Antibody-mediated blockade of IL-15 reverses the autoimmune intestinal damage in transgenic mice that overexpress IL-15 in enterocytes

Abstract

Celiac disease (CD) is an autoimmune inflammatory disease with a relatively high prevalence especially in the western hemisphere. A strong genetic component is involved in the pathogenesis of CD with virtually all individuals that develop the disease carrying HLA-DQ alleles that encode specific HLA-DQ2 or HLA-DQ8 heterodimers. Consumption of cereals rich in gluten triggers a chronic intestinal inflammation in genetically susceptible individuals leading to the development of CD. Emerging evidence has implicated a central role for IL-15 in the orchestration and perpetuation of inflammation and tissue destruction in CD. Therefore, IL-15 represents an attractive target for development of new therapies for CD. Transgenic mice that express human IL-15 specifically in enterocytes (T3(b)-hIL-15 Tg mice) develop villous atrophy and severe duodeno-jejunal inflammation with massive accumulation of NK-like CD8(+) lymphocytes in the affected mucosa. We used these mice to demonstrate that blockade of IL-15 signaling with an antibody (TM-beta1) that binds to murine IL-2/IL-15Rbeta (CD122) leads to a reversal of the autoimmune intestinal damage. The present study, along with work of others, provides the rationale to explore IL-15 blockade as a test of the hypothesis that uncontrolled expression of IL-15 is critical in the pathogenesis and maintenance of refractory CD.

Fig. 1.

T3^b-hIL-15 Tg mice manifest extensive intestinal villous atrophy and influx of lymphocytes. (A) H&E-stained section of proximal small intestine of a T3^b-hIL-15 Tg mouse showing villous atrophy and massive intraepthelial lymphocytic infiltration. Magnification, 10×. (B) Flow cytometry analysis of LPL and IEL isolated from the small intestine of T3^b-hIL-15 Tg or WT mice reveals the presence of large numbers of pathogenic CD8⁺ T cells with NKG2D expression in T3^b-hIL-15 Tg mice. Gated cells represent lymphocytes. (C) Detection of Rae-1 expression in the small intestines of T3^b-hIL-15 Tg mouse by immunohistochemistry where Rae-1 expression is indicated by the presence of reddish brown precipitants. In these immunohistochemistry experiments, an isotype control was included, but the background staining was minimal with this antibody. Each of the three untreated T3^b-hIL-15 Tg mice included in the study manifested the changes shown above.

Fig. 2.

TM-β1 antibody-mediated blockade of IL-15 activity reversed the abnormal peripheral blood lymphocytosis seen in T3^b-hIL-15 Tg mice in a dose- and a time-dependent manner. PBMC were isolated from WT, T3^b-hIL-15 Tg mice while undergoing antibody treatment longitudinally. The modulation of CD8+ T cell subsets with NK and activation markers were assessed by flow cytometry. (A) CD8α and NK1.1, (B) CD8α and CD44 expression. Gated cells represent lymphocytes. Data shown were at the termination of the experiment (6 months plus 8 weeks of therapy) from one representative mouse from each group of three animals and the other mice displayed similar profiles.

Fig. 3.

TM-β1 antibody-mediated blockade of IL-15 signaling reverses the macroscopic inflammatory pathologic lesions in T3^b-hIL-15 Tg mice. (A) Small intestine, and (B) spleen and mesenteric lymph node. Data shown are from one representative mouse from each group of three animals and the other mice displayed similar profiles.

Fig. 4.

TM-β1 antibody mediated blockade of IL-15 signaling reverses the microscopic inflammatory pathologic lesions in T3^b-hIL-15 Tg mice. H&E-stained sections of small intestine of WT, T3^b-hIL-15 Tg, and TM-β1-treated T3^b-hIL-15 Tg mice with original magnification at 20×. Data shown are from one representative mouse from each group of three animals at the end of the therapeutic study period and the other mice displayed similar profiles.

Fig. 5.

TM-β1-mediated blockade of IL-15 signaling virtually eliminated pathogenic and abnormal CD8+ T cell populations from small intestinal IEL and LPL of T3^b-hIL-15 Tg mice. LPL and IEL were isolated from the small intestines of WT, T3^b-hIL-15 Tg, and TM-β1-treated Tg mice, and subjected to surface phenotypic analysis by flow cytometry (A) Cell surface expression of CD44 and CD8α on LPL. (B) Cell surface expression of CD8α and NK1.1 on LPL. (C) Cell surface expression of CD3ε and CD8α on IEL were analyzed (Left), CD8α^high, CD3ε⁺ cells were gated and analyzed for CD8β coexpression (Right). (D) Cell surface expression of CD8α and NKG2D on LPL and IEL from small intestines of TM-β1 treated T3^b-hIL-15 Tg mice were analyzed by flow cytometry. Data shown are from one representative mouse from each group of three animals and the other mice displayed similar profiles.

Fig. 6.

Proliferation of lymphocytes from T3^b-hIL-15 Tg mice depended on IL-15, and showed polyclonality. (A) Splenocytes were isolated from WT and T3^b-hIL-15 Tg mice at the termination of the experiment (6 months plus 8 weeks of treatment), and these splenocytes were cultured in vitro with 50 ng/mL of human IL-15 for 72 h. Cell proliferation was analyzed by a colorimetric assay. (B) DNA PCR gel shows normal Jβ2 chain gene rearrangement on lymphocytes of T3^b-hIL-15 Tg mice. Lane 1, DNA marker; lanes 2–13, PBMC isolated from 12 different T3^b-hIL-15 Tg mice; lane 14, splenocytes from WT mouse; lane 15, splenocytes from T3^b-hIL-15 Tg mouse.