Allograft rejection remains an issue in long term success of allotransplantation and is also likely to negatively impact the function and longevity of tissue engineered grafts being explored as a possible solution to the shortage of available organs. In the direct pathway of allorecognition, rejection is typically initiated by activation of resting host T effector memory cells that cross-react with non-self MHC/peptide complexes, a process that requires graft cells to provide additional signals such as co-stimulators and cytokines. Graft cells capable of doing this are ?immunogenic?. Such T cells then differentiate into effector cells that are capable of killing cells that express the same non-self MHC/peptide complexes. Graft cell types that express the same alloantigens and can be recognized and killed by effector T cells but are unable to initiate the differentiation of resting effector memory into effector T cells are said to be ?antigenic? rather than immunogenic. Two immunogenic human cell types implicated in initiating rejection are graft dendritic cells (DCs) and graft endothelial cells (ECs). While DCs (and other passenger leukocytes) can be purged from a natural graft or left out of a bioengineered graft, ECs are essential for lining the blood vessels that sustain graft viability. We have proposed strategies to reduce rejection by limiting the immunogenicity of the graft to complement the current practice of suppressing the host immune response. It is unclear if removing DCs and altering ECs to be non- immunogenic will be sufficient to protect a graft from rejection. Here we propose to answer this question. Addressing this question requires that it be performed with human ECs because commonly used rodent ECs do not exhibit the same immunogenic capabilities as their human counterparts. To do so, we will bring to bear 5 novel technologies: a). genetic engineering of ECs to render them non-immunogenic and non-antigenic; b). nanomedicine to modulate the immunogenic capacity of ECs; c). 3D printing of a human skin composed of multiple different cell types as a target for rejection; d). a state-of-the-art human immune system mouse as a graft recipient; and e). cutting edge high dimensional serial immunofluorescence to determine the effects of our interventions on the rejection process. We will initially test our model mouse model and optimize our assays using natural human skin (aim 1) and then proceed to test our 3D skin constructs (aim 2) in which we will modify cell types to determine the role, if any, of human cells other than ECs and DCs. We acknowledge that there is some risk in a proposal that merges multiple technologies which have not been previously combined and therefore have chosen to use the R21 exploratory mechanism to support this research. However, the successful conduct of this project will not only provide an initial answer to an important question for human skin cells, but will also create a platform for further studies of allotransplantation with more complex human tissues and provide valuable insights for bioengineering of replacement organs.
Reducing the ability of an organ to activate the host?s immune system, whether transplanted from another individual or constructed in the laboratory from cells derived from another individual, can improve the long term success of transplantation while allowing reduction in dose and hence side effects from the drugs currently used to suppress the host?s immune system. This study is designed to identify which human cell types must be targeted in the graft to help it evade host immunity. Because these processes vary in crucial details from species to species, we will use cutting edge technologies to address this question including use of 3D printing to fabricate skin from different human cell types and transplanting these on to mice given a human immune system.
