Figure 5.

T-eff isolated from the acutely inflamed CNS are not suppressible by T-reg. Mononuclear cells from the spleen and the CNS of EAE mice were isolated at the peak of disease and FACS sorted for CD4⁺Foxp3/GFP⁻ (T-eff) and CD4⁺Foxp3/GFP⁺ (T-reg). (a) The T-reg populations were tested in MOG-specific suppression assays and in suppression assays driven by antibody to CD3 in a 1:1 ratio with naive MOG TcR-transgenic T cells (2D2) or with CNS-T-eff or splenic T-eff from EAE mice as indicated. The proliferation of responder cells was measured by 3[H]thymidine incorporation. Mean of triplicate assays (+ s.d.). *P < 2 × 10⁻⁹, **P < 3 × 10⁻⁴, ***P < 0.008, t-test. Supernatants were taken after 48 h and the cytokines IL-6 and TNF were analyzed by cytometric bead array. (b) Cytokine expression in T-eff from lymph nodes (LN), spleen (SPL) and the CNS at the peak of disease (day 14). Mononuclear cells were isolated as described and FACS purified based on the T-eff phenotype (CD4⁺Foxp3/GFP⁻). cDNA was prepared and quantitative PCR was performed for IL-6 and TNF. One of two independent EAE experiments. (c) The combination of IL-6 and TNF-α induced a complete block in T-reg–mediated inhibition of T-eff from unimmunized mice. CD4⁺Foxp3/GFP⁺ T-reg were isolated from naive Foxp3gfp.KI mice and tested in a ratio of 1:1 for suppression of CD4⁺Foxp3/GFP⁻ T-eff from unimmunized Foxp3gfp.KI mice upon polyclonal stimulation with antibody to CD3. Cytokines were added at a concentration of 25 ng/ml or titrated as indicated (in ng/ml). Proliferation was determined as 3[H]thymidine incorporation. Mean (+ s.d.) of triplicate cultures. Supernatants were collected after 48 h and assessed for IL-17 by ELISA (ND, not detectable).