IL-2 mutein selectively expands thymic memory Tregs with increased transendocytosis in healthy participants
Laura A. Cooney, Mitch Fahning, Liliane Khoryati, Anna Kus, Sheila Scheiding, Lori Blanchfield, Matt Lawrance, Basilin Benson, Kristina M. Harris, Gretchen A. Baltus, Shiuli Agarwal, Richard Wnek, Johannes F. Scheid, Kiki Cunningham-Bussel, Nancy D. Kim, S. Aubrey Stoch, Jyothsna Visweswaraiah, Nathan Higginson-Scott, Katalin Kis-Toth, Joanne L. Viney, Kevin L. Otipoby, Erik Sampson, Bridget Larkin, Daniel J. Campbell, S. Alice Long
Laura A. Cooney, Mitch Fahning, Liliane Khoryati, Anna Kus, Sheila Scheiding, Lori Blanchfield, Matt Lawrance, Basilin Benson, Kristina M. Harris, Gretchen A. Baltus, Shiuli Agarwal, Richard Wnek, Johannes F. Scheid, Kiki Cunningham-Bussel, Nancy D. Kim, S. Aubrey Stoch, Jyothsna Visweswaraiah, Nathan Higginson-Scott, Katalin Kis-Toth, Joanne L. Viney, Kevin L. Otipoby, Erik Sampson, Bridget Larkin, Daniel J. Campbell, S. Alice Long
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Research Article Clinical Research Immunology

IL-2 mutein selectively expands thymic memory Tregs with increased transendocytosis in healthy participants

Abstract

Tregs play an essential role in immune tolerance, and Treg-promoting therapies are in development for the treatment of many inflammatory disorders. IL-2–based therapies increase Treg frequency, but little is known about impacts on Treg heterogeneity and function. We extended analyses of an IL-2 mutein (MK-6194) single ascending dose trial in healthy human participants by comprehensively defining Treg subsets and gene expression changes in vitro and in vivo. We found highly specific and dose-dependent activation and expansion of Tregs in clinical and preclinical studies. Following a single subcutaneous dose in humans, thymus-derived Tregs were selectively activated and expanded, while peripherally induced Tregs were unaffected. Expanded Tregs had increased expression of genes and proteins consistent with activation, suppressor function, and homing to nonlymphoid tissue, as well as increased transendocytotic activity, as measured by CTLA-4–dependent capture of CD80 and CD86 from non-Tregs. These results shed light onto underlying mechanisms by which Treg-targeted therapy may promote immune tolerance.

Authors

Laura A. Cooney, Mitch Fahning, Liliane Khoryati, Anna Kus, Sheila Scheiding, Lori Blanchfield, Matt Lawrance, Basilin Benson, Kristina M. Harris, Gretchen A. Baltus, Shiuli Agarwal, Richard Wnek, Johannes F. Scheid, Kiki Cunningham-Bussel, Nancy D. Kim, S. Aubrey Stoch, Jyothsna Visweswaraiah, Nathan Higginson-Scott, Katalin Kis-Toth, Joanne L. Viney, Kevin L. Otipoby, Erik Sampson, Bridget Larkin, Daniel J. Campbell, S. Alice Long

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Figure 5

Preferential expansion of memory tTregs following IL-2 mutein treatment.

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Preferential expansion of memory tTregs following IL-2 mutein treatment....
(A) UMAP visualization of Treg (CD4+CD25hiFOXP3+) subclustering with heatmaps of selected subset-defining markers: green, high expression; blue, low expression. Includes integrated data from all cohorts at baseline and day 8. One cluster is defined as CD45RA+Helios+ naive tTregs (yellow), 2 clusters are defined as CD45RA–Helios+ memory tTregs (one with greater CLA expression, green; and the other with greater TIGIT expression, purple), and 1 cluster is defined as Helios– pTregs (blue). (B) Comparisons of Treg cluster frequencies between baseline and day 8. Contour plots, average ± SD of absolute counts for all cohorts across all individuals. CLA+ tTregs (time, P < 0.0001; dose, P = 0.0394) and TIGIT+ tTregs (time, P < 0.0001; dose, P = 0.0001) were significantly different by 1-way ANOVA. Mean ± SD of absolute cell counts for each Treg subset at baseline versus day 8. Dose indicated by color, Wilcoxon’s rank paired test. *P < 0.05. N as depicted in Figure 2A. (C) Comparison of CD25 mean metal intensity (MMI) and frequency of Ki67+ cells for all 3 tTreg clusters and 1 pTreg cluster. Data from the 3.5 mg and 10 mg cohorts combined. Repeated measures 1-way ANOVA with Bonferoni’s correction for multiple comparisons. **P < 0.01; ***P < 0.001; ****P < 0.0001. NS, no significant differences between any 2 groups (n = 11–12). (D) Healthy control human PBMCs were stimulated in vitro with MK-6194 (90 nM, 7.6 μg/mL) or recombinant wild-type human IL-2 (5 ng/mL) for 30 minutes, followed by flow cytometric analysis for induction of pSTAT5. Naive tTreg = CD45RA+Helios+, memory tTreg = CD45RA–Helios+, pTreg = CD45RA–Helios– (n = 5). *P < 0.05; **P < 0.01 by 2-way ANOVA with Tukey’s multiple-comparison test.