Antigenic Stimulation of Kv1.3-Deficient Th Cells Gives Rise to a Population of Foxp3-Independent T Cells with Suppressive Properties

Abstract

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS that has been linked with defects in regulatory T cell function. Therefore, strategies to selectively target pathogenic cells via enhanced regulatory T cell activity may provide therapeutic benefit. Kv1.3 is a voltage-gated potassium channel expressed on myelin-reactive T cells from MS patients. Kv1.3-knockout (KO) mice are protected from experimental autoimmune encephalomyelitis, an animal model of MS, and Kv1.3-KO Th cells display suppressive capacity associated with increased IL-10. In this article, we demonstrate that myelin oligodendrocyte glycoprotein-specific Kv1.3-KO Th cells exhibit a unique regulatory phenotype characterized by high CD25, CTLA4, pSTAT5, FoxO1, and GATA1 expression without a corresponding increase in Foxp3. These phenotypic changes result from increased signaling through IL-2R. Moreover, myelin oligodendrocyte glycoprotein-specific Kv1.3-KO Th cells can ameliorate experimental autoimmune encephalomyelitis following transfer to wild-type recipients in a manner that is partially dependent on IL-2R and STAT5 signaling. The present study identifies a population of Foxp3(-) T cells with suppressive properties that arises in the absence of Kv1.3 and enhances the understanding of the molecular mechanism by which these cells are generated. This increased understanding could contribute to the development of novel therapies for MS patients that promote heightened immune regulation.

Figure 1. MOG-specific Kv1.3 KO Th cells express significantly more CD25 and CTLA4 but less Foxp3 than WT cells

2D2-WT and 2D2-Kv1.3 KO Th cells were cultured with irradiated WT APCs and 10 μg/ml MOG 35-55 for 72 h. Expression of CD44, CD62L, CD25, CTLA4 and Foxp3 was analyzed by FACS analysis either directly ex vivo (a,c) or after 72 hours of stimulation (b,d). Plots are gated on CD4⁺ cells. Graphs below represent data from at least 3 independent experiments.

Figure 2. 2D2-Kv1.3 KO Th cells display increased IL-10, IL-4, and IL-2 secretion and increased expression of pSTAT5 accompanied by decreased IFN-γ and IL-17 secretion and decreased phosphorylation of p56lck and Erk1/2

2D2-WT and 2D2-Kv1.3 KO Th cells were stimulated with MOG 35-55 and irradiated APCs for 0 or 72 h. (a) Production of IFNγ, IL-17, IL-4, and IL-10 were analyzed by ELISA. (b) Production of IL-2 was analyzed by ELISA. (c) 2D2-Kv1.3 KO Th cells were CFSE labeled and stimulated with 10μg/ml MOG 35-55 + irradiated APCs for 72 h in the presence or absence of 20ng/ml rIL-2. CFSE dilution in CD4+ T cells was analyzed by FACS analysis. The percent of undivided cells is shown on the histograms. (d) STAT5, p56lck, β-Actin, and (e) Akt and Erk1/2 expression and phosphorylation were determined using western blotting. Numbers below bands indicate average densitometric ratio normalized to total protein (for phospho blots) or actin loading control.

Figure 3. Kv1.3 is not required for TCR crosslinking-induced elevation in cytoplasmic free Ca²⁺ by naïve or effector T cells

(a) WT (red line) and Kv1.3 KO (blue line) Th cells were loaded with the Ca²⁺ indicator dyes Fluo-4 and Fura Red, surface stained for CD4, and suspended in RPMI either directly ex vivo or 72 h after stimulation with anti-CD3/CD28-coated beads. Th cells were precoated with biotinylated anti-CD3ε antibody and treated with streptavidin (TCR) at the indicated time (arrow). Intracellular Ca²⁺ amounts were plotted as a median ratio of Fluo-4/Fura Red over time in order to minimize the variability in dye loading. (b) WT and Kv1.3 KO Th cells were stimulated with anti-CD3/CD28 beads for 24 hours. Nuclear and cytoplasmic extracts were prepared and NFATc1 expression and nuclear translocation were determined using western blotting. Numbers below bands indicate average densitometric ratio normalized to actin loading control.

Figure 4. FoxO1 nuclear localization and GATA1 expression are increased following stimulation of Kv1.3 KO Th cells in vitro

(a) WT and Kv1.3 KO Th cells were stimulated for 24 hours. Expression of FoxO1 and β-Actin was examined in cytoplasmic and nuclear extracts. (b) 2D2-WT and 2D2-Kv1.3 KO Th cells were stimulated with MOG and irradiated APCs for 0 or 72 hours. Expression of pFoxO1/pFoxO3a, FoxO1, and β-Actin was examined in total cell lysates by western blotting. (c) WT and Kv1.3 KO Th cells were stimulated for 24, 48, or 72 hours. Expression of GATA1 and β-Actin were evaluated by western blotting. Numbers below bands indicate average densitometric ratio normalized to actin loading control.

Figure 5. Kv1.3 KO Th cells maintain a stable phenotype in a highly inflammatory environment and consistently express increased FoxO1 and GATA1 transcription factors

(a) WT and Kv1.3 KO Th cells were stimulated with anti-CD3/CD28-coated beads for 72h in the presence of 10 ng/ml IL-12 (Th1). Production of IFN-γ and IL-17 was quantified by FACS analysis. Plots are gated on CD4⁺ cells and IFNγ MFI, normalized to WT from three independent experiments, is shown. (b) WT and Kv1.3 KO Th cells were stimulated for 72 hours in the presence of anti-IL-4, anti-IFN-γ, TGF-β, IL-6, and IL-23 (Th17). Proportions of IFN-γ and IL-17 positive cells were quantified by FACS analysis. Plots are gated on CD4⁺ and IL-17 MFI, normalized to WT from three independent experiments, is shown. (c) WT and Kv1.3 KO Th cells were stimulated and polarized to Th0, Th1, Th2, Th17, and Treg for 72 hours. Expression of GATA1, pFoxO1/pFoxO3a, FoxO1, pSTAT5, and β-Actin were examined in total cell lysates by western blotting. Numbers below bands indicate average densitometric ratio normalized to actin loading control. (d) WT (white bars) and Kv1.3 KO (black bars) Th cells were stimulated for 72 hours in vitro, rested and restimulated in the presence of 10 ng/ml IL-12 (Th1) for 72 hours. Secretion of IFN-γ, IL-17, and IL-10 were evaluated by ELISA. Graphs represent data from 2 independent experiments and error bars depict SEM. *** p < 0.001.

Figure 6. Adoptive transfer of activated 2D2-Kv1.3 KO Th cells ameliorates EAE but does not induce expansion of endogenous Foxp3⁺ Tregs

2D2-WT and 2D2-Kv1.3 KO Th cells were stimulated with MOG 35-55 and irradiated WT APCs for 72 hours. Th cells were isolated by ficoll gradient. (a) CD25 and Foxp3 expression were evaluated by FACS analysis. (b) Ficolled 2D2-WT or 2D2-Kv1.3 KO Th cells were injected into WT recipients at the time of immunization with MOG 35-55. (c) Kv1.3 KO and 2D2-WT Th cells were stimulated with MOG 35-55 and irradiated WT APCs for 72hours with or without 5ng/ml TGF-β. Following isolation by ficoll gradient Th cells were transferred to WT C57BL/6 recipients at the time of immunization. Animals were monitored for EAE clinical scores. (d) 2D2-WT or 2D2-Kv1.3 KO Th cells were stimulated as described above and transferred into CD45.1 congenic recipients at time of immunization. CD45.1⁺ CD4⁺ cells from recipient lymph nodes and spleen were analyzed by FACS analysis at peak of disease for the expansion of endogenous CD25⁺Foxp3⁺ Tregs. Graph is representative of 5 animals per group and 2 independent experiments.

Figure 7. Neutralization of IL-2 results in a partial loss of the regulatory phenotype in Kv1.3 KO Th cells

2D2-WT and 2D2-Kv1.3 KO Th cells were stimulated with MOG 35-55 and irradiated APCs for 72 hours in the presence of 10 μg/ml anti-IL-2 antibody or isotype control. CD25, Foxp3 (a), pSTAT5 and STAT5 expression (b), cytokine production (c), and FoxO1 expression (d) were examined. Numbers below bands indicate average densitometric ratio normalized to actin loading control. (e) Cells described above were transferred to C57BL/6 WT recipients at the time of immunization for EAE and clinical score was evaluated.