172.4 recognize CD100, a 150kDa protein expressed as a homodimer.

<p>(A) Surface radioiodinated YTS cell lysate was immunoprecipitated with mAb 172.4. The immunoprecipitate was analyzed first on non-reducing and then on reducing SDS-PAGE. The left lane is an aliquot of immunoprecipitated proteins resolved under reducing conditions. Molecular weight markers are as shown. The two forms of CD100 (150 and 120 kDa respectively) are indicated with arrows. (B, D) ELISA plates were coated with CD100-Ig and assayed for binding to the indicated antibodies as described in “Materials and Methods”. (C) ELISA plates were coated with CD99-Ig and assayed for binding to the indicated antibodies as described in “Materials and Methods”. The background binding to BSA was subtracted. Figure shows one representative experiment out of four performed. (E) IL-2 activated NK line was stained with either 172.4 or the commercial anti human CD100 antibody A8. FITC conjugated Goat F(ab') anti mouse IgG antibody was used as secondary Ab. Gray histogram is staining with secondary antibody only. Figure show one representative experiment out of 5 performed.</p

Expression of the ligand of 172.4 on lymphocyte sub-populations and NK line.

<p>Biotinylated 172.4 mAb was used in combination with other fluorescently labeled mAbs in a four color staining. 172.4 staining was detected using Cy-5 streptavidin. Cells were analyzed by flow cytometry. T cells were identified as CD3 positive, NK cells as CD56 positive, CD3 negative, and B cells as CD19 positive. Staining was performed on freshly isolated PBL (A, C and E) and on PBL that were cultured for 72 hr in the presence of IL-2 (100 u/ml) and human serum (B, D and F). Each dot blot shows a gated specific sub-population. An isotype matched antibody was used to determent the background staining for each antibody and is represented in the figure as the horizontal line. (H) Freshly isolated PBMC were incubated for 72 hr with 50 ng/ml of the indicated cytokines. Biotinylated 172.4 mAb was detected using Cy-5 streptavidin and used in combination with other fluorescently labeled mAbs in a four color staining. The PBL were analyzed for the expression of CD100 on NK cells and each dot plot represents a gated NK cells (CD3-, CD56+). Figure shows one representative experiment out of four performed.</p

The interaction between CD100 and CD72 leads to the phosphorylation of serine residues on proteins associate with CD100.

<p>(A) ³⁵S-labeled activated NK cells were incubated for 20 minutes with adherent BW or BW/CD72 cells. The wells were washed, the remaining cells were lysed and the level of radioactivity was measured in CPM units (counts per mint). The results presented after subtraction of NK cells only. (B) ³⁵S-labeled activated NK cells were incubated for 20 minutes with adherent BW/CD72 cells that were pre incubated with CD100-Ig or CD99-Ig for 2 hr prior to the incubation with NK cells. The wells were washed, the remaining cells were lysed and the level of radioactivity was measured. *p = 0.02. (C) Activated NK cells were incubated with target cells (BW or BW/CD72), in 37°C, for the indicated time periods. Cells were lysed and proteins were immunoprecipitated by mAb 172.4. The immunoprecipitated proteins were separated on a reducing SDS-PAGE. Phosphorylated proteins were detected in western blot by using rabbit anti phospho-serine polyclonal Ab. CD100 levels were detected in western blot by using the A8 anti human CD100. (D) Densitometrical analysis of the level of phosphorylation on serine residues of the three proteins indicated by arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000818#pone-0000818-g005" target="_blank">Figure 5C</a>. The levels of phosphorylation are relative to their level at time zero which was set as one. Figure shows one representative experiment out of two performed.</p

NK cytotoxicity and IFNγ secretion is enhanced after binding to CD72.

<p>(A) ³⁵S-labeled BW or BW/CD72 cells were tested for lysis by activated NK cells at different effectors to targets ratio as indicated in the figure. *p<0.005. (B) ³⁵S-labeled BW/CD72 cells were pre-incubated with the indicated fusion proteins and tested for lysis by activated NK cells at the indicated E:T ratios.*p<0.05. (C) ³⁵S-labeled BW cells were pre-incubated with the indicated fusion proteins and tested for lysis by activated NK cells at the indicated E:T ratios. The differences observed between the various treatments were not statistically significant. (D) NK cells were incubated with BW or BW/CD72 in the presence or absence of the various cytokines indicated in the figure and IFNγ secretion was measured by ELISA. Figure shows one representative experiment out of four performed. * p = 0.03, **p = 0.01</p

The effects of BW245C on the outcome of LVS infection.

<p>Mice infected with 10⁵ LVS were treated with BW245 diluted in carrier, or with carrier alone, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#s4" target="_blank">Materials and Methods</a>. To examine DC trafficking (A), MdLNs were collected 48 hrs post infection and analyzed for RTDC representation (CD11b^high/autofluorescence^low). Experiments were conducted on MdLN pools, each consisting of 6 organs, derived from infected or non-infected mice treated with either BW245C or carrier alone. Results are presented by bar diagrams as the averages of three independent experiments. * <i>P</i><0.05. Bacterial colonization of the MdLN (B) was examined 48 hrs post infection in 13 individual BW245C-treated and 13 mock-treated mice. ** <i>P</i><0.05. The effect of BW245 on mortality (C) was examined in groups of 10 mice comparing treated (open squares) and mock-treated mice (closed circles). The experiment presented represents one of three similar experiments. The effect of the drug on morbidity (D) was examined by monitoring weight loss for 5 consecutive days in groups of 10 mice each (8-week-old females). Groups consisted of non-infected BW245-treated mice (closed triangles), infected and treated mice (open squares), and infected mock-treated mice (closed circles). Data are presented as percentage of the weight determined on day zero and calculated separately for each individual mouse. *** <i>P</i>-values on days 2–5 range between <0.01 and <0.05.</p

The effects of FTY720 on accumulation of RTDC and LVS in the MdLN.

<p>Mice infected with 10⁵ LVS were treated with FTY720 diluted in carrier solution, or with carrier solution alone, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#s4" target="_blank">Materials and Methods</a>. To examine DC trafficking, MdLNs were collected 48 hrs post infection and analyzed for RTDC representation (CD11b^high/autofluorescence^low) as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#ppat-1000211-g003" target="_blank">Figure 3</a>. An experiment was conducted on MdLN pools, each consisting of 6 organs. MdLNs were derived from infected mice treated with either carrier alone (A) or with FTY720 (B). Non-infected, FTY720-treated mice (C) served as controls. Bacterial colonization of the MdLN (D) was examined 48 hrs post infection in each one of the 6 individual FTY720- treated and 6 mock-treated mice, <i>P</i><0.01. Three independent experiments, each conducted with groups of six mice, exhibited similar correlation between RTDC and LVS recruitment.</p

Cell recruitment to MdLN following airway infection with LVS.

<p>MdLNs were isolated at various time points after intra-nasal infection of mice. Single cell suspensions were analyzed by flow cytometry as indicated on the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#ppat-1000211-g003" target="_blank">Figure 3</a>. Number of AMΦ and RTDC at different time points post infection are presented as bar diagrams.</p

Interaction of BMDC and LVS in vitro Alter.

<p>(A) Propagation of LVS in J774A.1 cells and BMDC: Infection was performed as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#s4" target="_blank">Materials and Methods</a> section, and gentamicin was added 1 hr post pulsing with bacteria. Cells were harvested 2 or 24 hrs post infection, washed, and lysed with DOC. Lysates were diluted and plated for CFU enumeration. Each bar represents the average CFUs in three infection wells. The entire experiment represents one of three repetitions. For visualization of cell-associated bacteria by fluorescence microscopy, infected J774A.1 or BMDC cells were stained with anti-LVS antibodies 24 hrs post LVS infection. (B) Expression of CCR7 by BMDC: Cells were analyzed for CCR7 expression 24 hours post infection with the live or formaldehyde-killed LVS. Non-pulsed cells and cells pulsed with 1 µg/ml <i>E. coli</i> LPS served as negative and positive controls, respectively. Gray lines denote isotype-matched immunoglobulin controls. Fractions of CCR7^positive cells are indicated. (C) DC were pulsed as indicated above, and expression of co-stimulatory molecules was examined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#s4" target="_blank">Materials and Methods</a>. Geomeans of fluorescence intensities are presented as relative values, using the values obtained for each marker by pulsing with 1 µg/ml <i>E. coli</i> LPS as 100. Results are presented by bar diagrams as the averages of three independent experiments. (D) Migration of BMDC: Cells were pulsed as indicated above, 24 hrs later cells were examined for migration in Transwell chambers towards CCL19 or control medium. Non-pulsed cells and cells pulsed with LPS served as controls. Results are presented as percent cells migrating from the upper compartments to the lower compartment. Each bar represents the average migration in 3 Transwells. The experiment was repeated 3 times. Data presented in this figure were generated by using BALB/c BMDC. Results were confirmed with DC derived from C57BL6 mice.</p

Association of LVS with newly immigrating cells in infected lymph nodes.

<p>Airway cells of live mice were stained by instillation of CMTMR. Migration to lymph node was induced by intranasal infection with 10⁵ CFU of LVS. Single cell suspensions were prepared from pools of 8 lymph nodes, collected 48 hrs post infection. Cells were sorted by MACS with CD11b-microbeads. Bound and non bound cells were analyzed by flow cytometry (A) to evaluate the fraction of CMTMR⁺/CD11b⁺ cells. Sorted cells were examined by florescence microscopy for presence of the indicated cell phenotype (B). Large numbers of cells were screened to determine the proportion of the various cell populations (C). In addition, the average number of bacteria per cell in LVS-bearing CMTMR⁺ and CMTMR⁻ cells was determined (C, last column).</p

Association of LVS with a specific cell population in infected lymph nodes.

<p>Mice were infected intra-nasally with 10⁵ CFU of LVS. Forty-eight hours later MdLNs were collected from 10 animals and single cell suspensions were prepared and examined by several assays. (A) Association between viable bacteria and MdLN cells was assayed by examining non-washed (total LN suspension) and washed cells for presence of viable LVS prior or post gentamicin treatment. Bacterial counts are presented as CFU per a single MdLN. (B) Association between LVS and RTDC cells was assayed by subjecting the washed cell fraction (∼10⁸ cells) to sorting by magnetic microbeads coated with either anti-CD11b (B, top panel) or anti-CD11c (B, lower panel). Viable bacteria (no gentamicin treatment) were counted in the input cell suspension, in the bound cell fraction, as well as in cells that did not bind to the microbeads, and presented as CFU/10⁶ cells. The amounts of cells in the input and in the bound fractions were ∼10⁸/10 MdLNs and in the bound fractions only ∼10⁶/10 MdLNs. CD11b and CD11c sorted cells were also examined by fluorescence microscopy for presence of intracellular bacteria. Typical cells carrying bacteria are presented in the insets to (B). (C) The cell/bacteria association in the CD11b-sorted fraction was characterized by determining bacterial count in non-treated cells, in cells treated with gentamicin (genta), in cells treated with gentamicin followed by washing and saponin lysis (genta+sap), as well as in cells where saponin treatment preceded gentamicin treatment (sap+genta). All bacterial counts were performed in triplicates, error bars represent variation of these counts. Data presented in this figure were collected from a single experiment. Two additional CD11b sorting experiments and one additional CD11c sorting experiment were conducted, exhibiting similar enrichment of intracellular bacteria.</p