T-Cell-Mediated Suppression: From Bench to Bedside
Authors: Roncarolo Maria Grazia , Battaglia Manuela , Bacchetta Rosa , Levings Megan , Gregori Silvia 
Published: October 2019

Abstract
T regulatory cells (Tregs) play a pivotal role in promoting and maintaining tolerance. Several subsets of Tregs have been identified but, to date, the best characterized are the CD4+FOXP3+ Tregs (FOXP3+Tregs), thymic-derived or induced in the periphery, and the CD4+ IL-10-producing T regulatory type 1 (Tr1) cells. In the past decade much effort has been dedicated to develop methods for the in vitro induction and expansion of FOXP3+Tregs and of Tr1 cells for Treg-based cell therapy to promote and restore tolerance in T-cell mediated diseases, and for expanding antigen (Ag)-specific Tregs in vivo. FOXP3+Tregs constitutively express high levels of CD25 and of the transcription factor FOXP3. FOXP3+Tregs are distinguished from activated CD4+ T cells by the low expression of CD127, and by the DNA demethylation of a specific region of the FOXP3 gene called Treg-specific demethylated region (TSDR). FOXP3+Tregs suppress effector T-cell responses through cell-to-cell contact-dependent mechanisms and suppression requires activation via TCR and is IL-2 dependent. In vitro protocols to expand FOXP3+Tregs for adoptive transfer in vivo have been established. We demonstrated that rapamycin permits the in vitro expansion of FOXP3+Tregs while impairing the proliferation of non-Tregs. Moreover, rapamycin-expanded FOXP3+Tregs maintain their regulatory phenotype in a proinflammatory environment and Th17 cells do not expand in the presence of rapamycin. Despite the progress in FOXP3+Tregs expansion protocols, adoptive transfer of FOXP3+Tregs in humans remains a difficult experimental procedure due to the ability to expand a sufficient number of Ag-specific FOXP3+Tregs in vitro. To propagate a homogenous population of FOXP3+Tregs we developed a lentiviral vector (LV)-based strategy to ectopically express FOXP3 in CD4+ T cells. This method results in the development of suppressive cells that are super-imposable to FOXP3+Tregs. Conversion of effector T cells into FOXP3+Tregs upon LV-mediated gene transfer of wild-type FOXP3 was also obtained in CD4+T cells from immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. We also developed a LV platform, which selectively targets expression of the transgene in hepatocytes, to induce tolerance to self or exogenous Ags. Using this approach we showed that systemic administration of LV encoding for the gene of interest leads to the induction of Ag-specific FOXP3+ Tregs, which mediate tolerance even in pre-immunized hosts. Tr1 cells are identified by their cytokine profile (IL-10+TGF-b+IL-4-IL-17-). Tr1 cells express transiently FOXP3 upon activation; but FOXP3 expression never reaches the high levels characteristic of FOXP3+Tregs. Tr1 cell differentiation and function is independent of FOXP3 since suppressive Tr1 cells can be isolated or generated from peripheral blood of IPEX patients. Tr1 cells were first discovered in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplant (HSCT). Since their discovery, Tr1 cells have proven to be important in mediating tolerance in several immune-mediated diseases. The immuno-regulatory mechanisms of Tr1 cells have been studied over the years thanks to the possibility to generate these cells in vitro. Tr1 cells suppress T-cell responses via the secretion of IL-10 and TGF-β and by the specific killing of myeloid APC via Granzyme B and perforin. Tr1 cells can be induced in vitro in an Ag-specific manner in the presence of IL-10 or of DC-10. Proof-of-principle clinical trials in allogeneic HSCT demonstrated the safety of Treg-based cell therapy with these polarized Tr1 cells. We are currently planning a phase I/II trial using in vitro polarized Tr1 cells with DC-10 in patients after kidney transplantation. An alternative strategy for the induction of high numbers of human Tr1 cells is the LV-mediated gene transfer of human IL-10 into conventional CD4+ T cells. Stable ectopic expression of IL-10 leads to the differentiation of homogeneous populations of Tr1-like cells displaying potent suppressive functions both in vitro and in vivo. A major hurdle, which limited the studies and the clinical use of Tr1 cells, was the lack of specific biomarkers. By gene expression profiling of human Tr1 cell clones we identified two surface markers (CD49b and LAG-3), which are stably and selectively co-expressed on murine and human Tr1 cells induced in vitro or in vivo. The co-expression of CD49b and LAG-3 enables the isolation of highly suppressive Tr1 cells from in vitro IL-10-polarized Tr1 cells and allows tracking of Tr1 cells in peripheral blood of patients who developed tolerance after allogeneic HSCT. The identification of CD49b and LAG-3 as Tr1-specific biomarkers will facilitate the study of Tr1 cells in vivo in healthy and pathological conditions and the use of Tr1 cells for forthcoming therapeutic interventions. In conclusion, Tregs play a key role in maintaining immunological homeostasis in the periphery. Several open questions regarding FOXP3+ Tregs or Tr1 cell-based therapy in humans remain: how long do Tregs survive after transfer? Is their phenotype stable in pathological conditions and inflammatory environments? Is their mechanism of suppression in vivo Ag-specific? Carefully designed and standardized future clinical protocols reflecting a concerted action among different investigators will help to address these questions and to advance the field.

Disclosures:
No relevant conflicts of interest to declare.