1. What is the major problem being addressed by this study?
Cytomegalovirus (CMV) causes organ rejection and vascular disease in transplant recipients, potentially leading to death. CMV infects over half of the general population by adulthood and, like chicken pox, persists for life. It does not cause disease in healthy people, due in part to the action of white blood cells called T cells. These cells are blocked in transplant patients by the immunosuppression used to prevent organ rejection. Antiviral medication protects transplant patients from CMV, but is not completely effective and often causes side effects. Some patients have T cells that could protect against CMV if their immunosuppression were reduced. Our goal is to identify these patients by predicting the efficacy of T cell responses to CMV in heart, kidney, and lung transplant patients.

2. What specific questions are you asking and how will you attempt to answer them?
The long-term research question is to understand how to use T cells to protect transplant recipients from CMV without also attacking the donor organ. This proposal focuses on the question: how do transplant and immunosuppression change T cell responses to CMV? We will use innovative research techniques to study T cells shortly after transplantation and six months later. First, we will examine changes in T cell activity through analysis of small molecules that T cells use to function. We will also analyze the nature of T cell recognition of CMV by analyzing expression of genes that are key to T cell activity. Through these approaches we will gain understanding of the changes that occur in T cell immunity after transplantation, which is the first crucial step towards our long-term goal.

3. Overall, what is the potential impact of this work to the mission of the AHA?
The long-term impact of this research is to enable personalized treatment for transplant patients. CMV can cause severe disease in transplant patients, including vascular damage to a transplanted heart. For about 20% of patients, disease occurs even with preventive antiviral medication. Our studies could help develop diagnostic tools and fine-tune post-transplant therapies. For example, we may be able to predict if a transplant patient would benefit from reduced immunosuppression, without risking transplant rejection. In addition, understanding the role of a patient’s immune system in protection against CMV may contribute to treatment of other opportunistic infections. This study therefore offers great potential to improve therapeutic outcomes and quality of life after transplantation.

Immunosuppression makes organ transplantation possible, but increases the risk of opportunistic viral infections such as cytomegalovirus (CMV). CMV is a major cause of cardiac allograft vasculopathy (CAV) and rejection in heart transplant recipients. CAV is the leading cause of long-term graft failure after heart transplant.1 Antiviral medication protects against CMV, but does not eliminate disease. Reducing the dosage of immunosuppression in patients with protective T cell immunity to CMV may allow T cells to control the infection. However, both immunosuppression and exposure to CMV can change T cell immunity, potentially risking organ rejection. Transplanted organs also expose recipients to donor and viral antigens. Complicating matters further, some T cells cross-react with both CMV and alloantigen. Therefore, in order to use protective immunity to CMV to alter clinical care, it is critical to determine how post-transplant antigen exposure shapes T cell responses. Our preliminary studies demonstrate that the prevalence of CMV responsive T cells increases during the first-year post-transplant These changes may occur in response to subclinical reactivation or another stimulus such as alloantigen from the donor organ. Therefore, these cells could form protective responses to virus, pathogenic responses to donor, or both. We hypothesize that the post-transplant increase in CMV responsive T cells is caused by ongoing exposure to non-self, either viral proteins or alloantigen.

Specific Aim 1: Determine how specificity for CMV or alloantigen affects T cell function. To define the overlap of immunity to CMV and alloantigen, we will analyze blood from healthy volunteers for specificity and function of CD8 T cells responsive to CMV or alloantigen. We will assess function through expression of effector and phenotypic molecules, and repertoire through T cell receptor (TCR) sequencing. Innovative techniques include mass cytometry and single cell sequencing. Cross-reactive TCRs will be stimulated by both alloantigen and CMV.

Specific Aim 2: Determine how transplantation and immunosuppression affect CMV responsive T cells. To define changes in T cell responses to CMV and alloantigen, we will compare responses in transplant recipients during and at least 6 months after completion of antiviral prophylaxis. We will assess PBMC from recipients of heart, kidney, and lung transplant for T cell phenotype, function, and TCR repertoire. Antigen specific expansion will be defined as increased clonality of CMV- or allo-responsive T cells.

The immunosuppression that makes life-saving organ transplantation possible also increases a patient’s risk of opportunistic viral infection. One such infection is cytomegalovirus (CMV), which latently infects about 60% of adults. While protective immunity prevents CMV from causing disease in healthy individuals, immunosuppressed transplant patients can develop disease including damage to the transplant. In heart transplant patients, CMV induces vasculopathy, the leading cause of long-term transplant failure. T cells are an important component of the immune response to CMV that can protect transplant recipients from disease. Previous studies in this laboratory addressed changes in CMV responsive T cell populations after heart and kidney transplantation in CMV seropositive patients. All patients were immunosuppressed and treated with prophylactic antivirals; the kidney transplant patients also received lymphodepleting induction therapy. The relative abundance of CMV responsive T cells increased over a year posttransplant, even in patients who did not experience CMV disease. T cell expansion in the absence of disease could result from subclinical exposure to the virus, or exposure to a different antigen entirely, such as donor alloantigen. The goals of this proposal are to determine what the antigenic cause of the expansion is, and to identify the overlap of protective responses to CMV and pathogenic responses to alloantigen. These goals will be addressed through the following aims. Aim 1 is to determine how specificity of CMV responsive T cells affects function. Aim 2 is to determine how transplantation and immunosuppression affect CMV responsive T cells. These aims will be addressed by analyzing the functional properties of CMV-responsive T cells from blood of non-transplanted individuals and heart, kidney, and lung transplant recipients pre- and post-transplant. Analysis will focus on T cell responses to CMV polypeptides and alloantigen in order to correlate T cell phenotype with function. Approaches will take advantage of the unique resources available at Stanford and include high-throughput phenotypic analysis through mass cytometry and analysis of clonality of antigen specific T cells through single cell T cell receptor sequencing. These analyses will improve understanding of T cell immunity in transplant patients, with the long-term goal to extend transplant survival and to use immune based treatment to promote tolerance and prevent graft rejection.