PMC

Depletion of CD25⁺ Treg, but not Gr-1⁺ cells, before transplantation prevents prolongation of cardiac allograft survival by IL-33. Groups of WT BALB/c mice (n= 8/group) received anti-CD25 Ab to deplete CD4⁺ CD25⁺ Treg, anti-Gr-1 Ab to deplete Gr-1⁺ myeloid cells, or rat polyclonal IgG before receiving a B6 heterotopic heart transplant and IL-33 monotherapy as above. As a control, a fourth group received PBS only following transplantation.

Increased endothelial and myocardial cell expression of ST2 during acute allograft rejection. Cryostat sections from naïve B6 hearts, acutely-rejected B6 hearts (d10 post transplantation into BALB/c recipients), and BALB/c recipients’ native hearts were labeled with primary Abs to CD31 (red) or ST2 (green) that were then visualized with quantum dot-conjugated secondary Abs. Following nuclear staining with DAPI (blue), slides were scanned with a Mirax MIDI (3D Histech, Budapest, Hungary) and digital images generated with Panoramic Viewer (Version 1.14; 3D Histech). Images from a normal B6 and transplanted and native hearts from 2 representative graft recipients are presented at magnifications of 1X (left panels), 5X (right panels), and 40X (inset).

IL-33 monotherapy profoundly alters CD11b⁺ populations in cardiac transplant recipients, favoring Gr-1^int-expressing cells. Administration of IL-33 following heart transplantation promotes increased incidences of CD11b⁺ F4/80^lo Gr-1^int cells. On d 11, spleen cells from BALB/c mice given B6 heart grafts alone or with IL-33 monotherapy were compared by flow cytometry to normal animals or heart graft recipients receiving only PBS. A, The left panels depict the incidence of total CD45⁺ cells and the right panels indicate the incidence of those from the CD11b⁺ CD11c⁻ gate. Data are representative flow plots from 1 animal representative of the 3–4 assessed for each group. B, Data represent the mean frequency of the indicated population in CD45⁺ CD11b⁺ CD11c⁻ gated cells (n = 3–4/group). Error bars indicate 1SD and * = p<0.05 by Student’s t-test. C–D, IL-33 increases the number of splenic CD11b⁺ CD11c^lo F4/80^lo Gr-1^int cells. Data indicate the calculated total splenic numbers for the indicated populations. Error bars indicate 1SD and * = p<0.05 by Student’s t-test (n = 3–4/group).

Prolongation of cardiac allograft survival by IL-33 is dependent on host St2 expression. B6 heart survival is prolonged by IL-33 in WT, but not St2^−/− BALB/c recipients. BALB/c (St2^+/+ and St2^−/−) mice received heterotopic heart transplants and post-operative monotherapy of IL-33 (0.5 µg/d i.p. on d0, 1, 3, 6, 10, 13, 15, and 17). A–B, On d10 post-transplant, heart grafts from WT BALB/c mice treated with IL-33 displayed markedly reduced infiltrates of mononuclear cells in the myocardial interstitium and preserved tissue architecture compared to control mice receiving PBS. Data are from 2 animals representative of 4 in each group. A 20X and B 40X. C, Less CD3⁺ cell infiltrate is observed in IL-33-treated grafts. Data are from 1 animal per condition and representative of the group (n = 4). D, In St2^+/+, but not St2^−/− recipients, IL-33 significantly increased allograft survival (n = 5–8 per group).

IL-33 promotes the expansion of CD11b^lo CD11c⁺ MHC^lo and CD11b⁺ Gr-1^int cells in vivo. WT (St2^+/+; A) or St2^−/− BALB/c (B) mice were given IL-33 (0.5 µg/d) or Flt3L (10 µg/d) i.p. for 10 d (d0–d10). On d 11, total splenocytes were isolated, stained as described in the Materials and Methods, and analyzed by flow cytometric analysis. A and B, St2-dependent increase in CD11b^lo CD11c⁺ and CD11b⁺ Gr-1^int splenocytes following IL-33 administration. In A and B, data represent CD45⁺-gated (left panels) or CD45⁺ CD11b⁺ CD11c⁻ gated (right panels) cells from WT BALB/c St2^+/+ (A) or St2^−/− (B) mice and numbers on dot plots indicate % of gated cells. Data are from 1 experiment representative of 3 performed and depict 1 animal representative of the indicated treatment group. C, Mean incidence (top panels) for the indicated populations calculated across 3 independent experiments (n = 7–9/group). Total splenic numbers (lower panels) for the indicated populations from 1 experiment representative of the 3 independent experiments preformed (n =2–3/group). Error bars indicate 1 SD and * = p <0.05 by Student’s t-test. D, Decreased MHC class II and CD86 on CD11c⁺ and CD11b⁺ cells from mice treated with IL-33 or Flt3L. Freshly-isolated total splenocytes were assessed for CD86 and MHC class II (I-A^d/I-E^d). Shaded histogram depicts isotype control. Data depict 1 experiment representative of 3 experiments performed.

IL-33 preferentially expands CD11b⁺ F4/80^int cells containing Ly6G⁺ and Ly6C⁺ populations that potently inhibit allogeneic T cell responses. IL-33 facilitates the expansion of several CD11b and F4/80 expressing cell populations in the spleen. A, Flow cytometry plots, representative of at least 3 experiments performed, depict CD45⁺-gated total splenocytes from IL-33- or PBS-treated WT BALB/c mice. B, Panels present the incidence of Ly6G⁺ and Ly6C⁺ cells in CD11b^lo F4/80^hi (top panels), CD11b⁺ F4/80^int (middle panels), and CD11b^hi F4/80⁻ gated cells. Data are representative of more then 3 experiments performed. C, Ly6G or Ly6C cells were enriched from splenocytes pooled from each treatment group (n = 2 mice per group) and tested as suppressors (1:1) of purified B6 CD3⁺ T cell responders in MLR using CD11c⁺ BALB/c splenocytes (1:5) as stimulators. Data are from one experiment representative of three performed and are plotted as mean % suppression + 1SD and significances of differences determined by Student’s ‘t’ test. * = p <0.05 vs. T cells + stimulators only, * = p <0.05 vs. T cell and CD11c⁺ APC only; # = p <0.05 vs. PBS group; † = p <0.05 vs. IL-33 group. CPM measured for wells containing T cells only were 189±125 and for those with T cells and CD11c⁺-stimulators: 9823±2684 CPM. CPM for MLR containing indicated Ly6C or Ly6G enriched suppressors were: PBS Ly6G (9181±1434), IL-33 Ly6G (3197±47), Flt3L Ly6G (3157±528), PBS Ly6C (6934±601), IL-33 Ly6C (2637±199), and Flt3L Ly6C (834±79).

St2-dependent increase in suppressive CD4⁺ Foxp3⁺ cells, including a prominent ST2L⁺ subset, following IL-33 administration. IL-33-treated mice display an St2-dependent increase in splenic Foxp3⁺ CD4⁺ cells. A–B, Flow cytometric analysis of surface ST2L or intracellular Foxp3 expression by (A) WT BALB/c St2^+/+ or (B) BALB/c St2^−/− CD4⁺ T cells following treatment with PBS or IL-33 (0.5 µg/d: d0–d10). Data depict the % of CD45⁺ CD3⁺ CD4⁺-gated cells expressing ST2L (membrane bound ST2) and intracellular Foxp3 on d11. Flow cytometric plots are from 1 experiment representative of 3 performed. C, Mean incidence for the indicated population following IL-33 (0.5 µg/d: d0–d10) or Flt3L (10 µg/d: d0–d10) administration to WT BALB/c mice. Data are from 1 experiment (n = 2–3 mice per group) that is representative of 3 performed. Error bars indicate mean + 1SD and * = p <0.05 by Student’s t-test. D, Co-expression of ST2L by BALB/c splenic CD4⁺ CD25⁺ T cells. Data represent the % of splenic CD45⁺ CD3⁺ CD4⁺-gated cells expressing CD25⁺ (right panels) and CD4⁺ CD25⁺-gated (left panels) cells expressing ST2L and intracellular Foxp3 on d11 following PBS- or IL-33-treatment. Representative flow cytometric plots from 1 experiment representative of 4 performed. E–F, IL-33 expands B6 CD4⁺ Foxp3⁺ ST2L⁺ and STL2⁻ T cells displaying suppressive capacity. E, Flow cytometric analysis of C57BL/6 splenic CD3⁺ CD4⁺ T cells following treatment with PBS or IL-33 as above. Data depict the % of CD45⁺ CD3⁺ CD4⁺-gated cells and are representative of 3 independent experiments performed. F, FACS was used to isolated viable ST2L⁻ and ST2L⁺ CD4⁺ Foxp3⁺ (RFP⁺) cells from PBS- or IL-33-treated B6 Foxp3-reporter mice. Graded numbers of Foxp3⁺ (RFP⁺) cells were assessed for their capacity to suppress CD3/CD28-stimulated proliferation of CFSE-labeled B6 CD4⁺ CD25⁻ T cells. Data depict the Foxp3⁻ CD4⁺-gated cell proliferation profile, % divided, and division index as calculated via FlowJo Proliferation Platform. Data are from 1 experiment representative of 2 performed.

IL-33-treated allograft recipients display a Th2-type systemic response and increased intragraft Foxp3⁺ cells. A, Significantly decreased IL-6 and markedly increased circulating IL-5 and IL-13 levels observed in IL-33 recipients. The indicated serum cytokines were measured by Luminex assay on d 11 after transplantation of B6 hearts into WT BALB/c hosts which also received PBS or IL-33 as described in legend. Comparison to normal B6 serum is also shown. Data shown are the means + 1 SD. n = 3–4 mice/group. * = p <0.05; Student’s t-test. FGF, GM-CSF, IL-1α/β, IL-2, IL-17, CXCL1, MIP-1α, TNF-α and VEGF were not detected or significantly altered from normal levels. B, Following their isolation on d 11 from BALB/c mice given B6 heart transplants alone (PBS) or with IL-33 monotherapy, splenocytes were stained and assessed by flow cytometry after stimulation with PMA/ionomycin in the presence of Golgi Plug. The graph depicts mean % CD45⁺ positive cells + 1SD for cells in the indicated gate. n = 2–3/group. C, Also on d11, splenic CD4⁺ cells were purified from BALB/c mice given B6 heart transplants alone (control PBS treatment) or with IL-33 monotherapy. The frequency of directly-reactive CD4⁺ IL-5-secreting T cells was determined 3 d after stimulation with CD3-depleted BALB/c (donor) or C3H (3^rd party) splenocytes. Data are group means + SD, with n = 3/group. D–E, Assessment of splenic CD45⁺ CD8⁺ T cells in IL-33-treated versus control heart graft recipients revealed reduced incidences of CD3⁺ CD8⁺ T cells, particularly CD8⁺ IFN⁺ cells. D, Representative flow plots from 1 animal representative of the 3 analyzed. E, Graph depicts mean % CD45⁺ positive cells + 1SD for cells contained in the indicated gate. N = 3/group and * = p< 0.05, Student’s t-test. F, Confocal analysis of staining for ST2 (red), Foxp3 (green), and Hoescht (Blue) demonstrate that heart grafts from IL-33-treated recipients had increased Foxp3⁺ cells. The graph presents the mean number + 1SD of Foxp3⁺ cells per 40X field for each condition. Positive cells in five randomly selected 40X fields from 4 individuals per group were quantified through the use of ITCN ImageJ software. * = p< 0.05; Student’s t-test.