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.

Activation of TLR2 or TLR4 by endogenous ligands such as high mobility group box 1 (HMGB1) may mediate inflammation causing diabetic kidney injury. We determined whether blockade of HMGB1 signaling by: (1) supra-physiological production of endogenous secretory Receptor for Advanced Glycation End-products (esRAGE), a receptor for HMGB1; (2) administration of HMGB1 A Box, a specific competitive antagonist, would inhibit development of streptozotocin induced diabetic nephropathy (DN). Wild-type diabetic mice developed albuminuria, glomerular injuries, interstitial fibrosis and renal inflammation. Using an adeno-associated virus vector, systemic over-expression of esRAGE afforded significant protection from all parameters. No protection was achieved by a control vector which expressed human serum albumin. Administration of A Box was similarly protective against development of DN. To determine the mechanism(s) of protection, we found that whilst deficiency of TLR2, TLR4 or RAGE afforded partial protection from development of DN, over-expression of esRAGE provided additional protection in TLR2−/−, modest protection against podocyte damage only in TLR4−/− and no protection in RAGE−/− diabetic mice, suggesting the protection provided by esRAGE was primarily through interruption of RAGE and TLR4 pathways. We conclude that strategies to block the interaction between HMGB1 and its receptors may be effective in preventing the development of DN.

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

Aim: Donor brain death (BD) adversely affects organ quality. It has been associated with more rapid rejection of renal transplants and may also compromise the ability to achieve tolerance. Here, we aimed to establish a model of donor brain death and subsequent renal transplantation in mice. Methods: C57BL/6 mice were anaesthetised using Ketamine/Xylazine. A burr-hole was created, through which a 2F Fogarty balloon catheter was introduced. Pressure-controlled mechanical ventilation was commenced via a tracheostomy. Ventilator settings included 5cmH2O PEEP. Mean arterial pressure (MAP) was measured via a femoral cannula. MAP, HR, core temperature and O2 saturation were continuously monitored. BD was induced by controlled inflation of the balloon with 80-100μL at 5μl/min, and was confirmed by the absence of spontaneous respiration after ventilator disconnection. MAP was maintained ≥40mmHg for >3 hours using boluses of 0.25mL gelofusine iv. Samples were collected at 30 mins, 1 and 3 hrs after the induction of BD. Kidneys from some mice at 1 hr post-BD were grafted into syngeneic recipients. Samples were collected at d1 and 4 post-Tx. Serum HMGB1, S100B and cytokines were quantitated, and renal histology, cellular infiltration and gene expression determined. Results: A typical Cushing response was observed with a spike in MAP from 60mmHg to over 90mmHg, accompanied by tachycardia. Tachycardia persisted, whilst MAP slowly declined. Serum S100B (10163±2387pg/ml cf 1731±557, p=0.03), HMGB1 (25.0±3.2ng/ml cf 8.1±1.2, p<0.01), cytokines, and cytokine gene expression in kidneys were substantially increased by 1 hour after BD induction, notably IL-6 (105 pg/ml cf 18±15, p<0.01), KC (8830±1170pg/ml cf 97±29, p=0.02), and IL-1β (150±21pg/ml cf 3±0, p=0.02). The extent of neutrophil and macrophage infiltration was dramatically increased in kidneys from BD donors on day 4 post-Tx, compared with that in ctrl syngeneic grafts (nΦ 25.7±6.0/HPF cf 1.4±0.3, p<0.01 and F4/80 65.1±9.1/HPF cf 23.7±6.2, p<0.01). Gene expression for RAGE, TLR4, CXCL10, MCP-1, IL-6, TNF-α, TGF-β, IL-1β and HSP2 was considerably greater in kidneys from BD donors. Conclusions: A mouse model of brain death and subsequent renal transplantation was successfully established. Physiological and biochemical changes parallel those observed in clinical transplantation.

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: Previous studies have shown induction of specific tolerance and long term survival of fully allogeneic cardiac grafts when donor MHC class II genes were transferred to recipient bone marrow prior to transplantation. In our published data, expression of donor MHC class I (Kb) in recipient liver induced tolerance to Kb-expressing skin grafts. Here, we investigate the effect of high level recombinant Adeno-Associated Virus (AAV)-mediated expression of MHC class II in heart transplant setting. Methods: We developed an efficient liver-specific vector encoding H-2IAd (rAAV-IAd). C57BL/6 (B6, H-2b) WT and Foxp3GFPmice were injected with varying doses (5x108-5x1011 vector genome copies, vgc) to produce expression of H-2IAd on hepatocytes. Expression of IAd and costimulatory molecules was assessed by FACS on isolated hepatocytes, whilst expression of Invariant chain (Ii) and H-2M was determined by RT-PCR. Fully-allogeneic hearts from DBA/2 (H-2d) were transplanted into C57BL/6 at d7 or d100 post-transduction. Results: High level expression of H-2IAd on hepatocytes of B6 mice with doses of 5x1010 or 5x1011 rAAV-IAdwas observed.Figure: No Caption available.Foxp3+ CD4+ T cells were increased in the livers of mice receiving 5x1011 rAAV-IAd with 2±0.76% in normal ctrl, 2.4± 0.6% for 5x1010 IAd injected mice and 7.7±2.2% for 5x1011 IAd injected mice (p<0.03).Yet, the survival rate of hearts grafted into rAAV-IAd treated B6 mice was not improved compared to grafts in normal mice (MST 8d and 7d respectively). Hepatocytes from IAd-injected mice did not express CD40, CD80, CD86 or PD-L1. In addition, RT-PCR revealed minimal expression of H2-M and Ii, suggesting an impaired function in peptide editing of expressed IAd. Conclusion: High level expression of MHCII IAd can be achieved in recipient liver using rAAV vectors. However, initiating an immune response and tolerance to grafts bearing IAd might require supplying H2-M and invariant chain (Ii) genes for biosynthesis of fully functional MHCII IAd from hepatocytes.

Introduction. We previously used an adeno-associated viral (rAAV) vector to express donor MHC (H-2Kb) in recipient livers and induce donor-specific tolerance in a mouse skin transplant model. Kb expression in B10.BR mice led to impaired production of IFN-γ in response to Kb, but no substantial deletion of CD8 T cells, suggesting that tolerance was associated with functional silencing. Methods. To determine the role of direct recognition of class I by CD8-dependent T cells in tolerance induction, we generated rAAV encoding a mutant Kb (rAAV-D227K), where Asp at position 227 of the α3 domain is replaced with Lys, abrogating CD8 binding. Expression of Kb, D227K-Kb and PD-L1 on hepatocytes was assessed by FACS and IHC. Kb-bearing 178.3 skin was grafted onto uninjected recipients and mice injected with rAAV-D227K or rAAV-Kb ± PD-L1 blockade. Results. Uninjected B10.BR mice rejected 178.3 skin (MST=16 d, n=6), while grafts onto rAAV-Kb-injected mice survived long term (MST>250, n=5). Survival of 178.3 grafts onto mice injected with rAAV-D227K was only slightly prolonged (MST=27, n=6), suggesting that CD8 coreceptor engagement is needed for tolerance induction. Kb expression in B10.BR livers resulted in increased PD-L1, but this was not seen following Kb-D227K expression, or with Kb expression in C57BL/6 livers, suggesting that alloreactive CD8 T cell activation results in PD-L1 upregulation. Antibody blockade of PD-L1 plus rAAV-Kb treatment resulted in increased ALT levels (721±73) compared to treatment with rAAV-Kb (33±4) or anti-PD-L1 (35±2) alone. Experiments to determine the effect of PD-L1 blockade on skin graft survival are ongoing. Conclusion. Disruption of CD8 coreceptor engagement abolishes tolerance induction via liver-directed expression of donor MHC class I, indicating that direct recognition by CD8-dependent T cells is essential for the process. PD-L1 upregulation accompanied alloreactive CD8 T cell activation and blockade of PD-L1 resulted in hepatitis, suggesting that this molecule might play a role in the functional silencing of CD8 T cells in this model.

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.

The tolerogenic properties of the liver have long been recognised, especially in regard to transplantation. Spontaneous acceptance of liver grafts occurs in a number of experimental models and also in a proportion of clinical transplant recipients. Liver graft acceptance results from donor antigen-specific tolerance, demonstrated by the extension of tolerance to other grafts of donor origin. A number of factors have been proposed to be involved in liver transplant tolerance induction, including the release of soluble major histocompatibility (MHC) molecules from the liver, its complement of immunosuppressive donor leucocytes, and the ability of hepatocytes to directly interact with and destroy antigen-specific T cells. The large tissue mass of the liver has also been suggested to act as a cytokine sink, with the potential to exhaust the immune response. In this review, we outline the growing body of evidence, from experimental models and clinical transplantation, which supports a role for large tissue mass and high antigen dose in the induction of tolerance. We also discuss a novel gene therapy approach to exploit this dose effect and induce antigen-specific tolerance robust enough to overcome a primed T cell memory response.

Introduction: The model of kidney transplantation in mice is widely used in transplant immunobiology studies. The surgical procedure is difficult due to problems with the ureteric anastomosis. Two different ureteral reconstructions were used in our mouse kidney transplant model. The outcomes are retrospectively reviewed and compared here. Methods: Method 1. Patch: this involved anastomosis of the donor ureter together with a patch of donor bladder to recipient bladder. Method 2. Implant: this involved using a 5-0 suture to pull the ureter through the bladder wall. The ureter's peripheral tissue was then fixed to the bladder wall at the implant site using 10-0 microsutures. The initial success rate was defined as survival up to the third post-operative day. The donor and recipient times were recorded with a recent subgroup of concurrent transplants. Results: In animals transplanted with the patch method, the initial success rate was 79% (n=62), while 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, while 8.9% (9/101) for the implant method (p=0.21). The complications of limb paralysis, ureteric stenosis and hydronephrosis were not significant differences with the two methods respectively in terms of initial period (< 3 days), intermediate period (4 to 14 days) and long period (>14 days). Urine leak at the anastomosis site was 10.2% (5/49) with the patch method, while 1.1% (1/87) for the implant method (p=0.02). The average donor/recipient operation time with the implant method was 16±2minutes/64±5minutes (80minutes/transplant) while operation time with the patch method was 29±2minutes/79±2minutes (108minutes/transplant) (p< 0.001). Conclusion: 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.