The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

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.

The function of regulatory T cells (T reg cells) has been attributed to a growing number of diverse pathways, molecules and processes. Seemingly contradictory conclusions regarding the mechanisms underlying T reg cell suppressive activity have revitalized skeptics in the field who challenge the core validity of the idea of T reg cells as central immune regulators. However, we note that a consensus may be emerging from the data: that multiple T reg cell functions act either directly or indirectly at the site of antigen presentation to create a regulatory milieu that promotes bystander suppression and infectious tolerance. Thus, the versatility and adaptability of the Foxp3 + T reg cells may in fact be the best argument that these cells are 'multitalented masters of immune regulation'.Armed with the potential to destroy invading microorganisms and stop aberrant outgrowth of tumor cells, the immune system has extensive built-in mechanisms for preventing attack of healthy self tissues. The first line of such 'self-tolerance' is the elimination of selfreactive T lymphocytes and B lymphocytes during negative selection in the thymus and bone marrow, respectively. However, there has long been a belief that the immune system must have peripheral mechanisms in place to deal with immune cells that 'escape' central tolerance. For almost 40 years, immunologists have postulated the existence of suppressor T cells that police the immune system to avert unwanted immune responses 1-3 . However, that phenomenology was cast into doubt as various labs presented unique, hard-to-reproduce systems, each with complexities and idiosyncrasies that raised credibility issues. This situation was not unlike the early years of cytokine biology, during which dozens of activities were found in sera and cell supernatants without consistent molecular or biochemical 'signatures'. Fortunately, as biochemistry and the molecular biology revolution rescued the field of cytokine biology by identifying genes and biochemical structures tied to the varied biologic activities, the identification of a constellation of cell surface, transcriptional and biochemical markers that uniquely mark 'regulatory' T cells (T reg cells) has made possible a rebirth of the suppressor T cell field over the past decade.The realization that T reg cells have a unique surface expression profile incorporating CD25, CD62L and specific CD45 isoforms 4-6 , together with the identification of the T reg cellspecific transcription factor Foxp3, catapulted T reg cells from a rare CD4 + T cell subset to

The dynamics of CD4(+) effector T cells (Teff cells) and CD4(+)Foxp3(+) regulatory T cells (Treg cells) during diabetes progression in nonobese diabetic mice was investigated to determine whether an imbalance of Treg cells and Teff cells contributes to the development of type 1 diabetes. Our results demonstrated a progressive decrease in the Treg cell:Teff cell ratio in inflamed islets but not in pancreatic lymph nodes. Intra-islet Treg cells expressed reduced amounts of CD25 and Bcl-2, suggesting that their decline was due to increased apoptosis. Additionally, administration of low-dose interleukin-2 (IL-2) promoted Treg cell survival and protected mice from developing diabetes. Together, these results suggest intra-islet Treg cell dysfunction secondary to defective IL-2 production is a root cause of the progressive breakdown of self-tolerance and the development of diabetes in nonobese diabetic mice.

The in vivo mechanism of regulatory T cell (T(reg) cell) function in controlling autoimmunity remains controversial. Here we have used two-photon laser-scanning microscopy to analyze lymph node priming of diabetogenic T cells and to delineate the mechanisms of T(reg) cell control of autoimmunity in vivo. Islet antigen-specific CD4(+)CD25(-) T helper cells (T(H) cells) and T(reg) cells swarmed and arrested in the presence of autoantigens. These T(H) cell activities were progressively inhibited in the presence of increasing numbers of T(reg) cells. There were no detectable stable associations between T(reg) and T(H) cells during active suppression. In contrast, T(reg) cells directly interacted with dendritic cells bearing islet antigen. Such persistent T(reg) cell-dendritic cell contacts preceded the inhibition of T(H) cell activation by dendritic cells, supporting the idea that dendritic cells are central to T(reg) cell function in vivo.