Cbl-b-deficient mice express alterations in trafficking-related molecules but retain sensitivity to the multiple sclerosis therapeutic agent, FTY720

Abstract

The variable response to therapy in multiple sclerosis (MS) suggests a need for personalized approaches based on individual genetic differences. GWAS have linked CBLB gene polymorphisms with MS and recent evidence demonstrated that these polymorphisms can be associated with abnormalities in T cell function and response to interferon-β therapy. Cbl-b is an E3 ubiquitin ligase that regulates T cell activation and Cbl-b-deficient (Cbl-b(-/-)) mice show T cell abnormalities described in MS patients. We now show that Cbl-b(-/-) T cells demonstrate significant lymph node trafficking abnormalities. We thus asked whether the MS-approved drug, FTY720, postulated to trap T cells in lymphoid tissues, is less effective in the context of Cbl-b dysfunction. We now report that FTY720 significantly inhibits EAE in Cbl-b(-/-) mice. Our results newly document a role for Cbl-b in T cell trafficking but suggest nevertheless that MS patients with Cbl-b abnormalities may still be excellent candidates for FTY720 treatment.

Keywords: Casitas-B lineage lymphoma-b; Experimental autoimmune encephalomyelitis; FTY720; Multiple sclerosis; Sphingosine-1-phosphate receptor 1; T cell trafficking.

Figure 1

Decreased accumulation of Cbl-b^−/− CD4⁺ T cells in mLNs of RAG-1^−/− mice after adoptive transfer. 0.9–1.4 × 10⁶ WT or Cbl-b^−/− CD4⁺ CD25 T cells were injected i.p. into RAG-1^−/− mice and mLNs and pLNs were harvested on day 4 and day 8–11 for analysis. (A and B) Absolute number of transferred WT or Cbl-b^−/− CD4⁺ T cells in LNs of recipient RAG-1^−/− mice on day 4 (A), and day 8–11 (B) post transfer. (C) Frequency of transferred WT or Cbl-b^−/− CD4⁺ T cells in LNs of recipients on day 8–11 post transfer. (D) Frequency of WT and Cbl-b^−/− CD62L⁺ CD4⁺ T cells in mLNs and pLNs of recipients on day 8–11 post transfer. *p<0.05, ***p=0.0001, ****p<0.0001 by Student’s t-test. n=7–9/group for A, B, and C. n=6/group for D. The data shown are from three (A), five (B and C) and two (D) independent experiments.

Figure 2

Increased frequency of S1P₁⁺ CD4⁺ T cells in draining LNs of Cbl-b^−/− mice after CFA/MOG_35–55 immunization. WT and Cbl-b^−/− mice were immunized s.c. in the footpads with 150 µg of CFA and 80 µg of MOG_35–55 and the popliteal LNs were harvested 4–5 days later. (A) Percentage of CD4⁺ T cells (left) and CD8⁺ T cells (right) that are S1P₁⁺ in the popliteal LNs of WT and Cbl-b^−/− mice. (B) Percentage of CD4⁺ T cells (left) and CD8⁺ T cells (right) that are S1P₁⁺ in the mLNs of WT and Cbl-b^−/− mice. **p<0.01 by Student’s t-test. The data shown are from three independent experiments.

Figure 3

Decreased frequency of CD69⁺ CD4⁺ T cells in draining LNs of Cbl-b^−/− mice after CFA/MOG_35–55 immunization. WT and Cbl-b^−/− mice were immunized as in Figure 2 and the popliteal LNs were harvested 4–5 days later. (A) Percentage of CD4⁺ T cells (left) and CD8⁺ T cells (right) that are CD69⁺ in the popliteal LNs of WT and Cbl-b^−/− mice. (B) Percentage of CD4⁺ T cells (left) and CD8⁺ T cells (right) that are CD69⁺ in the mLNs of WT and Cbl-b^−/− mice. *p<0.05, ****p<0.0001 by Student’s t-test. The data shown are from three independent experiments.

Figure 4

CCR7 expression is unaltered in Cbl-b^−/− T cells after CFA/MOG_35–55 immunization. WT and Cbl-b^−/− mice were immunized, the popliteal LNs harvested as in Figure 2, and stained with human CCL19-Ig and anti-human IgG-Alexa Fluor 488. (A) MFI of CCR7 expression on CD4⁺ T cells (left) and on CD8⁺ T cells (right) in draining popliteal LNs of WT and Cbl-b^−/− mice. (B) MFI of CCR7 expression on CD4⁺ T cells (left) and on CD8⁺ T cells (right) in the mLNs of WT and Cbl-b^−/− mice. Results were analyzed using Student’s t-test. The data shown are from three independent experiments.

Figure 5

Equal lymphopenic response of WT and Cbl-b^−/− mice induced by the S1P lyase inhibitor, THI. WT and Cbl-b^−/− mice were administered 10 mg/kg of THI or vehicle (VC) via gavage, and percent of CD4⁺ and CD8⁺ T cells in the total blood leukocytes was determined by flow cytometry. (A) Percentage of blood CD4⁺ T cells and (B) Percentage of blood CD8⁺ T cells of WT or Cbl-b^−/− mice, 24 and 48 hrs after administration of VC or THI. Percentages represent mean percentage of T cells in THI-treated mice / mean percentage of T cells in VC-treated mice x100 at each time point. Results were analyzed using Student’s t-test. n=3–4/group. The data shown are from two independent experiments.

Figure 6

Equal lymphopenic response of WT and Cbl-b^−/− mice induced by the selective S1P₁ agonist, SEW2871. WT and Cbl-b^−/− mice were administered 20 mg/kg of SEW2871 or vehicle (VC) via gavage, and percentage of CD4⁺ and CD8⁺ T cells in the total blood leukocytes was determined by flow cytometry. (A) Percentage of blood CD4⁺ T cells and (B) Percentage of blood CD8⁺ T cells of WT or Cbl-b^−/− mice treated with VC or SEW2871 at 14, 22 and 38 hrs after treatment. Percentages represent mean percentage of T cells in SEW2871-treated mice / mean percentage of T cells in VC-treated mice x100 at each time point. **p<0.01, ***p<0.001 by Student’s t-test. n=4–7/group. The data shown are from five independent experiments.

Figure 7

Efficacy of FTY720 in inhibiting EAE in Cbl-b^−/− mice. EAE was induced in WT and Cbl-b^−/− mice by s.c. immunization with 165 µg of MOG_35–55 and 300 µg of CFA. 150 ng of Pertussis toxin was also given i.p. on day 0 and 2. On the day of EAE-induction, the mice were administered either 0.375 mg/kg of FTY720 (FTY) or vehicle (VC) via gavage. FTY720 or vehicle was then administered every five days until day 20. Clinical scores: 1 = tail flaccidity, 2 = abnormal gait, 3 = hind leg paralysis, 4 = front leg paralysis, 5 = death. The disease severity differed significantly between the VC-treated WT group vs. the VC-treated Cbl-b^−/− group, ***p=0.0004. In addition, the disease severity differed significantly between the VC-treated WT group vs. the FTY720-treated WT group, ****p<0.0001, and also between the VC-treated Cbl-b^−/− group vs. the FTY720-treated Cbl-b^−/− group, ****p<0.0001 by Mann-Whitney test. The data shown are from two independent experiments (n = 7–8/treatment group in total).