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

Selected examples of ligands for NK cell receptors that are not downregulated by HSV-2 miRNA.

<p>RKO cells were transduced with lentiviruses expressing GFP together with either HSV-2 miRNAs (black empty histogram, the miRNA are indicated on the top of the histograms) or with control miRNA (dark gray shaded histogram). Expression levels of MHC-I, MICB, ULBP2, ULBP3, PVR and ICAM1 were assessed by FACS using specific antibodies. Background levels (light grey shaded histogram) are the APC-conjugated Abs. The figure show one representative experiment out of three 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

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

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 downregulation of HLA-Cw6 by HSV-2 is dependent on the glutamic acid residue located in position 334.

<p>(A) Schematic representation of the cytoplasmic tails of various HLAs. Highlighted in red are the amino acid that are different between HLA-C and other HLA proteins. (B) The various 221 cells (expressing either wild type or mutants HLA proteins, indicated on top of the histograms) were infected with HSV-2 (MOI of 0.5). The levels of the various HLAs was determined by FACS (black empty histograms represent the infected cell staining). The dark gray shaded histograms represent staining of the corresponding uninfected cells. Background levels (light grey shaded histogram) are the APC-conjugated Abs. Figure show one representative experiment out of 3 performed.</p

HSV-2 specifically downregulates MHC-I expression.

<p>Time course for the expression of various NK cell ligands following HSV-2 infection of HeLa cells (MOI of 0.1). The expression levels of NK cells ligands (indicated on top of the histograms) were determined by staining with specific antibodies. Expression following infection is indicated by the black empty histograms. The dark gray shaded histograms represent staining of the corresponding uninfected cells. Background levels (light grey shaded histogram) are the secondary FITC-conjugated Abs staining. One representative experiment is shown out of three performed.</p

Infection of DCs by HSV-2 leads to HLA-C down regulation and consequently enhance NK cell killing.

<p>(A) DCs were infected with HSV-2 (MOI of 0.5), staining was performed 8 and 18 hours post infection (pi). The levels of HLA expression were determined by FACS (blue histograms, represent infected cells, red histograms represent uninfected cells). Top histograms: binding of KIR2DLI-Ig, middle histograms: binding of KIR2DL2-Ig, lower histograms; binding of the pan anti-MHC-I W6/32 antibody. Background levels (black empty histogram) are the APC-conjugated Abs. (B) Uninfected and HSV-2 infected DCs were incubated for 2 hours with primary bulk NK cells, 8 or for 18 hours post infection. NK cells were identified by staining for the expression of CD56 and the levels of NK cell degranulation was monitored by anti CD107a mAb. The percentages of NK cell degranulation are indicated. The staining was quantified by FACS. Figure show one representative experiment out of two performed.</p

Increased NK cell killing of HSV-2 infected HFF cells.

<p>HFF cells, either uninfected, infected with HSV-2 (MOI of 0.5) or infected with UV-inactivated HSV-2 (MOI of 0.5) were radioactively labeled and incubated with primary bulk NK cell cultures for 5 hours at the indicated effector to target ratios (E:T). The killing assays were performed 48 hours post infection. Shown are mean values ± SD. Statistically significant differences are indicated (_* p≤0.05, _** p≤0.005, by one-tailed t test). Error bars (SD) are derived from triplicates. One representative experiment is shown out of three performed.</p

Various NK cell ligands are not downregulated by HSV-2 miRNA.

<p>Various human cell lines indicated in the table were transduced with lentiviruses expressing GFP together with various HSV-2 miRNAs or with control miRNA. Expression levels of MHC-I, MICA, MICB, ULBP1, ULBP2, ULBP3, PVR and ICAM1 were assessed by FACS. The figure displays the human cell lines that were transduced with each of the 21 miRNAs of HSV-2. Plus indicate that the cell line was tranduced with the indicated miRNA and tested for expression of the indicated NK ligands.</p