Transfer of Kv1.3 Deficient T Cells with Stable Regulatory Properties Ameliorates EAE in a Manner That Is Partially Dependent on IL-2

Background: Kv1.3 is a voltage gated potassium channel known to be expressed on myelin reactive T cells of patients with multiple sclerosis (MS). Deletion of Kv1.3 in T cells protects mice from experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Characterization of T cells from Kv1.3^-/- mice identified a unique population of Th cells with regulatory properties that were not dependent on the expression of Foxp3. Antigenic stimulation of Kv1.3^-/- CD4⁺T cells results in upregulation of CD25 and CTLA4, and increased pSTAT5, FoxO1, and GATA1 expression.

Objectives: To determine the stability of the Kv1.3^-/- cell phenotype and to evaluate therapeutic potential of these cells in EAE.

Methods: To address the stability of this phenotype we polarized Kv1.3 ^-/- and WT T cells to Th1, Th2, Th17, and Treg subsets and measured IFNγ, GATA3, IL17, Foxp3, FoxO1, and GATA1 expression using flow cytometry or western blotting. To test whether Kv1.3 ^-/- T-cells could ameliorate EAE, they were stimulated in vitro and transferred into WT mice that were immunized to induce EAE. Behavioral scores and Foxp3 expression in host T-cells were evaluated. IL2 was evaluated by ELISA to determine its importance for Kv1.3 ^-/-T cell mediated suppression. IL2 was inhibited by anti-IL2 in vitro and CD25, pSTAT5, FoxO1 and cytokine expression were evaluated by flow cytometry, ELISA, and western blotting. T cells treated with anti-IL2 were also transferred into WT mice that were immunized for EAE and monitored daily.

Results: Kv1.3 ^-/- T-cells that were polarized to various subsets secreted less IFNγ and IL-17 whereas GATA3 and Foxp3 were unchanged compared to WT cells. Th1, Th2, and Treg subsets of Kv1.3^-/- T cells maintained increased expression of GATA1 and FoxO1. Mice that received Kv1.3 ^-/- T cells were protected from EAE compared to mice that received WT T cells and this suppression was not due to increased endogenous Foxp3+ T-regs. Kv1.3 ^-/- T cells produced more IL2 than WT T cells. Blockade of IL2 resulted in decreased CD25 and pSTAT5 expression in Kv1.3 ^-/-T cells while FoxO1 expression was not decreased. IL4 and IL10 production was decreased when IL2 was inhibited but IFNγ and IL17 production remained low in Kv1.3 -/- T-cells. Mice receiving cells cultured with anti-IL2 were partially protected against EAE.

Conclusions: Herein we demonstrate that Kv1.3 KO CD4+ T cells maintain a stable phenotype in the face of strong inflammatory signals, suppress EAE in WT animals and this effect is partially dependent on increased IL2 signaling, highlighting the therapeutic potential for this novel subset of regulatory T cells.