Interval of IL-2 Administration Has a Major Impact on Treatment Efficacy for Regulatory T Cell Expansion and Homeostasis
Authors: Kishi Yuriko , Meguri Yusuke , Iwamoto Miki , Asano Takeru , Yoshioka Takanori , Maeda Yoshinobu , Tanimoto Mitsune , Koreth John , Ritz Jerome , Matsuoka Ken-Ichi 
Published: October 2019

Abstract
IL-2 has a critical role in the immune homeostasis by expansion and maintenance of regulatory T cell (Treg). We previously demonstrated that low-dose IL-2 administration preferentially increased Treg in patients with active chronic GVHD and resulted in clinical improvement of the symptoms. In these years, the tolerogenic effects of IL-2 have been tested in the setting of various autoimmune-based diseases by many clinical trials. However, the schedules of IL-2 administration in each trial are different and the optimal strategy for expansion and maintenance of Treg is still unclear. To tackle this issue, we examined the impact of IL-2 dose intervals on Treg and the optimization for the induction and maintenance therapy by using murine IL-2 therapy model. CD4+CD25+Foxp3+ Treg were analyzed comparing with CD4+CD25-Foxp3- conventional CD4 T cells (Tcon) and CD8+ T cells. The expressions of Ki-67 and Bcl-2 in each subset were also examined. First, we performed the experimental allogeneic BMT, in which model 1x10E6 spleen cells (CD45.2) and 5x10E6 T cell depleted bone marrow cells (CD45.1) from C57BL/6 donors were transplanted into lethally-irradiated B6D2F1 recipients (CD45.2). To explore the optimum dose-interval for the increase of Treg from the baseline level as the induction therapy, we administered 5000 IU of IL-2 to recipients subcutaneously once (Induction-A), twice (Induction-B), four (Induction -C) or seven (Induction-D) times a week from day 35 to 49 after transplant. On day 50 after transplant, peripheral blood and spleen cells of mice from each group were harvested and CD45.1+H-2Kd- Donor bone-marrow derived lymphocytes were evaluated. The absolute number and %Treg of CD4 T cells, and the Ki-67 and Bcl-2 expression in Treg were compared with each frequency of administration. To increase Treg from baseline, the daily administration (Induction-D) provided the best Treg response among the tested groups and there was significant difference between control group and group I-D (%Treg: 10.5 % vs. 16.7 %, p<0.05). Treg proliferation was positively related to the frequency of IL-2 administration (%Ki-67+ cells: 17.7%, 17.2 %, vs. 9.9%, 9.0% and 7.8%, respectively, p<0.05). Secondly, we investigated the optimum dose-interval for the maintenance of expanded Treg level after the initial inductive IL-2 administration. We expanded Treg by daily administration of IL-2 to B6 mice for 2 weeks, thereafter we administrated IL-2 twice (Maintenance-A), four (Maintenance-B), or seven (Maintenance-C) times a week from day 14 to 21 for the maintenance. Of interest, to maintain expanded Treg level after the induction IL-2 therapy, Treg in cohorts of Maintenance-B and Maintenance-C were significantly higher than in cohort Maintenance-A (mean 9.7%, 10.2% vs 7.1%, respectively) and there was no significant differences between cohorts Maintenance-B and Maintenance-C, suggesting daily administration is not necessary and intermittent administration within the threshold may work for the maintenance of expanded Treg level after the intensive IL-2 administration. Treg proliferation of cohort Maintenance-C was significantly higher than that of the other cohorts (mean %Ki-67: 19.8 %, 20.5 % vs. 23.9 %, p<0.05). There were no significant difference in Bcl-2 expression in Treg among these cohorts but seemed negatively related to the frequency of IL-2 administration. These data suggested that daily administration of IL-2 seems to be optimal for expansion of Treg for induction therapy. In contrast, to maintain the expanded Treg, daily administration may not be required but less frequent times of administration within the threshold could be preferable. Taken together, the interval of IL-2 administration should be a major factor for Treg homeostasis as well as IL-2 dosage. Our data provide important information for developing therapeutic strategies to modulate Treg homeostasis in vivo and promote immune tolerance after transplant.

Disclosures
No relevant conflicts of interest to declare.