Journal: bioRxiv

Article Title: Oligodendrocyte Precursor Cells Are Co-Opted by the Immune System to Cross-Present Antigen and Mediate Cytotoxicity

doi: 10.1101/461434

Figure Lengend Snippet: PDGFRα-Cre ER x Rosa26-YFP bred to the C57BL/6 background were kept on a 0.2% CPZ diet for a total of 4 weeks. After 3 weeks CPZ, 4-hydroxytamoxifen (1 mg/mouse/day for 3 days) was injected to induce Cre recombination in PDGFRα expressing cells. Upon recombination PDGFRα expressing cells heritably express YFP regardless of differentiation status. Approximately 8-10 million MOG35-55 specific effector T-cells were isolated and purified from 2D2 TCR transgenic mice and were injected IP into recipient mice at 4 weeks. Simultaneously, the recipient mice were put back on a normal feed diet and were sacrificed 1-2 weeks after adoptive transfer. (a) 1-week (scale bar 400 μm) and (b) 2-week images of Black Gold myelin staining (top panel). Representative images of the corpus callosum (outlined by white dashed line) of brain sections. ( a, b 2 nd row ) stained with YFP ( ) and PDGFRα ( ) allowed tracking of recombined OPCs. Representative images of the corpus callosum of brain sections stained with YFP ( ) and CC1 ( ) identified recombined mature oligodendrocytes ( a, b 3 rd row ). Representative images of the corpus callosum of brain sections stained with CD3 ( ) amd MBP( ) show the distribution of lymphocytes and myelin (4 th row). (c-d) Quantification of 1-week (c) and two-week (d) immunohistochemistry data to identify different stages of oligodendrocyte differentiation using the markers YFP, PDGFRα, and CC1. Oligodendrocyte lineage populations were compared between groups; No CPZ ( ; n=6, 8), No CPZ + AT ( ; n=8, 8), CPZ ( ; n=7, 16), and CPZ + AT ( ; n=7, 11). Significance for quantified data (c-d) was assessed by one-way ANOVA analysis followed by Tukey’s multiple comparison analysis (α = 0.05, * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). (e) Quantification of immunohistochemistry staining of total CD3 in the corpus callosum at 1-week and 2-week post adoptive transfer ( left ). (f) Of the total CD3 positive percent CD4 ( ; n=9, 7) and CD8 ( ; n=9, 8) was quantified at 1-week ( middle ) and 2-week ( right ). (g) Representative confocal images of CD3 + /CD4 + T-cell interaction ( top ) and CD3 + /CD8 + T-cell interaction ( bottom ) with YFP + oligodendrocyte lineage cell.

Article Snippet: Surface staining using the following CD8 T-cell markers; APC-CD8a (1:100; eBioscience), PE-α-Vβ5 (1:100; Biolegend), and APC eFluor 780-α-CD3 (1:100; eBioscience) was completed for 30 minutes.

Techniques: Injection, Expressing, Isolation, Purification, Transgenic Assay, Adoptive Transfer Assay, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Oligodendrocyte Precursor Cells Are Co-Opted by the Immune System to Cross-Present Antigen and Mediate Cytotoxicity

doi: 10.1101/461434

Figure Lengend Snippet: (a) Timeline and experimental design. OPCs were cultured in PDGF to inhibit differentiation and stimulated with IFNγ (10 ng/mL) for 12 hours prior to Ovalbumin (500 μg/mL) protein addition. Both IFNγ and Ovalbumin protein were incubated with OPCs for a total of 8 hours prior to washing the cultures to remove unprocessed Ovalbumin. OT-1 CD8 + T-cells were isolated by magnetic sorting then stained with Cell Proliferation Dye eFluor 450 (10 μM) prior to initiation of CD8/OPC co-culture. 24-48 hours after the start of the co-culture CD8 were analyzed for activation. (b top left) CD8 percentage, at 24 hours after the coculture was initiated, was determined using Vβ5, CD3, and CD8, and subsequently used as a parent gate for all flow plots in the figure. (b) CD8 morphology ( top ), survival ( 2 nd row ), activation status using CD25 + /CD69 + ( 3 rd row ), and proliferation ( bottom ) with quantification (left to each panel) of OT-1s cultured with OPCs; no peptide ( ), Ovalbumin ( ), IFNγ + no peptide( ), and IFNγ + Ovalbumin ( ). (c) Timeline of experimental methodology and treatment times ( bottom ), No inhibitor (No I) or each inhibitor were added to OPC culture for two hours prior to the addition of OVA257-264 or Ovalbumin, and then left in wells during processing and presentation phase. Chloroquine, an endosomal maturation inhibitor was added at 100 μM. ONX-0914, β5i/PSMB8 inhibitor was added at 30 nM. Cathepsin S (protease involved in antigen processing) inhibitor was added at 10 nM. (top) 48 hrs after CD8 co-culture was initiated cell proliferation of the Vβ5, CD3, CD8a population of CD8s was analyzed for cell division and total percent divided. Significance for all quantified data was either assessed by two-way ANOVA analysis followed by a Tukey’s multiple comparison analysis (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bars in all graphs represent standard deviation from 3 experiments. (d) Cytokine and granular protein profiling of OT-1s; IFNγ ( top ), TNFα ( 2 nd row ), perforin ( 3 rd row ) and granzyme B ( bottom ). Significance for all quantified data was assessed by one-way ANOVA analysis followed by Tukey’s multiple comparison analysis (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bars represent standard deviation for 5 biological replicates.

Article Snippet: Surface staining using the following CD8 T-cell markers; APC-CD8a (1:100; eBioscience), PE-α-Vβ5 (1:100; Biolegend), and APC eFluor 780-α-CD3 (1:100; eBioscience) was completed for 30 minutes.

Techniques: Cell Culture, Incubation, Isolation, Staining, Co-Culture Assay, Activation Assay, Standard Deviation