Epicutaneous Immunization with Autoantigenic Peptides Induces T Suppressor Cells that Prevent Experimental Allergic Encephalomyelitis  Margaret S. Bynoe, J.Tori Evans, Christophe Viret, Charles A. Janeway  Immunity  Volume 19, Issue 3, Pages 317-328 (September 2003) DOI: 10.1016/S1074-7613(03)00239-5

Figure 1 ECi with Ac1-11 Induces Dominant Tolerance in MBP-TCR-Tg Mice (A) MBP-TCR-Tg mice were ECi with varying doses of Ac1-11: 1 mg, 100 μg, 10 μg, 1 μg, 0.1 μg, or PBS. (B) To determine whether protection is Ac1-11 specific, mice were ECi with Ac1-11 at 10 μg, with OVA at 10 μg and 100 μg concentrations, with MOG35-55 at 10 μg, with PLP139-151 at 10 μg, or with PBS for comparison. Note that the same group of mice treated with 10 μg of Ac1-11 per patch is shown in both (A) and (B). (C) MBP-TCR-Tg mice were ECi with 10 μg of Ac1-11 and given anti-IL4, anti-IL-10, anti-TGF-β monoclonal antibodies or their isotype-matched controls, or PBS prior to and during ECi with Ac1-11 as well as after EAE induction. (D) Irradiated naive B10.PL mice were adoptively transferred with 1 × 107 total splenocytes from Ac1-11 ECi or naive MBP-TCR-Tg mice (control group), or cotransferred with cells from both naive and Ac1-11 ECi mice. (E) Spleen and lymph node cells from Ac1-11 ECi mice are stained with anti-CD4 and the 3H12 clonotypic antibody (top left). Profiles for sorted CD4+/3H12+ cells (top right) and naive MBP-TCR-Tg Cα−/− splenocytes (bottom) are also shown. (F) 1 × 106 sorted Ac1-11-specific CD4 T cells from Ac1-11 ECi mice or naive MBP-TCR-Tg mice were adoptively transferred into MBP-TCR-Tg Cα−/− recipients. Immunity 2003 19, 317-328DOI: (10.1016/S1074-7613(03)00239-5)

Figure 2 Ts Cells Suppress Effector Cell Proliferation by Cell-to-Cell Contact and Are Functionally Altered (A) MBP-TCR-Tg Cα−/− mice were adoptively transferred with varying doses of sorted CD4+CD25+ T cells (0.25 × 106 [open square], 0.5 × 106 [open triangle], 1 × 106 [open circle]) or CD4+CD25− T cells (0.25 × 106 [closed square], 0.5 × 106 [closed triangle], 1 × 106 [closed circle]) from Ac1-11 ECi mice and monitored for EAE. (B) Sorted CD4 T cells from various Ac1-11 ECi mice (closed triangle, closed diamond, closed square, open circle), naive MBP-TCR-Tg mice (open triangle), or mice with end-stage EAE (closed circle) were stimulated in vitro with Ac1-11 to determine their proliferation status. (C) Sorted CD4 T cells from Ac1-11 ECi (open circle) or naive MBP-TCR-Tg mice (closed square) were cultured with APCs and varying concentrations of Ac1-11 alone; CD4 T cells from Ac1-11 ECi mice were also cultured with APCs, and fixed Ac1-11 (10 μg/ml), and varying concentrations of IL-2 (closed triangle). (D) Sorted CD4 T cells from Ac1-11 ECi mice, when cocultured with naive MBP-TCR-Tg T cells in the presence of peptide and APCs, could suppress naive MBP-TCR-Tg CD4 T cell proliferation in vitro in a dose-dependent manner. (E) Varying concentrations of anti-IL-4, anti-IL-10, anti-TGF-β mAbs, and their corresponding isotype-matched controls did not block the Ts cell-mediated suppression of naive CD4 T cell proliferation in the presence of fixed numbers of APCs, Ts cells, and antigen in vitro. (F) In a transwell assay, various numbers of Ts cells were cultured directly with fixed numbers of APCs, effector cells, and antigen (closed circle), or separated from effector cells by a membrane (closed diamond). No inhibition was observed when Ts cells were separated from effector cells, and inhibition was specific to Ts cells as CD4 T cells from nontransgenic littermates (open triangle) showed no inhibition of proliferation. Immunity 2003 19, 317-328DOI: (10.1016/S1074-7613(03)00239-5)

Figure 3 ECi with CNS Antigens (Ac1-11, PLP139-151) Suppress Spontaneous and R-R-EAE (A) Four- to six-week-old MBP-TCR-Tg Cα−/− mice when ECi with Ac1-11 show substantial protection from spontaneous EAE when compared to PBS controls. (B) Pooled spleen and lymph node cells collected 5.5 months after ECi from Ac1-11 ECi-sp mice (closed symbol) or from control mice (open symbol) were adoptively transferred into naive MBP-TCR-Tg Cα−/− recipients, and EAE was induced. (C) CD4 T cells from Ac1-11 ECi mice (closed symbol) or from naive MBP-TCR-Tg Cα−/− mice (open symbol) were transferred into naive MBP-TCR-Tg Cα−/− recipients that were monitored for spontaneous disease. (D) (SJL x PLJ)F1 mice were ECi with varying doses of PLP139-151 peptide. As controls, mice were ECi with OVA, PBS, or MOG135-155 peptide, another CNS antigen. (E) (B10.PL x SJL)F1 mice were ECi with Ac1-11 at 100 μg, 10 μg, or OVA as control. (F) Representative MHC class II/CD11c distribution for epidermal APCs prepared after epicutaneous immunization. (G) Epidermal APCs from mice ECi with Ac1-11 or Ac1-11-CFA were incubated with naive antigen-specific CD4 T cells. CD4 T cells were reisolated prior to stimulation with Ac1-11 and fresh APCs. (H) APCs from skin-draining lymph nodes of mice that were ECi with Ac1-11, Ac1-11-CFA, or PBS were cultured with naive antigen-specific CD4 T cells without exogenous antigen. (I) APCs from skin-draining lymph nodes of Ac1-11-CFA ECi mice were cultured with naive antigen-specific CD4 T cells and Ac1-11 plus anti-MHC class II (Y3JP) mAb or isotype control. Immunity 2003 19, 317-328DOI: (10.1016/S1074-7613(03)00239-5)

Figure 4 Ts Cell-Mediated Suppression of IFN-γ Secretion by Naive CD4 T Cells and Lack of CD4 T Cell Infiltration in the Brain Parenchyma of Disease-Resistant Mice (A) Sera from disease resistant (Ac1-11 ECi), diseased (EAE), and unmanipulated (naive) mice were tested for the presence of IFN-γ by ELISA prior to (week 0) and during ECi (weeks 1–2) as well as after EAE induction (weeks 3–6). (B) CD4 T cells from protected (Ac1-11 ECi) mice, normal MBP-TCR-Tg (naive) mice, or mice with active (score 1–3.5) EAE were stimulated in vitro with Ac1-11, and the supernatants were tested for IFN-γ production by ELISA. Three mice were analyzed in each group. (C) ELISA measurement of IFN-γ production in supernatants obtained from cultures in which Ts suppressed the proliferative response of naive MBP-TCR-Tg T cells to cognate stimulation. (D) No CD4 T cell infiltration is detectable in the brain parenchyma of protected mice by immunohistochemistry. Sections of perfusion-fixed brain tissue from Ac1-11 ECi mice protected from spontaneous EAE (a), induced EAE (b), naive mice (c), and mice with clinical signs of EAE (score of 1.5 [d] and 3 [e]). (E) Detailed summary of histological analysis. Immunity 2003 19, 317-328DOI: (10.1016/S1074-7613(03)00239-5)