Manipulating human regulatory T cells (Tregs) offers the opportunity to induce tolerance in a clinical setting. However, low numbers of antigen-specific Tregs and Treg instability upon prolonged expansion have hampered the implementation of Treg-based therapies. Chimeric antigen receptor (CAR) technology has greatly expedited the generation of tumor antigen-specific effector T (Teff) cells. CARs are synthetic receptors comprising an extracellular antigen-binding domain and an intracellular signaling domain. The latter is commonly a fusion of CD28 and CD3z, allowing for potent T cell activation directly downstream of antigen recognition. Adoption of the CAR platform for Treg engineering represents a promising strategy to generate custom-made antigen-specific Tregs for therapy. Yet, there are marked differences in function and signaling between Tregs and Teff cells. Here, we interrogated CAR-mediated signaling in human Tregs and Teff cells by systematically modifying the CAR cytoplasmic domain. We constructed CARs with three different CD28 mutants, abrogating binding to PI3K, ITK, and LCK. Interestingly, disrupting CAR-mediated PI3K signaling did not significantly alter activation or proliferation of either Treg or Teff cells. However, preliminary data show that loss of PI3K signaling impairs CAR-Treg suppressive capacity. In addition, CARs with three different CD3 chains downstream of CD28, namely CD3z, CD3d and CD3e were examined. Replacing CD3z with other subunits invariably decreased CAR-mediated activation. Altogether, our results demonstrate that PI3K signaling is crucial for CAR-Treg function and that CD3z is essential for optimal CAR-Treg signaling, suggesting that novel CAR signaling modules may maximize CAR-Treg therapies.
This work was supported by Juno Therapeutics, Inc.
University of California, San Francisco (UCSF)
Director, Hormone Research Institute
Director, Transplantation Research Laboratory