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.

Author notes

Disclosure: No relevant conflicts of interest to declare.