Low-Dose IL-2 Induces Bcl2 Expression and Resistance To Apoptosis In CD4 Regulatory T Cells
Authors: Murase Kazuyuki , Kawano Yutaka , Ryan Jeremy , Matsuoka Ken-ichi , Bascug Gregory , McDonough Sean , Smith Robert W , Cowens Kristen , Lazo-Kallanian Suzan , Daley John , Koreth John , Soiffer Robert J. , Letai Anthony , Ritz Jerome 
Published: October 2019

Abstract
CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the maintenance of self-tolerance and immune homeostasis. Interleukin-2 (IL-2) is critical for Treg development, expansion, activity and survival. Lack of either IL-2 or IL-2Rα (CD25) results in Treg deficiency and Treg impairment is associated with loss of tolerance, autoimmunity, and chronic GVHD. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo and clinical improvement of chronic GVHD (Koreth et al. NEJM 2011). We also reported that low-dose IL-2 enhanced Treg resistance to FAS-mediated apoptosis through the extrinsic pathway (Matsuoka et al. Sci Transl Med 2013). However susceptibility of Treg to apoptosis through the intrinsic pathway has not been examined. To examine the mechanisms whereby IL-2 affects susceptibility of Treg to apoptosis through the intrinsic pathway, we used a functional flow cytometry-based assay (BH3 profiling) to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). Mitochondria that are more sensitive to the BH3 peptides are more 'primed' for apoptosis, and more sensitive to pro-apoptotic signaling. This assay allowed us to compare “priming” which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization in Treg and Tcon. We also examined cytoplasmic expression of Bcl-2, BclxL, Mcl1, Bim, XIAP, Ki67 and Staurosporine (STS) induced apoptosis as additional measures of susceptibility to apoptosis and proliferation in each subset. In resting blood obtained from healthy donors (n=25), Treg were more “primed” than Tcon when exposed to several BH3 peptides (Bim, Bad, PUMA and BMF). This BH3 peptide pattern suggests that the anti-apoptotic protein, Bcl2 plays a primary role in the priming of these cells. Analysis of pro- and anti-apoptotic proteins revealed that Treg expressed higher levels Bim and BclxL and lower levels of Bcl2 and XIAP than Tcon, but there was no significant difference in Mcl1. Treg expressed higher levels of Ki67 and were more susceptible to both STS- and FAS-induced apoptosis than Tcon. Thus, Treg in healthy individuals have higher proliferative activity and are more susceptible to apoptosis than Tcon through both mitochondrial and death receptor pathways.
To examine the functional effects of IL-2 on T cell homeostasis, Treg and Tcon were purified by cell sorting and cultured with different concentrations of IL-2 (10, 100, 1000 IU/ml) with and without TCR stimulation (1ug/ml anti-CD3 + 1ug/ml anti-CD28). BH3 priming was measured after 3 days and results were compared to cells cultured in media alone. Low-concentration IL-2 (10 U/ml) lowered BH3 priming and increased Bcl2 expression in Treg. Similar effects were observed in Tcon, but higher concentrations of IL-2 (>100 U/ml) were required to increase Bcl2 expression and decrease BH3 priming in Tcon. TCR stimulation alone induced higher BH3 priming in Tcon but had little effect in Treg. The combination of TCR stimulation plus IL-2 increased BH3 priming in both Tcon and Treg. TCR stimulation increased proliferation only in Tcon, but the TCR + IL-2 combination increased proliferation in both Tcon and Treg. Thus, IL-2 alone reduced BH3 priming in unstimulated cells but the TCR + IL-2 combination induced higher proliferation and higher BH3 priming. STS-induced apoptosis assays revealed that low-dose IL-2 reduced susceptibility to apoptosis only in Treg. IL-2 did not change the expression of BclxL, Mcl1, and Bim but XIAP expression was increased in both Treg and Tcon. To confirm the effects of low-dose IL-2 on Treg priming were mediated by Bcl2, we examined the effect of ABT-199, a selective Bcl2 inhibitor on BH3 priming in Treg and Tcon. As shown in Figure 1, ABT-199 enhanced priming and spontaneous apoptosis of both Treg and Tcon. IL-2 had no effect on ABT-199-induced priming or apoptosis in Tcon. In contrast, IL-2 reversed the effects of ABT-199 on Treg priming and apoptosis providing further evidence that the inhibition of intrinsic pathway apoptosis mediated by IL-2 in Treg is dependent on Bcl2.

Disclosures:
Letai: AbbVie: Consultancy; Dana-Farber Cancer Institute: Patents & Royalties.