Cross-Reactive Antigen Expressed by B6 Splenocytes Drives Receptor Editing and Marginal Zone Differentiation of IgG2a-Reactive AM14 Vκ8 B Cells.

The AM14 BCR, derived from an autoimmune MRL/lpr mouse, binds autologous IgG2aa/j with low affinity, and as a result, AM14 B cells only proliferate in response to IgG2a immune complexes that incorporate DNA, RNA, or nucleic acid-binding proteins that serve as autoadjuvants. As such, AM14 B cells have served as a useful model for demonstrating the importance of BCR/TLR coengagement in the activation of autoreactive B cells. We now show that the same receptor recognizes an additional murine-encoded Ag, expressed by B6 splenocytes, with sufficient avidity to induce a TLR-independent proliferative response of BALB/c AM14 Vκ8 B cells both in vivo and in vitro.

Murine immune responses to virus-like particle-associated pre- and postfusion forms of the respiratory syncytial virus F protein.

Unlabelled: Virus-like particles (VLPs) built on the Newcastle disease virus (NDV) core proteins, NP and M, and containing two chimeric proteins, F/F and H/G, composed of respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective RSV neutralizing antibodies in mice. Here, we report the properties of VLPs constructed to contain mutant RSV F protein ectodomains stabilized in prefusion (pre-F/F) or postfusion (post-F/F) configurations. The structures of the chimeric proteins assembled into VLPs were verified immunologically by their reactivities with a conformationally restricted anti-F protein monoclonal antibody.

Modification of the respiratory syncytial virus f protein in virus-like particles impacts generation of B cell memory.

Unlabelled: Immunization with virus-like particles (VLPs) containing the Newcastle disease virus (NDV) core proteins, NP and M, and two chimera proteins (F/F and H/G) containing the respiratory syncytial virus (RSV) F- and G-protein ectodomains fused to the transmembrane and cytoplasmic domains of NDV F and HN proteins, respectively, stimulated durable RSV-neutralizing antibodies, F-protein-specific long-lived, bone marrow-associated plasma cells (LLPCs), and B cell memory, in striking contrast to RSV infection, which did not (M. R. Schmidt, L.

Apurinic/apyrimidinic endonuclease 2 regulates the expansion of germinal centers by protecting against activation-induced cytidine deaminase-independent DNA damage in B cells.

Activation-induced cytidine deaminase (AID) initiates a process generating DNA mutations and breaks in germinal center (GC) B cells that are necessary for somatic hypermutation and class-switch recombination. GC B cells can "tolerate" DNA damage while rapidly proliferating because of partial suppression of the DNA damage response by BCL6. In this study, we develop a model to study the response of mouse GC B cells to endogenous DNA damage.

Long-term and memory immune responses in mice against Newcastle disease virus-like particles containing respiratory syncytial virus glycoprotein ectodomains.

Although respiratory syncytial virus (RSV) is a significant human pathogen, no RSV vaccines are available. We have reported that a virus-like particle (VLP) RSV vaccine candidate stimulated, in mice, robust, protective anti-RSV glycoprotein T(H)1 biased immune responses without enhanced respiratory disease upon RSV challenge. We report here an analysis of long-term responses to these VLPs.

B cell receptor-mediated sustained c-Rel activation facilitates late transitional B cell survival through control of B cell activating factor receptor and NF-kappaB2.

Signaling from the BCR and B cell activating factor receptor (BAFF-R or BR3) differentially regulates apoptosis within early transitional (T1) and late transitional (T2; CD21(int)-T2) B cells during selection processes to generate mature B lymphocytes. However, molecular mechanisms underlying the differential sensitivity of transitional B cells to apoptosis remain unclear. In this study, we demonstrate that BCR signaling induced more long-term c-Rel activation in T2 and mature than in T1 B cells leading to increased expression of anti-apoptotic genes as well as prosurvival BAFF-R and its downstream substrate p100 (NF-kappaB2).

Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice.

The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival.

Expanded CD34+ human umbilical cord blood cells generate multiple lymphohematopoietic lineages in NOD-scid IL2rgamma(null) mice.

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation, we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells, cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice.

A new Hu-PBL model for the study of human islet alloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gamma chain gene.

Immunodeficient NOD-scid mice bearing a targeted mutation in the IL2 receptor common gamma chain (Il2rgamma(null)) readily engraft with human stem cells. Here we analyzed human peripheral blood mononuclear cells (PBMC) for their ability to engraft NOD-scid Il2rgamma(null) mice and established engraftment kinetics, optimal cell dose, and the influence of injection route. Even at low PBMC input, NOD-scid Il2rgamma(null) mice reproducibly support high human PBMC engraftment that plateaus within 3-4 weeks.

Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival.

We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism.

Bruton's tyrosine kinase mediates NF-kappa B activation and B cell survival by B cell-activating factor receptor of the TNF-R family.

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway.

BLyS and B cell homeostasis.

Naïve peripheral B cells survive in vivo because of active stimulation by the TNF superfamily ligand B lymphocyte stimulator (BLyS/BAFF). Although the survival promoting properties of BLyS are well known, the signal pathways and molecular effectors that characterize this stimulation are still being elucidated. In this communication, we discuss the signal cascades that effect BLyS dependent survival and the regulation of BLyS induced signaling.

Inducible DNA breaks in Ig S regions are dependent on AID and UNG.

Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cmu) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination.

Homeostatic proliferation of B cells.

Naïve B cells introduced into a lymphopenic host undergo antigen-independent proliferation which is inhibited in a cell dose dependent manner by feedback from mature B cells. Homeostatic proliferation is a generalized lymphocyte property with B cells sharing many of the inductive and regulatory characteristics established for naïve and memory CD4+ and CD8+ T cells and NK cells. In this communication we discuss the cytokine requirements for B cell HP, extend the murine studies to human cells, and propose the hypothesis that B cell HP may provide an antigen-independent mechanism for maintaining B cell memory.

B1b lymphocytes confer T cell-independent long-lasting immunity.

Many microbial pathogens employ antigenic variation as a strategy to evade the immune system, posing a challenge in vaccine development. To understand the requirements for immunity against such pathogens, we studied Borrelia hermsii, a relapsing fever bacterium. We found that mice deficient in T, follicular B, marginal zone B, or B1a lymphocytes resolved B.

The resolution of relapsing fever borreliosis requires IgM and is concurrent with expansion of B1b lymphocytes.

The rate of pathogen clearance is a critical determinant of morbidity and mortality. We sought to characterize the immune response responsible for the remarkably rapid clearance of individual episodes of bacteremia caused by the relapsing fever bacterium, Borrelia hermsii. SCID or Rag(-/-) mice were incapable of resolving B.

Naive B lymphocytes undergo homeostatic proliferation in response to B cell deficit.

Naive peripheral B cells are maintained in sufficient numbers and diversity to mount effective immune responses against infectious agents. However, the size and repertoire of this B cell pool is constantly diminished by normal cell turnover and Ag activation. Homeostatic (Ag-independent) proliferation in response to B cell depletion is one mechanism to compensate for this cell loss.

BCR engagement induces Fas resistance in primary B cells in the absence of functional Bruton's tyrosine kinase.

B cell susceptibility to Fas-mediated apoptosis is regulated in a receptor-specific fashion. CD40 engagement produces marked sensitivity to Fas killing, whereas surface Ig (sIg) engagement blocks Fas signaling for cell death in otherwise sensitive, CD40-stimulated B cell targets, and thus, induces a state of Fas resistance. The signaling mediator, Bruton's tyrosine kinase (Btk), is required for certain sIg-triggered responses, and Btk is reported to directly bind Fas and block Fas-mediated apoptosis.