In vivo intermittent bridging of IL-2R beta gamma and 4-1BB endodomains promotes robust T cell proliferation, memory  generation, and prevents exhaustion
T cell-based therapies, leveraging engineered cells or protein-based modulators, hold immense potential across various diseases. However, T cell exhaustion and impaired memory formation remain significant challenges. Through the design and investigation of various CAR constructs, our findings demonstrate that intermittent bridging of IL-2Rbeta gamma and 4-1BB effectively induces robust T-cell proliferation, enhances memory cell development, and prevents exhaustion. This effect is specifically dependent on the involvement of 4-1BB in the bridging process. While intermittent IL-2Rbeta gamma signaling alone or IL-2Rbeta gamma-CD3zeta bridging induces certain effector functions, it does not promote proliferation, memory cell generation, or prevent exhaustion. IL-2Ralpha is dispensable for the observed effects, whereas modulating IL-2/IL-2Rbeta gamma signaling strength substantially influences the magnitude and duration of IL-2Rbeta gamma-4-1BB bridging, thereby impacting T cell responses. Notably, a monomeric CAR-Enhancer remains highly effective, indicating that bridging IL-2Rbeta gamma and 4-1BB without necessitating their clustering is sufficient to achieve a robust synergistic effect. These findings provide novel insights for developing advanced immunotherapies.