IL-2/mAb complexes treat cGVHD but exacerbate aGVHD. (A-F) B10.BR mice were transplanted with either T-cell–depleted (TCD), B6 BM (BM Only), or B6 TCD BM with purified B6 Tcon to establish cGVHD. Mice given BM with Tcon were treated with 200 µL of phosphate-buffered saline (PBS) (cGVHD), or with 200 µL of IL-2/mAb complexes (0.5 µg IL-2/25 µg JES6-1 anti-IL-2 mAb) in PBS from days 28-56 after transplant. (A) Total number of splenic Tregs were measured on day 56 after transplant and found to be significantly higher in mice given IL-2/mAb complexes vs cGVHD mice. (B-C) Frequency of Tfr (CD4+Foxp3+PD1hiCXCR5hi) and Tfh (CD4+Foxp3-PD1hiCXCR5hi) out of total splenic CD4+ T cells were measured by flow cytometry on day 56 after transplant. (B) Tfr were found in a higher frequency, but (C) Tfh at a lower frequency in mice given IL-2/mAb complexes. (D-E) Histopathology scoring of the (D) liver and (E) lung based on H&E-stained cryopreserved sections of organs harvested on day 56 after transplant. Scores show reduced pathology in mice given IL-2/mAb complexes vs cGVHD. (F-H) Pulmonary function tests (PFTs) assessing (F) airway resistance, (G) total lung elastance, and (H) total lung compliance were performed on day 56 after transplant. PFTs demonstrate partial reversal of PFTs characteristic of cGVHD-associated BOS in mice given IL-2/mAb complexes. (A-F) Representative data from 2 independent experiments; n = 8-10 mice/group/experiment. Bar graphs show mean ± standard error of the mean (SEM). Multiple comparisons made using one-way ANOVA with Tukey’s posttest. Significance: *P < .05; **P < .01; ***P < .001; ****P < .0001. (I-M) BALB/c mice were transplanted with B6 BM (BM Only), BM with purified B6 Tcon (aGVHD), or BM, Tcon, and B6 Tregs (Tregs) on day 0. Groups were given 200 µL of PBS or 200 µL of IL-2/mAb complexes (0.5 µg IL-2/25 µg JES6-1 anti-IL-2 mAb) in PBS from days 0-3 after transplant. (I) Survival of recipient mice shows increased mortality rate in groups given IL-2/mAb complexes: aGVHD vs aGVHD/IL-2 complex (*); Tregs vs Tregs/IL-2 complex (***); aGVHD/IL-2 complex vs Tregs/IL-2 complex (*); aGVHD vs Tregs (**). (J) Recipient body weights show sharp declines for aGVHD/IL-2 complex group from days 3-7, and Treg/IL-2 complex group from days 13-17. Comparisons on graph: day 7: aGVHD vs aGVHD/IL-2 complex (***); day 17: Treg vs Treg/IL-2 complex (***); days 17-38: Treg vs aGVHD. (K) Clinical GVHD scores show sharp increases for aGVHD/IL-2 complex group from days 4-7, and for Treg/IL-2 complex group from days 11-14. Comparisons on graph: day 7: aGVHD vs aGVHD/IL-2 complex (****); day 17: Treg vs Treg/IL-2 complex (****); days 14-39: Treg vs aGVHD. (L-M) Flow cytometry analysis of splenic T cells on day 5 after transplant revealed a higher (L) frequency and greater (M) numbers of CD25hiCD4+Foxp3- and CD25hiCD8+Tcon in mice given IL-2 mAb complexes, compared with untreated groups. Survival differences analyzed by log-rank test. Bar graphs show mean ± SEM. Multiple comparisons made using one-way ANOVA with Tukey’s posttest. (I-M) Representative data from 3 independent experiments; n = 5-8 mice/group/experiment. Significance: *P < .05; **P < .01; ***P < .001; ****P < .0001.