Single-Cell Immune Profiling of Alloreactive T Cells Locally and Systemically in Patients Receiving Intestinal Transplantation

Background and Rationale: Abdominal injury is a leading cause of intestinal failure requiring intestinal transplantation, which is also a life-saving treatment for intestinal failure from other causes such as congenital abnormalities, ischemic injury, etc. Approximately 100-160 intestinal transplants (ITx) are performed in the US annually. However, patient survival rates are far from optimal, due to high rejection rates resulting from an immune attack of the recipient against the donor graft, termed host-versus-graft (HvG) reactivity. Additionally, intestinal grafts carry with them large numbers of donor lymphocytes that can attack the recipient in a disease called graft-versus-host disease (GVHD). The high levels of immune system-suppressing drugs used to prevent both rejection and GVHD come with a high risk of cancer and infections. Thus, there is an urgent need for a better understanding of patient immune responses to the donor graft to eventually develop a well-tolerated treatment strategy that controls rejection while reducing these risks. Scientists have demonstrated that high levels of mixed chimerism (coexistence of donor and recipient cells), especially >/= 4% donor T cell chimerism, commonly occurs in circulation without GVHD in ITx recipients, and that its presence correlates with reduced rejection rates. Using a previously established method involving sequencing of cellular DNA for identifying and tracking GvH- and HvG-reactive T lymphocytes, we have obtained evidence that higher levels of donor chimerism among circulating T cells post-ITx are associated with higher levels of GvH-reactive T cells in blood. Additionally, both GvH-reactive T cells and donor blood-forming stem and progenitor cells (hematopoietic stem and progenitor cells, HSPCs), derived from the intestinal graft as discovered recently, were detectable in recipient bone marrow (BM). This process is likely facilitated by a phenomenon termed 'lymphohematopoietic graft-vs-host responses (LGVHRs),' in which donor T lymphocytes from the ITx graft attack recipient blood-forming cells to make 'space' for their own engraftment into the BM, but do not induce GVHD. We now propose a study of single cell immune profiling of alloreactive T cells locally and systemically in patients receiving ITx to obtain a deeper understanding of the interplay between GvH- and HvG-reactive T cells in determining graft outcomes.

Topic Area: This proposal addresses the Immunomonitoring of Intestinal Transplants.

Objectives: We hypothesize that graft-derived GvH-reactive cells control HvG rejection responses locally and systemically through LGVHR, which facilitates a higher degree of blood chimerism and promotes 'immune tolerance,' a state of unresponsiveness of the immune system to substances or tissues that have the potential to induce an immune response. Given the advancement of single cell sequencing technology in recent years, we are now equipped with the ability to 'fingerprint' individual T cells to determine not only the specific T cell receptor sequences that mediate their response to foreign substances but also their functional gene expression profiles. This will be achieved by integrating a method for sequencing DNA from each specific T cell, with a method that identifies the genes expressed in that corresponding, respective cell. Patients will be monitored for GVHD, rejection, and mixed chimerism. More specifically, we will assess the functional gene expression patterns of circulating GvH-reactive T cells early post-Tx, and of graft-infiltrating HvG-reactive T cells during early rejection and late rejection, or quiescence. We will also monitor the interplay between GvH and HvG reactivity in recipient BM, if BM chimerism of T cells and blood-forming cells is confirmed.

Innovation, Application, and Impact: Single cell immune profiling of alloreactive T cells, key players in rejection after organ transplantation, is highly innovative and has not been performed before. Our proposed study in patients receiving ITx will provide novel insights into the role of LGVHR in promoting donor chimerism in the blood and BM, and into the mechanism behind the associations of these phenomena with reduced rejection and tolerance induction. We believe this may provide a window of opportunity to achieve durable chimerism and immune tolerance of the donor graft via infusion of additional blood-forming cells (HSPCs) from the donor without adding conditioning or risk of GVHD. Our findings thus far in ITx recipients also demonstrate the potential for the development of methods to determine real-time patient-specific immune responses to the organ graft, which would allow personalization of immunosuppression that could reduce both high rejection rates and the consequences of overimmunosuppression. Improved outcomes would increase ITx utilization as a life-saving, quality of life-improving procedure, and reduce the costs of caring for patients with intestinal failure from all causes, including abdominal injury.