Journal: Science Advances

Article Title: Epstein-Barr virus infection promotes T cell dysregulation in a humanized mouse model of multiple sclerosis

doi: 10.1126/sciadv.adu5110

Figure Lengend Snippet: ( A ) Experimental design: Donor PBMCs isolated from women with or without a history of EBV infection or an RRMS diagnosis were used to engraft immunocompromised NSG mice. Following a 3-week reconstitution period and confirmation of circulating human CD45 + cell repopulation, humanized NSG mice (HuPBMC) were immunized with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) antigens to induce EAE. ( B ) Donor serum IgG specific to acute EBV antigen viral capsid antigen (VCA). ( C ) Donor serum immunoglobulin G (IgG) specific to latent EBV antigen Epstein-Barr nuclear antigen 1 (EBNA-1). In (B) and (C), group data are shown as means with SEM and were curve fit with a one-site total binding equation. Statistical differences in titer curves were assessed by ordinary two-way analysis of variance (ANOVA). ( D ) Cell-associated EBV viral loads in donor PBMCs measured by BALF5 quantitative polymerase chain reaction (qPCR) assay. Data are shown as means with SEM and were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. In (B) to (D), n = 3 to 4 donors per group and the lower limit of detection (LOD) for each assay is represented by a dotted line. ( E ) Clinical disease scores post-induction for symptomatic HuPBMC EAE mice. Data are shown as means with SEM, and curves were analyzed by ordinary two-way ANOVA ( n = 17 to 25 mice per group derived from three to four donors per group). ( F ) Incidence of clinical EAE symptoms post-induction. Data are shown as percentage of the group, and curves were analyzed by log-rank (Mantel-Cox) test ( n = 54 to 62 mice per group derived from three to four donors per group). ( G ) Day of EAE symptom onset post-induction (DPI). Distribution of individual data is shown with median and quartiles (dashed lines) and was analyzed by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test ( n = 17 to 25 mice per group derived from three to four donors per group).

Article Snippet: Whole blood (80 ml) was processed for PBMC isolation by Lymphoprep (STEMCELL Technologies, no. 07801) gradient separation, according to the manufacturer’s instructions, within an hour of collection in K 2 -EDTA–coated vacutainer tubes (BD, no. 366643).

Techniques: Isolation, Infection, Biomarker Discovery, Recombinant, Binding Assay, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Science Advances

Article Title: Epstein-Barr virus infection promotes T cell dysregulation in a humanized mouse model of multiple sclerosis

doi: 10.1126/sciadv.adu5110

Figure Lengend Snippet: PBMC donor demographics, disease characteristics, and serology. All RRMS and healthy donors were female. RRMS participants were treatment naïve and in clinical remission at the time of donation. HD, healthy donor; EDSS, expanded disability status scale; VCA, viral capsid antigen; EBNA-1, Epstein-Barr nuclear antigen 1; CMV, cytomegalovirus; NA, not applicable.

Article Snippet: Whole blood (80 ml) was processed for PBMC isolation by Lymphoprep (STEMCELL Technologies, no. 07801) gradient separation, according to the manufacturer’s instructions, within an hour of collection in K 2 -EDTA–coated vacutainer tubes (BD, no. 366643).

Techniques:

Journal: Science Advances

Article Title: Epstein-Barr virus infection promotes T cell dysregulation in a humanized mouse model of multiple sclerosis

doi: 10.1126/sciadv.adu5110

Figure Lengend Snippet: ( A ) Demyelination of the spinal cord in the HuPBMC EAE model. Perfused spinal cords were obtained days 19 to 25 post-induction (5 to 8 days post–symptom onset) from HuPBMC EAE mice and days 15 to 25 post-induction from NOD EAE mice (5 to 15 days post–symptom onset). Eriochrome cyanine–stained sections (top) obtained from the lower thoracic region of the spinal cord show representative myelination indices (MI) for each of the respective group means. Individual data points represent averages of serial sections sampled from four to six equidistant regions along the entire length of the spinal cord ( n = 18 regional points from three unengrafted NSG control mice; n = 22 to 36 regional points from five to six mice per group for EAE-induced NOD and HuPBMC groups). Distribution of individual data is shown with median and quartiles (dashed lines) and was analyzed by Kruskal-Wallis with Dunn’s multiple comparisons test. ( B to D ) Human CD8 + cells infiltrate the CNS of HuPBMC EAE mice engrafted with RRMS donor PBMCs. Representative images of lumbar spinal cord (B) and cerebellar (C and D) sections from an unengrafted control NSG mouse (left) and a symptomatic HuPBMC EAE mouse (right) derived from a donor with RRMS. Perfused tissues were collected day 15 post–EAE induction (day 4 post–symptom onset). Sections were labeled with FluoroMyelin (green), NeuroTrace 530/615 (red), 4′,6-diamidino-2-phenylindole (DAPI; blue), anti-hCD8 (yellow), and anti–Iba-1 (light blue). Example hCD8 + cells are indicated by white arrowheads (B and C), and hCD8 + cells in proximity to Iba-1 + cells by red arrowheads (D). Scale bars indicate size as specified per panel, showing (A) 200 μm; (B) 200 μm and (insets) 50 μm; (C) 500 μm and (insets) 50 μm; and (D) 100 μm and (inset) 50 μm.

Article Snippet: Whole blood (80 ml) was processed for PBMC isolation by Lymphoprep (STEMCELL Technologies, no. 07801) gradient separation, according to the manufacturer’s instructions, within an hour of collection in K 2 -EDTA–coated vacutainer tubes (BD, no. 366643).

Techniques: Staining, Control, Derivative Assay, Labeling

Journal: Science Advances

Article Title: Epstein-Barr virus infection promotes T cell dysregulation in a humanized mouse model of multiple sclerosis

doi: 10.1126/sciadv.adu5110

Figure Lengend Snippet: ( A ) Total human CD45 + immune cell counts, ( B ) hCD19 + B cell counts, ( C ) hCD3 + T cell counts, ( D ) EBV genome copies in splenic DNA, ( E ) hCD3 + CD4 + T cell counts, and ( F ) hCD3 + CD8 + T cell counts in whole brains, spinal cords, and spleens of recipient HuPBMC EAE mice at endpoint, grouped by PBMC donor EBV serostatus and RRMS diagnosis. Perfused organs were collected days 14 to 27 post–EAE induction (average 5 to 10 days post–symptom onset). For total immune cell quantification, n = 29 to 35 mice per group derived from two to three donors per group. For viral load quantification, n = 28 to 53 mice per group derived from two to four blood donors per group; n = 5 replicates for control EBV + B95-8 cell line, and assay lower limit of detection is represented by a dashed line. All data are shown as means with SEM and were analyzed by Kruskal-Wallis with Dunn’s multiple comparisons test.

Article Snippet: Whole blood (80 ml) was processed for PBMC isolation by Lymphoprep (STEMCELL Technologies, no. 07801) gradient separation, according to the manufacturer’s instructions, within an hour of collection in K 2 -EDTA–coated vacutainer tubes (BD, no. 366643).

Techniques: Biomarker Discovery, Derivative Assay, Control

Journal: Science Advances

Article Title: Epstein-Barr virus infection promotes T cell dysregulation in a humanized mouse model of multiple sclerosis

doi: 10.1126/sciadv.adu5110

Figure Lengend Snippet: Figure shows whole brain and spinal cord infiltration and spleen reconstitution in recipient HuPBMC EAE mice at endpoint, grouped by PBMC donor EBV serostatus and RRMS diagnosis. ( A ) Concatenated flow cytometric plots of IFN-γ and IL-17A expression, showing the mean frequency of hCD3 + CD4 + cells ± SD, as well as corresponding total ( B ) IFN-γ + (IL-17A − ), ( C ) IL-17A + (IFN-γ − ), and ( D ) IFN-γ + IL-17A + hCD4 + T cell counts in each tissue. ( E ) Concatenated flow cytometric plots of IFN-γ and GzmB expression, showing the mean frequency of hCD3 + CD8 + cells ± SD, quantified as ( F ) %IFN-γ + (GzmB − ), ( G ) %GzmB + (IFN-γ − ), and ( H ) %IFN-γ + GzmB + of hCD3 + CD8 + cells in each tissue. Perfused organs were collected days 14 to 27 post–EAE induction (average 5 to 10 days post–symptom onset). Isolated immune cells were stimulated with PMA and ionomycin for cytokine detection ( n = 9 to 20 mice per group derived from one to two donors per group). All plotted data are shown as means with SEM and were analyzed by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test or by Kruskal-Wallis with Dunn’s multiple comparisons test.

Article Snippet: Whole blood (80 ml) was processed for PBMC isolation by Lymphoprep (STEMCELL Technologies, no. 07801) gradient separation, according to the manufacturer’s instructions, within an hour of collection in K 2 -EDTA–coated vacutainer tubes (BD, no. 366643).

Techniques: Biomarker Discovery, Expressing, Isolation, Derivative Assay

Journal: Science Advances

Article Title: Epstein-Barr virus infection promotes T cell dysregulation in a humanized mouse model of multiple sclerosis

doi: 10.1126/sciadv.adu5110

Figure Lengend Snippet: The proportion of hCD3 + CD4 + T cells expressing FOXP3 in ( A ) freshly isolated donor PBMC ( n = 3 to 4 donors per group), ( B ) in the peripheral blood of engrafted HuPBMC mice at 3 weeks post–PBMC injection ( n = 57 to 62 mice per group derived from three to four donors per group), and in the ( C ) brain, ( D ) spinal cord, and ( E ) spleen of HuPBMC EAE mice at endpoint ( n = 30 to 35 mice per group derived from two to three donors per group). The ratio of infiltrating ( F ) hCD4 + IFN-γ + (T H 1) and ( G ) hCD8 + IFN-γ + GzmB + (T c ) to regulatory hCD4 + FOXP3 + (T reg ) cells per tissue in recipient HuPBMC EAE mice at endpoint, grouped by PBMC donor EBV serostatus and RRMS diagnosis ( n = 7 to 20 mice per group from one to two donors per group). Cells were isolated from perfused organs collected days 14 to 27 post–EAE induction (average 5 to 10 days post–symptom onset) and, for cytokine detection, stimulated with PMA and ionomycin. Data are shown as means with SEM and were analyzed by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (A) or by Kruskal-Wallis with Dunn’s multiple comparisons test [(B) to (G)].

Article Snippet: Whole blood (80 ml) was processed for PBMC isolation by Lymphoprep (STEMCELL Technologies, no. 07801) gradient separation, according to the manufacturer’s instructions, within an hour of collection in K 2 -EDTA–coated vacutainer tubes (BD, no. 366643).

Techniques: Expressing, Isolation, Injection, Derivative Assay, Biomarker Discovery

Journal: Science Advances

Article Title: Epstein-Barr virus infection promotes T cell dysregulation in a humanized mouse model of multiple sclerosis

doi: 10.1126/sciadv.adu5110

Figure Lengend Snippet: Previously frozen, whole PBMC samples from EBV + RRMS, EBV + HD, and EBV − HD blood donors were incubated with anti-CD3/CD28–coated beads for 96 hours to stimulate T cells in the absence of a specific antigen. Figure shows ( A ) the proliferation index determined by CFSE staining, ( B ) the proportion of hCD4 + T cells having undergone a specified number of cellular divisions by CFSE staining, ( C ) Ki-67 expression, ( D ) Tumor necrosis factor–α (TNFα) expression, and ( E ) IFN-γ and IL-17A expression on hCD3 + CD4 + T cells, as well as ( F ) the proliferation index determined by CFSE staining, ( G ) the proportion of hCD8 + T cells having undergone a specified number of cellular divisions by CFSE staining, ( H ) Ki-67 expression, ( I ) TNFα expression, and ( J ) IFN-γ expression on hCD3 + CD8 + T cells. Concatenated flow plots indicate the sum proportion of marker positive cells for all donors in each group. The colored symbol legend is applicable to all comparisons ( n = 3 to 4 blood donors per group). All plotted data are shown as means with SEM and were analyzed by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test or by Kruskal-Wallis with Dunn’s multiple comparisons test.

Article Snippet: Whole blood (80 ml) was processed for PBMC isolation by Lymphoprep (STEMCELL Technologies, no. 07801) gradient separation, according to the manufacturer’s instructions, within an hour of collection in K 2 -EDTA–coated vacutainer tubes (BD, no. 366643).

Techniques: Incubation, Staining, Expressing, Marker