Journal: Nature

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease.

doi: 10.1038/s41586-019-1895-7

Figure Lengend Snippet: Fig. 1 | Peripheral CD8+ TEMRA cells are increased in AD and are negatively associated with cognition. a, Four cohorts were used to assess adaptive immunity in AD. b, Representative SPADE trees of PBMCs from healthy individuals and patients with MCI or AD in cohort 1 show an increased abundance of a CD8+ cluster (cluster 63) in patients with MCI or AD. Background tree nodes are sized according to cell counts. Insets are coloured according to CD8 expression. c, Quantification of cluster 63 as a percentage of total PBMCs. The percentage of cluster 63 cells is significantly higher in patients with MCI or AD than healthy control individuals. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. d, Marker expression analysis of cluster 63 corresponds to a CD3+CD8+CD45RA+CD27− TEMRA population. Data in c, d were pooled from seven independent experiments with similar results. e, Linear regression showing the inverse correlation between cognitive score and the percentage of CD8+ TEMRA cells in individuals from cohort 2. Pearson’s correlation r values are shown for each group. The significance of the difference between the two data sets was measured by ANCOVA. f, Stimulation with PMA and ionomycin (stim.) induces increased expression of IFN-γ in CD8+ T cells from patients with MCI or AD. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. g, Differential expression analysis (scRNA-seq) of CD8+ TEMRA cells from healthy individuals (n = 7) and patients with MCI or AD (n = 6) shows upregulated TCR signalling. Model-based analysis of single-cell transcriptomics (MAST) differential expression test with Benjamini–Hochberg correction. h, Pathway analysis of differentially expressed genes in CD8+ TEMRA cells from patients with MCI or AD (n = 6 subjects) versus healthy individuals (n = 7 subjects) shows increased antigenic stimulation of CD8+ TEMRA cells in patients with MCI or AD. Fisher’s exact test with Benjamini–Hochberg correction. Pathways (circles) with positive z-scores are coloured red; those with negative z-scores are coloured blue. The size of the circle corresponds to the size of the z-score (two-sided).

Article Snippet: Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam).

Techniques: Expressing, Control, Marker, Quantitative Proteomics, Single-cell Transcriptomics

Journal: Nature

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease.

doi: 10.1038/s41586-019-1895-7

Figure Lengend Snippet: Fig. 2 | CD8+ T cells enter the brain in patients with AD. a, Confocal imaging of cerebral amyloid angiopathy (CAA) in the post-mortem brain of a patient with AD from cohort 3 shows CD8+ T cells in the perivascular space of Aβ+ blood vessels with cerebral amyloid angiopathy in three AD-affected hippocampi. Arrowheads indicate CD8+ T cells; asterisks indicate blood vessel lumen. Scale bars, 20 μm. b, Higher numbers of CD8+ T cells were detected in AD-affected than control hippocampi. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. c, A CD8+ T cell is shown associated with MAP2+ neuronal processes. d, CD8+ T cells are localized to the leptomeninges and adjacent to hippocampal Aβ plaques. Scale bar, 100 μm. Data in c, d were replicated in three independent experiments.

Article Snippet: Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam).

Techniques: Imaging, Control

Journal: Nature

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease.

doi: 10.1038/s41586-019-1895-7

Figure Lengend Snippet: Fig. 3 | Clonal expansion of CD8+ TEMRA cells in the CSF of patients with AD. a, Plate-seq and drop-seq methods used for scTCR-seq and scRNA-seq of immune cells of the CSF in patients from cohort 4. b, CD8+ TCRαβ clonality (plate-seq) in the CSF of patients with AD and healthy control individuals. c, The top (most expanded) clone in AD had a marker expression profile of CD8+CD45RA+CD27− TEMRA cells. Data were replicated in two independent experiments. d, CSF cells analysed by drop-seq and clustered by multidimensional reduction with t-SNE, showing populations of immune cells that include CD8+ TEMRA cells (n = 9 healthy control individuals (10,876 cells); n = 9 patients with MCI or AD (10,391 cells)). e, Marker expression of CSF clusters, including CD8+ TEMRA cells. CD62L is also known as SELL; CD11c is also known as ITGAX. Data were pooled from three independent experiments. f, Concentration of clonal cells in locations of CD8+ T cell clusters (n = 9 subjects per group). g, Representative plots of CD8+ TCRαβ clonality (drop-seq) in age-matched subjects shows enhanced clonal expansion and more highly expanded clones in AD. Clones are coloured by proportion of the total TCRαβ sequences. h, Quantification of maximum clones (% TCRαβ sequences) shows a

Article Snippet: Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam).

Techniques: Control, Marker, Expressing, Concentration Assay, Clone Assay

Journal: Nature

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease.

doi: 10.1038/s41586-019-1895-7

Figure Lengend Snippet: Fig. 4 | Antigen identification of clonally expanded TCRs in the CSF of patients with AD. a, Unweighted network analysis of CD8 TCRαβ sequences combined from plate-seq and drop-seq experiments. Group node IDs with individual TCRαβ clones are depicted as circles and sized according to the proportion of total sequences of each clone. Arrow indicates a shared clonal TCRαβ sequence with specificity for EBV EBNA3A. Note that several healthy control (HC) subjects have no clones. b, Shared TCRαβ sequences among patients with MCI or AD. Three patients had identical TCRβ chains with specificity for EBV EBNA3A. The antigen specificity of TCRβ is shown below19. c, Differential expression of EBV-specific clones in MCI and AD (from n = 3 subjects) versus all CSF T cells shows enhanced expression of cytotoxic effector genes. MAST differential expression test with Benjamini–Hochberg correction. d, Workflow for antigen identification of CSF TCRs. GLIPH was applied to TCR sequencing to derive homologous TCR sequences between patients. GLIPH identified two patients with AD who had identical TCRβ chains

Article Snippet: Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam).

Techniques: Clone Assay, Sequencing, Control, Quantitative Proteomics, Expressing

Journal: International Journal of Nanomedicine

Article Title: Surface engineered polyanhydride-based oral Salmonella subunit nanovaccine for poultry

doi: 10.2147/IJN.S185588

Figure Lengend Snippet: OMPs-specific cell-mediated immune response in OMPs-F-PNPs orally inoculated and Salmonella -challenged chickens. Notes: ( A ) Flow cytometry analyses of CD8 + /CD4 + cell ratio. Splenocytes were immunostained with fluorochrome-labeled mouse anti-chicken CD4 and mouse anti-chicken CD8α antibody. The frequency of CD4 + and CD8 + lymphocytes in the spleen was examined, and the result was expressed as CD8 + /CD4 + cell ratio. ( B ) Serum IFN-γ levels estimated by ELISA. ( C ) OMPs-specific lymphocytes’ proliferation was measured as stimulation index values in PBMCs by using a calorimetric assay. Each bar is the mean ± SEM of 8–10 chickens, and the data were analyzed by nonparametric Kruskal–Wallis test followed by P -value differences in between the groups determined by Mann–Whitney test. Asterisk refers to statistical difference between two indicated groups ( * P <0.05). OMPs-F-PNPs, OMPs and F-protein-entrapped and surface F-protein-coated PNPs. Abbreviations: Ch, challenge; F, flagellar; OMPs, outer membrane proteins; IFN-γ, interferon gamma; PBMCs, peripheral blood mononuclear cells; PNPs, polyanhydride nanoparticles; SEM, standard error of the mean.

Article Snippet: Splenocytes (1×10 6 cells/well) were seeded in 96-well plate and incubated with pretitrated 1:200 dilution of fluorescein isothiocyanate-conjugated mouse anti-chicken CD4 (Southern Biotech, Birmingham, AL, USA), pretitrated 1:400 dilution of phycoerythrin-conjugated mouse anti-chicken CD8α (Southern Biotech), or 1:100 diluted mouse IgG specific isotype control antibody for 20 minutes at 4°C.

Techniques: Flow Cytometry, Labeling, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Membrane

Journal: Vaccines

Article Title: Construction and Evaluation of the Immunogenicity and Protective Efficacy of Recombinant Replication-Deficient Human Adenovirus-5 Expressing Genotype VII Newcastle Disease Virus F Protein and Infectious Bursal Disease Virus VP2 Protein

doi: 10.3390/vaccines11061051

Figure Lengend Snippet: Comparison of cellular immunity in different groups: ( A ) Statistical analysis of the peripheral blood lymphocyte stimulation index in the ND groups. Under ConA stimulation, the SI values of peripheral blood lymphocytes in the PBS group were significantly lower than those of the rDHN3-mF, rAd5-F, and rAd5-VP2-F2A-F groups ( p < 0.001). The SI values of peripheral blood lymphocytes in the rAd5-EGFP group were significantly lower than those in the rDHN3-mF, rAd5-F, and rAd5-VP2-F2A-F groups ( p < 0.01). However, there was no significant difference between the rDHN3-mF, rAd5-F, and rAd5-VP2-F2A-F groups ( p > 0.05), and no significant differences between the PBS and rAd5-EGFP groups ( p > 0.05). Under inactivated NDV stimulation, the SI values of peripheral blood lymphocytes in the PBS and rAd5-EGFP groups were significantly lower than those in the rDHN3-mF, rAd5-F, and rAd5-VP2-F2A-F groups ( p < 0.0001). SI values in the rAd-VP2-F2A-F group were significantly different from those of the rDHN3-mF and rAd5-F groups ( p < 0.05); no significant differences between the rDHN3-mF and rAd5-F groups ( p > 0.05), and there was no significant difference between the PBS and rAd5-EGFP groups ( p > 0.05) (ns—non-significant; * p < 0.05); ( B ) Statistical analysis of the peripheral blood lymphocyte stimulation index in IBD groups. From ( D ) above, it can be seen that the SI values of peripheral blood lymphocytes in the HVT-VP2 vector vaccines group, rAd5-VP2 group, and rAd5-VP2-F2A-F group were not significantly different from each other regardless of ConA stimulation or inactivated IBDV stimulation in the IBD group ( p > 0.05). However, the SI values of the above three groups were significantly higher than those of the rAd5- EGFP and the PBS groups ( p < 0.05). There was no significant difference between the PBS and rAd5-EGFP groups ( p > 0.05) (ns—non-significant); ( C , D ) Statistical analysis of the percentage of CD4+ and CD8+ T lymphocytes in the peripheral blood in ND groups. It can be seen from these two pictures that the percentages of CD4+ and CD8+ T lymphocytes in the rAd5-F, rDHN3-mF, and rAd5-VP2-F2A-F groups were significantly higher than those in the PBS group ( p < 0.05). There was no significant difference between the above three vaccine groups ( p > 0.05); ( E , F ) Statistical analysis of the percentage of CD4+ and CD8+ T lymphocytes in the peripheral blood in the IBD groups. It can be seen from these two pictures that the percentage of CD4+ and CD8+ T lymphocytes in the rAd5-VP2 group, the HVT-VP2 vector vaccines, and rAd5-VP2-F2A-F groups were significantly higher than in the PBS group ( p < 0.05). There was no significant difference between the three vaccine groups ( p > 0.05).

Article Snippet: The T-lymphocyte subpopulations were analyzed by flow cytometry (Beckman Coulter, Carlsbad, CA, USA) with the following antibodies: mouse anti-chicken CD3, mouse anti-chicken CD4, and mouse anti-chicken CD8α (Southern Biotech, Birmingham, AL, USA).

Techniques: Comparison, Plasmid Preparation, Vaccines