LIMLE mRNA expression in transplantation models, cell subtypes and organs.

<p>LIMLE mRNA expression in (A) cardiac grafts and PBMC of rat syngenic recipients (Syng), allograft recipients developing acute (AR) or chronic rejection (CR), or allograft tolerant recipients (Tol) at day 5 or 100 after transplantation (n = 5, *p<0.05 **p<0.01 ***p<0.001), (B) Splenic DCs, alveolar macrophage lineage cells NR8383 (Mac), B cells, T cells and EC, stimulated (stim) or not (NS) with LPS, IFNγ, CpG, anti-CD3/CD28 or IFNγ respectively (for 12 hours) (n = 3), (C) rat BMDCs stimulated or not (NS) with LPS, IFNγ, CpG, Poly(I:C) or IL-10 for 6, 12, 24 or 48 hours (n = 3, *p<0.05), (D) Human DCs stimulated or not (NS) with LPS for 12 hours (n = 3, **p<0.01) (E) mRNA expression of LIMLE in different organs from naive rats as indicated (n = 5). (A–E) Quantitative RT-PCR results were expressed in Arbitrary Units (AU) of LIMLE/HPRT transcript ratio ± SEM. Statistical evaluation was performed using Student's <i>t</i> test for unpaired data, and results were considered significant if <i>p</i> values were <0.05.</p

The role of LIMLE in the stimulatory properties of BMDCs.

<p>(A) a) LIMLE mRNA expression in rat BMDCs transfected with control or one of two LIMLE specific RNAi. Quantitative RT-PCR results were expressed in Arbitrary Unit (AU) of LIMLE/HPRT transcript ratio ± SEM (n = 3 *p<0.05 ***p<0.001); b) V5 tagged LIMLE protein expression in eukaryote cells transfected with a plasmid encoding rat LIMLE and V5 and either a control or one of two LIMLE specific RNAi. Data were expressed in Arbitrary Units (AU) of % of V5⁺ cells assessed by flow cytometry compared to control RNAi  = 100 (n = 6, **p<0.01) (B) Proliferation of allogeneic T cells stimulated with control or one of two LIMLE specific RNAi transfected BMDCs (MLR) for 4 days as follow a) ³H incorporation (cpm) for bulk T cells, b) CFSE loss from CD4⁺CD25^neg and c) CFSE loss from CD4⁺CD25^high T cells (n≥3, *p<0.05). (C) IFNγ, IL-6 and IL-12 quantification by ELISA of MLR supernatants (n = 3, *p<0.05). (D) Representative histograms (flow cytometry) of mouse BMDC transfection efficiency (with red fluorescent control RNAi (plain line) or untransfected control (dotted line)). (E) LIMLE mRNA expression in mouse BMDCs transfected with control or one of two LIMLE specific RNAi. Quantitative RT-PCR results were expressed in Arbitrary Units (AU) of LIMLE/GAPDH transcript ratio ± SEM (n = 3, *p<0.05 ***p<0.001). (F) Proliferation of OT-I CD8⁺ T cells stimulated for 4 days with OVA_257–264 peptide (0.1 ng/ml) (n = 3) or OVA protein (500 μg/ml) (n = 4) loaded mouse BMDCs (transfected with control or one of two LIMLE specific RNAi). Data were expressed in AU of CFSE dilution compared to control = 100 (*p<0.05 **p<0.01 ***p<0.001). Statistical evaluation was performed using the Student's <i>t</i> test for unpaired data, and results were considered significant if <i>p</i> values were <0.05.</p

Cellular localization of LIMLE protein.

<p>Representative pictures of immuno-fluorescence staining of (A and B) COS or (C) BMDCs transfected with a plasmid encoding full length rat LIMLE protein and containing the V5 tag (red) with DAPI (blue) and and phalloidin (polymerized actin) (green). Original magnification ×1200 (Plan Apo N.A: 1.4 zoom 2.). (Bi,ii) Representative histograms of fluorescence intensity (AU) in COS cells detected in pixels with the indicated box, graphed with the linescan function of Metamorph Image Processing Software. (Ci) Representative picture of LIMLE overlapping with actin in DCs, processed with Metamorph Image Processing Software. Pictures were representative of three independent experiments.</p

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

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

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