Combinatorial immunotherapy induces tumor-infiltrating CD8⁺ T cells with distinct functional, migratory, and...

Programmed death (ligand) 1 (PD-(L)1) blockade and OX40/4-1BB costimulation have been separately evaluated in the clinic to elicit potent antitumor T cell responses. The precise mechanisms underlying single agent activity are incompletely understood. It also remains unclear if combining individual therapies leads to synergism, elicits novel immune mechanisms, or invokes additive effects.

We performed high-dimensional flow cytometry and single-cell RNA sequencing-based immunoprofiling of murine tumor-infiltrating lymphocytes (TILs) isolated from hosts bearing B16 or MC38 syngeneic tumors. This baseline infiltrate was compared to TILs after treatment with either anti-PD-(L)1, anti-OX40, or anti-4-1BB as single agents or as double and triple combinatorial therapies. Fingolimod treatment and CXCR3 blockade were used to evaluate the contribution of intratumoral versus peripheral CD8⁺ T cells to therapeutic efficacy.

We identified CD8⁺ T cell subtypes with distinct functional and migratory signatures highly predictive of tumor rejection upon treatment with single agent versus combination therapies. Rather than reinvigorating terminally exhausted CD8⁺ T cells, OX40/4-1BB agonism expanded a stem-like PD-1^loKLRG-1⁺Ki-67⁺CD8⁺ T cell subpopulation, which PD-(L)1 blockade alone did not. However, PD-(L)1 blockade synergized with OX40/4-1BB costimulation by dramatically enhancing stem-like TIL presence via a CXCR3-dependent mechanism.

Our findings provide new mechanistic insights into the interplay between components of combinatorial immunotherapy, where agonism of select costimulatory pathways seeds a pool of stem-like CD8⁺ T cells more responsive to immune checkpoint blockade (ICB).