Lineage-specific immune checkpoint dynamics define a context-dependent regulatory framework with translational relevance in human γδ T cells

Abstract

Immune checkpoint receptors (ICRs) are widely used as markers of T-cell exhaustion, yet their interpretation remains context-dependent and is poorly described in γδ T cells. Here, we investigated the dynamics, functional impact, and differentiation-associated distribution of ICRs across human γδ and αβ T-cell subsets. Peripheral blood mononuclear cells from healthy donors were stimulated in vitro to assess ICR dynamics in γδ and αβ T-cell subsets by flow cytometry. Functional effects of PD-1 and TIM-3 blockade were evaluated through proliferation, cytokine production, and degranulation assays. In parallel, previously published single-cell RNA-sequencing datasets of tumor-infiltrating γδ and CD8 T cells were subjected to secondary bioinformatic analysis to characterize differentiation-associated ICR expression patterns and their modulation following immune checkpoint blockade (ICB) therapy. ICR expression was regulated in a receptor-, lineage- and context-dependent manner. Polyclonal stimulation induced broad ICR upregulation, whereas phosphoantigen-driven Vδ2 T cell expansion resulted in a selective profile with sustained TIM-3 expression and transient modulation of PD-1 and TIGIT. Checkpoint distribution was structured across differentiation states, with TIGIT enriched in antigen-experienced subsets, LAG-3 and TIM-3 in naive compartments, and PD-1 broadly expressed. PD-1 blockade was associated with donor-dependent trends toward increased proliferation and cytokine production, particularly under IL-15 stimulation, whereas TIM-3 inhibition showed variable effects and combined blockade did not produce a consistent additive response. Single-cell transcriptomic analyses revealed that tumor-infiltrating γδ T cells displayed heterogeneous ICR expression across differentiation states, whereas CD8 T cells showed a more progressive pattern of checkpoint expression. Because classical exhaustion-associated transcriptional and epigenetic programs were not evaluated, these patterns neither establish nor exclude the presence of exhausted γδ T-cell subsets. ICB therapy was associated with persistence or upregulation of alternative checkpoints. These findings indicate that ICR expression in γδ T cells is dynamically shaped by activation, differentiation, and environmental context. However, ICR expression alone is insufficient to define or exclude a state of T-cell exhaustion.