receptor heavy chain (VH J558) in mice can overcome the lack of anti-polysaccharide antibody responses in young mice even in the absence of an IL-7-dependent B lymphopoiesis

YY1 is required for germinal center B cell development and immunoglobulin class switching.

<p><b>(A)</b> Spleen cells from non-immunized <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were stained with various antibodies to identify total B cells (CD19⁺AA4.1⁺, upper panel) and germinal center B cells (GC-B, DUMP^-IgD^-GL7^hiCD95^hi, lower panel). Percentages and number of <b>(B)</b> total B cells, and <b>(C)</b> GC-B cells per spleen of <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice. Fig A-C are from three independent experiments (<i>n</i> = 3 mice for each genotype). <b>(D)</b> We used ELISA to detect various isotypes of serum immunoglobulins from <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice. The concentration of IgM, IgA, total IgG, as well as IgG subclasses, IgG1, IgG2 and IgG3 were measured from sera samples that were obtained from four experiments (<i>n</i> ≥ 4 mice for each genotype). Asterisks indicate p<0.001.</p

YY1 is required for antigen-specific germinal center development and for generation of antigen-specific IgG1.

<p><b>(A)</b> Splenocytes from NP-CGG immunized <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were harvested at 14 days after immunization and stained with various antibodies, as well as PNA to detect GC B cells. We gated on CD4⁻CD8⁻F4/80⁻Gr1⁻(DUMP⁻) IgD^- cells that were subdivided into PNA⁺B220⁺ GC-B cells. GC-B cells were gated and further subsetted into NP-specific (NP⁺B220⁺) GC-B cells. Representative results are from three independent experiments. <b>(B)</b> Numbers of NP-specific (NP⁺B220⁺) GC-B cells per spleen of immunized mice (<i>n</i> = 3). <b>(C)</b> <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were immunized with NP-CGG, and 14 days later spleen sections were stained with anti-GL7, anti-IgD and anti-TCRβ antibody. GL7-rich regions demarcate germinal center B cells. <b>(D, E)</b> Serum from NP-CGG immunized <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were collected at 14 days after immunization and NP-specific serum Igs were analyzed using ELISA. <i>D</i>. The concentration of low affinity (NP26, left panel) and high affinity (NP4, right panel) IgG1 in the serum. <i>E</i>. Titer of NP-specific total IgM in the sera of immunized mice. Data are derived from sera samples that were obtained from three experiments. Asterisks indicate p<0.001.</p