PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

Class-switch recombination (CSR) and somatic hypermutation (SHM) occur during the differentiation of germinal center B cells (GCBs). Activation-induced cytidine deaminase (AID) is responsible for both CSR and SHM in GCBs. Here, we show that ablation of PTEN through the Cγ1-Cre mediated recombination significantly influences the CSR and SHM responses. The GCs fail to produce the IgG1 B cells, the high affinity antibodies and nearly lost the dark zone (DZ) in Ptenfl/flCγ1Cre/+ mice after immunization, suggesting the impaired GC structure. Further mechanistic investigations show that LPS- and interleukin-4 stimulation induced the transcription of Cγ1 in IgM-BCR expressing B cells, which efficiently disrupts PTEN transcription, results in the hyperphosphorylated AKT and FoxO1 and in turn the suppression of AID transcription. Additionally, the reduced transcription of PTEN and AID is also validated by investigating the IgM-BCR expressing GCBs from Ptenfl/flCγ1Cre/+ mice upon immunization. In conclusion, PTEN regulated AID transcription in GCBs is essential for the CSR and IgG antibody responses

Splenic red pulp macrophages produce type I interferons as early sentinels of malaria infection but are dispensable for control.

Type I interferons (T1IFNs) are among the earliest cytokines produced during infections due to their direct regulation by innate immune signaling pathways. Reports have suggested that T1IFNs are produced during malaria infection, but little is known about the in vivo cellular origins of T1IFNs or their role in protection. We have found that in addition to plasmacytoid dendritic cells, splenic red pulp macrophages (RPMs) can generate significant quantities of T1IFNs in response to P. chabaudi infection in a TLR9-, MYD88-, and IRF7-dependent manner. Furthermore, T1IFNs regulate expression of interferon-stimulated genes redundantly with Interferon-gamma (IFNG), resulting in redundancy for resistance to experimental malaria infection. Despite their role in sensing and promoting immune responses to infection, we observe that RPMs are dispensable for control of parasitemia. Our results reveal that RPMs are early sentinels of malaria infection, but that effector mechanisms previously attributed to RPMs are not essential for control

Critical coordination of innate immune defense against Toxoplasma gondii by dendritic cells responding via their Toll-like receptors

Toll-like receptors (TLRs) play an important role in host defense against a variety of microbial pathogens. We addressed the mechanism by which TLRs contribute to host defense against the lethal parasite Toxoplasma gondii by using mice with targeted inactivation of the TLR adaptor protein myeloid differentiation primary response gene 88 (MyD88) in different innate cell types. Lack of MyD88 in dendritic cells (DCs), but not in macrophages or neutrophils, resulted in high susceptibility to the T. gondii infection. In the mice deficient in MyD88 in DCs, the early IL-12 response by DCs was ablated, the IFN-γ response by natural killer cells was delayed, and the recruited inflammatory monocytes were incapable of killing the T. gondii parasites. The T-cell response, although attenuated in these mice, was sufficient to eradicate the parasite during the chronic stage, provided that defects in DC activation were compensated by IL-12 treatment early after infection. These results demonstrate a central role of DCs in orchestrating the innate immune response to an intracellular pathogen and establish that defects in pathogen recognition by DCs can predetermine sensitivity to infection

Requirement for MyD88 Signaling in B Cells and Dendritic Cells for Germinal Center Anti-Nuclear Antibody Production in Lyn-Deficient Mice

The intracellular tyrosine kinase Lyn mediates inhibitory receptor function in B cells and myeloid cells, and Lyn(-/-) mice spontaneously develop an autoimmune and inflammatory disease that closely resembles human systemic lupus erythematosus. TLR-signaling pathways have been implicated in the production of anti-nuclear Abs in systemic lupus erythematosus and mouse models of it. We used a conditional allele of Myd88 to determine whether the autoimmunity of Lyn(-/-) mice is dependent on TLR/MyD88 signaling in B cells and/or in dendritic cells (DCs). The production of IgG anti-nuclear Abs, as well as the deposition of these Abs in the glomeruli of the kidneys, leading to glomerulonephritis in Lyn(-/-) mice, were completely abolished by selective deletion of Myd88 in B cells, and autoantibody production and glomerulonephritis were delayed or decreased by deletion of Myd88 in DCs. The reduced autoantibody production in mice lacking MyD88 in B cells or DCs was accompanied by a dramatic decrease in the spontaneous germinal center (GC) response, suggesting that autoantibodies in Lyn(-/-) mice may depend on GC responses. Consistent with this view, IgG anti-nuclear Abs were absent if T cells were deleted (TCRβ(-/-) TCRδ(-/-) mice) or if T cells were unable to contribute to GC responses as the result of mutation of the adaptor molecule SAP. Thus, the autoimmunity of Lyn(-/-) mice was dependent on T cells and on TLR/MyD88 signaling in B cells and in DCs, supporting a model in which DC hyperactivity combines with defects in tolerance in B cells to lead to a T cell-dependent systemic autoimmunity in Lyn(-/-) mice

<i>P. chabaudi</i> infection induces IFNB production in pDCs and RPMs.

<p>(A) No splenocytes are YFP⁺ in mock-infected samples, but some splenocytes become YFP⁺24 h after <i>P. chabaudi</i> infection of C57BL/6 <i>Ifnb</i> reporter mice. 2.5×10⁶ events are depicted in each dot plot. (B) pDCs and RPMs constitute over 90% of YFP⁺ events in C57BL/6 mice. (C) Both pDCs and RPMs contribute to splenic IFNB production in 129Sv mice. pDCs were depleted with a single 500 µg dose of anti-mPDCA1 antibody 18 h before infection with <i>P. chabaudi</i>. Grey dots represent individual mice, with horizontal bars representing the mean (B) or geometric mean (C).</p

Strong hydrophobicity enables efficient purification of HBc VLPs displaying various antigen epitopes through hydrophobic interaction chromatography

Hepatitis B core virus -like particle (HBc-VLP) has become a carrier for expression and presentation of foreign epitopes as vaccine candidates. Efficient purification is necessary for preparation of HBc-VLP and its derivatives with various foreign epitopes. In previous reports, HBc-VLP was mainly purified with ion exchange chromatography. In this study, we developed a platform purification technique based on hydrophobic interaction chromatography (HIC). The underlying principle is the strong hydrophobicity on the surface of HBc-VLP, which was found by mathematical calculation and experimental measurement. Based on HIC, a complete downstream process, from feedstock supernatant to the final pure product, was developed involving a heat pretreatment, an HIC, an ultrafiltration concentration and a size exclusion chromatography. The total recovery of HBc-VLP was 41.92% with nearly 100% purity. HIC was also applicable to three HBc-based vaccine candidates displaying epitope from nuclear protein (NP) and matrix protein 2 (M2e) of the influenza A virus, as well as from ovalbumin (OVA). Optimal HIC condition for these three recombinant VLPs was rationally designed by analysis on their surface hydrophobicity, which was influenced upon insertion of the foreign epitope. By applying the same process developed for HBc-VLP, satisfactory results were achieved for product recovery, purity and host cell DNA removal, thus it is possible to become a platform technique for various HBc-VLP based vaccine candidates.</p

Immunization (i.n.) with live GAS strain M1 provided cross-serotype protection.

<p>Mice were i.n. immunized three times with live M1 strain (0.5−1.0×10⁸/mouse). 10 days after the last immunization mice were challenged with a sublethal dose of GAS serotypes M1 (<i>A</i>), M49 (<i>B</i>), or M28 (<i>C</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107638#s2" target="_blank">Material and Methods</a>). NALTs were taken 24 hr after challenge and homogenized for CFU counting. Data are geometric means of two independent experiments.</p