In Situ-Targeting of Dendritic Cells with Donor-Derived Apoptotic Cells Restrains Indirect Allorecognition and Ameliorates Allograft Vasculopathy

Chronic allograft vasculopathy (CAV) is an atheromatous-like lesion that affects vessels of transplanted organs. It is a component of chronic rejection that conventional immuno-suppression fails to prevent, and is a major cause of graft loss. Indirect allo-recognition through T cells and allo-Abs are critical during CAV pathogenesis. We tested whether the indirect allo-response and its impact on CAV is down-regulated by in situ-delivery of donor Ags to recipient's dendritic cells (DCs) in lymphoid organs in a pro-tolerogenic fashion, through administration of donor splenocytes undergoing early apoptosis. Following systemic injection, donor apoptotic cells were internalized by splenic CD11chi CD8α+ and CD8− DCs, but not by CD11cint plasmacytoid DCs. Those DCs that phagocytosed apoptotic cells in vivo remained quiescent, resisted ex vivo-maturation, and presented allo-Ag for up to 3 days. Administration of donor apoptotic splenocytes, unlike cells alive, (i) promoted deletion, FoxP3 expression and IL-10 secretion, and decreased IFN-γ-release in indirect pathway CD4 T cells; and (ii) reduced cross-priming of anti-donor CD8 T cells in vivo. Targeting recipient's DCs with donor apoptotic cells reduced significantly CAV in a fully-mismatched aortic allograft model. The effect was donor specific, dependent on the physical characteristics of the apoptotic cells, and was associated to down-regulation of the indirect type-1 T cell allo-response and secretion of allo-Abs, when compared to recipients treated with donor cells alive or necrotic. Down-regulation of indirect allo-recognition through in situ-delivery of donor-Ag to recipient's quiescent DCs constitutes a promising strategy to prevent/ameliorate indirect allorecognition and CAV

Dendritic Cells Expressing Transgenic Galectin-1 Delay Onset of Autoimmune Diabetes in Mice

Type 1 diabetes (T1D) is a disease caused by the destruction of the β cells of the pancreas by activated T cells. Dendritic cells (BC) are the APC that initiate the T cell response that triggers T1D. However, DC also participate in T cell tolerance, and genetic engineering of DC to modulate T cell immunity is an area of active research. Galectin-1 (gal-1) is an endogenous lectin with regulatory effects on activated T cells including induction of apoptosis and down-regulation of the Th1 response, characteristics that make gal-1 an ideal transgene to transduce DC to treat T1D. We engineered bone marrow-derived DC to synthesize transgenic gal-1 (gal-1-DC) and tested their potential to prevent T1D through their regulatory effects on activated T cells. NOD-derived gal-1-DC triggered rapid apoptosis of diabetogenic BDC2.5 TCR-transgenic CD4+ T cells by TCR-dependent and -independent mechanisms. Intravenously administered gal-1-DC trafficked to pancreatic lymph nodes and spleen and delayed onset of diabetes and insulitis in the NODrag1 -/- lymphocyte adoptive transfer model. The therapeutic effect of gal-1-DC was accompanied by increased percentage of apoptotic T cells and reduced number of IFN-γ-secreting CD4+ T cells in pancreatic lymph nodes. Treatment with gal-1-DC inhibited proliferation and secretion of IFN-γ of T cells in response to β cell Ag. Unlike other DC-based approaches to modulate T cell immunity, the use of the regulatory properties of gal-1-DC on activated T cells might help to delete β cell-reactive T cells at early stages of the disease when the diabetogenic T cells are already activated. Copyright © 2005 by The American Association of Immunologists, Inc

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

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

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

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