Graft rejection in immune competent vertebrates can utilize both T and NK cell responses to hematopoietic cell transplants [HCT] and tissue grafts. Graft-versus-host disease (GVHD), a T-cell mediated syndrome, develops following HCT to immunosuppressed hosts, even when MHC [HLA humans, H2 mice] between donor and recipient are identical. Although GVHD is attributed to T-cell responses to non-HLA minor antigens, the possible contribution by the innate immune system has not been well studied. We will study the role of the innate histocompatibility system in allogeneic HCT in protochordates mice and humans. In Botryllus schlosseri (a protochordate closely related to vertebrates), a single gene locus, the FuHC, controls graft acceptance or rejection. Botryllus colonies that share one allele form chimeras, colonies that do not, reject. Botryllus chimera formation utilizes a system analogous to NK cells. However, like the mammalian MHC, the FuHC gene is highly polymorphic and populations carry thousands of alleles. We hypothesize that studying the mechanism of stem cell rejection or induced tolerance in Botryllus will provide novel insights into the pathways that lead to tolerance induction in mammals. We have identified, mapped, and cloned the FuHC. The FuHC shares domain homology to genes highly expressed on mammalian stem cells (Tie1 and Tek), and sequence and structural homology to genes involved in NK cell signaling and cell adhesion (CD155 and Igsf4) implicating NK-like cells as mediators of this primitive recognition system. In Botryllus chimeras, circulating cells of one partner can compete and replace the germline and/or soma of the other partner. The host tissues'replacement follows genetic hierarchies for somatic and germline stem cell competitive potential (SCP) of "winner" vs. "loser" strains. Our studies have defined these predatory cell lineages as prospectively isolatable stem cells and showed that the FuHC locus and germline competition inheritance operate at the level of transplanted, purified stem cells. Utilizing the Botryllus SCP trait we propose to investigate the evolutionary molecular mechanisms that regulate allogeneic stem cell competition in a host. We plan to: (i) identify genes that may regulate stem cell competition in Botryllus, (ii) investigate the role of selected genes in stem cell transplantation engraftment and competitive repopulation of tissues, and (iii) investigate the potential role of genes homologous to genes altering stem cell competition potential in Botryllus in mammalian transplantation immunity. Using high throughput sequencing platforms we plan to study the molecular regulation of SCP by comparing differential expression of genes between "winner" and "loser" strains. Boolean analysis will be carried out on this data to identify candidate SCP genes. The expression patterns of selected genes will be validated via qRT-PCR and in situ hybridization, and their function evaluated by knockdown and in vivo engraftment studies. Allelic polymorphism of mouse and human genes, with homology to genes that alter stem cell competition potential in Botryllus will be analyzed;if found, we will determine whether these are related to transplantation outcomes.
For future use of hematopoietic chimerism in therapies, many fundamental issues related to the molecular regulation of cellular chimerism need to be elucidated. Here we plan and suggest to search for genes that regulate stem cell activity in a protochordate model organism that demonstrate genetic hierarchies for somatic and germline stem cell competition in a chimera. We will then seek to determine if mouse and human genes with homology to genes that prevent stem cell competition in this protochordate, can be related to mammalian hematopoietc stem cell transplantation immunity. Since protochordates are believed to be the link between vertebrates and invertebrates, this study has the potential to discover an ancestral recognition system regulating blood based chimerism that may still be present in humans.
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