The response to anti–PD-1 and anti–LAG-3 checkpoint blockade is associated with regulatory T cell reprogramming

Immune checkpoint blockade (ICB) has revolutionized cancer treatment; however, many patients develop therapeutic resistance. We previously identified and validated a pretreatment peripheral blood biomarker, characterized by a high frequency of LAG-3⁺ lymphocytes, that predicts resistance in patients receiving anti-PD-1 (aPD-1) ICB. To better understand the mechanism of aPD-1 resistance, we identified murine tumor models with a high LAG-3⁺ lymphocyte frequency (LAG-3^hi), which were resistant to aPD-1 therapy, and LAG-3^lo murine tumor models that were aPD-1 sensitive, recapitulating the predictive biomarker we previously described in patients. LAG-3^hi tumor-bearing mice were sensitive to aPD-1 + anti-LAG-3 (aLAG-3) therapy, and this benefit was CD8⁺ T cell dependent. The efficacy of combination therapy was enhanced in LAG-3^hi (but not LAG-3^lo) mice with depletion of CD4⁺ T cells. Furthermore, responses to aPD-1 + aLAG-3 correlated with regulatory T cell (T_reg) phenotypic plasticity in LAG-3^hi mice, suggesting a specific role for T_regs in response to aPD-1 + aLAG-3 treatment. Using T_reg fate-tracking <i>Foxp3^GFP-Cre-ERT2</i> × ROSA^YFP reporter mice, we demonstrated that expanded populations of unstable T_regs correlated with improved response to combination therapy in LAG-3^hi mice. Complementing these preclinical data, an increased proportion of unstable T_regs also correlated with higher response rate and improved survival after aPD-1 + aLAG-3 therapy in a cohort of patients with metastatic melanoma (<i>n</i> = 117). These data indicate that T_reg phenotypic plasticity affects aPD-1 + aLAG-3 responsiveness, which may represent a biomarker to aid patient selection and a rational therapeutic target for a subset of PD-1-refractory patients.