Objective
Assuming that the potential incompatibility between the KIRs expressed on the recipient's NK cells and the HLA molecules expressed on the graft cells can lead to an unfavorable outcome of the transplant, this project proposes to: a) determine which specific KIR / HLA combinations are actually correlated with an increased likelihood of progressive loss of graft function or acute rejection and, on the other end which ones favor the organ/islet engraftment b) to evaluate the use of monoclonal antibodies which, by targeting molecules of the activating axes of NK cells, are able to decrease NK reactivity against the graft when KIR / HLA combinations are particularly unfavorable.
To achieve these two objectives, we aim to: 1) perform gene association study between different KIR / HLA pairs and organ function over time of kidney (as control), kidney + pancreas and islet transplant recipients; 2) generate both cellular and mouse models recapitulating NK reactivity mechanisms in favorable and unfavorable KIR / HLA combinations; 3) test the consequence on graft survival of anti-B7-H3 and anti-CD155 monoclonal antibodies in the same cellular and mouse models.
Background Rationale
Despite progresses in insulin analogues and closed-loop insulin pump technology, treatment of T1D with exogenous insulin remains hindered by side effects such as high or dangerously low blood sugar concentrations. Therefore, the transplantation of whole-organ pancreas or islets purified from pancreas of deceased organ donors has become a viable therapeutic option to reverse T1D and achieve normal blood sugar levels. However, both pancreas and islet transplantation are limited by paucity of cadaveric donors. For this reason, advances in stem cell technology and xenotransplant (transplant using nonhumans organs) field led to identification of new sources, potentially infinite, of insulin-producing cells. Nevertheless, autoimmune-mediated insulin-producing cell loss and allograft rejection continue to represent one of the most important concerns forcing patients to undergo lifelong immunosuppressive therapy, with a strong reduction in terms of life quality. Despite immunosuppression, insulin independence beyond ten years is only reached by less than 50% of transplanted patients. Early loss of islets is due to different causes, including toxicity of immunosuppressive drugs and innate immune responses, which represents the first form of immune reaction that is triggered in our body towards a foreign agent. Natural Killer (NK) are immune cells that play a critical role in the early immune responses and it has been demonstrated that are directly implicated in the loss of islets after intraportal allogeneic transplantation. Cytotoxic activity of NK cells significantly increases after islet transplantation, leading to progressive loss of graft function and failure. In human, cytotoxic activity of NK cells is regulated by fine balance of both activating and inhibitory signals that depend on killer immunoglobulin-like receptors (KIR), a set of heterogenous activating and inhibitory receptors whose main interactors are the human leukocyte antigen (HLA) class I molecules, a group of highly variable proteins that are expressed on surface of the majority of the somatic cells. When inhibitory KIRs recognize and tightly bind the HLA class I molecules, NK cells do not destroy cells that express such HLA molecules. Conversely, when KIRs and HLAs are unable to bond themselves, due to differences that make them structurally incompatible, NK cells become prone to attack those cells which they recognize as strangers. In kidney transplant it has been recently reported that incompatibility between KIR and HLA is capable to negatively affect long-term outcome of transplantation. Analogously, a link between chronic rejection and compatibility of KIR/HLA was reported also in lung and liver transplantation. We can state that a KIR/HLA mismatch results in low level of inhibitory signals that may shift the balance towards activating signals leading to NK-mediated immune response. Activation of NK cells is triggered by a plethora of activating receptors that recognize several activating molecules on graft cells. In particular, we found that two of these activating molecules, B7-H3 and CD155, are particularly enriched on surface of islet as well as of stem cell-derived beta cells, that are recently proposed by several clinical trials as promising source of insulin-secreting cells for cell therapy of T1D. Moreover, we found that when these cells lose HLA class I molecules that prevent the binding to the inhibitory KIRs, they become susceptible to NK killing. However, by blocking or genetically disrupting both B7-H3 and CD155, we prevented beta cells recognition and death, suggesting that both molecules may affect immunogenicity of pancreas or islets as well and may represent the activating signaling responsible of loss of the graft function when inhibitory KIRs expressed on the recipient's NK cells and the HLA molecules expressed on the graft cells fail to interact.
Description of Project
Type 1 diabetes (T1D) is a chronic debilitating disease associated with several life-threatening complications. Maintaining normal blood sugar concentrations is crucial for preventing these complications. Currently, intensive insulin regimes have contributed to maintenance of normal blood glucose levels. However, despite insulin therapy, some T1D patients may experience wide and unpredictable fluctuations in blood sugar levels, greatly impacting on their quality of life and putting them at great risk for severe complications such as kidney failure.. Currently, the only option, that T1D patients have over insulin therapy consists of the whole pancreas (in the 70% of cases together with kidney) or islet alone (region of the pancreas that contains insulin-producing cells called beta cells) transplantation. However, application of these transplant therapies is limited to a small number of patients due to both the paucity of donors and the need to establish to a lifelong immunosuppression therapy to prevent graft rejection. Research is ongoing to develop replenishable sources of insulin-producing cells, including stem cell-derived beta cells and porcine islets, to allow expansion of transplant options to a larger group of patients. However, although the identification of alternative cell sources represents an important step forward, transplantation is still hindered by short graft survival and complications related to immune response against donor cells. One of the cell types involved in immune response against graft is represented by Natural Killer (NK) cells, that are involved in killing cells infected by viruses, cancer cells or other kind of foreign cells just like those of a transplanted organ. Whether or not the NK cell kills these cells depends on a balance of signals from activating and inhibitory receptors on the NK cell surface. The most important NK receptors are the killer immunoglobulin-like receptors (KIR), that recognize molecules named human leukocyte antigen (HLA) class I. Normal cells express HLA class I molecules, which mark these cells as “self” and “safe”. NK cells recognize HLAs by inhibitory KIRs, and this “switches off” the NK cells, preventing it from killing. However, interindividual variation between human makes that, in some cases, the KIRs of the recipient may be incompatible with the HLA of the donor. When this occurs is likely that NK cells lose inhibition signals and become activated. Up to now, a correlation between specific KIR/HLA combinations and the risk of rejection in kidney, lung and liver transplantation has been demonstrated. Moreover, in our preliminary study, we demonstrated that the loss of interaction between KIR and HLA is the cause of activation of NK cells against stem cell-derived beta cells, through the recognition of two specific molecules called B7-H3 and CD155. By destroying these two molecules, however, we were able to prevent NK attack. Based on evidence reported in literature and on our preliminary data, we then hypothesized that loss of islets or other sources of insulin-producing cells may be the consequence of “unsafe” KIR/HLA combination leading to loss of inhibitory signals needed for counterbalance activating ones in NK. In light of this, we aim to investigate the impact of KIR/HLA combination on pancreas and islet transplant survival, identifying unsafe combinations that lead to an increased risk of graft loss. Moreover, we will test the use of anti-B7-H3 and anti-CD155 antibodies in cellular and murine models of unfavorable KIR/HLA combinations. The use of monoclonal antibodies may be a feasible alternative strategy to prevent NK cell activation and killing when incompatibility between KIRs and HLAs occurs. Taken together, findings of our research could have a clear and direct translational path to the clinical application, improving the prospects also offered by the transplantation of the new beta cell sources.
Anticipated Outcome
Investigating graft function of a large group of transplanted patients with 5-years follow-up we expect to identify the KIR/HLA combinations that are either protective (ensuring the long-term survival of the graft) or unsafe (associated with reduced organ function or a higher risk of acute rejection). This study will therefore allow to create a predictive model that, on the basis of the KIR/HLA pair, can quantify the risk of loss of the organ function over time. We expect to provide a new tool for the choice of the donor a priori, or, when alternative sources of beta cells become available, to offer the patient those with lower risk. Furthermore, through the development of cellular and mouse models, we expect to pinpoint the molecular mechanisms that guide the process of recognition of the graft by NK cells. In particular, we expect to model both incompatible and perfect match conditions between KIR and HLA,for quantifying the immune response as a function of the degree of their compatibility. This will allow to test the therapeutic use of antibodies that are able to prevent activation of NK cells, minimizing the negative effects deriving from the incompatibility between KIR and HLA. Encouraged by our preliminary data, we expect to observe both in cellular and murine models a strong reduction in loss of graft function and survival after treatment with anti-B7-H3 and anti-CD155 antibodies. This could be the starting point for further investigations that might lead to the definition of new clinical protocols in the transplant field.
Relevance to T1D
Evidence that certain recipient/donor KIR/HLA pairs influence long-term graft survival have been reported in literature. If we will succeed in proving that incompatibility between KIRs and HLAs belongs to the early events influencing pancreas/islet or human stem cell-derived beta cell transplant outcome, categorizing transplantation according to KIR/HLA pairs will assume clinical importance for determining graft long-term survival, acting as pivot for personalized medicine in T1D treatment. Thus far, only HLA matching is currently considered in the selection of organ donors. Implementation of protective HLA/KIR combinations could improve the rate of graft success. However, we also believe that favorable KIR/HLA pairs may be necessary but not sufficient condition to ensure long-term survival of the graft. Consequently, if successful, our strategy to use monoclonal antibodies against both B7-H3 and CD155, abolishing the activating signals on NK cells involved in the graft rejection, may represent a potential pharmacological therapy aid to ameliorate success rate with fewer side effects and risks for the patient's health. As a matter of fact, the use of these antibodies would be temporally limited, but could produce long-term NK modulation and self-tolerance effects. Furthermore, the time needed for clinical development of this therapeutic approach could also be shorted, since phase 1 and 2 studies on the use of monoclonal antibodies against these immune checkpoints are already underway.