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Background Interleukin-2 (IL-2) has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs). Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs) to prevent autoimmune diseases, such as type 1 diabetes (T1D). Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target. Aldesleukin (Proleukin; recombinant human IL-2), which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs. Methods and Findings To define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D), a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low) from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39) found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = −0.052, 0.254) and 0.497 × 106 IU/m2 (SE = 0.092, 95% CI = 0.316, 0.678), respectively. On analysis of secondary outcomes, using a highly sensitive IL-2 assay, the observed plasma concentrations of the drug at 90 min exceeded the hypothetical Treg-specific therapeutic window determined in vitro (0.015–0.24 IU/ml), even at the lowest doses (0.040 × 106 and 0.045 × 106 IU/m2) administered. A rapid decrease in Treg frequency in the circulation was observed at 90 min and at day 1, which was dose dependent (mean decrease 11.6%, SE = 2.3%, range 10.0%–48.2%, n = 37), rebounding at day 2 and increasing to frequencies above baseline over 7 d. Teffs, natural killer cells, and eosinophils also responded, with their frequencies rapidly and dose-dependently decreased in the blood, then returning to, or exceeding, pretreatment levels. Furthermore, there was a dose-dependent down modulation of one of the two signalling subunits of the IL-2 receptor, the β chain (CD122) (mean decrease = 58.0%, SE = 2.8%, range 9.8%–85.5%, n = 33), on Tregs and a reduction in their sensitivity to aldesleukin at 90 min and day 1 and 2 post-treatment. Due to blood volume requirements as well as ethical and practical considerations, the study was limited to adults and to analysis of peripheral blood only. Conclusions The DILT1D trial results, most notably the early altered trafficking and desensitisation of Tregs induced by a single ultra-low dose of aldesleukin that resolves within 2–3 d, inform the design of the next trial to determine a repeat dosing regimen aimed at establishing a steady-state Treg frequency increase of 20%–50%, with the eventual goal of preventing T1D. Trial Registration ISRCTN Registry ISRCTN27852285; ClinicalTrials.gov NCT01827735, Frank Waldron-Lynch and colleagues investigate immune cell biomarker responses in patients with type 1 diabetes following a single dose of recombinant interleukin-2., Author Summary Why Was This Study Done? Insulin replacement, the only approved therapy for the treatment of type 1 diabetes (T1D), treats the symptoms but not the underlying cause of the disease, namely, immune-mediated destruction of the insulin-producing β cells of the pancreas. Aldesleukin, recombinant interleukin-2 (IL-2), was selected as a candidate immunotherapy to prevent or delay autoimmunity because previous genetic and phenotypic analyses indicate a major role for the IL-2 pathway in the development of T1D. IL-2 is critical for maintaining the function of the regulatory T cells (Tregs) that prevent autoimmunity. Therefore, the first step in our approach to T1D prevention was to determine the single doses of aldesleukin that increase Treg numbers within the physiological range, aiming to mimic the protection against T1D afforded by the risk-reducing alleles of the IL-2 pathway. What Did the Researchers Do and Find? We successfully employed a state-of-the-art dose-finding, open label, adaptive clinical trial design in 40 participants with T1D to determine the doses of aldesleukin needed to raise Treg frequencies by 10% and 20%. Increased Treg frequencies induced by aldesleukin were preceded by a peak of aldesleukin in the blood at 90 minutes that caused a transient dose-dependent decrease of Tregs and other cell subsets in the circulation. Following treatment with a dose of aldesleukin, Tregs had a decreased sensitivity to IL-2 that returned to baseline on day 3 after treatment. What Do These Findings Mean? The DILT1D trial showed that it is possible to conduct an adaptive dose-finding trial to estimate two drug doses based on an immune cell biomarker as a primary outcome. Partial desensitisation of Tregs might help explain why some participants in previous trials with daily dosing regimens did not respond to aldesleukin, suggesting that in future trials the drug should not be administered on a daily basis.