CD137-mediated immunotherapy for allergic asthma

Abstract

The prevalence of asthma continues to increase. Asthma is caused by a Th2 cell-driven immune response. Its optimal treatment remains a challenge, and a sufficient immunotherapeutic approach to treating asthma has yet to be found. Using a murine asthma model, we show that a single injection of an anti-CD137 (4-1BB) mAb prevents the development of airway hyperreactivity, eosinophilic airway inflammation, excessive mucus production, and elevated IgE during the observation period of 7 weeks. Most importantly, even established disease is completely reversed by anti-CD137 mAb administration. The protection is associated with markedly reduced Th2 cytokine production and increased secretion of the Th1 cytokine IFN-gamma. While B lymphocytes are partly depleted, the number of CD8+ T cells is increased. Blockade of IFN-gamma and depletion of CD8+ T cells during treatment with anti-CD137 mAb reduces in part but does not abrogate the protective effect of CD137 mAb. In contrast, CD137 mAb-mediated CD4+ T cell anergy is critical for the observed effects, since transfer of CD4+ T cells from CD137 mAb-treated mice conveyed protection. These data demonstrate, for the first time to our knowledge, the capacity of anti-CD137 mAb to ameliorate allergic asthma, and they indicate CD137 as a possible target for therapeutic intervention in this disease.

Figure 1. Immunization and antibody treatments.

Prevention protocol: Mice were immunized with OVA i.p. on days 1 and 14, followed by intranasal (i.n.) OVA challenges on days 14–16, 28–30, and 42–44. Anti-CD137 mAbs were injected i.p. 1 day before the first OVA immunization. AHR was measured on days 17 and 45, and mice were sacrificed on day 18 (acute phase) or 46 (chronic phase). Reversal protocol I: Mice were immunized with OVA i.p. on day 1, followed by intranasal OVA challenges on days 8–10. To confirm the induction of AHR by OVA sensitization, AHR was measured the following day. Anti-CD137 mAb or control mAb was injected i.p. 12 hours after AHR measurement on day 11. Intranasal OVA challenge was repeated on days 15–17, and AHR was measured on day 18. Mice were sacrificed the following day. Reversal protocol II: Mice were immunized with OVA i.p. on day 1, followed by intranasal OVA challenges on days 8–10, 14–16, 21–23, and 29–31. To confirm the induction of AHR by OVA sensitization, AHR was measured the following day. Anti-CD137 mAb or control mAb was injected i.p. 12 hours after AHR measurement on day 32. Intranasal OVA challenge was repeated on days 39–41, and AHR was measured on day 42. Mice were sacrificed the following day (indicated by cross).

Figure 2. Agonistic anti-CD137 mAb prevents AHR and eosinophilic airway inflammation in OVA-immunized BALB/c mice.

(A) Methacholine-induced AHR was measured 17 and 45 days after antibody injection. At both time points, anti-CD137 mAb completely inhibited AHR in OVA-immunized mice (measured by enhanced pause [Penh]). (B) The increased cell number in the BAL fluid of OVA-immunized mice was almost completely abrogated by anti-CD137 mAb at both observation points. (C) Lung tissues from BALB/c mice sacrificed on day 46 (H&E; magnification, ×100) revealed dense peribronchiolar mononuclear cell infiltrates consisting mainly of lymphocytes and eosinophils in the OVA-immunized group. Inflammation was strongly reduced by anti-CD137 mAb. (D) Computer-based quantification of lung inflammation clearly demonstrated the significant antiinflammatory effect of anti-CD137 mAb. (E) PAS-stained lung sections (magnification, ×100) revealed mucus hypersecretion in OVA-immunized mice compared with controls, which was almost abrogated by anti-CD137 mAb. (F) Computer-based quantification of mucus production clearly verified inhibition of mucus production by anti-CD137 mAb. (G) Anti-CD137 mAb inhibited Th2 and induced Th1 cytokine production. This effect was still significant on day 46. (H) Anti-CD137 mAb almost completely inhibited proliferation of spleen cells of OVA-immunized BALB/c mice. (I) Anti-CD137 mAb significantly reduced OVA-specific IgE and IgG₁ serum levels on days 18 and 46 in OVA-immunized mice. *P < 0.05, OVA plus anti-CD137 mAb versus OVA plus control antibody by Student’s t test. (A–F) n ≥ 9 animals per group and data point; data are expressed as mean ± SEM from 3 independent experiments. (G–I) n ≥ 4 animals per group; here representative results from 1 of 3 experiments a shown.

Figure 3. Therapeutic effect of anti-CD137 mAb on the established asthma phenotype.

BALB/c mice were immunized with OVA on day 1 and challenged with OVA intranasally on days 8–10. After treatment with anti-CD137 mAb or control mAb on day 11, mice were challenged again with OVA on days 15–17. (A) OVA immunization resulted in methacholine-induced AHR as measured 1 day after the last intranasal OVA challenge (day 10). Injection of anti-CD137 mAb 12 hours after the first AHR measurement (day 11) completely inhibited this already established AHR as demonstrated 7 days later. (B) After the last challenge with OVA (day 17), AHR was assessed invasively on day 18, and results are presented as lung resistance (R_L) and dynamic compliance (Cdyn). (C) Mice were sacrificed on day 18, and BAL was performed. Anti-CD137 mAb almost completely abrogated the total cell number in the BAL fluid of OVA-immunized BALB/c mice. (D) Anti-CD137 mAb converted an established Th2 to a Th1 cytokine response in OVA-immunized BALB/c mice. (E) Anti-CD137 mAb inhibited proliferation of spleen cells from OVA-immunized BALB/c mice. (F) Anti-CD137 mAb reduced OVA-specific IgE and IgG₁ serum levels in OVA-immunized BALB/c mice. *P < 0.05, OVA plus anti-CD137 mAb versus OVA plus control antibody by Student’s t test. (A and B) n ≥ 9 animals per group and data point; data are expressed as mean ± SEM from 3 independent experiments. (c–e) n ≥ 4 animals for each group; here representative results from 1 of 3 experiments a shown.

Figure 4. Therapeutic effect of anti-CD137 mAb in a chronic asthma model.

BALB/c mice were immunized with OVA on day 1 and challenged with OVA several times intranasally during the following 31 days. Lung function was measured on day 32. Anti-CD137 mAb was injected the same day, followed by further intranasal challenges with OVA (days 39–41). (A) OVA immunization resulted in methacholine-induced AHR as measured 1 day after the last intranasal OVA challenge (day 32). Injection of anti-CD137 mAb 12 hours after the first AHR measurement completely inhibited this already established AHR as demonstrated 10 days later. (B) Mice were sacrificed on day 43, and BAL was performed. Anti-CD137 mAb almost completely abrogated the total cell number in the BAL fluid of OVA-immunized BALB/c mice. (C) Anti-CD137 mAb reduced OVA-specific IgE serum levels in OVA-immunized BALB/c mice. (D) Anti-CD137 mAb converted an established Th2 to a Th1 cytokine response in culture supernatants of splenocytes and lung cells in OVA-immunized BALB/c mice. (E) Quantification of lung inflammation and mucus production by a computer-based image-analyzing program clearly demonstrated the inhibitory effect of anti-CD137 mAb. *P < 0.05, OVA plus anti-CD137 mAb versus OVA plus control antibody by Student’s t test. (a, b, and e) n ≥ 8 animals per group and data point; data are expressed as mean ± SEM from 2 independent experiments. (c and d) n ≥ 4 animals for each group; here representative results from 1 of 2 experiments a shown.

Figure 5. Therapeutic effect of anti-CD137 mAb on airway remodeling.

BALB/c mice were immunized with OVA on day 1 and challenged with OVA intranasally several times according to reversal protocol II (Figure 1), described in Methods. After application of anti-CD137 mAb or control mAb on day 32, mice were challenged again with OVA on days 39–41. On day 43, mice were sacrificed. (A) MSB-stained lung tissues (magnification, ×400) from BALB/c mice revealed increased peribronchiolar ECM deposition, which was reduced by anti-CD137 mAb. (B) For quantification of ECM deposition, digital photographs of 4 bronchioles per tissue section were taken and analyzed with a computer-based image-analyzing program, demonstrating the protective effect of anti-CD137 mAb. (C) Total lung collagen was increased in OVA-immunized mice compared with control animals. This effect was partly abrogated (62%) by anti-CD137 mAb application. *P < 0.05, OVA plus anti-CD137 mAb versus OVA plus control antibody by Student’s t test. n ≥ 8 animals per group; data are expressed as mean ± SEM from 2 independent experiments.

Figure 6. The anti-CD137 mAb–mediated therapeutic effect depends partly on CD8⁺ T cells and IFN-γ.

(A) CD8⁺ T cell depletion and (B) anti–IFN-γ mAb application significantly increased AHR (measured on day 18) in anti-CD137 mAb–treated OVA-immunized BALB/c mice compared with control mice. (C) Depletion of CD8⁺ T cells and (D) application of anti–IFN-γ mAb before anti-CD137 mAb treatment increased the total cell number and the number of eosinophils in BAL fluid of anti-CD137–treated OVA-immunized mice. (E) Depletion of CD8⁺ T cells and (F) application of anti–IFN-γ mAb had no effect on the decreased levels of Th2 cytokines after anti-CD137 mAb treatment, whereas the CD137-mediated increase in IFN-γ production was abrogated. *P < 0.05, OVA plus anti-CD137 mAb versus OVA plus anti-CD137 mAb plus anti-CD8 mAb; ***P < 0.001, OVA plus anti-CD137 mAb versus OVA plus anti-CD137 mAb plus anti–IFN-γ mAb by Student’s t test. (a–d) Data are expressed as mean ± SEM; n ≥ 9 animals per group and data point for 3 independent experiments. (E and F) Data are expressed as mean ± SEM; n ≥ 4 animals for each group, and representative results from 1 of 3 experiments are shown.

Figure 7. Anti-CD137 mAb induces CD4⁺ T cell anergy.

BALB/c mice were immunized with OVA in Alum i.p. on day 1 and treated with anti-CD137 mAb or control mAb on day 3. Mice were sacrificed 5 days later, and CD4⁺ T cells were isolated and restimulated in vitro with OVA. (A) Transfer of CD4⁺ T cells from OVA-immunized mice into SCID mice induced AHR in response to methacholine. This effect was abrogated when the OVA-immunized mice were treated with anti-CD137 mAb before cell transfer. Data are expressed as Penh (mean ± SEM; n ≥ 7 animals per group and data point, 2 independent experiments). (B) CD4⁺ T cells that were separated from OVA-immunized BALB/c mice proliferated in response to OVA. In contrast, CD4⁺ T cells separated from OVA-immunized BALB/c mice that had received anti-CD137 mAb did not proliferate. CD4⁺ T cells separated from OVA-immunized mice that had received anti-CD137 mAb did proliferate again when treated with IL-2. Data are expressed as mean cpm ± SEM; n ≥ 4 animals for each group. Representative results from 1 of 2 experiments are shown. (C) IL-2 abrogated anti-CD137 mAb–mediated inhibition of Th2 cytokine production in cell culture supernatants of OVA-immunized BALB/c mice. Data are expressed as mean ± SEM; n ≥ 4 animals for each group. Representative results from 1 of 2 experiments are shown. *P < 0.05, OVA versus OVA plus anti-CD137 mAb; ^#P < 0.05, OVA plus anti-CD137 mAb versus OVA plus anti-CD137 mAb plus IL-2 by Student’s t test.