Regulation of the systemic anti-donor response by donor apoptotic splenocytes.

<p>A) Assessment by IFN-γ ELISPOT assay of the indirect T cell response elicited in the spleens of B6 mice, naïve (control), or transplanted 60 days before with syngeneic (control) or BALB/c aortic grafts. Numbers indicate the number of spots. Each group included 4 mice. B) Detection by FACS of circulating alloAb in B6 recipients of syngeneic or BALB/c aortic grafts under different conditions and analyzed 60 days after surgery. Results are representative of 6 mice per group. Numbers indicate percentages of cells. Numbers in parenthesis represent mean fluorescence intensity. C) Evaluation of the direct T cell response by IFN-γ ELISPOT assay in the spleen of B6 mice, naïve (control) or 60 days after being transplanted with syngeneic (control) or BALB/c aortas, under different conditions. Each group included 4 mice per group. * p<0.001.</p

Splenic DCs remain quiescent following interaction with donor apoptotic cells in vivo.

<p>A) B6 mice were left untreated or injected i.v. with PKH67-labeled BALB/c apoptotic splenocytes. Histograms show the expression of surface markers by splenic CD11c^hi DCs (i) with internalized PKH-67⁺ apoptotic cell fragments (thick line), (ii) without PKH-67⁺ fragments from the same mice (gray), and (iii) from control non-injected mice (dotted line). One representative out of 3 individual experiments is illustrated. B) Eighteen h after i.v. administration of PKH-67-labeled BALB/c apoptotic splenocytes in B6 mice, splenic CD11c^hi DCs with internalized PKH-67⁺ fragments (R2) or not (R1) were FACS-sorted and cultured in medium with GM-CSF and LPS. After 24 h, surface expression of MHC class-II Ag and CD86 was analyzed by flow cytometry in the FACS-sorted splenic DCs that had internalized PKH-67⁺ fragments (gray histograms) or not (thick line histograms), and compared to the phenotype of freshly-isolated splenic CD11c^hi DCs (dotted line histogram). One representative out of 3 experiments is shown. C) CFSE-labeled 1H3.1 CD4 T cells were adoptively transferred (i.v.) into B6 mice at different times after administration of BALB/c splenocytes (alive, apoptotic or necrotic) and proliferation of 1H3.1 T cells was evaluated based on CFSE dilution assessed by FACS, 3 days after T cell transference. Presentation of BALB/c allopeptides to splenic 1H3.1 CD4 T cells (based on CFSE-dilution) decreased drastically by 7 days. Numbers represent percentages of dividing cells. One representative experiment with 3 mice per group and time point is shown.</p

Donor apoptotic cells are recognized by indirect CD4 T cells.

<p>A) Analysis by FACS of proliferation and phenotype of CFSE-labeled 1H3.1 CD4 T cells transferred into B6 mice that were then treated (i.v.) with BALB/c splenocytes, alive or apoptotic. As control, to promote 1H3.1 T cell activation/proliferation, a group of B6 mice was treated with BALB/c apoptotic cells plus agonistic CD40 mAb. Numbers in dot plots represent percentages of cells. Numbers in parenthesis indicate the mean fluorescence intensity of the left quadrants combined. One representative experiment out of 6 is shown. B) ELISPOT analysis for IFN-γ of splenocytes from B6 mice reconstituted (or not, control) with 1H3.1 CD4 T cells and then injected i.v. with B6 (control) or BALB/c apoptotic splenocytes (alone or with agonistic CD40 mAb), or with BALB/c splenocytes alive. Three days later, splenocytes from the host B6 mice were cultured for 36 h in ELISPOT plates alone, or with the BALB/c peptide IEα_52–68. Each group included 6 mice. C) Assessment by FACS of the percentages of CD4 1H3.1 T cells (Thy1.1⁺) in tissues of host B6 mice (Thy1.2⁺) previously reconstituted with 1H3.1 cells and then treated with B6 (control) or BALB/c apoptotic splenocytes (the latter alone or with agonistic CD40 mAb), or with BALB/c splenocytes alive. Results represented values pooled from 6 mice per group. * p<0.01.</p

Administration of donor apoptotic cells regulates the indirect T cell responses.

<p>A) Allo-Ag presentation and ELISPOT assays of splenocytes from B6 mice reconstituted (or not, control) with 1H3.1 CD4 T cells and then injected i.v. with B6 (control) or BALB/c apoptotic splenocytes (alone or with agonistic CD40 mAb), or with BALB/c splenocytes alive. Fourteen days later, splenocytes from the host B6 mice were cultured alone or with the BALB/c peptide IEα_52–68 during 72 h for assessment of T cell proliferation by [³H]TdR incorporation, or for 36 h in ELISPOT plates for quantification of IFN-γ and IL-10 secretion. Each group included 6 mice. B) Analysis by FACS of the percentage of CD4 1H3.1 T cells (Thy1.1⁺) expressing FoxP3 in the spleen of host B6 mice (Thy1.2⁺) 14 days after receiving i.v. B6 (control) or BALB/c apoptotic splenocytes, or BALB/c splenocytes alive. Numbers indicate percentages of cells. One representative of 6 individual experiments is shown. * p<0.01. C) Assessment by in vivo killing assays of the Ag-specific lytic activity of B6 mice reconstituted with 2C CD8 T cells and treated (day 1) with 10⁷ MHC-I^−/− B6 apoptotic or alive splenocytes loaded (or not) with the H2K^b-retricted SYGL peptide. Mice were challenged (or not) on day 6 with SYGL plus IFA (i.p.). On day 9, mice were i.v. injected with a 1/1 mixture of CFSE^hi BALB/c×B6 (F1) (target) and CFSE^lo B6 (control) splenocytes. Five h later, the ratio of CFSE^hi vs. CFSE^lo cells in the spleen was determined by FACS. Numbers indicate percentages of cells. The specific lysis was calculated and is shown as the mean±SD of 3 mice per group.</p

Impact of donor apoptotic splenocytes on lymphocyte infiltration and allo-Ab deposition in aortic grafts.

<p>Detection by fluorescence microscopy of infiltrating CD4 and CD8 T cells, and IgG and C3d deposited in aortic BALB/c grafts, 60 days after transplant in syngeneic (control) or B6 recipient mice, left untreated or injected with BALB/c splenocytes, apoptotic, necrotic, or alive. Cell nuclei were stained blue with DAPI. Asterisks indicate the vessel lumen. ×200.</p

Blood-borne donor apoptotic cells are captured and presented to T cells by splenic CD11c^hi DCs.

<p>A) PKH67-labeled BALB/c apoptotic cells were injected i.v. in B6 mice and the entrapment of blood-borne PKH-67⁺ (green) apoptotic cell fragments by splenic CD11c^hi CD45RA⁻ DCs (CD8α⁺ or CD8α⁻) and CD11c^int CD45RA⁺ pDCs was analyzed by FACS, 18 h later. B) WT and CD11c-DTR-eGFP B6 mice (both Thy1.2⁺) were reconstituted with CFSE-labeled 1H3.1 CD4 T cells (Thy1.1⁺), then treated (or not, control) with DT and injected i.v. with BALB/c apoptotic cells. Injection of DT in CD11c-DTR-eGFP mice deleted selectively CD11c^hi DCs and spared CD11c^int pDCs in the spleen (upper dot plots). In the absence of splenic pDCs, CD11c-DTR-eGFP mice were unable to present BALB/c apoptotic cell-derived allopeptides and induce proliferation of the adoptively transferred 1H3.1 CD4 T cells, evaluated 48 h later by CFSE-dilution by FACS-analysis (bottom dot plots). One representative out of 5 (in A) and 3 (in B) individual experiments is shown.</p

Effect of donor apoptotic splenocytes on CAV.

<p>A) Microscopic analysis of cross-sections of aortic BALB/c grafts procured 60 days after transplantation from syngeneic or allogeneic (B6) recipient mice left untreated, or injected (i.v., day -7) with BALB/c splenocytes apoptotic, necrotic or alive, or with C3H (third-party) apoptotic splenocytes. Sections were stained with H&E or to identify collagen deposition, elastic fibers, or α-sm actin⁺ cells. The sections shown are representative of 6 independent aortic transplants per group. B) Method employed to quantify morphometrically intimal thickening in aortic grafts by means of a microscope equipped with an imaging system and image analyzing software. C) Comparison of the intimal thickening of aortic BALB/c grafts, 60 days after transplantation in syngeneic or B6 recipient mice left untreated or injected with BALB/c splenocytes apoptotic, necrotic, or alive, or with C3H (third-party) apoptotic splenocytes. Results represented values pooled from 6 mice per group.</p

SDC2 is over-expressed in IGFBP-3 expressing human skin.

<p>Human skin explants were infected with either control adenovirus (cAd; <b>A</b> and <b>C</b>) or adenovirus encoding IGFBP-3 (Ad-3; <b>B</b> and <b>D</b>) and maintained in culture for 7 d and sections of paraffin embedded tissue were analyzed for the expression of SDC2 by immunohistochemistry. <b>B</b> and <b>D</b> are histological images showing the expression of SCD2 in dermal fibroblasts following infection with Ad-IGFBP-3. A and B, magnification = 100x. C and D, magnification = 400x.</p

A) TGFβ induction of SDC2 gene expression is IGFBP-3-dependent. Normal fibroblasts were transfected with siRNA targeting IGFBP-3 (siBP3), then stimulated with TGFβ (10 ng/ml) for 48 hours.

<p>RT-PCR was used for the detection of SDC2 in 100 ng equivalent of template. β-actin was used as control. Experiments were done in triplicate. B) TGFβ induction of SDC2 protein is IGFBP-3-dependent. Primary human lung fibroblasts were transfected with siRNA targeting IGFBP-3 or IGFBP-5 as a related protein control. Fibroblasts were stimulated with TGFβ (10 ng/ml). Cellular lysates were analyzed by immunoblotting for SDC2 protein after 72 hrs. Efficiency of silencing was assessed by detecting IGFBP-3. GAPDH was used as internal control.</p

TGFβ and IGFBP-3 induce SDC2 gene expression.

<p>Normal lung fibroblasts were stimulated with recombinant TGFβ (10 ng/ml), IGFBP-3 (BP-3; 250 ng/ml) or both (B+T) for 48 hours. SDC2 mRNA was detected by RT-PCR using 100 ng template. β-actin was used as control.</p