Michela Perego

Human natural regulatory CD4(+) T cells comprise 5-10% of peripheral CD4(+)T cells. They constitutively express the IL-2Ralpha-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(high)Foxp3(+) T cells. This CD4(+)CD25(high)Foxp3(+)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(high)Foxp3(+)LAG-3(+) T cells are functionally active cells that release the immunosuppressive cytokines IL-10 and TGF-beta1, but not IL-2. An in vitro suppression assay using CD4(+)CD25(high)LAG-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(high)Foxp3(+) regulatory T cell subset whose frequency is enhanced in the PBMCs of patients with cancer and is expanded at tumor sites.

Tumor recurrence years after seemingly successful treatment of primary tumors is one of the major causes of mortality in patients with cancer. Reactivation of dormant tumor cells is largely responsible for this phenomenon. Using dormancy models of lung and ovarian cancer, we found a specific mechanism, mediated by stress and neutrophils, that may govern this process. Stress hormones cause rapid release of proinflammatory S100A8/A9 proteins by neutrophils. S100A8/A9 induce activation of myeloperoxidase, resulting in accumulation of oxidized lipids in these cells. Upon release from neutrophils, these lipids up-regulate the fibroblast growth factor pathway in tumor cells, causing tumor cell exit from the dormancy and formation of new tumor lesions. Higher serum concentrations of S100A8/A9 were associated with shorter time to recurrence in patients with lung cancer after complete tumor resection. Targeting of S100A8/A9 or β2-adrenergic receptors abrogated stress-induced reactivation of dormant tumor cells. These observations demonstrate a mechanism linking stress and specific neutrophil activation with early recurrence in cancer.

Abstract Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34 + cells and implanting autologous thymus in immune-deficient NOD- scid IL2Rγ null (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3 + Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8 + /Granz B + T cells homeostasis are observed in tumor regions where FOXP3 + Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.