Engineered T Regulatory Type 1 Cells for Clinical Application

Abstract

T regulatory cells, a specialized subset of T cells, are key players in modulating antigen (Ag)-specific immune responses in vivo. Inducible T regulatory type 1 (Tr1) cells are characterized by the co-expression of CD49b and lymphocyte-activation gene 3 (LAG-3) and the ability to secrete IL-10, TGF-β, and granzyme (Gz) B, in the absence of IL-4 and IL-17. The chief mechanisms by which Tr1 cells control immune responses are secretion of IL-10 and TGF-β and killing of myeloid cells via GzB. Tr1 cells, first described in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplantation, have been proven to modulate inflammatory and effector T cell responses in several immune-mediated diseases. The possibility to generate and expand Tr1 cells in vitro in an Ag-specific manner has led to their clinical use as cell therapy in patients. Clinical grade protocols to generate or to enrich and expand Tr1 cell medicinal products have been established. Proof-of-concept clinical trials with Tr1 cell products have demonstrated the safety and the feasibility of this approach and indicated some clinical benefit. In the present review, we provide an overview on protocols established to induce/expand Tr1 cells in vitro for clinical application and on results obtained in Tr1 cell-based clinical trials. Moreover, we will discuss a recently developed protocol to efficient convert human CD4⁺ T cells into a homogeneous population of Tr1-like cells by lentiviral vector-mediated IL-10 gene transfer.

Keywords: IL-10; T regulatory cell-based therapy; T regulatory type 1 (Tr1) cells; gene transfer; tolerance.

Figure 1

T regulatory type 1 (Tr1)-mediated suppression in vivo. In steady-state condition, Tr1 cells reside in the spleen and circulate in the periphery. During inflammation, Tr1 cells are recruited to the site of tissue injury (i.e., after infections, autoimmune reactions, or transplantation) and are activated by professional antigen-presenting cells [APCs; dendritic cells (DCs)] via their T cell receptor, thus by their cognate antigen (Ag). Upon activation, Tr1 cells secrete IL-10 and TGF-β and (1) directly inhibit effector T cell (i.e., Th1 and Th17 cells) proliferation and pro-inflammatory cytokines production and (2) indirectly inhibit effector T cells by modulating professional APCs (i.e., downregulation of costimulatory and HLA class II expression and inhibition of pro-inflammatory cytokine secretion). (3) Tr1 cells can suppress effector T cells by cell-to-cell contact-mediated mechanisms, (4) suppress CD8⁺ T cell responses (i.e., proliferation and IFN-γ production), and (5) mediate bystander suppression by specifically killing professional APCs [DC or macrophages (M)], thus preventing naive T (Tn) cell priming and reactivation of effector T cells (i.e., Th1 and Th17 cells). Concomitantly, (6) Tr1 cells via IL-10 and TGF-β promote the induction of tolerogenic DC and anti-inflammatory macrophages (M2), which in turn promote de novo induction of Tr1 cells and T regulatory cells (Tregs), restoring tissue homeostasis and promoting long-term tolerance.

Figure 2

Protocols to generate/expand T regulatory type 1 (Tr1) cells. (A) Tr1-enriched cell lines. Donor-derived PBMC or CD4⁺ T cells are stimulated with host-derived monocytes in the presence of recombinant human IL-10 for 10 days. Alternatively, PBMC or CD4⁺ T cells are cultured for 10 days with allogenic dendritic cell (DC)-10, differentiated in vitro from peripheral blood monocytes with GM-CSF/IL-4/IL-10, in the presence of recombinant human IL-10 for 10 days (45). To generate T-allo10 cells, donor-derived T cells are cultured with host-derived DC-10 (Bacchetta and Roncarolo, unpublished data), whereas to induce host-derived T10 cells, T cells are stimulated with donor-derived DC-10 (46). (B) Tr1 cell clones. PBMC are stimulated with antigen (Ag; i.e., ovalbumin or collagen II) in the presence of IL-2 and IL-4 to enrich/expand Ag-specific T cells, followed by T cell cloning and expansion of the T cell clones using Schneider cells (4). (C) Tr1-like cell lines. Human CD4⁺ T cells are preactivated for 48 h with soluble anti-CD3/CD28 mAbs and IL-2 and then transduced with lentiviral vector (LV)-IL-10 overnight. Transduced T cells are isolated and expanded in feeder mixture (47). To generate allospecific IL-10-transduced cells, naive CD4⁺ T cells are stimulated with allogeneic DC and transduced with LV-IL-10 upon secondary stimulation. After selection, IL-10-trasduced cells are expanded in vitro with feeder mixture (48).