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

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

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

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

High incidence of AFN in PV+LCMV double immune mice following PV re-challenge.

<p>(A) Naïve, PV-immune, and (PV+LCMV WT) double immune mice were re-challenged with PV, sacrificed 3 days PI, and the severity of AFN in the visceral fat pads was assessed. (*) indicates <i>p</i>&lt;.05 in frequency of AFN using the Kruskai-Wallis test (one-way ANOVA non-parametric). (B), (C), and (D) represent experiments performed using the LCMV clone 13 system and its naturally derived V207A mutant. (B) Domination of NP205-specific CD8 T cells in PV+Clone 13 LCMV WT double immune mice. PBL were collected from double-immune mice, before the final challenge with PV, and stimulated with peptides <i>ex vivo</i> in a standard ICS assay. These are representative frequencies of the IFNγ positive CD8α+ T cells from 4 independent experiments using 5 mice per group. (C) Incidence of AFN after PV challenge. Naïve, (PV+Clone 13 LCMV WT), and (PV+Clone 13 LCMV NP-V207A) double immune mice re-challenged with PV were sacrificed 4 days PI, and the severity of AFN in the visceral fat pads was assessed. Compilation of data from 4 independent experiments. (*) and (***) indicate <i>p</i>&lt;.05 and <i>p</i>&lt;.0001, respectively. (D) Domination of cross-reactive NP205-specific CD8 T cells isolated from the visceral fat pad of (PV+Clone 13 LCMV WT) double immune mice following PV re-challenge. Standard ICS and FACs analyses were performed. Numbers are representative frequencies of IFNγ+, CD8α+ T cells from two similar experiments.</p

Lack of MHC stabilization by LCMV NP L212A.

<p>MHC stabilization assays for LCMV WT (NP205) and mutant (L212A) peptides. RMA-S cells were incubated with different concentrations of peptides and stained against H2K^b to detect its stabilization on the cell surface.</p

Abrogation of heterologous immunity by point mutation in NP205.

<p>Immunologically naïve control or LCMV-immune mice were challenged with 2×10⁷ PFU of PV and tested for PV PFU in spleens or abdominal fat pads 4 days post-infection. Exp. 1 is representative of three experiments using WT LCMV Clone 13 and its naturally derived V207A mutant. Exp. 2 is representative of two experiments using rescued recombinant LCMV Armstrong and its rV207A mutant. Exp. 3 is representative of two experiments using rescued recombinant LCMV Armstrong and its rL212A mutant. n = 5 per group. All comparisons of WT LCMV-immune to naïve mice are <i>p</i>&lt;0.05 as indicated by one-way ANOVA analysis and <i>p</i>≤0.02 by Students t-test. There was no statistically significant difference in PFU in PV-challenged naïve mice vs. challenged NP205 mutant LCMV-immune mice.</p

Analysis and comparisons of NP205-K^b structures.

<p>(A), (B) and (C): superposition of LCMV (pink) with PV (blue) structures, with the peptide in stick representation and the MHC H2K^b in grey cartoon. The tip of the α2-helix is colored accordingly to the peptides bound by the H2K^b molecules, representing the section from residue 150 to 156 of the α2-helix (B &amp; C). (C) shows, with a different orientation, the residues that change conformation between the peptide-MHC complexes, namely Serine-99, Glutamine-114, Leucine-156, Glutamate-152 as well as Glycine-151, for which the Cα atom is represented by a sphere. (D) superposition of the LCMV (pink) with LCMV-V207A (green) structures, with peptide in stick representation and MHC in grey cartoon. (E) and (F): comparison of LCMV (pink) and LCMV-V207A (green) mutant peptide, both bound to the H2K^b molecule (grey cartoon) in the same orientation. The P3 residues are colored in yellow. Arginine-155, Glutamate-152 and Alanine-151 of the H2K^b molecule are represented as grey stick to show the different interaction of their side chains between both structures. The red dashed lines represent the hydrogen bond made between the residues.</p

Analysis of immune response and immunopathology with the LCMV-Armstrong rL212A anchoring amino acid mutant.

<p>(A) Diminished cross-reactive NP205 CD8 T cell responses in the (PV+rV207A) double immune mice. PV-immune mice were immunized with either rWT or rV207A variant Armstrong strain LCMV. After six weeks, PBL were collected and stimulated with LCMV-specific CD8 T cell peptides. The data represent average frequencies of the IFNγ-positive, CD8α+ T cells. This is representative of 3 experiments, with n = 5/group. (B) Complete elimination of cross-reactive NP205 CD8 T cell responses in (PV+rL212A) double immune mice. PV-immune mice were immunized with either rWT or rL212A LCMV Armstrong. After six weeks, PBL were collected and stimulated with LCMV-specific CD8 T cell peptides. Data represent average frequencies of IFNγ-positive, CD8α+ T cells. This is representative of two experiments, with n = 5/group. (C) Prevention of AFN by the rL212A anchoring mutant. Naïve, PV-immune, (PV+rWT) and (PV+rL212A) double immune groups were re-challenged with PV. Four days later fat pads were harvested and AFN scores evaluated. This is a compilation of two similar experiments. (***) indicates <i>p</i>&lt;.0001. (D) Photographs of abdominal fat pads and tissue histology sections. Abdominal fat pads were harvested, photographed (top), and then fixed in 10% neutral buffered formaldehyde and embedded in paraffin at the UMMS histology core facility. Thin tissue sections (5 µm) were stained with hemotoxylin and eosin (bottom). The digital photographs of the sections were taken using a Nikon Eclipse E300 microscope system.</p

Correlation of frequencies of CD8 T cells in double-immune mice with pathology after PV re-challenge.

<p>Linear regression analyses comparing the frequencies of antigen-specific CD8 T cells in the (PV+WT LCMV) double immune mice with the severity of AFN following PV re-challenge. These represent data compiled from four independent experiments using LCMV Clone 13 virus. (A) LCMV NP205-specific CD8 T cell response. (B) PV NP38-specific CD8 T cell response. (C) Ratio of LCMV NP205 to PV NP38. (D) LCMV GP33 and the ratio of GP33/PV NP38.</p