Resolution of psoriasis upon blockade of IL-15 biological activity in a xenograft mouse model

Abstract

Psoriasis is a chronic inflammatory disease of the skin characterized by epidermal hyperplasia, dermal angiogenesis, infiltration of activated T cells, and increased cytokine levels. One of these cytokines, IL-15, triggers inflammatory cell recruitment, angiogenesis, and production of other inflammatory cytokines, including IFN-gamma, TNF-alpha, and IL-17, which are all upregulated in psoriatic lesions. To investigate the role of IL-15 in psoriasis, we generated mAb's using human immunoglobulin-transgenic mice. One of the IL-15-specific antibodies we generated, 146B7, did not compete with IL-15 for binding to its receptor but potently interfered with the assembly of the IL-15 receptor alpha, beta, gamma complex. This antibody effectively blocked IL-15-induced T cell proliferation and monocyte TNF-alpha release in vitro. In a human psoriasis xenograft model, antibody 146B7 reduced the severity of psoriasis, as measured by epidermal thickness, grade of parakeratosis, and numbers of inflammatory cells and cycling keratinocytes. These results obtained with this IL-15-specific mAb support an important role for IL-15 in the pathogenesis of psoriasis.

Figure 1

Recognition of receptor-bound IL-15 by mAb 146B7 and inhibition of IL-15–induced effects. (a) Biotinylated mAb 146B7 (filled squares) showed dose-dependent binding to IL-15 bound to IL-15Rα, which was coated onto an ELISA plate, whereas biotinylated 404E4 (filled triangles) did not show binding. Polyclonal huIgG1 (open squares) served as a control. This experiment was repeated two times, yielding similar results. (b) Dose-dependent binding of biotinylated mAb 146B7 (filled squares) to IL-15 bound to Raji lymphoma cells expressing IL-15Rα was shown by flow cytometry. Polyclonal huIgG1 (open squares) was used as a control. This experiment was repeated five times, yielding similar results. MFI, mean fluorescence intensity. (c) Effect of mAb 146B7 on IL-15–induced proliferation. Human PBMCs were incubated with IL-15 (12.5 ng/ml) in combination with mAb 146B7 (filled squares), mAb 404E4 (filled inverted triangles), or with huIgG1 (open squares) for 72 hours. BrdU incorporation was measured to assay proliferation. Representative data of 9 individual experiments are shown. (d) mAb 146B7 inhibits IL-15– but not IL-2–induced TNF-α production. Human PBMCs were incubated with IL-15 (0, 25, 100 ng/ml) or with IL-2 (100 ng/ml) in combination with mAb 146B7 at various concentrations for 72 hours. The amounts of TNF-α produced were measured by ELISA. A representative experiment from a series of 11 experiments is shown.

Figure 2

mAb 146B7 inhibits protection of apoptosis by IL-15. Human PBMCs were incubated with IL-15 (5 ng/ml) in culture medium alone (filled squares) or in combination with 10 μg/ml mAb 146B7 (open circles) or with 10 μg/ml isotype control antibody (filled triangles) for 5 days. Apoptosis was measured using annexin V conjugated with FITC and PI. Live (a and d), early (b and e), and late (c and f) apoptotic cells were defined by annexin V-FITC^negPI^neg, annexin V-FITC^posPI^neg, and annexin V-FITC^posPI^pos staining by flow cytometry, respectively. Representative data of three individual experiments with six different PBMC donors are shown. (a–c) mAb 146B7 inhibits IL-15–induced survival of monocytes. (d–f) No significant differences in comparison were found in the fraction of live and apoptotic T cells. *P < 0.05, mAb 146B7 versus no antibody treatment. **P < 0.005, mAb 146B7 versus no antibody treatment, and mAb 146B7 versus anti-KLH.

Figure 3

Epitope specificity of human anti-IL-15 antibodies. mAb 146B7 (a) and mAb 404E4 (b) were tested for binding to human IL-15 (filled squares), IL-15 mutant Q108S (filled inverted triangles; Gln at residue 108 was replaced by Ser; a mutation in the γ-chain interaction site), and mutant D8SQ108S (filled triangles; Gln at residue 108 was replaced by Ser, and Asp at position 8 was substituted for Ser; mutations in both the β and γ-chain interaction sites of IL-15 with its receptor) (34).

Figure 4

mAb 146B7 effectively alleviates psoriasis in a psoriasis SCID mouse xenograft model. SCID mice engrafted with human psoriatic skin were treated i.p. with PBS, mAb 146B7, or CsA. Skin grafts were harvested after treatment, embedded in paraffin, and stained with H&E (upper panels) or for Ki-67 nuclear antigen (lower panels). The tissue samples shown are from the same patient and are representative experiments with psoriatic skin obtained from four different patients.

Figure 5

Effect of mAb 146B7 on psoriasis in a psoriasis SCID mouse xenograft model. Quantitative representation of data on the effect of mAb 146B7 shown in Figure 4. SCID mice engrafted with human psoriatic skin were treated i.p. with PBS (n = 15), mAb 146B7 (n = 15), or CsA (n = 16). Skin grafts were harvested after treatment, embedded in paraffin, stained with H&E or for Ki-67 nuclear antigen, and evaluated as indicated for epidermal thickness (a), numbers of inflammatory mononuclear cells in upper dermis (b), numbers of Ki-67⁺ cycling keratinocytes (c), and grades of parakeratosis (d). Values shown in a–c represent mean ± SEM. Values shown in d represent percentages of mice in each treatment group obtaining the score 0 (white), 1 (light gray), 2 (dark gray), or 3 (black), respectively. The reductions induced by mAb 146B7 compared with PBS were statistically significant for all measured parameters (*P < 0.05, **P < 0.005).

Figure 6

mAb 146B7 results in a reduction in the number of CD57⁺, CD68⁺, CD4⁺, and CD8⁺ cells in the psoriasis SCID xenograft model. SCID mice engrafted with human psoriatic skin were treated i.p. with PBS (n = 15), mAb 146B7 (n = 15), or with CsA (n = 16). Skin grafts were harvested after treatment, embedded in paraffin, and stained for CD57⁺ NK cells (a), CD68⁺ macrophages (b), CD4⁺ T cells (c), and CD8⁺ T cells (d), and evaluated for number of positively stained cells. Values shown represent mean ± SEM. *P < 0.05 (PBS compared with mAb 146B7).

Figure 7

A recent paper by Dubois et al. (40) provided new insight in the mechanism of action of IL-15. The formation of IL-15/IL-15Rα complexes on cell surfaces enables transendosomal recycling of IL-15, leading to persistence of surface-bound IL-15 (a). IL-15/IL-15Rα complexes may induce signaling via IL-15Rβ− and γ-chains expressed on the same cell in cis (b) or expressed on a distinct cell in trans (c). Antibody 146B7 binds at the interaction site of IL-15 with the γ-chain (d) and may effectively block signaling through the γ-chain both in cis and in trans (e).