Journal: Nature
Article Title: Opposing T Cell Responses in Experimental Autoimmune Encephalomyelitis
doi: 10.1038/s41586-019-1467-x
Figure Lengend Snippet: C57BL/6J mice were immunized with an emulsion containing MOG 35–55 + CFA + PTX, MOG 35–55 + SP + CFA + PTX, or MOG 35–55 + Flu peptide + CFA + PTX. Spleen and LN cells were harvested from D10 PI mice, enriched for either CD4 + , CD8 + T cells, or antigen presenting cells (APC) using Miltenyi cell enrichment kits followed by FACS. CellTraceTM Violet (CTV) Dye labelled CD4 + T cells from MOG immunized mice were co-cultured with APC from MOG immunized mice either in the (a) absence of CD8 + T cells or in the presence of CD8 + T cells from (b) MOG + SP, (b) WT, (d) CFA + PTX, (e) MOG + flu, or (f) CD8 + T cells from Perforin knockout mice immunized with MOG + SP. (g) CTV labelled CD4 + T cells from MOG 35–55 + CFA + PTX immunized mice were co-cultured with CD8 + T cells from MOG 35–55 + SP + CFA + PTX in the presence of anti-Qa-1b antibody (10 ug/ml). In addition (h) CTV labelled CD4 + T cells from OVA 329–337 CFA + PTX were co-cultured with CD8 + T cells from MOG 35–55 + SP + CFA + PTX immunized mice. 7 days post co-culture, cells were washed and stained with surface markers and analyzed for CD4 + T cell proliferation (CTV Dye dilution). Representative data from two independent experiments. C57BL/6J mice were immunized with an emulsion containing MOG 35–55 + SP + CFA + PTX (n = 10) and 10 days post-immunization spleen and lymph node cells were isolated, stained, enriched for CD8 + T cells with Miltenyi cell enrichment kit followed by FACS to CD44 + CD122 + Ly49 + (Ly49 + ) and CD44 + CD122 + Ly49 − (Ly49 − ) cells. Sorted Ly49 + and Ly49 − cells were adoptively transferred (8 million cells/animal) to C57BL/6J mice (n = 5 mice/group) at the time of MOG 35–55 + CFA + PTX immunization. (i) The clinical scores following adoptive transfer of Ly49 + and Ly49 − cells and MOG 35–55 + SP + CFA + PTX immunization were recorded, and the significance of differences between clinical courses was calculated by regression analysis with the two-way ANOVA followed by Bonferroni post hoc multiple comparison test. **** p < 0.0001. Data are shown as mean ± SEM. Representative data from 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 (interphotoreceptor binding protein) 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 and retina outer nuclear layer photoreceptors (black arrow). (l) Following subcutaneous injection of both IRBP + 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). (RGC, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal plexiform layer). Five C57BL/6J mice were examined for each condition. EAU was induced in mice and mice were euthanized on day 21 post-immunization. Mouse eyes were enucleated, fixed, and pupil-optic nerve sections were examined by histology. C57BL/6J mice were immunized with an emulsion containing IRBP + CFA + PTX or IRBP + SP + CFA + PTX. Spleen and LN cells were harvested from Day 10 post-immunized mice, enriched for either CD4 + , CD8 + T cells, or antigen presenting cells (APC) using Miltenyi cell enrichment kits followed by FACS. CellTraceTM Violet (CTV) Dye labelled CD4 + T cells from IRBP immunized mice were co-cultured with APC from IRBP immunized mice with purified (n) CD8 + CD44 + CD122 + Ly49 + [Ly49 + ], (o) CD8 + CD44 + CD122 + Ly49 − [Ly49 − ] or (m) without CD8 + T cells from IRBP + SP immunized mice 7 days post co-culture, cells were washed and stained with surface markers and analyzed for CD4 + T cell proliferation (CTV Dye dilution).
Article Snippet: On the day of immunization and 2 days post-immunization, each mouse received 200 ng of PTX (List Biological Laboratories, 180) by intraperitoneal injection.
Techniques: Cell Culture, Knock-Out, Co-Culture Assay, Staining, Isolation, Adoptive Transfer Assay, Injection, Binding Assay, Purification
![C57BL/6J mice were immunized with an emulsion containing MOG 35–55 + CFA + <t>PTX,</t> MOG 35–55 + SP + CFA + PTX, or MOG 35–55 + Flu peptide + CFA + PTX. Spleen and LN cells were harvested from D10 PI mice, enriched for either CD4 + , CD8 + T cells, or antigen presenting cells (APC) using Miltenyi cell enrichment kits followed by FACS. CellTraceTM Violet (CTV) Dye labelled CD4 + T cells from MOG immunized mice were co-cultured with APC from MOG immunized mice either in the (a) absence of CD8 + T cells or in the presence of CD8 + T cells from (b) MOG + SP, (b) WT, (d) CFA + PTX, (e) MOG + flu, or (f) CD8 + T cells from Perforin knockout mice immunized with MOG + SP. (g) CTV labelled CD4 + T cells from MOG 35–55 + CFA + PTX immunized mice were co-cultured with CD8 + T cells from MOG 35–55 + SP + CFA + PTX in the presence of anti-Qa-1b antibody (10 ug/ml). In addition (h) CTV labelled CD4 + T cells from OVA 329–337 CFA + PTX were co-cultured with CD8 + T cells from MOG 35–55 + SP + CFA + PTX immunized mice. 7 days post co-culture, cells were washed and stained with surface markers and analyzed for CD4 + T cell proliferation (CTV Dye dilution). Representative data from two independent experiments. C57BL/6J mice were immunized with an emulsion containing MOG 35–55 + SP + CFA + PTX (n = 10) and 10 days post-immunization spleen and lymph node cells were isolated, stained, enriched for CD8 + T cells with Miltenyi cell enrichment kit followed by FACS to CD44 + CD122 + Ly49 + (Ly49 + ) and CD44 + CD122 + Ly49 − (Ly49 − ) cells. Sorted Ly49 + and Ly49 − cells were adoptively transferred (8 million cells/animal) to C57BL/6J mice (n = 5 mice/group) at the time of MOG 35–55 + CFA + PTX immunization. (i) The clinical scores following adoptive transfer of Ly49 + and Ly49 − cells and MOG 35–55 + SP + CFA + PTX immunization were recorded, and the significance of differences between clinical courses was calculated by regression analysis with the two-way ANOVA followed by Bonferroni post hoc multiple comparison test. **** p < 0.0001. Data are shown as mean ± SEM. Representative data from 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 <t>subcutaneous</t> <t>injection</t> of IRBP (interphotoreceptor binding protein) 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 and retina outer nuclear layer photoreceptors (black arrow). (l) Following subcutaneous injection of both IRBP + 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). (RGC, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal plexiform layer). Five C57BL/6J mice were examined for each condition. EAU was induced in mice and mice were euthanized on day 21 post-immunization. Mouse eyes were enucleated, fixed, and pupil-optic nerve sections were examined by histology. C57BL/6J mice were immunized with an emulsion containing IRBP + CFA + PTX or IRBP + SP + CFA + PTX. Spleen and LN cells were harvested from Day 10 post-immunized mice, enriched for either CD4 + , CD8 + T cells, or antigen presenting cells (APC) using Miltenyi cell enrichment kits followed by FACS. CellTraceTM Violet (CTV) Dye labelled CD4 + T cells from IRBP immunized mice were co-cultured with APC from IRBP immunized mice with purified (n) CD8 + CD44 + CD122 + Ly49 + [Ly49 + ], (o) CD8 + CD44 + CD122 + Ly49 − [Ly49 − ] or (m) without CD8 + T cells from IRBP + SP immunized mice 7 days post co-culture, cells were washed and stained with surface markers and analyzed for CD4 + T cell proliferation (CTV Dye dilution).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5319/pmc07145319/pmc07145319__nihms-1566265-f0012.jpg)