Th2.rapa Cell Treatment of Established Murine Acute GVHD Is Abrogated by IL-2 Therapy and T Regulatory Cells.
Authors: Foley Jason E. , Mariotti Jacopo , Amarnath Shoba , Han Soo , Eckhaus Michael , Fowler Daniel H. 
Published: October 2019

Abstract
Rapamycin-generated donor Th2 cells attenuate established acute murine GVHD (Foley et al, JI, 2005) and are dependent in part upon IL-4 and IL-10 secretion (ASBMT Meeting, 2007). That is, Th2.rapa cell recipients (Th2 infusion, d 14 post-BMT) had increased survival relative to GVHD controls (post-BMT survival, median days; 33.7±0.4 vs. 24.8±1.2; p=0.0002) whereas recipients of IL-4 or IL-10 knockout Th2.rapa cells did not have increased survival (28.9±0.3 and 24.6±0.2 days, respectively; p=NS). These data indicate that Th2.rapa cells operate through a Th2-type mechanism rather than a Treg cell mechanism; in addition, we found that Th2.rapa cells expressed low levels of the Treg cell transcription factor, Foxp3 (<5% CD4+Foxp3+). Additional experiments were performed to further investigate a potential role of Treg cell biology to Th2.rapa cell therapy. First, we hypothesized that IL-2 therapy may promote Th2.rapa cell expansion and efficacy in a manner analogous to IL-2 promotion of Treg cell responses in vivo. Second, we hypothesized that enrichment of the Th2.rapa cell product with natural (unmanipulated) Treg cells may enhance an anti-GVHD effect. Contrary to our first hypothesis, we found that IL-2 therapy (50,000 IU bid; d14–18 post-BMT) reduced the number of splenic Th2.rapa cells at d 19 post-BMT (CD90.1-marked cells, million [M]/spleen; 5.0±0.4 [no IL-2] vs. 2.3±0.4 [+IL-2]; p=<0.001) and increased the number of unmanipulated donor CD4+ cells (CD45.2-marked cells, M/spleen; 16.6±0.7 [no IL-2] vs. 26.6±2.4 [+IL-2]; p=0.004) and CD8+ cells (15.9±1.7 [no IL-2] vs. 23.9±2.4 [+IL-2]; p=0.03). IL-2 therapy also inhibited Th2.rapa cell-mediated cytokine polarization (d 19 post-BMT, pg/ml; IL-4 reduced from 3501±179 [no IL-2] to 1116±261 [+IL-2], p=<0.0001; IL-10 reduced from 707±56 [no IL-2] to 288±37 [+IL-2], p=0.0002; and IFN-γ increased from 81±22 [no IL-2] to 320±97 [+IL-2], p=0.042). Importantly, for Th2.rapa cell recipients, IL-2 therapy reduced post-BMT survival (d post-BMT; 42.0±0.5 [no IL-2] vs. 33.8±1.0 [+IL-2], p=<0.0001). With regard to our second hypothesis, we found that addition of Treg cells to the Th2.rapa cell product (Treg to Th2.rapa cell ratio, 1:10) reduced the number of Th2.rapa cells at d 19 post-BMT (M/spleen; 16.6±1.3 [no Treg] vs. 7.9±1.2 [+ Treg], p=0.0012) and increased the number of unmanipulated donor CD4+ cells (M/spleen; 12.4±0.7 [no Treg] vs. 20.7±0.8 [+Treg], p=<0.0001) and CD8+ cells (M/spleen; 8.4±0.8 [no Treg] vs. 16.1±1.4 [+Treg], p= 0.0014). Treg cell co-infusion also inhibited Th2.rapa cell-mediated cytokine polarization (d 19 post-BMT, pg/ml; IL-4 reduced from 497±47 [no Treg] to 100±15 [+ Treg]; p=<0.0001); IL-10 reduced from 160±32 [no Treg] to 27±7 [+Treg]; p= 0.004; and IFN-γ increased from 83±5 [no Treg] to 230±40 [+Treg]; p=0.006). Finally, for Th2.rapa cell recipients, co-infusion of Treg cells reduced post-BMT survival (median d post-BMT; 44.2±1.1 [no Treg] vs. 30.7±1.3 [+Treg]; p=0.0002). In conclusion, interventions that promote Treg cell responses, namely infusion of IL-2 and co-administration of natural Treg cells, reduce Th2.rapa cell promotion of IL-4 and IL-10 post-BMT and reduce the Th2 cell-mediated survival advantage against established GVHD. Because IL-4 and IL-10 are required for Th2.rapa cell therapy of GVHD in this model, these new data indicate that Treg cells abrogate Th2.rapa cell therapy by inhibiting the Th2 cell effector response.