Project 2

Artificial APCs as Stimuli of Polyclonal and Anitgen-specific Regulatory T cells

There are recent exciting advances in the biology of T regulatory cells that indicate their potential for the therapy of autoimmune disorders. Present methods of ex vivo expansion of Tregs are difficult to scale up and all of the current approaches have limitations to feasibility and FDA approval. Our long-term goal is to develop second generation culture systems for Tregs that will be useful for adoptive immunotherapy for autoimmune diabetes, and more specifically, to develop artificial APCs as stimuli of polyclonal and antigen-specific regulatory T cells. The central theme of this application is that improved T cell culture systems can enable successful adoptive transfers of T cells by improving engraftment, persistence and function. A major hypothesis of this project is that cellbased artificial APC (aAPC) can be constructed to activate and expand Tregs with equal or better efficiency
than DC or antibody coated magnetic beads. A related hypothesis is that human TREG subsets have distinct growth requirements and differing intrinsic replicative potentials. In this project, we will apply novel cell culture systems to study the replicative capacity effector functions of newly recognized human TREG cell subsets. The following three specific aims will test the central hypothesis that human TREG cell subsets have distinct costimulatory and cytokine requirements for long term growth and for activation of effector function by:

1. To develop enhanced cell-based artificial APC (aAPC) for TREG adoptive immunotherapy. Here we will take advantage of an aAPC based on the K562 leukemia line that we have previously developed and optimized for CD8 T cell growth, and modify the aAPC for TREG activation and expansion. We have constructed a library of lentiviral vectors that express most known costimulatory molecules and cytokines, and we will express these in combinatorial sets in the aAPC to optimize TREG activation and growth.
2. To determine the replicative capacity and functional attributes of ex vivo expanded human TREG cell subsets. Using the cell-based aAPCs developed above, we will carry out in vitro experiments to determine the replicative capacities of natural, adaptive and antigen specific TREG subsets. Tregs from normal donors and autoimmune donors will be tested. We will focus our efforts on identifying aAPC that expand TREG subsets to eliminate or reduce the ex vivo growth delay of TREG that has been observed by other investigators in this program and in the field.
3. To adapt cell-based aAPC for clinical use in phase I pilot trials by testing candidate cell-based aAPCs for their relative ability to activate, expand polyclonal and antigen specific TREG, and retain functional capabilities. The optimal cell-based aAPC will be selected and transferred to Core A where the culture process will be scaled up for clinical grade manufacturing to support the filing of an IND.

In summary, a non-toxic immunotherapy and immunoprevention for T1D is poised to become a reality with the advent of improved understanding of the cell biology of Tregs and the development of efficient culture systems to enable the routine expansion of Tregs with potent function. Our focus will be on developing the first culture system optimized for human TREG activation and expansion. Below is a description of a comprehensive approach necessary to bring Tregs to the clinic for safety and feasibility testing in T1D.