WASH prevents the localization of MHCII into lysosomes following endocytosis.

<p>BMDCs derived from (A) <i>Vav-Cre</i> and (B) <i>WASH^f/f Vav-Cre</i> were cultured with an antibody against MHCII following the endocytosis assay then fixed and labeled with antibodies against WASH and LAMP1 for microscopic analysis. (C) Images from A and B were analyzed for MHCII co-localization with LAMP1 using Pearson's co-localization coefficient in ZEN (Carl Zeiss). Zoomed images are demarcated by the white box and dashed lines in the adjacent images. For each condition, >20 individual cells were imaged. Images were collected with 100× oil objective. Scale bars, 10 µm. Bars represent mean ≥ SEM. Horizontal lines indicate statistical comparison between indicated groups, *<i>p</i>≤0.05. (D) Ubiquitinated MHCII was detected in BMDCs by immunoprecipitation of total MHCII followed by immunoblot for ubiquitin (Ub). Blots were subsequently stripped and reprobed with I-A^b antibody as a loading control.</p

VPS35 localizes with MHCII and Vps35 is required in MHCII cell surface retention.

<p>(A) BMDCs were transduced with shRNA constructs targeting VPS35 (shVPS35) or luciferase (shLuc) as a control. Cells were treated with the Golgi transport inhibitor Brefeldin-A for a 5-hour chase, and MHCII cell surface expression was determined by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098606#s2" target="_blank">Materials and Methods</a> section for details). The percent of initial MHCII remaining on the plasma membrane after chase was calculated for three independent samples and plotted as mean ±s.d. To confirm efficient knockdown, VPS35 expression relative to β-actin was determined by quantitative PCR. (B) BMDCs were fixed and labeled with antibodies against MHCII and VPS35 for microscopic analysis. (C) Following the MHCII endocytosis assay, BMDCs were fixed and labeled with anti-VPS35. Zoomed images are demarcated by the white box and dashed lines in the adjacent image. For each condition, >20 individual cells were imaged. Images were collected with 100× oil objective. Scale bars, 10 µm and 1 µm.</p

WASH is required for efficient antigen presentation and T cell priming.

<p>(A) BMDCs from <i>WASH^f/f LysM-Cre</i> mice and control <i>LysM-Cre</i> mice were cultured with OT-II T cells and ovalbumin-derived peptide antigen at the indicated doses. After three days in culture, T cell proliferation was determined by flow cytometry and cell count. (B) Alternatively, BMDCs were first pulsed with peptide antigen, then washed and cultured at the indicated cell numbers with OT-II T cells. T cell proliferation was determined by flow cytometry and cell count after three days. (C) <i>WASH^f/f CD11c-Cre</i> mice and control <i>WASH^f/f</i> mice were immunized by subcutaneous injection of ovalbumin peptide in CFA. Seven days later, draining lymph nodes were harvested and restimulated <i>in vitro</i> with ovalbumin peptide. Antigen-specific T cells producing IL-2 upon restimulation were enumerated by ELISPOT.</p

WASH-deficiency impairs priming of autoreactive T cells and attenuates disease progression in EAE.

<p>(A) <i>WASH^f/f CD11c-Cre</i> mice and control <i>WASH^f/f</i> mice were immunized by subcutaneous injection with MOG peptide in CFA to induce experimental autoimmune encephalitis (EAE). Disease progression was monitored over time using the following scoring system: grade 1 = Tail weakness, grade 2 =  hind limb weakness sufficient to impair righting, grade 3 =  one limb plegic, grade 4 =  hind limb paralysis, grade 5 =  moribund. (B) Mice were immunized with MOG peptide as above and sacrificed at day seven. Draining lymph nodes were harvested and restimulated <i>in vitro</i> with MOG peptide to enumerate antigen-specific T cells by IL-2 ELISPOT.</p

ITAM signaling negatively regulates the antigen response of CD8 T cells and clearance of WNV <i>in vivo.</i>

<p>(A) DF and WT mice were immunized with SIINFEKL and CFA. Seven days later endogenous CD8 T cell response from draining popliteal lymph nodes was measured either with ELISPOT or bead-based ELISA. Data are representative from three independent experiments including in total 30 mice (n = 15 in each group). (B) Mice were infected with WNV and antigen-specific CD8 T cell response was followed by virus clearance and analyzed seven days later. Data are representative from 3 independent experiments. *<i>P</i><0.01, **<i>P</i><0.05.</p

DC-ITAM deficiency show exaggerated production of IL-12 after TLR ligation.

<p>(A) Bone marrow from DF and WT mice were cultured in GM-CSF and at day 10 stimulated with CpG1826 (1 µM) for 6 hrs to evaluate levels of intracellular IL-12. (B) Bone marrow from DF and WT mice were cultured in GM-CSF and at day 10 pre-sorted CD11c⁺CD11b⁺ were stimulated with LPS (10ng/ml). Cells then were lysed and RT-PCR was used to evaluate IRF8 and IL-12 mRNA levels (normalized to β-actin). (C) Pre-sorted CD11c⁺ from draining lymph nodes of immunized DF and WT mice were stimulated <i>in vitro</i> with CpG1826 (1 µM) to evaluate intracellular IL-12 expression in CD11c⁺CD11b⁺Ly6C⁺MHCII⁺ cells. ICS – intracellular cytokine staining. Data are representative from three independent experiments including 3 mice per each investigated group.</p

Defects in migration of naïve CD4⁺ T cells from old mice into inflamed LN.

<p><b>A.</b> Scheme of adoptive transfer studies. 2 x 10⁶ FACS-sorted naïve (CD44^- CD62L⁺) CD4⁺ T cells from adult or old mice were differentially labeled with fluorescent dyes, mixed in a 1:1 ratio, and transferred to recipient adult or old mice that had been infected with WNV-KUN 48 hours earlier via a subcutaneous route. <b>B-C</b>. One hour later, draining popliteal LN (<b>B</b>) and spleen (<b>C</b>) were harvested. Cells were counted and the frequency of adult and old donor cells was determined by flow cytometry. In each tissue, the data was normalized to the levels of adult donor cells in the adult recipient organ and is a composite of four independent experiments. Asterisks indicate statistical significance (*, <i>P</i> < 0.05; Mann-Whitney test). <b>D-E</b>. Time-lapse two-photon intravital imaging of diapedesis of labeled donor naïve CD4⁺ T cells (adult = green; old = blue) in the HEV from the DLN of a recipient WNV-KUN-infected mouse. Images are individual frames from a continuous time-lapse movie (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005027#ppat.1005027.s007" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005027#ppat.1005027.s008" target="_blank">S2</a> Movies</b>). Relative time is displayed in min:sec. Panel <b>D</b> shows a picture of a Q-dot labeled vessel in a LN with multiple old (blue) and adult (green) CD4⁺ T cells adhering to the endothelium. The white dotted line is drawn to highlight the edge of the vessel lumen. The white box shows a zoomed in view of an extravasation "hot spot" that is then presented as a 4 time-lapse images in panel <b>E</b>. Two T cells (cells 1 and 2) from adult mice are shown undergoing diapedesis, whereas two representative T cells (cells 3 and 4) from old mice remain in the lumen over the same time frame. <b>F</b>. Cellular deformation of donor naïve CD4⁺ T cells in the HEV from the DLN of a recipient WNV-KUN-infected mouse as determined by analysis of two-photon microscopic images. <b>G.</b> Time-lapse two-photon imaging of movement of labeled donor naïve CD4⁺ T cells (adult = green; old = blue) in explanted DLN of a recipient WNV-KUN-infected mouse (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005027#ppat.1005027.s009" target="_blank">S3 Movie</a></b>). The figure presents representative cell tracking for adult and old CD4⁺ T cells followed by evaluation of displacement. White opaque dots represent cells with tracked paths (green or blue). Red arrow indicates cell displacement. Scale bar (white): 40 μm. <b>H-L.</b> Analysis of movement parameters of adult and old donor naïve CD4⁺ T cells in explanted LN 6 to 8 hours post-transfer to recipient mice infected with WNV-KUN 48 hours earlier. Individual cells were tracked and (<b>H</b>) speed (μM/min), (<b>I</b>) cell displacement factor (μm/min^1/2), (<b>J</b>) mean square cell displacement over time (μm²), (<b>K</b>) randomness of migration (Hotelling’s test of directionality) and (<b>L</b>) the predicted time of search efficiency for antigen. For panel <b>K,</b> the migration directionality was of a small magnitude, and no significant difference was observed between the cells from adult and old mice. For panel <b>L</b>, to judge the impact that the observed motility defects of old T cells would have on their ability to search antigen in DLN, we used a mathematical model [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005027#ppat.1005027.ref030" target="_blank">30</a>] to predict the time needed by adult and old T cells to first reach increasingly distant locations (first passage time). The first passage time is proportional to the motility coefficient, which we estimated from the mean square displacement data. The predicted time to reach a location 500 μm away is ~20 hours for adult T cells, but ~50 hours for old T cells. The data in <b>F</b>, <b>H and I</b> are shown as a scatter plot and reflects three independent experiments. Asterisks indicate statistical significance (***, <i>P</i> < 0.001, ****, <i>P</i> < 0.0001; Mann-Whitney test).</p

Decreased accumulation of CD4⁺ T cells and CD19⁺ B cells in DLN of old mice after immunization with ovalbumin.

<p><b>A-C</b>. Adult and old mice were immunized with ovalbumin (OVA) complexed with complete Freund’s adjuvant in the footpad. At the indicated days post infection, the draining popliteal LN was harvested, and cells were counted (<b>A</b>). Antibody staining detected specific lymphocyte populations including CD4⁺ T cells (<b>B</b>) and CD19⁺ B cells (<b>C</b>). The results were pooled from two independent experiments with a total of 5 mice per group and data is expressed as the mean ± the standard error of the mean (SEM). <b>D</b>. Numbers of IL-2 secreting CD4⁺ T cells as judged by ELISPOT assay following OVA_323–337 peptide stimulation in the DLN of adult and old mice at day 7 after immunization. <b>E-F.</b> Draining popliteal LN cells were analyzed by flow cytometry at 6 days after infection, and the numbers of GC B cells (CD19⁺ Fas⁺ GL7⁺) (<b>E</b>) and T_FH cells (CD4⁺ PD1⁺ CXCR5⁺) (<b>F</b>) were quantified. Each data point represents and individual mice and the results are pooled from two independent experiments. Asterisks indicate statistical significance (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; unpaired t test).</p

Humoral immune response of donor bone marrow into irradiated recipient mice.

<p><b>A.</b> Scheme of reconstitution studies with bone marrow from adult or old mice. 10⁷ bone marrow cells from adult or old mice were mixed with 10⁷ bone marrow cells from μMT (B cell deficient) mice and transferred into irradiated recipient CD45.1 mice. After 12 weeks, mice were infected with WNV (New York strain) and serum was harvested 5, 8, and 15 dpi. <b>B.</b> After 12 weeks post transfer, blood was sampled and tested for reconstitution efficiency. The vast majority (>95%) of circulating CD19⁺ B cells was derived from the CD45.2 donor mice. <b>C-D</b>. IgM (<b>C</b>) and IgG (<b>D</b>) levels were measured by ELISA for reactivity with WNV E protein. Data is plotted as the reciprocal log₁₀ titer and represents data from 5 mice per group.</p

Increased expansion of Mo-DCs under inflammatory conditions is GM-CSF derived.

<p>(A) Mice were immunized in the footpad with SIINFEKL and CFA and frequencies and total number of Mo-DCs were evaluated at the indicated time points. (B) Mice were immunized in the footpad with SIINFEKL and CFA and the transcript levels of GM-CSF in the draining lymph nodes were evaluated at day 7 post-immunization. Data are representative from three independent experiments including 3 mice per each investigated group. *<i>P</i><0.01</p