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. 2009 Apr 13;206(4):751-60.
doi: 10.1084/jem.20082824. Epub 2009 Mar 30.

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression

Kylie E Webster  1 , Stacey Walters, Rachel E Kohler, Tomas Mrkvan, Onur Boyman, Charles D Surh, Shane T Grey, Jonathan Sprent
Affiliations

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression

Kylie E Webster et al. J Exp Med. .

Abstract

Via a transcription factor, Foxp3, immunoregulatory CD4(+)CD25(+) T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2-IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1-2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2-IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex-incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

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Figures

Figure 1.
Figure 1.
Rapid and widespread proliferation of T reg cells after IL-2–JES6-1 treatment. Mice were injected i.p. daily for 3 d (days 0, 1, and 2) with JES6-1 mAb and/or IL-2 and analyzed on day 3 (A and B), day 5 (C–E), day 6 (F), or where indicated (G and H), usually in the spleen. Doses were 1 µg IL-2 and 5 µg mAb unless otherwise specified. (A) The effect of varying the ratio of IL-2 and JES6-1 on CD4+CD25+Foxp3+ (T reg cell) expansion was analyzed by injecting titrated doses (1–50 µg) of JES6-1 mAb, plus a fixed concentration (1 µg) of rmIL-2. The arrow indicates a 1:2 molar ratio of JES6-1 mAb to IL-2. (B) The effect of titrating the dose of IL-2 plus JES6-1 mAb (molar ratio constant at 2:1) was analyzed by varying the total dose from 0–24 µg per day. (C) The total numbers of T reg cells in the spleen were enumerated after treatment with JES6-1, IC mAb (rat IgG2a), IL-2, IL-2–IC mAb, or IL-2–JES6-1 mAb. Horizontal bars represent means. (D) Expansion of T reg cells in the spleen, MLN, liver, BM, PP, and small intestinal LP after injection of IL-2–JES6-1. (E) Number of Foxp3+ cells in the CD4+ single-positive and CD4+CD8+ double-positive thymocyte populations after IL-2–JES6-1 treatment. (F) Proliferation, as measured by CFSE dilution, of adoptively transferred CD4+CD25+ or CD4+CD25− Ly5.1+ T cells in LNs of mice injected with IL-2–JES6-1. Percentages of donor cells divided (as determined by CFSE incorporation) are shown. (G) Kinetics of T reg cell expansion in the spleen after three injections of IL-2–JES6-1. Percentages (left) and total cell numbers (right) are shown. The percentage of Foxp3+ cells after 200 µg PC61 i.p. (day −1) followed by three injections of IL-2–JES6-1 is shown (arrows). (H) Expansion of cells with the βγ IL-2R, MP CD8+ T cells and NK cells, compared with T reg cells (αβγ IL-2R) after IL-2–JES6-1 treatment. Data are shown as a fold change for each cell type compared with untreated mice over 9 d (the dashed line indicates a fold change of 1). Data in A, B, D, and G (left) are shown as percentages of CD4+ T cells. Data are representative of two to five independent experiments. Data are means ± SEM. Flow cytometry plots depict log10 fluorescence.
Figure 2.
Figure 2.
Phenotype of IL-2–JES6-1–expanded T reg cells. Mice were treated on days 0, 1, and 2 with 1 µg/5 µg IL-2–JES6-1, 1 µg IL-2, or PBS, and the phenotype of the expanded T reg cells was analyzed on day 3 or 7, as described. (A) Splenic CD4+CD25+Foxp3+ T reg cells were analyzed on day 3 by flow cytometry for expression of cell-surface molecules, CD25, GITR, TGF-β, ICOS, CD44, CD103, ICAM-1, and PD-1, and intracellular molecules, Foxp3, and CTLA-4. (B) Quantification of IL-10 and TGF-β mRNA after in vitro restimulation of FACS-sorted CD4+CD25+ or CD4+CD25− T cells isolated from mice on day 3 after treatment (reference gene, 18s RNA). (C) In vitro suppression of CD4+CD25− T cells (effectors) by FACS-sorted CD4+CD25+ T reg cells (suppressors; in indicated ratios) isolated from mice on day 3 or 7 after treatment. Proliferation of effectors was measured by incorporation of [3H]thymidine. (D) In vivo suppression of homeostatic proliferation of CD4+CD25− Thy1.1+ T cells adoptively transferred into RAG−/− hosts in a 1:1 ratio with CD4+CD25+ T reg cells isolated from C57BL/6 mice on day 3 after treatment. Thy1.1+ cell numbers were determined on day 7 after transfer. Data are representative of two to three independent experiments. Data are means ± SEM. Flow cytometry plots depict log10 fluorescence.
Figure 3.
Figure 3.
Impaired induction of EAE in mice treated with IL-2–JES6-1. EAE was induced on day 0 by immunization of MOG35-55 in CFA. Mice were treated with combinations of 1 µg IL-2, 5 µg JES6-1, and 1 mg/kg rapamycin, as described (n = 8–10 per group). (A) Disease score of mice treated on days −3, −2, and −1 with PBS, IL-2, or IL-2–JES6-1. (B) Disease score of mice treated with PBS, IL-2, or IL-2–JES6-1 at the onset of symptoms (typically days 7, 8, and 9). (C) Disease score of mice treated with PBS, IL-2–JES6-1 (days 2, 3, and 4), rapamycin (days 2, 3, and 4), or IL-2–JES6-1 (days 2, 3, and 4) plus rapamycin (days 2, 3, and 4). The treatment groups in C were further analyzed in D and E. (D) The number of CD4+IL-17+ (triangles) and CD4+IFN-γ+ (diamonds) in the draining LNs and spinal cords of mice at day 12 after MOG immunization. For the draining LNs, the percentage of cytokine-producing CD4+cells for the four groups ranged from a mean of 1.5–2% for IL-17A+ cells and 2.5–3% for IFN-γ+ cells; for the spinal cord, the mean percentage of positive cells in the control group was 11% for IL-17A+ cells and 43% for IFN-γ+ cells; and in the rapamycin group the mean was 5% for IL-17A+ cells and 30% for IFN-γ+ cells (n = 8–9 per group). (E) T reg cells quantified as cell numbers (top) and percentages (bottom) of CD4+ T cells in the draining LNs and spinal cords of mice at days 7 and 12 after MOG immunization (n = 5 per group). Data are representative of two to three independent experiments. Data are means ± SEM (A–C). Symbols represent individual mice and horizontal bars represent means in D and E. #, mice euthanized for ethical reasons.
Figure 4.
Figure 4.
Long-term allograft survival in IL-2–JES6-1–treated mice. (A) Streptozotocin-induced diabetic C57BL/6 (H2b) mice were treated with PBS (n = 10), 1 µg IL-2 (n = 4), or 1 µg/5 µg IL-2–JES6-1 (n = 34) on three consecutive days (days −3, −2, and −1). On day 0, mice were transplanted with BALB/c (H2d) islets, and BGLs were monitored as a measure of graft function and survival. Grafts were considered rejected after two consecutive BGLs >16 mmol/liter after a period of normoglycemia. Cumulative data from nine independent experiments are shown. (B) Representative hematoxylin-eosin–stained islet graft under the kidney capsule (>100 POD; left), islet graft stained for insulin (middle), and graft with minor peri-islet mononuclear accumulation (arrow; right; n = 5). (C) Mixed lymphocyte reaction using T cells from long-term engrafted mice or control naive mice as responders, and syngeneic C57BL/6, donor BALB/c, or third party CBA/Ca-irradiated splenocytes as stimulators. The proliferation of responders was measured by incorporation of [3H]thymidine. Data are means ±SEM. (D) Survival of BALB/c islet grafts in B6.RAG−/− hosts after adoptive transfer of 2 ×107 splenocytes i.v. from long-term engrafted mice (n = 9) or control naive C57BL/6 mice (n = 3). (E) Survival of BALB/c islet grafts in C57BL/6 hosts after challenge with BALB/c T-depleted splenocytes at either <100 d (n = 6) or >100 d (n = 5) after grafting. Data are shown as a Kaplan-Meier graft survival (left) or blood glucose measurement (the horizontal gray line indicates a BGL of 16 mmol/liter, the threshold for rejection; right). (F) Representative immunohistochemical staining for Foxp3+ cells in peri-islet mononuclear accumulation (inset; n = 5). Foxp3+ cells are denoted by horseradish peroxidase staining (arrowheads). I, islet graft. Data are representative of three independent experiments unless otherwise specified. Bars: (B) 100 µm; (F, left) 750 µm; (F, right) 20 µm.
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