Cortisol-Driven CD8+ T Cell Dysfunction in Colorectal Cancer and Engineering Glucocorticoid-Resistant CAR-T Cell Therapies

Abstract

Glucocorticoids (GCs) are key regulators of T-cell fate, yet their role in cancer immunity remains incompletely understood. In particular, how the endogenous GC cortisol shapes human CD8⁺ T cells has remained unclear. This work shows that sustained cortisol exposure shifts glucocorticoid receptor (GR) binding away from canonical response elements toward RUNX motif–enriched sites, consistent with non-canonical, RUNX3-tethered programmes that drive dysfunction. Chromatin and transcriptomic analyses revealed induction of non-classical immunoregulatory genes such as SNX9, AREG and PIK3IP1, uncovering novel mechanisms of cortisol-driven suppression. In colorectal cancer (CRC), cortisol reactivation rather than de novo synthesis underpins steroid availability in the tumour microenvironment. Steroid profiling revealed systemic suppression of GC levels in murine models, while in human CRC, analysis showed impaired cortisol inactivation and accumulation of 20β dihydrocortisol (20β-DHF) in female tumours. Single-cell RNA-sequencing (scRNA-seq) identified HSD11B1 upregulation in cancer-associated fibroblasts (CAFs) from female mismatch repair–deficient (MMRd) tumours, driving local cortisol regeneration. CBR1, which converts cortisol into 20β-DHF, was broadly expressed across cell types and further elevated in female MMRd tumours. Consistent with these findings, survival analysis of large CRC cohorts showed that high HSD11B1 expression predicted poor outcomes in female patients, whereas the GC-inactivating enzyme HSD11B2 was protective. To overcome this immunosuppressive barrier, the non-viral Sleeping Beauty (SB) platform was optimised to generate CEACAM5-targeted CAR-T cells efficiently. This refinement addressed the long-standing inefficiency of SB in human T cells, resulting in robust CAR expression and a favourable stem-like memory T cell (TSCM) phenotype without inducing exhaustion. On this foundation, CRISPR-mediated deletion of GR (NR3C1) conferred resistance to cortisol, preserving cytotoxicity and cytokine production under steroid pressure while preventing CAR loss, NK-like transition and exhaustion. Together, these findings redefine GC control of CD8⁺ T cells as a RUNX3-tethered programme under sustained cortisol exposure, identify a sex-specific CAF-driven niche of GC reactivation in CRC and establish a translational framework for steroid-resistant CAR-T therapy in solid tumours.