Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.

cells FIGURE 3. Liver CD-DCs markedly sppress donor-reactive host T cel proliferation in CFSE-MLRSpleen (Sp) or liver CD-DCs or nCD-DCs iso lated from graft recipients on 7 POD were cocultured with responde (C3H (host) spleen T cells) and stimulators (B6 (donor) spleen DC) in CFSE-MLR (T cells: DC (each) =10:1). Liver CD-DCs, markedly sup pressed host T cell proliferation. % T cell proliferation in indicated. Data are representative of 3 experiments that gave similar results. FIGURE 2. Infiltrating host DCs and CD-DCs peak on 7 POD and PD-L1 CD-DCs persist in the graft for at least 300 POD. (A) Chronological changes in (left) the incidence of total DC and (right) the incidence of CD-DC (as % total DC) in the graft. (B) Expression of PD-L1 by CD-DCs and non (n)CD-DCs in the graft. PD-L1 expression by CD-DCs was significally higher than of nCD-DC, especially on 7POD. 3-6 mice per time point *: P<0.05, ***: P<0.001 © 2017 Wolters Kluwer Abstracts S31

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37 ° C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

Introduction: Donor brain death (BD) adversely affects organ quality. Inflammation mediated by engagement of TLR4 and RAGE may contribute to inferior outcomes. Endogenous secretory (es)RAGE is a soluble decoy receptor which sequesters HMGB1 and other RAGE ligands blocking binding to TLRs 2 and 4, as well as to RAGE. Aim: To determine whether in vivo expression of esRAGE can reduce inflammation in syngeneic KTx from BD donors. Methods: Donor and recipient C57BL/6 mice received 5×1011 VG rAAV-esRAGE or ctrl vector 7 days pre-transplant. Donor kidneys were grafted 1 hr after BD induction, and sampled at d1 and 4 post-Tx. Cellular infiltration, proliferation, and gene expression were determined. Serum and kidneys were also collected 3 hrs post-BD. Results: Serum HMGB1 levels in esRAGE-BD mice (˜10ng/ml) were significantly less than those in untreated BD mice (>25ng/ml, p=0.02). There was a trend towards reduced expression of CXCL10, MCP-1, MIP-2, CCL22, IL-10, IL-6, IL-1β and TNF-α in the kidneys of esRAGE-BD mice, but this was not consistently observed after KTx. Neutrophil and macrophage infiltrate density was substantially reduced by esRAGE (nΦ 9.1±2.0/HPF cf 23.1±6.0, p=0.03; F4/80 27.3±3.2/HPF cf 65.1±9.1, p<0.01).Figure: No Caption available.Proliferation of parenchymal cells (PCNA staining) was enhanced in esRAGE-treated mice (7.6±0.5/HPF cf 4.2±1.2, p=0.05).Figure: No Caption available.Conclusions: esRAGE significantly reduced neutrophil and macrophage infiltration following syngeneic renal Tx from BD donors. RAGE is a binding partner for the integrin MAC-1, and reduced leucocyte infiltration may reflect inhibition of this interaction. esRAGE increased proliferation of renal parenchymal cells after Tx from BD donors, similar to documented effects of soluble RAGE in liver injury. These effects may be translated into improved graft function.

BACKGROUND:Murine kidney transplantation is an important model for studies of transplantation immunobiology. The most challenging aspect of the difficult surgical procedure is the ureteric anastomosis.METHODS:Two different approaches to ureteric reconstruction are compared here. Method 1, Patch: this involves anastomosis of the donor ureter together with a patch of donor bladder to recipient bladder. Method 2, Implant: this utilizes a 5-0 suture to pull the ureter through the bladder wall. The ureter's peripheral tissue is then fixed to the bladder wall at the implant site with 10-0 micro-sutures.RESULTS:In animals transplanted with the patch method, the initial success rate, defined as survival up to the third post-operative day, was 79% (n = 62), whereas the initial success rate for the implant method was 86.1% (n = 101; P = 0.28). The death rate from unknown and/or unspecified causes in the initial period was 16.1% (10/62) for the patch method, and 8.9% (9/101) for the implant method (P = 0.21). The average donor/recipient operation time with the implant method was 14.8 ± 2.2/61.4 ± 4.7 min (76 min per transplant), whereas operation time with the patch method was 28.3 ± 2.4/77.8 ± 5.5 min (106 min per transplant; P < 0.001). The ureteric implant method resulted in a lower rate of urinary leak compared with the patch method (1.1% versus 10.2%; P = 0.02).CONCLUSIONS:The ureteric implant method for mouse kidney transplantation is a reliable approach with at least as high a success rate as the bladder patch method and with a shorter operation time.

BACKGROUND:The liver has long been recognized as having tolerogenic properties. We investigated whether recombinant adenoassociated virus (rAAV)-mediated expression of donor major histocompatibility complex in recipient livers could induce tolerance to donor-strain grafts. METHODS:Naive B10.BR (H-2) or B10.BR recipients primed with a H-2K-expressing (K) skin graft were injected with rAAV-expressing H-2K (rAAV-K) to induce K expression on hepatocytes 7 days before challenge with a K skin graft. K-specific responses were measured by interferon (IFN)-γ ELISpot and flow cytometric assessment of directly H-2K reactive cells. Fully allogeneic grafts from C57BL/6 (H-2) donors were transplanted onto longstanding B10.BR recipients of K skin to test for linked epitope suppression. RESULTS:rAAV-K-treated B10.BR mice accepted K skin grafts with increased median survival time (MST) more than 169 days compared to uninoculated (MST=18.5 days) and rAAV-K-treated controls (MST=19 days). rAAV-K-treated B10.BR animals primed with K skin grafts also accepted secondary K skin grafts in the long term (MST>100 days) compared to accelerated rejection in primed, uninoculated mice (MST=12 days). Treatments did not induce liver pathology, assessed by serum alanine aminotransferase levels and histology. IFN-γ ELISpot analysis of splenocytes from rAAV-K-treated mice indicated reduced responses to donor K antigen, but protection was not extended to fully allogeneic C57BL/6 skin or heart grafts, even in recipients that had accepted K skin grafts in the long term. CONCLUSIONS:High-level expression of donor major histocompatibility complex in recipient livers promotes tolerance to skin allografts, even in animals primed to produce a memory response. This provides proof of concept for an approach using liver-targeted gene delivery for tolerance induction to donor antigen.