Introduction: Sarcoidosis is a granulomatous disease of unknown etiology that primarily affects the lungs and is associated with T helper (Th)1 and Th17 inflammation. We previously found decreased expression of the co-inhibitory marker cytotoxic T-lymphocyte antigen 4 (CTLA-4) in regulatory T cells (Tregs) in lung-draining lymph nodes (LN) of patients. CTLA-4 expression is essential for Treg suppressive function. However, whether reduced CTLA-4 expression in Tregs contributes to granulomatous inflammation remains unclear.
Objectives: This study examined the role of Treg-specific CTLA-4 haploinsufficiency on immune activation and granuloma formation in vivo.
Materials and Methods: Using the Cre–LoxP system, we generated Treg-specific CTLA-4 haploinsufficient mice. Immune phenotyping was conducted using flow cytometry, and transcriptomic profiles were generated from sorted Tregs and CCR6⁺ T helper (Th17 lineage) cells. Lung granulomas were induced using 6,6‑trehalose dimycolate (TDM) and evaluated by histology and flow cytometry.
Results: Treg-specific CTLA-4 haploinsufficiency resulted in increased Treg proliferation and activation, reflected by elevated Ki67 and ICOS expression respectively, without changes in cytokine production (Figure 1A-C). In mesenteric LN, total CD4⁺ Th cell numbers were increased. Among these, CCR6⁺ Th cells showed the strongest activation, characterized by increased expression of ICOS and Ki67 and reduced expression of CTLA-4 (Figure 1D). Transcriptomic profiling of sorted Tregs revealed signatures consistent with enhanced IFN‑y responsiveness, reduced Treg stability, and impaired suppressive function, while CCR6⁺ Th cells displayed a shift toward a pro‑inflammatory Th17.1‑like profile (Figure 2A). Reduced CTLA-4 expression in Tregs and Th17 cells, together with a shift towards a Th17.1 phenotype resembled findings in lung draining lymph nodes in sarcoidosis patients. Nevertheless, this activated immune phenotype did not appear to enhance TDM-induced granuloma formation in the lungs of CTLA-4Foxp3-Hz mice (Figure 2B).
Conclusions: Treg-specific CTLA-4 haploinsufficiency was associated with significant changes in Treg gene expression and enhanced Th17.1 differentiation but was not sufficient to induce or enhance granuloma formation.