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Analysis of anti-human CD22 human mAbs affinity and specificity on human PBMCs. a) Affinity determination of anti-human CD22 mAbs using SPR by flowing various concentration of CD22 antibody over CD22 chip-bound. b) Flow cytometry analysis of CD22 mAbs on human PBMC. Human PBMC were labeled with anti human CD19 (APC) and purified CD22 mAbs (clone γ1λ1, γ3λ3-5, γ23κ5-2 or γ27λ26) coupled with Alexa Fluor 568 fluorochrome or with a commercial mouse mAb anti-human CD22 (Ms anti-human CD22). (PPT 666 kb

Adoptive transfer of B cells transfers tolerance.

<p>Cells were sorted by FACS Aria <b>(sorting strategy displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119686#pone.0119686.s003" target="_blank">S3D and E Fig.</a>)</b> from the spleen of tolerant rats (>100 days after the graft) that had received a transfer of splenocytes from a previously tolerant recipient and adoptively transferred to sub-lethally irradiated recipients the day before the transplant. <b>(A)</b> B cells (CD45RA⁺, n = 3), T cells (TCR⁺, n = 4), CD8⁺ Tregs (CD8⁺CD45RC^low, n = 2), pDCs (mAb 85C7⁺ n = 3) Groups are compared with each other and to irradiated animals transferred with naive splenocytes (naive splenocytes, n = 5) by Log-rank (Mantel-Cox) Test <i>p</i> <0.05*; p<0.01**; p<0.001***. <b>(B)</b> Wild type (WT) and B cell-deficient <i>Igm</i> knockout (KO) rats were treated with AAV-FGL2 (n = 8 and 3, respectively), AAV-null (n = 5) or untreated (NT, n = 3), and analyzed for graft survival. <b>(C)</b> Splenocytes from adoptively-transferred tolerant rats were depleted in CD45RA⁺ B cells (CD45RA⁻ cells) or not (splenocytes) and transferred to new irradiated recipients. Log-rank (Mantel-Cox) Test p<0.01**. <b>(D) Left:</b> A fraction of the transferred tolerogenic CD45RA⁺ B cells was tested for inhibition of CFSE-labeled CD4⁺CD25⁻ T cell proliferation in response to allogeneic LEW.1W cDCs, pDCs (stimulator/effector ratio of 1:4) or anti-CD3 at day 6 of culture. Shaded grey: naive CD45RA⁺ B cells n = 3, black line: tolerogenic CD45RA⁺ B cells n = 4. <b>Right</b>: Representative histogram of one proliferation assay of CD4⁺CD25⁻ T cells with allogeneic pDCs and CD45RA⁺ B cells from naive (shaded grey) or splenocyte-transferred tolerant rats (black line). <b>(E)</b> Graft infiltrating cells were analyzed for the presence of CD45RA⁺ cells in graft of rats transferred with B cells, at days 100 after the graft, as compared with syngeneic grafts (n = 3).</p

Over-expression of FGL2 <i>in vivo</i> prolongs cardiac allograft survival.

<p><b>(A)</b> Cardiac graft recipients received intravenously 3x10¹²vector genomes/kg of AAV-FGL2 (▲ n = 8), or non coding AAV (▼ n = 5, 2 different experiments), and received a heterotopic transplant 30 days later. Graft survival was evaluated by palpation through the abdominal wall. Log-rank (Mantel-Cox) test ***<i>p<0</i>.<i>001</i> for AAV-FGL2 vs. AAV null controls. <b>(B)</b> Left: Relative proportion of dividing CD4⁺CD25⁻ T cells at day 6 in the presence of different concentrations of recombinant human FGL2-GST was evaluated by CFSE dilution by gating first on DAPI^- live cells and then on TCR⁺CD4⁺ cells <b>(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119686#pone.0119686.s002" target="_blank">S2B Fig.</a>).</b> The negative control was purified rat IgG at 10 μg/ml (n = 4, ** <i>p</i><0.01). Right: Representative histogram of relative proportion of dividing CD4⁺T cell in the presence of 10μg/ml FGL2-GST protein (black line) or IgG control (grey).</p

Splenocytes from AAVFGL2-treated rats with long-term surviving grafts transfer donor alloantigen-specific long-term graft survival in an iterative manner.

<p>Splenocytes from long-term AAV-FGL2-treated recipients were injected <i>i</i>.<i>v</i>. into sub-lethally irradiated recipients (LEW.1A) the day before heart allotransplantation (LEW.1W). Graft survival was evaluated by palpation through the abdominal wall. Total splenocytes (1x10⁸ cells) from long-term (≥120 days) AAV-FGL2-treated rats were adoptively transferred (1^st-transferred, n = 5), and then total splenocytes (10⁸ cells) were iteratively transferred to 2^nd- (n = 6), 3rd (n = 4), 4^th (n = 3), 5^th (n = 3) and 6^th (n = 3) LEW.1A recipients receiving LEW.1W hearts. Third-party grafts were from Brown-Norway origin and adoptive transfer of splenocytes from LEW.1W-transplanted animals did not inhibit acute rejection (third party, n = 3, performed in animals that received a second adoptive transfer). Splenocytes from naive non-transplanted rats did not inhibit acute rejection (naive splenocytes, n = 5) and non-irradiated non-transferred recipients (no treatment, n = 6) also showed acute rejection. Irradiation alone without cell transfer delays graft survival but does not prevent graft from rejection (irradiated, n = 5). All groups were compared to irradiated animals transferred with naive splenocytes by Log-rank (Mantel-Cox) Test (<i>p value</i> ***<0.001).</p

Alloantibody production was suppressed after AAV-FGL2 treatment and adoptive transfer of splenocytes.

<p>Sera were collected from naive rats, or at the moment of rejection from rats treated with AAV-control or AAV-FGL2 (rejecting at < 30 days or > 120 days after transplantation) or receiving adoptive transfers (> 120 days after transplantation). Levels of donor-specific IgG1, IgG2a, and IgG2b antibodies were evaluated by cytofluorimetry and normalized to serum from naive rats (MFI / MFI syngeneic). Two way Anova, Bonferroni post test <i>p value</i> * <0.5; ** <0.01; ***<0.001.</p

Reduced progression of the viral replication cycle by the multiplex strategy.

<p>Transduced and control U-251 MG cells infected with HCMV were harvested eight days pi. a) Relative viral genome quantification normalized to HCMV-infected control U-251 MG cells (dash line) (n = 3 independent experiments, +/- SD). One-way ANOVA and the multiple comparison test were performed and significant differences in the comparison to the control are mentioned. b) Cells were FACS-stained for total gB expression. Representative dot plots of total gB expression after infection with Toledo. c) gB expression normalized to HCMV-infected control U-251 MG cells (dashed line) (triangles: unsp. gRNA; dots: singleplex; diamonds: multiplex) (n = 4 independent experiments). One-way ANOVA and the multiple comparison test were performed.</p

Decrease in IE expression by HCMV-targeting CRISPR/Cas9 systems.

<p>Control and transduced U-251 MG cells were infected with HCMV and harvested at two or eight days pi. a) Representative FACS histograms of intranuclear IE expression eight days pi are shown for all U-251 MG cell lines and three different viral strains. The gray histogram represents uninfected U-251 MG cells. b) IE expression in the different U-251 MG cell lines normalized to the HCMV-infected control U-251 MG cells (dashed line) (n = 4 to 5 independent experiments). One-way ANOVA and multiple comparison tests were performed to compare the results within the different cell lines and are presented in the table under each graph. Mann-Whitney tests were performed to analyze each cell line over time (day 2 pi <i>vs</i>. day 8 pi). The only significant difference is noted in the graph. c) Western blot analysis of protein extraction obtained using the TriPrep kit eight days pi (one representative western blot out of 3 independent blots is shown for each virus strain as well as for the uninfected control U-251 MG cells).</p

Mutations in the <i>UL122/123</i> gene induced by the CRISPR/Cas9 anti-HCMV in U-251 MG cell line.

<p>Control and transduced U-251 MG cells were infected with HCMV (Toledo, MOI of 1) and cultured for eight days. Viral DNA was extracted and PCR amplified. a) A T7-assay was performed on the exon 2 PCR amplicon to detect indels induced by the singleplex strategy. b) Electrogram for the T7 assay from the Caliper LabChip analysis for the unsp. gRNA and singleplex strategies. c) Large deletions induced by the multiplex strategy were highlighted by analyzing the whole <i>UL122/123</i> gene amplicon. d) Electrogram for the PCR products from the Caliper LabChip analysis identify a major 500 bp amplicon and a smear above with the multiplex strategy. Arrows highlight the indels (singleplex) and larger deletions (multiplex) induced by the anti-HCMV CRISPR/Cas9 strategies. One representative experiment out of three is shown for Toledo, and similar data were found with TB40GFP and VR1814 (n = 3 independent experiments per virus strain). LM, lower marker; UL, upper marker. e) Sequence analysis of the mutations induced by the singleplex strategy in the viral genome four days pi. Black: protospacer + <u>PAM</u>; bold: start codon; gray: insertions; gray-white: substitution.</p

Design of the HCMV-targeting gRNAs.

<p>Scheme for the targeted <i>UL122/123</i> gene and its major splice variants for IE1 and IE2 with the position of the three designed gRNAs anti-HCMV and their corresponding sequences (Scissors: gRNA/Cas9 complex).</p

The anti-HCMV CRISPR/Cas9 system induces mutations resulting in a decrease in IE protein expression in primary fibroblasts.

<p>MRC5 cells were transduced with one of the three type 1 LVs and selected by puromycin treatment (2 μg/mL) for 2 days. Control (untransduced) and puromycin-resistant MRC5 cells were subcultured prior to infection with Toledo (MOI of 0.1). Two days pi, proteins and DNA were extracted from the infected cells via TriPrep Kit. a) Viral DNA extracts were PCR-amplified at the target region. Amplicons were subsequently subjected to T7 endonuclease to detect the indels induced by the singleplex strategy. b) PCR amplicons of the whole IE gene were analyzed to detect larger deletions induced by the multiplex strategy. The arrows highlight the indels (singleplex) and larger deletions (multiplex) induced by the anti-HCMV CRISPR/Cas9 strategies (one out of three independent experiments is shown). c) Western blot analysis of IE and Cas9 expression 2 days pi (one representative western blot out of 3 independent experiments is shown). d) At each passage, the proteins were extracted with the TriPrep Kit and the Cas9 expression was assessed by Western blot. e) Relative quantification of Cas9 expression based on the Western blot (d) normalized to the housekeeping protein actin. Pt, post-transduction.</p