Regulatory T (T reg) cells are critical regulators of immune tolerance. Most T reg cells are defined based on expression of CD4, CD25, and the transcription factor, FoxP3. However, these markers have proven problematic for uniquely defining this specialized T cell subset in humans. We found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4+ T cells in peripheral blood. We demonstrate that the majority of these cells are FoxP3+, including those that express low levels or no CD25. A combination of CD4, CD25, and CD127 resulted in a highly purified population of T reg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact, cells separated based solely on CD4 and CD127 expression were anergic and, although representing at least three times the number of cells (including both CD25+CD4+ and CD25−CD4+ T cell subsets), were as suppressive as the “classic” CD4+CD25hi T reg cell subset. Finally, we show that CD127 can be used to quantitate T reg cell subsets in individuals with type 1 diabetes supporting the use of CD127 as a biomarker for human T reg cells.

Type 1 diabetes (T1D) is an autoimmune disease that occurs in genetically susceptible individuals. Regulatory T cells (Tregs) have been shown to be defective in the autoimmune disease setting. Thus, efforts to repair or replace Tregs in T1D may reverse autoimmunity and protect the remaining insulin-producing β cells. On the basis of this premise, a robust technique has been developed to isolate and expand Tregs from patients with T1D. The expanded Tregs retained their T cell receptor diversity and demonstrated enhanced functional activity. We report on a phase 1 trial to assess safety of Treg adoptive immunotherapy in T1D. Fourteen adult subjects with T1D, in four dosing cohorts, received ex vivo–expanded autologous CD4+CD127lo/−CD25+ polyclonal Tregs (0.05 × 108 to 26 × 108 cells). A subset of the adoptively transferred Tregs was long-lived, with up to 25% of the peak level remaining in the circulation at 1 year after transfer. Immune studies showed transient increases in Tregs in recipients and retained a broad Treg FOXP3+CD4+CD25hiCD127lo phenotype long-term. There were no infusion reactions or cell therapy–related high-grade adverse events. C-peptide levels persisted out to 2+ years after transfer in several individuals. These results support the development of a phase 2 trial to test efficacy of the Treg therapy.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes using recombinant viral vectors, which do not target transgenes to specific genomic sites. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Regulatory T cells (Tregs) constitute an attractive therapeutic target given their essential role in controlling autoimmunity. However, recent animal studies provide evidence for functional heterogeneity and lineage plasticity within the Treg compartment. To understand better the plasticity of human Tregs in the context of type 1 diabetes, we characterized an IFN-γ–competent subset of human CD4+CD127lo/−CD25+ Tregs. We measured the frequency of Tregs in the peripheral blood of patients with type 1 diabetes by epigenetic analysis of the Treg-specific demethylated region (TSDR) and the frequency of the IFN-γ+ subset by flow cytometry. Purified IFN-γ+ Tregs were assessed for suppressive function, degree of TSDR demethylation, and expression of Treg lineage markers FOXP3 and Helios. The frequency of Tregs in peripheral blood was comparable but the FOXP3+IFN-γ+ fraction was significantly increased in patients with type 1 diabetes compared to healthy controls. Purified IFN-γ+ Tregs expressed FOXP3 and possessed suppressive activity but lacked Helios expression and were predominately methylated at the TSDR, characteristics of an adaptive Treg. Naive Tregs were capable of upregulating expression of Th1-associated T-bet, CXCR3, and IFN-γ in response to IL-12. Notably, naive, thymic-derived natural Tregs also demonstrated the capacity for Th1 differentiation without concomitant loss of Helios expression or TSDR demethylation.

OBJECTIVE— Regulatory T-cells (Tregs) have catalyzed the field of immune regulation. However, translating Treg-based therapies from animal models of autoimmunity to human clinical trials requires robust methods for the isolation and expansion of these cells—a need forming the basis for these studies. RESEARCH DESIGN AND METHODS— Tregs from recent-onset type 1 diabetic patients and healthy control subjects were isolated by fluorescence-activated cell sorting and compared for their capacity to expand in vitro in response to anti-CD3–anti-CD28–coated microbeads and IL-2. Expanded cells were examined for suppressive function, lineage markers and FOXP3, and cytokine production. RESULTS— Both CD4 + CD127 lo/− and CD4 + CD127 lo/− CD25 + T-cells could be expanded and used as Tregs. However, expansion of CD4 + CD127 lo/− cells required the addition of rapamycin to maintain lineage purity. In contrast, expansion of CD4 + CD127 lo/− CD25 + T-cells, especially the CD45RA + subset, resulted in high yield, functional Tregs that maintained higher FOXP3 expression in the absence of rapamycin. Tregs from type 1 diabetic patients and control subjects expanded similarly and were equally capable of suppressing T-cell proliferation. Regulatory cytokines were produced by Tregs after culture; however, a portion of FOXP3 + cells were capable of producing interferon (IFN)-γ after reactivation. IFN-γ production was observed from both CD45RO + and CD45RA + Treg populations. CONCLUSIONS— The results support the feasibility of isolating Tregs for in vitro expansion. Based on expansion capacity, FOXP3 stability, and functional properties, the CD4 + CD127 lo/− CD25 + T-cells represent a viable cell population for cellular therapy in this autoimmune disease.