Lung tumor–infiltrating T _reg have divergent transcriptional profiles and function linked to checkpoint blockade response

Abstract

Regulatory T cells (T reg ) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating T reg (TIL-T reg ) from anti–PD-1–treated and treatment-naive non–small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)–specific T reg derived from a murine tumor model. We identified 10 subsets of human TIL-T reg , most of which have high concordance with murine TIL-T reg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in T reg suppression, including LAG3 . Functionally, the OX40 hi GITR hi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-T reg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-T reg –expressing T cell receptors that are specific for TAA fully develop a distinct T H 1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 ( Tbet) , IFNG , and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific T H 1-like T reg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti–PD-1–responding tumors. These findings demonstrate that TIL-T reg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-T reg may positively contribute to antitumor responses.

Spatial proteomic analysis of a lung cancer model reveals regulatory T cells attenuate KRAS-G12C inhibitor-induced immune responses

“…7f). The Tregs in p1_C27 were highly expressed all tested markers except GITR, an immune checkpoint that was recently described to mark the most immune suppressive Treg subset in NSCLC and associated with PD-1 resistance ( 21 ). Some of the other communities expressed similar or higher levels of GITR on the Tregs ( p1_C26 , p1_C23 , p1_C6 , p1_C17 ) and showed above average expression of immune checkpoints on the CD4 + and CD8 + T cells ( p1_C7 , p1_C23 , p1_C17 , p1_C26) , more similar to the mouse T/DC community.…”

“…Yet, Chen et al similarly identified immune hubs associated with response to PD-1 blockade that were rich in stem-like (progenitor exhausted) TCF7 + PD-1 + CD8 + T cell and mregDCs, also containing Tregs ( 57 ). This indicates that a careful definition of the Treg communities, including cell states ( 21 ) and local cytokine environments, will be required in order to identify accurately predictive biomarkers for resistance to PD-1/PDL1 blockade. Spatial analysis of pre-treatment biopsies from cohorts such as ARCTIC( 58 ), MYSTIC( 59 ), and CHECKMATE 227,( 60 ) comparing anti-PDL1/PD-1 as monotherapy or in combination with anti-CTLA-4, could help to better define the Treg communities that confer resistance to blocking the PD-1/PD-L1 axis alone, yet may be responsive to the addition of Treg targeting.…”

Depletion of effector regulatory T cells drives major response to induction dual immune checkpoint blockade

“…A decrease in 4-1BB + Tregs was similarly associated with MPR to neoadjuvant CTLA-4 and PD-1 in HPV-negative HNC profiled by scRNA-seq 52 . The eTreg population, highly expressive of both GITR and OX40 , may be the Tregs subset that restricts response to PD-1 in lung cancer 53 . Our data are consistent with murine models that showed that higher CTLA-4 expression on Tregs explained their preferential depletion by CTLA4 ICB 54 , and that tumor response was dependent upon Fc competence to mediate ADCC and Treg depletion 34,35,54,55 .…”

Potential anti-tumor effects of regulatory T cells in the tumor microenvironment: a review

“…However, Tregs were discovered in an ICIs responsive mouse tumor model to acquire a Th1-like transcriptional signature upon tumor entry, i.e., upregulation of T-bet and certain pro-inflammatory disease-related genes. Homology analysis revealed the existence of a similar subpopulation in human lung cancer, which is also enriched in tumors that respond to ICIs [ 162 ]. The study found that disruption of the CARD9-BCL10-MALT1 (CBM) signaling complex in TI-Tregs from mice resulted in loss of their inhibitory function and a shift to a pro-inflammatory, IFN-γ-secreting phenotype, leading to enhanced efficacy of anti-PD-1/PD-L1 antibodies [ 163 ].…”