Engagement of the CD137 (4-1BB) costimulatory molecule inhibits and reverses the autoimmune process in collagen-induced arthritis and establishes lasting disease resistance

Abstract

Agonistic antibodies against CD137 act as costimulators in the activation of CD8 T cells. They enhance the immune response against syngeneic tumour grafts and suppress T cell-dependent humoral immune responses in vivo. The present study was undertaken to determine whether suppression of antibody production by anti-CD137 mAb affects the development of collagen-induced arthritis (CIA). Male DBA/1J mice were immunized with bovine collagen II (CII) and treated with an agonistic anti-CD137 mAb or an isotype-matched control mAb. Mice were assessed regularly for macro- and microscopic signs of arthritis and for the appearance of collagen-specific antibody production. Interferon (IFN)-gamma determination, FACS analysis of splenocytes and histopathological joint examinations were performed after the animals were killed. Administration of anti-CD137 mAb at the time of collagen immunization blocked the development of disease and inhibited the humoral immune response against CII. Agonistic anti-CD137 mAb exhibited therapeutic efficacy even after the immune response to CII had succeeded and the disease became apparent. Furthermore, it induced a protective memory in the animals, enabling resistance to subsequent challenges with the pathogenic antigen. Our results suggest a key role for CD137 in the pathogenesis of CIA. This model provides insights into immunoregulatory conditions that control the pathogenesis of autoimmune diseases.

Figure 1

Prevention of CIA and inhibition of humoral immune response against CII in anti-CD137-treated mice. DBA/1J mice (n = 15) were immunized twice with CII (days 0 and 21) and were not treated (n = 5; circle), treated three times (days 0, 6 and 21) with isotype-matched control mAb (n = 5; diamond) or with anti-CD137 antibody (n = 5; triangle). Shown are the mean arthritis scores±SEM (a), the the mean thickness of the paws + SEM (c) and the mean of anti-CII-Ab concentration±SEM (d). Anti-CD137 treatment prevents the induction of CIA as shown in the determination of the arthritis score (a), the increase of thickness of the paws (c, percentage compared to day 0) and serum titres of anti-CII antibodies (d). (b) Pictures from left forepaws of different DBA/1J mice, which were not immunized and untreated (left), immunized with bCII only (middle, score 3) or immunized and treated with anti-CD137 antibody (right, score 0).The results of immunization with bCII with/without three injections of anti-CD137 are representative of three independent experiments.

Figure 2

Anti-CD137 treatment induces a long-lasting protection against bCII. DBA/1J mice were immunized with bCII (days 0 and 21) and treated with anti-CD137 mAb once on day 0 (n = 5, c, d) or three times on days 0, 6 and 21 (n = 4, a, b). After 112 days mice were re-challenged with bCII (vertical line). Arthritis scores (a, c) and serum concentration of anti-CII-antibodies (b, d) are shown. Each symbol represents the follow-up of one mouse.

Figure 3

Delayed treatment with anti-CD137 antibody protects mice from the development of CIA. DBA/1J mice were immunized with bCII (days 0 and 21) and treated (triangles) or not treated (circles) with a single injection of anti-CD137 mAb on day 14 after primary immunization. Shown are the mean arthritis scores±SEM (a, c) and the mean of anti-CII-Ab concentration±SEM (b, d) of two independent experiments (a, b: n = 3 and c, d: n = 4).

Figure 4

Treatment with anti-CD137 mAb at onset of the disease influences the course of CIA. DBA/1J mice (n = 5) were immunized (bCII on days 0 and 21) and treated with a single injection of anti-CD137 mAb when the first clinical signs of arthritis appeared (score = 1). Arthritis scores (a) and concentration of antibCII antibodies (b) are represented for each animal. One mouse was treated with anti-CD-137 mAb on day 27, three mice were injected on day 35 and one mouse on day 37.

Figure 5

Anti-CD 137 treatment protects joints from destruction through CIA. DBA/1J mice were killed on day 89 after priming and histology sections were prepared from knee joints and stained with haematoxylin/eosin from mice which were not immunized/not treated (a), immunized with bCII (day 0 and 21,b) and immunized with bCII and treated three times with anti-CD137 antibody, as described in Figure 1 (bCII on days 0 and 21, anti-CD137 mAb on days 0, 6 and 21,c). No abnormalities in either (a) nor (c) were seen. In contrast, in (b) the cartilage shows an irregular surface and chondrocyte proliferation. In the surrounding tissue signs of fibroblast proliferation and a minor degree of proliferation of synovial epithelium are evident (arrow). Magnification × 90. C = cartilage/chondrocytes, JS = joint space, S = synovialis.

Figure 6

Treatment with anti-CD137 antibody inreases the numbers of CD8⁺, CD4⁺ T cells and of CD4⁺ CD25⁺, CD3⁺ CD45RB⁺ and decreases the numbers of B cells. Twenty-eight days post-priming (bCII on days 0 and 21, anti-CD137 mAb or control mAb on days 0, 6 and 21) DBA/1J mice were killed and single splenocytes suspensions were recovered. FITC- or PE-conjugated antibodies against CD4, CD8, CD25, CD45RB, CD3 and CD19 surface markers were used to determine their expression using flow cytometry. Splenocytes of non-treated mice (control), of mice immunized with bCII (bCII), immunized and injected with three doses of anti-CD137 mAb (bCII + anti-CD137) and mice injected with anti-CD137 mAb alone (anti-CD137) were analysed. Numbers in each quadrant represent the percentage of cells as assayed by quadrant statistical analysis.

Figure 7

Anti-CD137 mAb treatment in DBA/1J mice induced splenomegaly and IFN-γ production in spleen cells. DBA/1J mice (each group n = 3) were immunized with bCII on days 0 and 21, treated with anti-CD137 mAb (days 0, 6 and 21) and killed on days 7, 21 and 28. (a) Splenocytes were recovered and subjected to ELISpot analysis to determine the production of IFN-γ. Spleens were cultured with (grey columns) or without (black columns) CII (0·2 mg/ml). Mean values±SEM of counted IFN-γ spots per group are represented. Control: untreated mice; bCII: mice treated with bCII alone; bCII + anti-CD 137: mice treated with bCII and anti-CD 137; anti-CD 137: mice treated with anti-CD 137 alone. (b) Spleens of killed mice on day 28 were measured, weighed and photographed. A representative spleen of each group is shown. Spleen weights of each mouse are indicated.