Extended Data Fig. 6: CD8+ T cells elicited after MOG + SP immunization are specific, their suppression is mediated by perforin, adoptive transfer of CD122+CD44+Ly49+ abrogates EAE, and SP triggers a more severe, inflammatory retinal uveitis than IRBP peptide alone.
From: Opposing T cell responses in experimental autoimmune encephalomyelitis
a–g, C57BL/6J mice were immunized with an emulsion containing MOG35–55, CFA and PTX (a), MOG35–55, CFA, PTX and SP (b), or MOG35–55, CFA, PTX and influenza peptide (e). Spleen and LN cells were isolated from mice ten days after immunization, and cells were enriched for CD4+ or CD8+ T cells or APCs by FACS. CD4+ T cells from MOG-immunized mice were labelled with CellTrace Violet (CTV) and co-cultured with APCs from MOG-immunized mice in the absence (a) or presence of CD8+ T cells from wild-type mice (c) or mice immunized with MOG plus SP (b), CFA plus PTX (d), MOG plus influenza peptide (e), or CD8+ T cells from perforin-knockout (PFNKO) mice immunized with MOG plus SP (f). g, CTV-labelled CD4+ T cells from mice immunized with MOG35–55, CFA plus PTX were co-cultured with CD8+ T cells from mice immunized with MOG35–55, CFA, PTX plus SP in the presence of anti-Qa-1b antibody (10 μg ml−1). h, CTV-labelled CD4+ T cells from mice immunized with OVA329–337, CFA plus PTX were co-cultured with CD8+ T cells from mice immunized with MOG35–55, CFA, PTX plus SP. Seven days after co-culture, cells were washed and stained with surface markers and analysed for CD4+ T cell proliferation (CTV dilution). Representative data are from two independent experiments. i, C57BL/6J mice were immunized with MOG35–55, CFA, PTX plus SP (n = 10) and ten days after immunization spleen and LN cells were isolated, stained and enriched for CD8+ T cells followed by FACS for Ly49+ and Ly49− cells. Sorted Ly49+ and Ly49− cells were adoptively transferred (8 million cells per mouse) to C57BL/6J mice (n = 5 mice per group) at the time of immunization. The clinical scores after adoptive transfer and immunization are shown. ****P < 0.0001, regression analysis with two-way ANOVA followed by Bonferroni post hoc multiple comparison test. Data are mean ± s.e.m. and representative of two independent experiments. j, In the wild-type, untreated mouse eye, the retina shows a normal laminar pattern and there are no leukocytes in the vitreous. k, After subcutaneous injection of IRBP peptide antigen, there was only a mild inflammatory response in 40% of eyes with activated leukocyte invasion of the vitreous (red arrow) and mild disruption of the retina outer nuclear layer photoreceptors (black arrow). l, After subcutaneous injection of both IRBP and SP there was a severe inflammatory response in 80% of eyes with activated leukocyte invasion of the vitreous (red arrows) and severe disruption of the retina photoreceptors (black arrows). INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RGC, retinal ganglion cell layer; RPE, retinal plexiform layer. Five C57BL/6J mice were examined for each condition. EAU was induced, and mice were euthanized on day 21 after immunization. Mouse eyes were enucleated, fixed and pupil-optic nerve sections were examined by histology. m–o, C57BL/6J mice were immunized with IRBP, CFA and PTX with or without SP. Spleen and LN cells were isolated from mice 10 days after immunization, and cells were enriched for CD4+ or CD8+ T cells, or APCs by FACS. CTV-labelled CD4+ T cells from IRBP-immunized mice were co-cultured with APCs from IRBP-immunized mice and purified Ly49− T cells (n), Ly49+ T cells (o) or without CD8+ T cells (m) from mice immunized with IRBP and SP. Seven days after co-culture, cells were washed and stained with surface markers and analysed for CD4+ T cell proliferation.
