LAG-3 expression defines a subset of CD4+CD25 highFoxp3+ regulatory T cells that are expanded at tumor sites

Human natural regulatory CD4⁺ T cells comprise 5-10% of peripheral CD4⁺T cells. They constitutively express the IL-2Rα - chain (CD25) and the nuclear transcription Foxp3. These cells are heterogeneous and contain discrete subsets with distinct phenotypes and functions. Studies in mice report that LAG-3 has a complex role in T cell homeostasis and is expressed in CD4⁺CD25⁺ T regulatory cells. In this study, we explored the expression of LAG-3 in human CD4 ⁺ T cells and found that LAG-3 identifies a discrete subset of CD4⁺CD25^highFoxp3⁺ T cells. This CD4 ⁺CD25^highFoxp3⁺LAG-3⁺ population is preferentially expanded in the PBMCs of patients with cancer, in lymphocytes of tumor-invaded lymph nodes and in lymphocytes infiltrating visceral metastasis. Ex vivo analysis showed that CD4⁺CD25^highFoxp3 ⁺LAG-3⁺ T cells are functionally active cells that release the immunosuppressive cytokines IL-10 and TGF-β1, but not IL-2. An in vitro suppression assay using CD4⁺CD25^highLAG-3 ⁺ T cells sorted from in vitro expanded CD4⁺CD25 ^high regulatory T cells showed that this subset of cells is endowed with potent suppressor activity that requires cell-to-cell contact. Our data show that LAG-3 defines an active CD4⁺CD25^highFoxp3 ⁺ regulatory T cell subset whose frequency is enhanced in the PBMCs of patients with cancer and is expanded at tumor sites.