Development of a Lentiviral Display System for High-Throughput T-Cell Ligand Deorphanization and Specificity-Based Reprogramming
Abstract
This research proposal addresses the Peer Reviewed Medical Research Program topic area of Emerging Infectious Disease. New or unknown pathogens are often incredibly difficult to treat or vaccinate against. This is largely because it takes significant time (years or even decades) and effort to determine how the immune system defends against them, and what parts of the pathogens, or antigens, are used to alert the immune system to the pathogen’s presence. T cells are key effectors of the adaptive immune response, which is theoretically capable of defending against nearly any pathogen upon first recognition while also retaining “memory” of the attack that bolsters the immune defense against subsequent exposures. To accomplish this, before being released to patrol the body, each T cell produces a unique antigen receptor, termed the T cell receptor (TCR). T cells circulate throughout the body, and upon detection of a foreign peptide via the TCR, activate a host of defense mechanisms designed to eliminate the pathogen from which it originated. Everyone possesses on the order of 10 million unique T cells, and each one recognizes a distinct set of peptide antigens bound to specialized molecules, major histocompatibility complexes (MHCs). To ensure all possible pathogens can be recognized, it has been calculated that each TCR in an individual must recognize on the order of 100,000 unique peptide-MHC (pMHC) combinations. However, T cells must achieve this cross-reactivity in a way controlled enough to not have T cells inadvertently induce autoimmune responses by mistakenly recognizing peptides from one’s own body as foreign. Understanding the basis of how TCRs can balance the need to recognize all potential antigens while not producing an autoimmune response is an area of intense research interest. Understanding what T cells recognize during the course of a successful immune response and how to more effectively induce successful immune responses is of particular interest for Emerging Infectious Disease, the topic area for this proposal. Unless it is a well-studied and/or simple pathogen, there is presently no robust or rapid way to take T cells from an infected or vaccinated patient and determine what those T cells recognize. Present techniques used to accomplish these goals either rely upon significant previous knowledge of the potential disease or can only be done on a handful of TCRs at a time. To overcome this obstacle, this proposal aims to create a technology able to rapidly match TCRs with the antigens they recognize in a single experiment. To achieve these goals, the system must be able to operate with single-cell resolution, but also be readily adaptable to large and complex mixtures of T cells, such as those sampled from an infected or vaccinated patient. This proposal is for the development of a system to allow for DNA sequencing to detect the TCR-antigen pairs. This approach leverages the fact that lentiviruses can be targeted to enter specific cells based on their surface proteins and, once inside, permanently encode a DNA cargo into the target cell. Lentiviruses will be adapted to display pMHCs on their surface, which will permit them to use the pMHC-TCR interaction as a means of entry into T cells that may be key players in the immune response to an emerging pathogen. The lentiviruses will then encode the sequence of the pMHC they display directly into the T cell’s genome. Previously developed single cell analysis techniques will then be able to simultaneously detect the sequences encoding each cell’s TCR as well as any the pMHCs able to be recognized by that cell. Modern biotechnology enables the concurrent generation of many unique viruses, each displaying a different pMHC combination. The approach in this proposal would exploit this ability to screen thousands or even millions of antigens against potentially tens or hundreds of T cells simultaneously. This approach has two key application
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Oct 29, 2018
- Source ID
- W81XWH1810208
Entities
People
- Michael E Birnbaum
Organizations
- Massachusetts Institute of Technology
- United States Army