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

Abstract

BackgroundProgrammed 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.MethodsWe 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.ResultsWe 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-1loKLRG-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.ConclusionsOur 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).

Stem-like T cells and niches: Implications in human health and disease

“…Success clinical practice have been released in solid organ and HSC transplantation, type 2 diabetes treatment, and neurodegenerative diseases. Similar results can be achieve by using cytokines such as IL-7, IL-15, and IL-21 or by activating proper costimulatory receptors like 4-1BB, OX40 and CD27 ( 21 , 204 ). Combined these studies, it shows that pharmacological, cytokine and costimulatory signal involved stemness can be used to generate antigen-specific stem-like T cells ( Figure 5A ).…”

“…(A) Progress of T cells differentiation toward effector T (T EFF ) cell pool depends on the strength of stimulatory signals. Differentiating progress of primed T N cells can be delayed or suppressed by targeted inhibitors (such as mTOR, GSK3β, MEK, AKT inhibitor); or by using cytokines, such as interleukin‐21 (IL-21), interleukin-7 (IL-7), interleukin-15 (IL-15), or by using proper costimulatory signal; or by taming metabolic modulators; or by curtailing TCR signaling ( 16 , 21 , 109 , 201 – 204 ). (B) Direct reprogramming methods using naïve- or stem-associated factors or miRNAs ( 205 , 206 ).…”

Single-cell level temporal profiling of tumour-reactive T cells under immune checkpoint blockade

“…The apparent short-life of KLRG1 hi PD-1 int CD8 T cells by Tocky analysis is compatible with the evidence that KLRG1 is a marker for short-lived effector CD8 T cells in viral infections 46 and that effector CD8+ T cells acquire KLRG1 expression when infiltrating tissues 47 . In cancer, the combination of aOX40 and anti-4-1BB increases KLRG1 hi PD-1 int CD8 T cells in M38bearing mice 48 . On the other hand, the high frequency of KLRG1 lo PD-1 hi CD8 T cells at the Persistent locus implies that these T cells are found in both tdLN and tumour, actively interacting with antigen recognition presenting cells.…”

Immune Checkpoint Proteins, Metabolism and Adhesion Molecules: Overlooked Determinants of CAR T-Cell Migration?

“…In this regard, data have been reported showing the presence of specific niches within tumors for several subsets of T cells attracted by specific cues [ 27 , 28 ]. Transcriptomic analysis performed on T cells purified from tumors indicates that TSCM cells express a motility-associated gene expression suggesting active migration [ 29 ]. However, that does not necessarily mean that exhausted T cells are defective in their ability to migrate and reach cancer cells.…”