IL-3 Is a Marker of Encephalitogenic T Cells, but Not Essential for CNS Autoimmunity

Abstract

Identifying molecules that are differentially expressed in encephalitogenic T cells is critical to the development of novel and specific therapies for multiple sclerosis (MS). In this study, IL-3 was identified as a molecule highly expressed in encephalitogenic Th1 and Th17 cells, but not in myelin-specific non-encephalitogenic Th1 and Th17 cells. However, B10.PL IL-3-deficient mice remained susceptible to experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Furthermore, B10.PL myelin-specific T cell receptor transgenic IL-3^-/- Th1 and Th17 cells were capable of transferring EAE to wild-type mice. Antibody neutralization of IL-3 produced by encephalitogenic Th1 and Th17 cells failed to alter their ability to transfer EAE. Thus, IL-3 is highly expressed in myelin-specific T cells capable of inducing EAE compared to activated, non-encephalitogenic myelin-specific T cells. However, loss of IL-3 in encephalitogenic T cells does not reduce their pathogenicity, indicating that IL-3 is a marker of encephalitogenic T cells, but not a critical element in their pathogenic capacity.

Keywords: GM-CSF; IL-3; Th17 cells; Th1 cells; experimental autoimmune encephalomyelitis; multiple sclerosis.

Figure 1

IL-3 is highly expressed in encephalitogenic Th1 and Th17 cells. Splenocytes from naïve MBP-specific T cell receptor Tg mice were differentiated into Th1 cells with plate-bound anti-CD3/28 Ab plus IL-12 or antigen-presenting cell (APC)/Ag plus IL-12. Th17 cells were differentiated with APC/Ag plus IL-6 + TGF-β or APC/Ag plus IL-6 + anti-IL4/IL12/IFNγ. (A,C) Cells were collected and transferred into B10.PL mice (5 × 10⁶ cells/mouse). Experimental autoimmune encephalomyelitis clinical scores were monitored daily. Data are representative of three independent experiments (mean ± SEM). **p < 0.01, Mann–Whitney U test. (B,E) Supernatants were analyzed by ELISA for IL-3. (D) Flow cytometric analysis of IL-17 and IFNγ in Th17 cells. (F) IL-3 and (H) GM-CSF were stained intracellularly and analyzed by flow cytometry (gated on CD4⁺ cells). (G) Illustration of IL-3 and GM-CSF loci in mouse and human chromosomes.

Figure 2

IL-3 is not required for T cell encephalitogenicity. (A) Splenocytes from naïve Il3^+/+, Il3^+/−, and Il3^−/− MBP-specific T cell receptor (TCR) Tg mice were activated with MBP Ac1-11 and feeder cells without exogenous cytokines in vitro for 3 days. Supernatants were collected and analyzed by ELISA for IL-3 and GM-CSF. (B) Il3^+/+ or Il3^−/− TCR Tg splenocytes were cocultured with Il3^+/+ or Il3^−/− feeder cells in the presence of MBP Ac1-11 peptide in vitro for 3 days. Encephalitogenic Th1 cells were differentiated with IL-12, and encephalitogenic Th17 cells were differentiated with IL-6 + anti-IL4/IL12/IFNγ. Supernatants were analyzed by ELISA for IFNγ, IL-17A, and GM-CSF. *p < 0.05, unpaired Student’s t-test. (C) Experimental autoimmune encephalomyelitis (EAE) was induced by immunization of B10.PL Il3^+/+ Il3^+/− and Il3^−/− mice with MBP Ac1-11/CFA. Disease incidence is in parentheses. Day of disease onset and maximum clinical scores for each mouse are shown for two experiments. (D,E) Splenocytes from naïve Il3^+/+ and Il3^−/− TCR Tg mice were activated with MBP Ac1-11 peptide under encephalitogenic Th1 and Th17 conditions for 3 days. (D) Th1 cells and (E) Th17 cells were collected and adoptively transferred into B10.PL mice (5 × 10⁶ cells/mouse). EAE clinical scores were monitored daily (mean ± SEM). Day of disease onset and maximum clinical scores for each mouse are shown for all replicate experiments. (F–H) Splenocytes from naïve MBP-specific TCR Tg mice were activated with MBP Ac1-11 peptide under encephalitogenic Th1 and Th17 conditions in the presence of anti-IL-3 or isotype Ab for 3 days. (F) Supernatants were analyzed by ELISA for IFNγ, IL-17A, and GM-CSF. (G) Th1 cells and (H) Th17 cells were collected and adoptively transferred into naive B10.PL mice (5 × 10⁶ cells per mouse).