LAG-3 and 4-1BB identify dysfunctional antigen-specific T cells in the tumor microenvironment and combinatorial LAG-3/4-1BB targeting gives synergistic tumor control

Abstract

Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous anti-tumor response, immune regulatory pathways can subvert the effector phase and enable tumor escape. One possible negative regulatory pathway is T cell-intrinsic anergy. Recently, we have shown that the transcription factor Egr2 is criti-cal in controlling the anergic state using an in vitro model system. Gene expression profiling and Egr2 ChIP-Seq analysis revealed multiple Egr2-driven cell surface proteins in T cell anergy, including the inhibitory recep-tor LAG-3, but also the costimulatory receptor 4-1BB. We examined whether these surface proteins identify dysfunctional tumor-reactive CD8 + T cells. A major population of CD8 + TILs co-expressing 4-1BB and LAG-3 was observed in the " progressor " tumor models B16 and C1498 but not the spontaneously rejected " regressor " MC57.SIY model. Kinetic analysis showed the appear-ance and persistence of this population over time only in the tumor. Upon ex vivo stimulation the 4-1BB + LAG-3 + as well as CD8+Egr2GFP + TILs exhibited a significant reduction in IL-2 production compared to their negative counterpart indicating TIL dysfunction. TCR repertoire analysis showed CDR3b skewing in the 4-1BB + LAG-3 + compartment, indicating oligoclonality. In addition, CD8 + TIL specific for the model antigen SIY expressed LAG-3 and 4-1BB. Further, 2C T cells transferred into a tumor bearing host upregulated LAG-3 and 4-1BB within 3 days after entering the tumor. These data confirmed that the vast majority of tumor-reactive CD8 + TILs express 4-1BB and LAG-3. Although the 4-1BB + LAG-3 + TIL population lacked the ability to produce IL-2, high levels of IFN-g, GzmB, Perforin, IL-10 as well as the Treg-recruiting che-mokines CCL1 and CCL22 were produced by this popu-lation. These data argue that this population may exert negative immunomodulatory functions and not be com-pletely inert. Therapeutic targeting of this population by agonistic 4-1BB and blocking LAG-3 mAb combination resulted in increased tumor control and an overall shift towards short term effector KLRG1 hi IL7R lo phenotype in CD8 + TILs as well as decreased expression of several inhibitory receptors including PD-1 and 2B4. Interest-ingly, ex vivo stimulation of CD8 + TILs after treatment resulted in restoration of the ability to produce IL-2. Our results suggest that the co-expression of LAG-3 and 4-1BB identifies a critical subpopulation of dysfunctional tumor antigen-specific CD8 + TILs and may contribute to an immune suppressive tumor microenvironment. Inhibit-ing LAG-3 while agonizing 4-1BB resulted in an increased anti-tumor response and a significant change in phenotype and function in the CD8 + TIL compartment.