Targeting the IL-15 receptor with an antagonist IL-15 mutant/Fc gamma2a protein blocks delayed-type hypersensitivity

Abstract

Owing to shared receptor components, the biologic activities of IL-15 are similar to those of IL-2. However, the patterns of tissue expression of IL-2/IL-2R alpha and IL-15/IL-15R alpha differ. The development of agents targeting the receptor and signaling elements of IL-15 may provide a new perspective for treatment of diseases associated with expression of IL-15/IL-15R. We designed, genetically constructed, and expressed a receptor site-specific IL-15 antagonist by mutating glutamine residues within the C terminus of IL-15 to aspartic acid and genetically linked this mutant IL-15 to murine Fc gamma2a. These mutant IL-15 proteins specifically bind to the IL-15R, competitively inhibit IL-15-triggered cell proliferation, and do not activate the STAT-signaling pathway. Because the receptor site-specific antagonist IL-15 mutant/Fc gamma2a fusion proteins had a prolonged t(1/2) in vivo and the potential for destruction of IL-15R+ leukocytes, we examined the immunosuppressive activity of this agent. An IL-15 mutant/Fc gamma2a fusion protein markedly attenuated Ag-specific delayed-type hypersensitivity responses and decreased leukocyte infiltration within the delayed-type hypersensitivity sites. These findings suggest that 1) IL-15/IL-15R+ cells are crucial to these T cell-dependent immune responses, and 2) treatment with IL-15 mutant/Fc gamma2a protein may ameliorate T cell-dependent immune/inflammatory diseases.

FIGURE 1

Western analysis of IL-15 mutant/Fcγ2a fusion protein. SDS-PAGE of IL-15 mutant/Fcγ2a fusion protein under reducing (lanes 1 and 3) and nonreducing (lanes 2 and 4) conditions shows homodimerization of single species of protein. This protein is immunoreactive with both anti-IgG2a Ab (lanes 1 and 2) and anti-IL-15 Ab (lanes 3 and 4).

FIGURE 2

IL-15 mutant/Fcγ2a protein specifically binds to the IL-15R. IL-3-sensitive BAF-BO3 cells were washed and incubated with medium alone (dotted line) or medium supplemented with IL-15 mutant/Fcγ2a protein (bold line), followed by interaction with FITC-conjugated goat anti-mouse Fc Ab (A). The stained IL-15R⁺ cells were analyzed by flow cytometry. This binding was blocked by molar excess of rhIL-15 (B). Excess amounts of rhIL-2 or 4G3/3E12 rat anti-mouse IL-2Rγ Ab did not inhibit the binding of IL-15 mutant/Fcγ2a protein to its receptors (C and D). The data presented are representative of results achieved in three separate experiments.

FIGURE 3

IL-15 mutant/Fcγ2a protein competitively blocks IL-15, but not IL-2- or IL-3-rich medium-triggered cell proliferation. IL-2Rβ⁺ BAF-BO3 cells were incubated with rhIL-15 (10 ng/ml) in the presence of different concentrations of IL-15 mutant/Fcγ2a protein (filled circle) for 48 h, followed by 6 h of [³H]TdR pulse, and cell-incorporated radioactivity was counted in a scintillation counter. The IL-15 mutant/Fcγ2a protein blocked rhIL-15-driven BAF-BO3 cell proliferation in dose-dependent manner. But rhIL-2 (open square)- or IL-3-rich medium (open triangle)-dependent cell growth was not lessened in the presence of IL-15 mutant/Fcγ2a proteins. The data = mean ± SD of triplicate experiments. Similar results were obtained in three separate experiments.

FIGURE 4

IL-15 mutant/Fcγ2a protein fails to trigger tyrosyl phosphorylation of STAT3/STAT5. IL-2Rβ⁺ BAF-BO3 cells were washed, and restimulated with buffer alone, rhIL-2 (50 U/ml), or 10 ng/ml of either human rIL-15 or IL-15 mutant/Fcγ2a protein for 2 min at 37°C. Followed by SDS-PAGE and transfer onto membrane, immunostaining with phosphospecific STAT3 Ab (first row), anti-STAT3 Ab (second row), anti-phosphotyrosine Ab (third row), and anti-STAT5 Ab (fourth row) showed lack of phosphorylation of STAT3 and STAT5 in case of stimulation by IL-15 mutant/Fcγ2a protein. Cell lysates were obtained after stimulation with media (A), rhIL-2 (B), IL-15 mutant/Fcγ2a (C), or rhIL-15 (D).

FIGURE 5

IL-15 mutant/Fcγ2a protein has prolonged circulating t_1/2 (6 h). The time-related serum concentration was determined following a single bolus i.v. dose (6 µg) of the fusion protein. Blood samples were obtained by retroorbital bleeding at the indicated intervals. The levels of mutant fusion protein were detected by ELISA with rat anti-human IL-15 Ab as capture Ab and horseradish peroxidase-conjugated rat anti-mouse IgG2a Ab as the detection Ab. Four mice were used for determining the t_1/2. Data = mean ± SD.

FIGURE 6

Immunohistochemical staining for the presence of macrophages/monocytes (12 h after Ag rechallenge) with anti-mouse F4/80 IgG2b. The arrows denote for F4/80⁺ cells in specimens obtained from mice undergoing: mouse IgG treatment (a), IL-15 mutant/Fcγ2a protein treatment (b), mouse IgG and CsA treatment (c), and IL-15 mutant/Fcγ2a and CsA treatment (d). The decrease in footpad swelling and cell infiltration in IL-15 mutant/Fcγ2a ± CsA-treated groups is parallel.

FIGURE 7

IL-15 mutant/Fcγ2a treatment decreased the infiltration of CD4⁺ T cells in the footpads. Immunohistochemical staining was done against CD4⁺ T cells (12 and 24 h after Ag rechallenge). Ten randomly selected fields (each 100 µm²) were scored on ethanol-fixed tissue sections. Numbers of cells are expressed in mean ± SE.