Fig 1.
IL-2 complexes promote BM rejection.
The ability of IL-2 complexes to replace Treg therapy was tested in different BMT models. Donor chimerism was analyzed in blood 14 days after transplantation by staining the BALB/c specific marker H2-Dd on myeloid (Mac-1) cells. (A) IL-2 complexes were less effective than Treg therapy in promoting BM engraftment. Naïve C57BL/6 mice were grafted with 20×106 unseparated BALB/c BM cells (d0) under the cover of costimulation blockade (α-CD154, CTLA4Ig) and a short course of rapamycin. The recipients were additionally treated with either in vitro activated Tregs (3×106) (n = 4), IL-2 complex expanded Tregs (3×106) (n = 4) or IL-2 complexes (5μg IL-2 / 25μg α-IL-2; d-4, d-3, d-2) (n = 6). Two color flow cytometry plots are shown from representative BMT recipients (left). Each dot of the scatter diagram represents one mouse from one experiment (right) [in vitro Tregs vs. in vivo Tregs: p = 0.0341; in vitro Tregs vs. IL-2 complexes: p = 0.0187]. (B) IL-2 complexes enhanced BM rejection. Naïve C57BL/6 mice received a total body irradiation of 1Gy, costimulation blockade (α-CD154, CTLA4-Ig), as well as 20×106 unseparated BALB/c BM cells (d0) with varying doses (1μg IL-2 / 5μg α-IL-2, n = 8; 0.5 μg IL-2 / 2.5μg α-IL-2, n = 5, 0.25 μg IL-2 / 1.25μg α-IL-2, n = 5) of IL-2 complexes; (d3, d5) or without IL-2 complexes (n = 11). Two-color flow cytometry plots are shown from representative BMT recipients (left). Each dot in the scatter diagram depicts one mouse from two individual experiments (right) [no IL-2 complexes vs. 1μg IL-2 / 5μg α-IL-2 p = 0.0004; no IL-2 complexes vs. 0.5μg IL-2 / 2.5μg α-IL-2: p = 0.7769, no IL-2 complexes vs. 0.25μg IL-2 / 1.25μg α-IL-2: p = 0.8966]. (C) Omission of CTLA4-Ig did not reverse the detrimental effect of IL-2 complexes. Naïve C57BL/6 mice were irradiated with 1Gy TBI before receiving costimulation blockade (α-CD154) and 20×106 unseparated BALB/c BM cells (d0) with (1μg IL-2 / 5μg α-IL-2; d3, d5) (n = 6) or without IL-2 complexes (n = 6). Two-color flow cytometry plots are shown from representative BMT recipients (left). Each dot in the scatter diagram shows one mouse from one experiment (right) [p = 0.0627]. (D) IL-2 complexes increased the reactivity of CD8 T cells and NK cells toward donor antigens. Splenocytes from untreated mice or mice treated with IL-2 complexes were stimulated in vitro with irradiated BALB/c (allogeneic) or C57BL/6 (syngeneic) BM cells. The proliferation of CD8 T and NK cells was assessed by measuring the proliferation marker Ki67. Each symbol represents 2 mice from one experiment. (F) 4×105 responder splenocytes from congenic CD45.1 mice were stimulated in vitro with α-CD3 and co-cultured with equal number of either in vitro activated or IL-2 complex expanded Tregs.
Fig 2.
IL-2 complexes induce Treg proliferation in primary and secondary lymphoid tissues.
The effectivity of IL-2 complexes to expand and activate Tregs in vivo was assessed. Female C57BL/6 mice were injected i.p. for 3 days (d0, d1, d2) with IL-2 complexes (5μg IL-2 / 25μg α-IL-2) or left untreated. Indicated tissues were analyzed 2 days after the last administration (d4) by flow cytometry. (A) IL-2 complexes increased the proportion of Foxp3+ Tregs within CD3+ CD4+ (upper panels) and CD3+ CD8+ T cells (lower panels) in blood, spleen, lymph nodes and thymus. Histograms display representative mice (n = 6). (B) Application of IL-2 complexes (n = 4) increased the intracellular expression of Foxp3 in comparison with untreated mice (n = 4) as determined by the MFI of Foxp3-APC [Ki67+: p = 0.0012; Ki67−: p = 0.0304]. We compared Foxp3 expression between Ki67+ proliferating and Ki67− resting CD4 T cells to ensure that the measured effect was truly intrinsic and did not result from the increased cell mass of the dividing cells. Data are pooled from two independent experiments (left). The increase of Foxp3 was also evident by epifluorescence microscopy of CD4+ CD25+ separated cells purified from Foxp3-mRFP reporter mice (magnification 63x). Representative pictures are shown (right). (C) In vivo expanded Tregs exhibited an activated phenotype. IL-2 complexes increased the surface expression of CD69, PD1, ICOS, GITR and CD25 on CD4+ Tregs, as well as their intracellular expression of CTLA4. Representative mice are shown (n = 4). (D) CD4+ Tregs primarily expand through proliferation in the blood, spleen, lymph nodes and thymus upon IL-2 complex treatment as determined by their intracellular expression of the proliferation marker Ki67. Representative mice were chosen for histograms (n = 4).
Fig 3.
IL-2 complexes preferentially induce the proliferation of Helios+ Nrp1+ Tregs.
To determine the effect of IL-2 complexes (5μg IL-2 / 25μg α-IL-2) on thymus derived Tregs, naïve C57BL/6 mice received three successive injections (d0, d1, d2) and Treg specific markers were measured in the spleen two days (d4) after the last injection. (A) IL-2 complexes (n = 6) slightly raised the amount of thymus derived Helios+ Nrp1+ Tregs compared to untreated mice (n = 6). Representative mice are displayed in two color dot plots (left). Data are pooled from three independent experiments (right) [p = 0.0287]. (B) Helios+ Nrp1+ Tregs exhibited a higher degree of proliferation than Helios− Nrp1− Tregs as measured by their expression of the proliferation marker Ki67 both before and after treatment with IL-2 complexes. Histograms show representative mice (left). The bars compare the expression of Ki67 between Helios+ Nrp1+ (n = 6) and Helios− Nrp1− (n = 6) Tregs upon IL-2 complex treatment (right) [p<0.0001]. Data are pooled from three independent experiments. (C) Helios+ Nrp1+ Tregs exhibit a higher surface expression of CD25. Representative mice were selected for histograms (left). The bars show the MFI of CD25-PE-Cy7 on Helios+ Nrp1+ and Helios- Nrp1- Tregs before (n = 3) and after (n = 3) IL-2 complex treatment (right) [untreated: p = 0.0003; IL-2 complexes: p = 0.0003]. The bars represent the mean of three mice from one experiment. (D) Blockade of MHC class II molecules decreased the amount and absolute cell number of CD4+ Tregs in the spleen compared to single giving of IL-2 complexes. Two color dot plots illustrate representative mice (left). Each bar shows the mean of 3 mice from one experiment (right) [p = 0.0031]. (E) Co-administration of α-MHC-II did not change the proportion of Helios+ Nrp1+ Tregs after IL-2 complex treatment. Representative mice are shown in the two color dot plots (n = 3).
Fig 4.
IL-2 complexes also expand lymphocyte populations other than Tregs.
Distinct lymphocyte populations were analyzed in the spleen following IL-2 complex treatment to determine their specificity. Naïve C57BL/6 mice received IL-2 complexes (5μg IL-2 / 25μg α-IL-2) on three consecutive days (d0, d1, d2) and absolute cell numbers were analyzed 2 (d4) and 4 (d6) days after the last administration. (A) IL-2 complexes increased the absolute number of splenocytes [p = 0.0009 for d4; p = 0.0027 for d6] and splenic CD4+ Tregs [p <0.0001 for d4; p = 0.0001 for d6] 2 (d4) and 4 (d6) days after the last injection. The average increase in total splenocytes exceeds the mean increase of Tregs indicating that IL-2 complexes also expand other lymphocytes. Both groups consisted of 4 mice from 2 independent experiments at each time point. (B) IL-2 complexes also expand splenic CD3+ CD4+ Foxp3− T cells [p = 0.0491 for d4; p = 0.0028 for d6], CD3+ CD8+ T cells [p = 0.0118 for d4; p = 0.0016 for d6], CD3− NK1.1+ NK cells [p = 0.0009 for d4, p = 0.0004 for d6], CD3+ NK1.1+ NK cells [p < 0.0001 for d4, p = 0.0012 for d6] and CD19+ B cells [p = 0.0016 for d4, p = 0.008 for d6]. Both groups consisted of 4 mice respectively at each time point. Data are pooled from 2 independent experiments. (C) Fold change of distinct lymphocyte populations in the spleen at d4 and d6.
Fig 5.
IL-2 complexes relocate B cells from the bone marrow to secondary lymphoid organs.
The mechanisms of B cell expansion in secondary lymphoid organs were investigated. Naive C57BL/6 mice received IL-2 complexes (5μg IL-2 / 25μg α-IL-2) for three days (d0, d1, d2) and distinct cell populations were analyzed in indicated tissues 2 days (d4) after the last dose. (A) CD3+ CD4+ Foxp3− T cells, CD3+ CD8+ T cells, CD3− Nk1.1+ NK cells and CD3+ NK1.1+ cells but not CD19+ B cells proliferated upon IL-2 complex treatment in the spleen as assessed by their expression of Ki67. Histograms are shown from representative mice (n = 4). (B) IL-2 complexes (n = 4) reduced the proportion and absolute number of CD19+ B cells within CD45+ leukocytes in the BM as compared with untreated mice (n = 4). Two color dot plots illustrate representative mice (left). Data are pooled from 2 independent experiments (right) [p = 0.014].
Fig 6.
Rapamycin reduces the expansion and activation of CD8 effector cells.
IL-2 complexes were combined with selected agents to alleviate the unintended expansion and activation of CD8 T cells. IL-2 complexes (5μg IL-2 / 25μg α-IL-2) were administered to female C57BL/6 mice three times in succession (d0, d1,0 d2) together with specified agents. The effect of combined therapy was determined on splenic CD8+ T cells and CD4+ Foxp3+ Tregs 2 (d4) and 4 (d6) days after the last injection. (A) Co-administration of rapamycin (n = 4) reduced the absolute cell number of splenic CD8+ T cells at both measured time points compared to mice treated only with IL-2 complexes (n = 4) [p = 0.0142 for d4; p = 0.0244 for d6]. Data are pooled from two independent experiments. Groups receiving α -IL-6 and IL-15-Fc consisted of 2 mice from one experiment at each time point. (B) Rapamycin (n = 4) but not α-IL-6 (n = 2) or IL-15-Fc (n = 2) considerably decreased the amount of effector memory CD8+ T cells (d6) defined as CD44hi CD62lo compared to IL-2 complex treatment alone (n = 4). Two color dot plots depict representative mice. (C) Addition of α-IL-6 (n = 2) decreased the fraction of Foxp3+ cells within splenic CD4 T cell population while rapamycin (n = 4) and IL-15-Fc (n = 2) had no profound effect (d6). Histograms illustrate representative mice of each group.