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.

PURPOSE: To determine the immunogenicity and antitumor activity of a vaccine consisting of autologous, tumor-derived heat shock protein gp96-peptide complexes (HSPPC-96, Oncophage; Antigenics, Inc, Woburn, MA) in metastatic (American Joint Committee on Cancer stage IV) melanoma patients.PATIENTS AND METHODS: Sixty-four patients had surgical resection of metastatic tissue required for vaccine production, 42 patients were able to receive the vaccine, and 39 were assessable after one cycle of vaccination (four weekly injections). In 21 patients, a second cycle (four biweekly injections) was given because no progression occurred. Antigen-specific antimelanoma T-cell response was assessed by enzyme-linked immunospot (ELISPOT) assay on peripheral blood mononuclear cells (PBMCs) obtained before and after vaccination. Immunohistochemical analyses of tumor tissues were also performed.RESULTS: No treatment-related toxicity was observed. Of 28 patients with measurable disease, two had a complete response (CR) and three had stable disease (SD) at the end of follow-up. Duration of CR was 559+ and 703+ days, whereas SD lasted for 153, 191, and 272 days, respectively. ELISPOT assay with PBMCs of 23 subjects showed a significantly increased number of postvaccination melanoma-specific T-cell spots in 11 patients, with clinical responders displaying a high frequency of increased T-cell activity. Immunohistochemical staining of melanoma tissues from which vaccine was produced revealed high expression of both HLA class I and melanoma antigens in seven of eight clinical responders (two with CR, three with SD, and the three with long-term disease-free survival) and in four of 12 nonresponders.CONCLUSION: Vaccination of metastatic melanoma patients with autologous HSPPC-96 is feasible and devoid of significant toxicity. This vaccine induced clinical and tumor-specific T-cell responses in a significant minority of patients.