*Purpose: Promoting immune regulation through expansion of regulatory T cell (Treg) may allow immunosuppression minimization and improve long-term graft outcomes. Low-dose IL-2 is known to expand Tregs but its short half-life and off-target expansion of NK and CD8 T cells limits its clinical applicability. Here, we show the effects of a novel mutein IL-2 (mIL-2) that has increased Treg selectivity and improved pharmacokinetics through conjugation with an antibody Fc portion.

*Methods: First, we treated mouse splenocytes in vitro with mIL-2, WT Fc-IL-2 or negative control and measured the levels of phosphorylated STAT5 (pSTAT5), a downstream effector of IL-2 receptor in various immune cells. Next, we studied in vivo effects of single-dose and sustained-dose (twice a week for 3 weeks) mIL-2 treatment in B6 mice. Splenocytes from these mice were evaluated for Treg functional markers and cytokine production by flow cytometry. Lastly, we tested mIL-2 in two minor mismatch skin transplant models: male to female B6 and OVA to B6; as well as rechallenged with allo or third-party grafts. Recipients were subcutaneously treated twice a week with 0.5 mg/kg of mIL-2 or control IgG.

*Results: mIL-2 increased levels of pSTAT5 selectively in Tregs in a wide dose range but not in NK or CD8 T cells. In vivo, single dose mIL-2 increased circulating Tregs up to 40% of CD4⁺ T cells by day 4 with an effect lasting for 7 days. Sustained mIL-2 treatment led to stable expansion of circulating Tregs at 40-50 % without expanding effector cells and a 5.3-fold rise in splenic Tregs (Fig.1A). Regarding functional suppressive markers, mIL-2 increased % of CTLA-4⁺, TGF-b⁺, and IL-10⁺ splenic Tregs at day 4 after single injection (Fig.1B). In male to female skin transplant model, mIL-2 led to a sustained Treg expansion without affecting NK cells (Fig.1C) or CD8 T cells (not shown). It also prolonged median graft survival (MGS) to >185 days vs 34.5 days (p=0.0004) in the control group (Fig.1D). Treatments were stopped, and recipients were challenged with a similar allo-skin or third-party skin (male B6 or OVA B6, respectively). Second male skin grafts survived long-term while third-party grafts rejected early, indicating antigen-specific tolerance (MGS >65 vs 17 days, respectively, p<0.001). Lastly, mIL-2 also prolonged graft survival in OVA to B6 recipient mice (MGS 13 vs >30 days, p=0.01).

*Conclusions: mIL-2 selectively expands Tregs, increases CTLA-4⁺ Tregs, and prolongs skin graft survival in mice.

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