Phenotypes of the residual circulating regulatory T cells at day 1.

<p>(A and B) CD25 expression was increased on mTregs (average baseline CD25 MFI on mTreg = 7,412, SE = 181, range 5,119–9,393, <i>n</i> = 37). (C and D) Concurrently, there was a dose-dependent reduction in CD122 on mTregs in blood (baseline CD122 MFI on mTreg = 444.2, SE = 14.0, range 288.0–616.0, <i>n</i> = 33). (E) There was a reduction in pSTAT5 levels in mTregs incubated with a saturating concentration of aldesleukin (1,000 IU/ml) in vitro when assessing blood obtained 90 min after dosing of aldesleukin. (F) At day 1 post-dosing, there was a dose-dependent reduction in the percentage of mTregs that were pSTAT5⁺ following incubation with 0.4 IU/ml aldesleukin in vitro (percent of pretreatment time point mTregs that were pSTAT5⁺ following aldesleukin incubation: 56.25%, SE = 1.60%, range 43.23%–71.03%, <i>n</i> = 22). (G) There was a reduction in pSTAT5 levels in nTregs assessed 90 min post-dosing when the cells were incubated with a saturating dose of aldesleukin (1,000 IU/ml) in vitro. (H) At day 1 post-dosing, there was not a consistent change from baseline in the percentage of nTregs that were pSTAT5⁺ following incubation with 0.4 IU/ml aldesleukin in vitro (baseline percent of nTregs that were pSTAT5⁺ following incubation with 0.4 IU/ml aldesleukin: 58.01%, SE = 1.65%, range 40.83%–69.88%, <i>n</i> = 21). (A) and (C) show averaged response plots across the five dose groups. (B), (D), and (E) show the best fitted models with 95% CIs. MFI, mean fluorescence intensity; mTreg, memory regulatory T cell; nTreg, naïve regulatory T cell.</p

Regulatory T cell primary endpoint.

<p>(A) Percentage of Tregs was defined as the percentage of CD3⁺CD4⁺CD25^highCD127^low cells within the CD3⁺CD4⁺ gate measured. (B) Individual participant dose allocations and dose groups showing convergence of the study to doses that achieve the two defined Treg targets. (C) A cubic model described the Treg dose response to aldesleukin best, with dashed lines showing the 10% and 20% Treg targets and doses. The shaded areas represent 95% CIs. Baseline, or pretreatment, Treg (percent of CD4⁺ T cells): 6.60% (SE = 0.25%, range 3.50%–10.70%, <i>n</i> = 39). SSC-A, side-scattered light-A; Treg, regulatory T cell.</p

Lymphocyte responses to a dose of aldesleukin.

<p>(A) Average response curves of the absolute change in lymphocyte count across the five dose groups (average baseline lymphocyte count 1.78 × 10⁹/l, SE = 0.08, range 0.95–3.84, <i>n</i> = 39). (B) Three-dimensional plot of dose, baseline lymphocyte count, and change in lymphocyte count on day 1, with lines representing the vertical projections of points (coloured by dose) on the dose/baseline lymphocyte count axis. The surface grid represents the regression model for change in lymphocyte count on day 1 (colour scale), showing that the decrease in lymphocytes depends both on dose and pretreatment count.</p

Effects of aldesleukin on NK CD56^bright cell number, phenotypes, and proliferation.

<p>(A and B) NK CD56^bright cells showed a rapid dose-dependent decline, with the majority of cells not in circulation at 90 min (NK CD56^bright cells percent of lymphocytes: 0.41%, SE = 0.03%, range 0.09%–0.96%, <i>n</i> = 38). (C) Concurrent with this decline is a dose-dependent increase in NK CD56^bright cell pSTAT5 levels (baseline pSTAT5 MFI = 16.55, SE = 0.70, range 9.51–27.87, <i>n</i> = 37). (D and E) There was a sustained dose-dependent reduction in expression of CD25 (MFI = 642, SE = 32, range 255–1,148, <i>n</i> = 38) on NK CD56^bright cells and (F) a transient reduction in CD122 at 90 min (G) followed by a linear dose-dependent increase on day 1 (baseline CD122 MFI = 6,605, SE = 213, range 3,786–9,554, <i>n</i> = 38). (H) The outcome of treatment was increased proliferation of NK CD56^bright cells (baseline percentage of Ki-67⁺ NK CD56^bright cells = 9.9%, SE = 0.9%, range 3.35%–25.9%, <i>n</i> = 30). MFI, mean fluorescence intensity; NK, natural killer.</p

Eosinophil response depends on baseline counts and aldesleukin dose.

<p>(A) Eosinophil counts showed an initial transient decrease at 90 min in a hyperacute response to aldesleukin followed by a dose-dependent increase on day 1, with a return to baseline by day 3–4 (average baseline eosinophil count 0.15 × 10⁹/l, SE = 0.03, range 0.04–0.86, <i>n</i> = 39). (B) Three-dimensional plot of dose, baseline eosinophil count, and change in eosinophil count on day 1, with lines representing the vertical projections of points (coloured by dose) on the dose/baseline eosinophil count axis. The change in eosinophil count is affected by both dose and baseline eosinophil count using a linear dose-response model, with the grid showing the regression model (colour scale) for increase in eosinophils on day 1 (colour scale) (absolute change in eosinophil count on day 1 = −0.0058 + [0.0693 × dose] + [0.1748 × baseline]).</p

Summary of non-serious adverse events and reactions (safety population).

<p>Summary of non-serious adverse events and reactions (safety population).</p

DILT1D study profile and adaptive design.

<p>(A) Flow chart showing the allocation of participants to the three predefined study populations: evaluable, safety, and analysis. (B) The study was conducted in two phases, a learning phase (140 d) and an adaptive phase (240 d). Individual participants are represented by a horizontal line, the length corresponding to the time from treatment until their final visit. In the learning phase, the first ten participants received a 0.04 × 10⁶, 0.16 × 10⁶, 0.60 × 10⁶, 1.00 × 10⁶, or 1.50 × 10⁶ IU/m² dose of aldesleukin (colour represents dose allocated) in ascending order, with each dose being administered to two participants before escalation. In the adaptive phase, the DDC (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1002139#pmed.1002139.s030" target="_blank">S3 Materials</a>) met on 19 occasions to review the interim safety data and allocate doses based on the analysis of the accumulated Treg data (shaded area) from all treated participants at that time. (C) Schematic of the study design illustrating that each participant who passed screening was administered a single dose of aldesleukin and followed for 60 d. During the adaptive phase, Treg data for every participant up to day 7 was included in an interim analysis and then further doses were allocated to the next participants. Treg, regulatory T cell; DDC, Dose Determining Committee.</p

Hyperacute regulatory T cell response to aldesleukin.

<p>(A) Treg proportions as a percent of CD4⁺ T cells (average Treg level 6.6%, SE = 0.2%, range 3.5%–10.7%, <i>n</i> = 39) and (B) Treg counts following injection of aldesleukin as measured by the clinical grade FACS assay in conjunction with the BD Multitest TBNK assay are shown (average baseline Treg count 0.06 × 10⁹/l, SE = 0.01, range 0.02–0.14, <i>n</i> = 39). (C) Tregs as a percent of CD4⁺ T cells were measured in the mechanistic FACS assay (average Treg level 6.99%, SE = 0.27%, range 3.93%–10.74%, <i>n</i> = 37). (D) The decline of Tregs in blood on day 1 fits a cubic model (shaded area presents the 95% CI, <i>n</i> = 37). (E) Plasma IL-2 levels following aldesleukin dosing. The dotted grey lines mark the 0.015 and 0.24 IU/ml concentrations that are the threshold levels of aldesleukin at which Tregs, and Teffs and NK CD56^bright cells, respond, respectively. (F) Relationship between the dose of aldesleukin administered and the plasma concentration of IL-2 at day 1 in vivo. (G) Aldesleukin dose-response curves generated in whole blood from DILT1D participants for pSTAT5 responses within individual cell populations on day 60 post-treatment (mean with bars showing 95% CI, <i>n</i> = 39). (A–C) show averaged response plots across the five dose groups. (D) and (F) show the best fitted models with 95% CI. Teff, effector T cell; Treg, regulatory T cell.</p

In vivo regulatory T cell phenotypes and functional responses to aldesleukin.

<p>(A and B) mTregs had their maximum pSTAT5 response to treatment at 90 min, and a detectable response was sustained for up to 4 d at the higher doses and was dose dependent on day 1 with a cubic dose response (average baseline pSTAT5 MFI = 7.36, SE = 0.33, range 4.64–12.53, <i>n</i> = 36). (C–F) Following activation, mTregs had a dose-dependent increase in CTLA-4 and FOXP3 expression, returning to baseline by day 3–4 post-dosing (mTreg CTLA-4 MFI = 1,539, SE = 65, range 877–2,411, <i>n</i> = 32; and mTreg FOXP3 MFI = 1,174, SE = 68, range 580–2,009, <i>n</i> = 34). (G) Concurrent with these changes on day 1, there was an increase in proliferation of mTregs in blood (baseline Ki-67⁺ mTreg = 15.27%, SE = 0.86%, range 7.10%–30.20%, <i>n</i> = 33). (H) Intracellular staining of Tregs from whole blood for FOXP3 showed an increase in FOXP3⁺ Tregs on day 3 (FOXP3⁺ Tregs/CD4⁺ T cells = 6.44%, SE = 0.25%, range 4.03%–10.30%, <i>n</i> = 37). (I) Analysis of FOXP3 gene demethylation on total Tregs and CD62L^low (effector memory) and CD62L^high (central memory) CD4⁺ memory T cells sorted from whole blood at pretreatment, post-treatment (day 3), and the last visit (day 60) showing stability of this Treg phenotype. (K) Tregs expanded in vivo at day 3 post-aldesleukin suppressed in vitro proliferation of autologous Teffs equivalently to Tregs at day in a suppression assay across the dose range (Treg:Teff ratio) tested. Error bars in (I) and (K) represent SEs. (J) Predictive cubic models based on the study data for CD25, pSTAT5, and Treg responses at the doses identified to increase Tregs by 10% and 20%. The error bars present the 95% confidence intervals around the predictions by these models. CM, central memory; EM, effector memory; MFI, mean fluorescent intensity; mTreg, memory regulatory T cell.</p

Effects of aldesleukin on effector T cell number, phenotypes, and proliferation.

<p>(A) mTeffs were responsive to aldesleukin, with their frequencies as a percentage of non-regulatory CD4⁺ T cells altered in circulation (B), resulting in opposing effects, with lower doses leading to higher mTeff frequencies and higher doses leading to reduced frequencies (average baseline mTeff percent of non-regulatory CD4⁺ T cells: 61.1%, SE = 1.9%, range 38.6%–87.4%, <i>n</i> = 37). (C and D) There was increased pSTAT5 in mTeffs (mTeff pSTAT5 MFI = 7, SE = 0.3, range 4–13, <i>n</i> = 36). Concurrently there was a dose-dependent decrease in CD25 (average baseline mTeff CD25 MFI = 1,005, SE = 31, range 676–1,436, <i>n</i> = 37) (E and F) and in CD122 (MFI = 137, SE = 7, range 59–248, <i>n</i> = 33) (G and H). (I and J) There was a dose-dependent increase in proliferation of mTeffs as measured by an increase in Ki-67⁺ mTeffs over the 7 d following treatment (baseline Ki-67⁺ Teffs = 2.92%, SE = 0.32%, range 0.75%–10.40%, <i>n</i> = 33). MFI, mean fluorescence intensity; mTeff, memory effector T cell; Treg, regulatory T cell.</p