Involvement of IFN-γ and perforin, but not Fas/FasL interactions in regulatory T cell-mediated suppression of experimental autoimmune encephalomyelitis

Abstract

Autoaggressive, myelin-reactive T cells are involved in multiple sclerosis and its prototype experimental autoimmune encephalomyelitis (EAE) in mice. A peripheral negative feedback mechanism involving regulatory CD4+ and CD8+T cells (Treg) operates to suppress disease-mediating T cell responses. We have recently characterized a novel population of Qa-1a-restricted, TCR-peptide-reactive CD8αα+TCRαβ+ Treg that induce apoptotic depletion of the encephalitogenic Vβ8.2 cells in vivo and provide protection from EAE. Here we have used mice deficient in perforin, Fas/FasL and IFN-γ molecules to investigate their role in Treg-mediated regulation of EAE. Data show that Fas/FasL interactions are not involved, but regulation mediated by Treg is dependent on the presence of IFN-γ and the perforin pathway. These data provide a molecular mechanism of Treg-mediated killing of the pathogenic T cells and have important implications in the design of immune interventions for demyelinating disease.

Figure 1. Vaccination with p42–50 protects B10.PL.lpr mice from active EAE

Groups (4 mice in each) of wildtype B10.PL., B10.PL.lpr and B10.PL.gld mice were vaccinated i.p with 10 µg p42–50/IFA or PBS/IFA. 7 days later EAE was actively induced following immunization with MBPAc1–9/CFA/PTx. Mice were monitored daily for signs of clinical disease, and mean disease score (+/− SEM) of each group is shown. Data are representative of 3 independent experiments.

Figure 2. Vaccination with p42–50 protects B10.PL.gld mice from passive EAE

Groups (4 mice in each) of B10.PL.gld mice were vaccinated i.p with 10 µg p42–50/IFA or PBS/IFA. 7 days later, mice received 1.85×10⁶ MBP-reactive CD4+ T cells i.v. Mice were monitored daily for signs of clinical EAE disease. Mean score (+/− SEM) of each group is shown. Data are representative of two independent experiments.

Figure 3. The anti-MBP T cell response is suppressed in mice vaccinated with p42–50

Groups (4–5 mice in each) of mice were vaccinated i.p with 10 µg p42–50. 8 days later mice were injected with 100 µg MBPAc1–20/CFA/PTx. 10 days later DLN were harvested, and proliferation (upper panel) and IFN-γ secretion (lower panel) in response to an in vitro recall at an optimal concentration of 10 µg/ml MBPAc1–20 was analyzed. Data are representative of two independent experiments.

Figure 4. Successful TCR-peptide vaccination requires a Th1 environment

Groups (6–8 mice in each) of mice were nasally instilled with 10 µg B5 either without cytokines or with IL-4 (10 pg/mouse) or IL-12 (0.3 µg/mouse) to deviate the response towards a Th2 or Th1 direction, respectively (top panel). Active EAE was induced five days later with MBP/CFA/PTx. Mean disease score of each group is shown. IFN-γ is required for p42–50 to protect against EAE (lower panel). Groups (6–8 mice in each) of B10.PL IFN-γ knockout mice were vaccinated with PBS/IFA or p42–50/IFA as indicated. 7 days later mice were immunized with 200 µg MBPAc1–9/CFA/PTx for the active induction of EAE. Mean clinical disease score is shown.

Figure 5. Vaccination with p42–50 in a Th2-like environment potentiates EAE in B10.PL.wt and B10.PL.lpr mice

Groups (6–8 mice in each) of mice were vaccinated with 10 µg p42–50 in the presence of IL-4 (10 pg/mouse) to create a Th2 environment. Seven days later EAE was actively induced by MBPAc1–9/CFA/PTx immunization. Mean disease score of each group is shown.