Screening self-peptides for recognition by mouse alloreactive CD8+ T cells using direct ex vivo multimer staining.

Here, we present a protocol to identify immunogenic self-peptide/allogeneic major histocompatibility complex (MHC) epitopes. We describe the generation of enriched alloreactive CD8+ T cells by priming mice with a skin graft expressing the allogeneic MHC class I molecule of interest, followed by boosting with a liver-specific AAV vector encoding the heavy chain of that donor MHC allomorph. We then use a peptide-exchange approach to assemble a range of peptide-MHC (pMHC) multimers for measuring recognition of the various epitopes by these alloreactive T cells. For complete details on the use and execution of this protocol, please refer to Son et al. (2021).1.

Interleukin-5 (IL-5) Therapy Prevents Allograft Rejection by Promoting CD4+CD25+ Ts2 Regulatory Cells That Are Antigen-Specific and Express IL-5 Receptor.

CD4+CD25+Foxp3+T cell population is heterogenous and contains three major sub-groups. First, thymus derived T regulatory cells (tTreg) that are naïve/resting. Second, activated/memory Treg that are produced by activation of tTreg by antigen and cytokines. Third, effector lineage CD4+CD25+T cells generated from CD4+CD25- T cells' activation by antigen to transiently express CD25 and Foxp3. We have shown that freshly isolated CD4+CD25+T cells are activated by specific alloantigen and IL-4, not IL-2, to Ts2 cells that express the IL-5 receptor alpha. Ts2 cells are more potent than naïve/resting tTreg in suppressing specific alloimmunity. Here, we showed rIL-5 promoted further activation of Ts2 cells to Th2-like Treg, that expressed foxp3, irf4, gata3 and il5. In vivo, we studied the effects of rIL-5 treatment on Lewis heart allograft survival in F344 rats. Host CD4+CD25+T cells were assessed by FACS, in mixed lymphocyte culture and by RT-PCR to examine mRNA of Ts2 or Th2-like Treg markers. rIL-5 treatment given 7 days after transplantation reduced the severity of rejection and all grafts survived ≥60d whereas sham treated rats fully rejected by day 31 (p<0.01). Treatment with anti-CD25 or anti-IL-4 monoclonal antibody abolished the benefits of treatment with rIL-5 and accelerated rejection. After 10d treatment with rIL-5, hosts' CD4+CD25+ cells expressed more Il5ra and responded to specific donor Lewis but not self. Enriched CD4+CD25+ cells from rIL-5 treated rats with allografts surviving >60 days proliferated to specific donor only when rIL-5 was present and did not proliferate to self or third party. These cells had more mRNA for molecules expressed by Th2-like Treg including Irf4, gata3 and Il5. These findings were consistent with IL-5 treatment preventing rejection by activation of Ts2 cells and Th2-like Treg.

The self-peptide repertoire plays a critical role in transplant tolerance induction.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: a comprehensive resource for in vitro studies of hepatic metabolism.

The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.

Blockade of HMGB1 Attenuates Diabetic Nephropathy in Mice.

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.

ACE2 Therapy Using Adeno-associated Viral Vector Inhibits Liver Fibrosis in Mice.

Angiotensin converting enzyme 2 (ACE2) which breaks down profibrotic peptide angiotensin II to antifibrotic peptide angiotensin-(1-7) is a potential therapeutic target in liver fibrosis. We therefore investigated the long-term therapeutic effect of recombinant ACE2 using a liver-specific adeno-associated viral genome 2 serotype 8 vector (rAAV2/8-ACE2) with a liver-specific promoter in three murine models of chronic liver disease, including carbon tetrachloride-induced toxic injury, bile duct ligation-induced cholestatic injury, and methionine- and choline-deficient diet-induced steatotic injury. A single injection of rAAV2/8-ACE2 was administered after liver disease has established. Hepatic fibrosis, gene and protein expression, and the mechanisms that rAAV2/8-ACE2 therapy associated reduction in liver fibrosis were analyzed. Compared with control group, rAAV2/8-ACE2 therapy produced rapid and sustained upregulation of hepatic ACE2, resulting in a profound reduction in fibrosis and profibrotic markers in all diseased models. These changes were accompanied by reduction in hepatic angiotensin II levels with concomitant increases in hepatic angiotensin-(1-7) levels, resulting in significant reductions of NADPH oxidase assembly, oxidative stress and ERK1/2 and p38 phosphorylation. Moreover, rAAV2/8-ACE2 therapy normalized increased intrahepatic vascular tone in fibrotic livers. We conclude that rAAV2/8-ACE2 is an effective liver-targeted, long-term therapy for liver fibrosis and its complications without producing unwanted systemic effects.

Analysis of Post-Liver Transplant Hepatitis C Virus Recurrence Using Serial Cluster of Differentiation Antibody Microarrays.

BACKGROUND: Hepatitis C virus (HCV) reinfection of the liver allograft after transplantation is universal, with some individuals suffering severe disease recurrence. Predictive markers of recurrent disease severity are urgently needed. In this study, we used a cluster of differentiation (CD) microarray to predict the severity of HCV recurrence after transplantation. METHODS: The CD antibody microarray assays of live leukocytes were performed on peripheral blood taken in the first year after transplantation. The results were grouped into phases defined as; Pre-transplant (day 0), Early (day 3 to week 2), Mid (week 4 to week 10), and Late (week 12 to week 26). Hepatitis C virus severity was based on fibrosis stages in the first 2 years (F0-1 mild and F2-4 severe). RESULTS: Serial blood samples from 16 patients were taken before and after liver transplantation. A total of 98 assays were performed. Follow-up was 3 years or longer. Comparing recurrence severity, significantly greater numbers of CD antigens were differentially expressed on the pretransplant samples compared to any posttransplant timepoints. Five differentially expressed CD antigens before transplantation (CD27 PH, CD182, CD260, CD41, and CD34) were significantly expressed comparing severe to mild recurrence, whereas expression of only CD152 was significant in the late phase after transplantation. No relationship was observed between the donor or recipient interleukin-28B genotypes and HCV recurrence severity. CONCLUSIONS: This study shows that circulating leukocyte CD antigen expression has utility in assessing recurrent HCV disease severity after liver transplantation and serves as a proof of principle. Importantly, pretransplant CD antigen expression is most predictive of disease outcome.

Safety, feasibility, and effect of remote ischemic conditioning in patients undergoing lung transplantation.

BACKGROUND: Primary graft dysfunction (PGD) remains a significant problem after lung transplantation. Data from animal and clinical studies suggest that remote ischemic conditioning (RIC) may reduce ischemia-reperfusion injury in solid organ transplantation. METHODS: A pilot randomized controlled trial of 60 patients undergoing bilateral sequential lung transplantation assessed the utility of RIC in attenuating PGD. Treated recipients underwent 3 cycles of lower limb ischemic conditioning before allograft reperfusion. The primary outcome measure was a comparison of the partial pressure of arterial oxygen/fraction of inspired oxygen ratio (P/F ratio) between treatment groups. RESULTS: No adverse effects of tourniquet application were observed. The mean lowest P/F ratio during the first 24 hours after transplantation was 271.3 mm Hg in the treatment arm vs 256.1 mm Hg in the control arm (p = 0.46). PGD grade and severity and the rate of acute rejection also showed a tendency to favor the treatment arm. Sub-group analysis demonstrated a significant benefit of treatment in patients with a primary diagnosis of restrictive lung disease, a group at high risk for the development of PGD. RIC was not accompanied by systemic release of high-molecular-weight group box 1. Levels of cytokines, high-molecular-weight group box 1, and endogenous secretory receptor for advanced glycation end products peaked within 2 hours after reperfusion and likely reflected donor organ quality rather than an effect of RIC. CONCLUSIONS: RIC did not significantly improve P/F ratios or PGD in this randomized controlled trial. However, encouraging results in this small study warrant a large multicenter trial of RIC in lung transplantation.

A modified method for heterotopic mouse heart transplantion.

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.

Optimized method for ureteric reconstruction in a mouse kidney transplant 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.

Liver transplant tolerance and its application to the clinic: can we exploit the high dose effect?

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.

Gene therapy for tolerance: high-level expression of donor major histocompatibility complex in the liver overcomes naive and memory alloresponses to skin grafts.

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.

Expression of common gamma chain signalling cytokines and their receptors distinguishes rejection from tolerance in a rat organ transplant model.

BACKGROUND: Signalling through the cytokine common γ chain (γc) is crucial for survival of activated T cells. In its absence, severe combined immunodeficiency ensues and transplanted tissues are not rejected. METHODS: To determine whether differences in the availability of γc signalling cytokines correlate with rejection or acceptance, we examined expression of all γc signalling components in organs transplanted between PVG donors and DA recipients. In this combination hearts or kidneys are rejected in <10 days while livers survive >100 days. Expression of the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 and their receptors γc, IL-2Rα, IL-2Rß/IL-15Rß, IL-4Rα, IL-7Rα, IL-9Rα, IL-15Rα and IL-21Rα was determined by real-time PCR pre-transplant and on days 3, 5 and 7 after transplantation. RESULTS: Most increased after transplantation, although there were significantly lower levels of IL-2, IL-2Rα, IL-4 and IL-15Rα in tolerant livers compared to rejecting hearts or kidneys. IL-9 was only expressed in normal kidneys and decreased during rejection. IL-15 was constitutively expressed and did not change after transplantation. IL-21 and IL-21R increased in all transplanted organs to a similar extent. IL-7Rα in liver was considerably increased compared with heart or kidney, consistent with its known inverse relationship to global levels of γc signalling. CONCLUSIONS: In transplanted livers, acceptance is associated with low levels of all γc cytokines or receptors except IL-21. This is consistent with "dilution" of γc cytokines from a finite clone size of alloreactive T cells in livers, which are ten times larger than kidneys or hearts.

Analysis of human liver disease using a cluster of differentiation (CD) antibody microarray.

BACKGROUND: A CD antibody microarray has been previously developed allowing semi-quantitative identification of greater than 80 CD antigens on circulating leucocytes from peripheral blood samples. This assay, which uses a live cell-capture technique, enables an extensive leucocyte immunophenotype determination in a single analysis and to date this has been used successfully to characterise diseases including human leukaemias and HIV infection. AIMS: To determine CD antigen expression profiles for patients with various liver diseases and to look for preserved disease-specific signatures. METHODS: Three liver disease groups including hepatitis C (HCV) (n = 35), non-alcoholic steatohepatitis (NASH) (n = 21) and alcohol-related liver disease (n = 14) were compared with a normal group (n = 23). Hierarchal Clustering (HCL) and Principal Component Analysis (PCA) of the data revealed distinct binding patterns for patients with and without cirrhosis. RESULTS: Patients with cirrhosis and portal hypertension compared with those without cirrhosis had significantly reduced expression of several markers of T-cell function including CD45, CD8, CD28 and TCR α/ß. Disease prediction algorithms based on the expression data were able to discriminate cirrhotics from non-cirrhotics with 71% overall success, which improved to 77% when only patients with HCV were considered. CONCLUSIONS: These results demonstrate disease-specific consensus patterns of expression of CD antigens for patients with chronic liver disease, suggesting that the CD antibody array is a promising tool in the analysis of human liver disease, and with further refinement may have future research and clinical utility.

Approaching the promise of operational tolerance in clinical transplantation.

Long-term acceptance of transplanted organs without requirement for indefinite immunosuppression remains the ultimate goal of transplant clinicians and scientists. This clinical state of allograft acceptance termed "operational tolerance" has been elusive in routine practice. However, there are published reports of recipients where immunosuppression has been discontinued, by intention or patient noncompliance, in which the outcome is a nondestructive immune response and normal function. The question now arises how clinical operational tolerance might be achieved in the majority of recipients. This review provides an overview of current approaches to achieve operational tolerance, including the use of donor bone marrow and depletion of recipient T cells and the resistance of liver transplants to rejection. It also describes the key role of clinical immune monitoring and future approaches to tolerance induction including inhibition of T-cell signaling, manipulation of costimulatory pathways, and expansion of regulatory T cells. The principles of these experimental approaches may ultimately be extended to provide safe and effective control of transplant rejection and induction of clinical operational tolerance.

Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments.

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8:CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG-/- donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-ß administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.

Donor IL-4-treatment induces alternatively activated liver macrophages and IDO-expressing NK cells and promotes rat liver allograft acceptance.

Most approaches to transplant tolerance involve treatment of the recipient to prevent rejection. This study investigates donor treatment with IL-4 for its effect on subsequent rat liver allograft survival. Rat orthotopic liver transplants were performed in rejecting (PVG donor to Lewis recipient) or spontaneously tolerant (PVG to DA) strain combinations. Donors were untreated or injected intraperitoneally with IL-4 (30,000U/day) for 5days. Tissue infiltrates and gene expression were examined by immunohistochemistry and real-time quantitative PCR. IL-4 induced a marked leukocyte infiltrate in donor livers prior to transplant. Macrophages comprised the major population, although B cells, T cells and natural killer (NK) cells also increased. IL-4-induced liver macrophages had an alternatively activated phenotype with increased expression of mannose receptor but not inducible nitric oxide synthase (NOS2). IL-4 also induced IDO and IFN-gamma expression by NK cells. Donor IL-4-treatment converted rejection to acceptance in the majority of Lewis recipients (median survival time >96days) and did not prevent acceptance in DA recipients. Acceptance in Lewis recipients was associated with increased donor cell migration to recipient spleens and increased splenic IL-2, IFN-gamma and IDO expression 24h after transplantation. Donor IL-4-treatment increased leukocytes in the donor liver including potentially immunosuppressive populations of alternatively activated macrophages and IDO-expressing NK cells. Donor treatment led to long-term acceptance of most livers in association with early immune activation in recipient lymphoid tissues.

NKG2D and its ligands.

NKG2D is an activating immunoreceptor, first recognized on NK cells but subsequently found on gammadelta T cells, CD8(+) alphabeta T cells and macrophages. In NK cells, inhibitory signals are generally dominate over activating signals. However, activating signals mediated through engagement of NKG2D by its ligands on target cells can bypass signals transmitted through inhibitory NK receptors, allowing NKG2D to function as a "master-switch" in determining the activation status of NK cells. NKG2D is important for T cell and NK cell-mediated immunity to viruses and tumours, and has roles in autoimmune disease, allogeneic transplantation, and xenotransplantation. Depending upon the situation, development of strategies to either block or to enhance the interactions between NKG2D and its ligands may have important implications for human health and disease.

Gene therapy in transplantation.

Gene therapy is an exciting and novel technology that offers the prospect of improving transplant outcomes beyond those achievable with current clinical protocols. This review explores both the candidate genes and ways in which they have been deployed to overcome both immune and non-immune barriers to transplantation success in experimental models. Finally, the major obstacles to implementing gene therapy in the clinic are considered.