M2 expression is predominantly associated with a germinal center phenotype following intranasal infection.

<p>Mice were infected at 1000 PFU via the intranasal route with the parental MHV68 H2bYFP and MHV68 H2bYFP M2-Thy1.1 viruses. Splenocytes were harvested and cell surface markers were analyzed by flow cytometry at 16 dpi. Each data point represents one animal and the horizontal bar represents the mean. Statistics were determined by two-tailed unpaired t test with Welch’s correction. The frequency of B220⁺YFP⁺ latently infected B cells (A) exhibiting a germinal center (B) or plasma cell (C) phenotype were identified by flow cytometry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006543#ppat.1006543.g008" target="_blank">Fig 8</a>. The B220⁺YFP⁺Thy1.1⁺ (M2 reporter) population was identified as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006543#ppat.1006543.g007" target="_blank">Fig 7</a> and subphenotyped using surface markers consistent with a germinal center B cell (E) and plasma cell (F).</p

M2-transduced B cells establish splenic plasma cell and germinal center B cell populations upon adoptive transfer in naïve mice.

<p>Primary splenic B cells isolated by negative selection from naïve CD45.1+ donors were stimulated with LPS and subsequently transduced with M2- or M2.stop- retroviruses prior to adoptive transfer into naïve C57BL/6 mice (CD45.2+). Splenocytes from adoptive transfer recipients were harvested at D1 and D5 post transfer and cell surface markers were analyzed by flow cytometry. Data was compiled from two independent experiments with 3 mice/group. Each data point represents one animal and the horizontal bar represents the mean. Statistics were determined by two-tailed unpaired t test with Welch’s correction. (A) Representative flow plots depicting gating strategies to identify and subphenotype the transduced adoptively transferred B cell population (CD45.1+Thy1.1+) for plasma cell (B220^LoCD138^Hi) or germinal center B cell (B220+GL7^HiCD95^Hi) cell surface markers. Absolute numbers of splenic CD45.1+ B cells (B), CD45.1+Thy1.1+ B cells(C), CD45.1+B220+Thy1.1+ B cells (D), CD45.1+Thy1.1+ B cells displaying a plasma cell phenotype, and (E) CD45.1+B220+Thy1.1+ B cells displaying a germinal center phenotype (F).</p

M2 antigen expression modulates splenic B cell activation and differentiation in vivo.

<p>The transduced adoptively transferred B cell populations from M2 and M2.stop animals were analyzed for cell surface marker expression as a function of transgene expression. Data is representative of two independent experiments with 3 mice/group. A) Representative histogram depicting Thy1.1 expression within the adoptively transferred (CD45.1+) population in M2 and M2.stop animals at D5 post transfer. Thy1.1 expression in the CD45.1+ fraction was divided into Thy1.1 –, Thy1.1 Lo/Int (or Thy1.1 +), and Thy1.1 Hi populations. Representative histograms displaying B cell surface marker expression and intracellular IL10 production corresponding to Thy1.1 expression intensities are displayed for M2 (B) and M2.stop (C) transduced B cells.</p

Generation of recombinant MHV68 M2 reporter viruses.

<p>(A) The M2-Thy1.1 or M2-mCherry reporter construct (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006543#ppat.1006543.g001" target="_blank">Fig 1A</a>) was incorporated at the M2 locus in the MHV68 H2bYFP background under the control of the native M2 promoter. C57BL/6 mice were infected at 1000PFU via the intraperitoneal route and splenocytes were harvested at 14 dpi. Statistics were determined by two-tailed unpaired t test with Welch’s correction. (B and C) Representative flow plots (left) and quantitation (right) of B220⁺YFP⁺ frequency in latently infected splenocytes were obtained from infections comparing the parental MHV68 H2bYFP virus and two (2) independent clones of either the MHV68 H2bYFP M2-mCherry (B) or M2-Thy1.1 (C) virus. Each data point represents one animal and the horizontal bar represents the mean.</p

M2-mCherry reporter construct recapitulates IL10-dependent expansion of M2-transduced primary B cells in vitro.

<p>(A) Retroviruses were derived from replication defective murine stem cell virus (MSCV) vectors that drive constitutive expression of the wild type M2, M2.stop (M2 ORF containing a translational stop codon at amino acid 13), or M2-mCherry upstream of the IRES-Thy1.1 cassette. (B) Primary B cells were isolated from naïve mice by negative selection and stimulated with LPS prior to retroviral transduction. Cell cultures were harvested at the indicated time points and cell surface Thy1.1 expression was analyzed by flow cytometry. (C) Supernatants from retrovirally transduced B cell cultures were analyzed for IL10 secretion by ELISA. Mean and standard deviation values for (B) and (C) were derived from compiling data from two independent experiments containing three replicates per condition per time point.</p

Detection of M2 reporter activity in activated splenic B cells.

<p>(A) Representative images of transduced primary B cells obtained from brightfield and TRITC filters at day 3 post-transduction. (B) Representative flow cytometry histograms obtained at day 3 post-transduction compare mCherry expression in Thy1.1 positive (red) and Thy1.1 negative (grey) populations as determined by FACS analysis. (C) Frequencies of cell surface Thy1.1 and intracellular mCherry expression of M2-transduced (left panel) and M2-mCherry-transduced (right panel) B cell cultures were monitored over the time course by flow cytometry. Mean and standard deviation values were derived from compiling data from two independent experiments containing three replicates per condition per time point.</p

M2 reporter viruses efficiently reactivate virus from latently infected splenocytes ex vivo.

<p>Mice were infected at 1000PFU IP with the parental MHV68 H2bYFP virus and two independent clones of either the MHV68 H2bYFP M2-mCherry (A) or M2-Thy1.1 (B) viruses. Infected splenocytes harvested at 14 dpi were pooled from 3–5 mice/condition and serial dilutions were plated onto feeder cells as described in materials and methods. Cytopathic effect (CPE) was scored at 14–21 days post-explant to determine the frequency of cells that are capable of reactivation infectious virus ex vivo.</p

M2 protein is detected in the germinal center but not in the plasma cell compartment during latency.

<p>Mice were infected at 1000PFU via the intraperitoneal route with the parental MHV68 H2bYFP and second generation MHV68 H2bYFP M2 reporter viruses. Splenocytes were harvested and analyzed by flow cytometry at 14 dpi. Each data point represents one animal and the horizontal bar represents the mean. Statistics were determined by one way analysis of variance (ANOVA) followed by Bonferonni’s multiple comparisons post tests. (A) Representative flow plots (left) and quantitation (right) of latently infected B220⁺YFP⁺ population (top panel) and B220⁺YFP⁺M2-reporter⁺ cells (bottom panel) exhibiting a germinal center phenotype (GL7^HiCD95^Hi). (B) Representative flow plots (left) and quantitation (right) of YFP+ (top panel) and YFP⁺M2-reporter⁺ cells (bottom panel) exhibiting a plasma cell phenotype (B220^LoCD138^Hi).</p

Altered GC B cell populations in IL-21R-/- mice.

<p>Mice were infected intranasally with 1,000 pfu of MHV68-H2bYFP and splenocytes were harvested at the indicated time points. (A) Representative flow plots showing germinal center B cells populations. Plots are gated on YFP^- or YFP⁺ germinal center B cells (CD3^-, CD4^-, CD8^-, B220⁺, CD95^hi, GL7^hi). Centroblasts are defined as CXCR4^hi, CD86^lo-neg and centrocytes are defined as CXCR4^lo-neg, CD86^hi. Quantitation of (B) uninfected and (C) infected centroblast and centrocyte populations. *, p<0.05; **, p<0.01; ***, p<0.001; ****,p<0.0001.</p

Model of M2 mediated IL-10.

<p>Upon BCR engagement, multiple protein kinases including Src family kinases (Lyn,Fyn,Src,Blk,Yes), Syk, Btk get activated leading to the activation of PLCγ2.Activated PLCγ2 catalyzes the hydrolysis of phosphatidylinositol-4,5-biphosphate [(PtdIns(4,5)P2] to DAG and IP3. Binding of IP3 to its receptor on the ER membrane leads to release of ER Ca²⁺ stores. However, this increase in Ca²⁺ is only transient and following depletion of ER Ca²⁺ stores, an influx of extracellular Ca²⁺ occurs (termed as SOCE-Store Operated Calcium Entry). This prolonged and sustained increase in Ca²⁺ leads to activation of the Calcineurin-NFAT pathway. In the case of M2, <i>Miranda et al</i> have shown that M2 can interact with Fyn and PLCγ2. Our model suggests that interaction of M2 with proximal membrane signaling molecules such as Src kinase and/or PLCγ2 potentially mimics events that occur upon BCR engagement resulting in the Ca²⁺ mediated activation of the NFAT pathway. Induction of IRF4 by M2 is dependent, at least partially by the activation of NFAT pathway. IRF4 can activate regulatory elements in the IL-10 promoter locus (termed CNS-Conserved Noncoding Sequences) in T cells and in our case, in B cells as well. These series of events leads to eventual increase in the levels of IL-10 observed upon induction of M2.</p