Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Blood and tonsil mononuclear cells pregated on CD8 T-cells by flow cytometry for perforin and granzyme B. B . Expression intensity of CD69, CD103, CXCR5, CD127, PD-1, Perforin and granzyme B in multidimensional tSNE space for CD8 + T-cells, gated from total CD45 + T-cells. Peripheral blood (blue), tonsil tissue (orange). C . Percentage of blood and tonsil CD8 + T-cells expressing indicated markers. D . Schematic of protocol for isolate of HIV + CD8 + T cells from peripheral blood and tonsil tissue by scRNA-seq using Seq-Well v3 (top). UMAP of CD8 + T cells coloured by tissue source (bottom). E . Heatmap of z-scored gene expression of CD8 + T cells illustrating differentially expressed genes between blood and tonsil. Selective specifically expressed genes are marked alongside. F . Violin plots showing selected genes for cytolytic, transcription factors, tissue-resident memory and immune suppressive markers, expressed in CD8 + T cells. FDR-adjusted p<0.05; full results can be found in Supplementary Table S1. G . Fluorescent immunohistochemistry of whole tonsil section (left) from HIV uninfected donor shown by CD8, CD4 and merged panels (middle) with quantification using 10 unrelated areas identified outside follicles (outside GCs) and within follicular germinal centers ( inside GCs). P-values by Kruskal-Wallis multi comparisons.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Flow Cytometry, Expressing, Gene Expression, Immunohistochemistry

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Percentage of blood (circles) and tonsil (triangles) CD8 + T cells expressing CD69, CD103, CXCR5, CD27, CD127, PD-1, granzyme B and perforin markers between HIV − and HIV + . Red data point indicates viremic (detectable HIV RNA) and red/black data point indicates suppressed (undetectable HIV RNA). P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated with p-value. B . Representative FACS plot of CD8 + T-cells from HIV-(grey triangles) and HIV + (red triangles includes both ART suppressed and viremic individuals) tonsils for co-expression of CD69/CD103 and PD-1/CD127 with cumulative data shown with horizontal bars representing median values and p-values by Kruskal-wallis multiple comparisons. C . CD8, CD69, CD103 fluorescent immunohistochemistry of whole tonsil sections zoomed in at individual GCs for three independent donors from HIV − (left), HIV + ART + (middle) and HIV + ART − (right) with individuals markers shown. D . Same as in C but for CD8, HIV-p24 and PD-1.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Representative FACS plot of the gating strategy of CD8 + T-cells from tonsil cells to detect naïve, T N (CCR7 + CD45RA + ), central memory (T CM , CCR7 + CD45RA − ), effector memory (T EM , CCR7 − CD45RA − ) and T EMRA (CCR7 − CD45RA + ). B . Different memory subset distribution within blood and tonsil CD8 + T-cells for central memory, T CM (blue), Effector memory, T EM (red), transitional, T EMRA (orange), T Naive (grey) with cumulative memory subset distribution of CD8 + T cells for blood (circles, left) and tonsil (triangles, right). C . Distribution of blood central memory (T CM ), transitional memory (T EMRA ), effector memory (T EM ) and naïve subsets within CD127, CD69, PD-1, perforin and granzyme B expressing CD8 + T-cells cumulative for all study participants in HIV − (grey) and HIV + (red). D . Same as in C but showing data from tonsil CD8 + T-cells. P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated above. E . The frequency of CD103, CD69, CD127, perforin, and granzyme B (Granz B) cells measured on PD-1 + (left) and PD-1 − (right) CD8 + T-cells from blood in HIV + (red) and HIV − (grey) individuals. F . Same as in E but showing data from tonsil CD8 + T-cells. P-values calculated using Paired Student’s t test. Horizontal bars represent median values.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Representative flow plots showing CMV-(top) and HIV-specific (bottom) tetramer stains of blood (left) and tonsil tissue (right) from the same participant. B . Heat map showing expression frequencies for the indicated markers among CD8 + T cells from CMV tetramer and HIV tetramer specific CD8 + T-cells in blood (top) and tonsil (bottom) gated CD8 + T-cells with frequencies for each tetramer population indicated in the bar below from blue (0%) to red (100%). C . Frequencies of CD69, CD103, PD-1 and CD127 from HIV-, CMV-, and non-specific (‘CD8’) CD8 + T-cells within blood (left) and tonsil (right) tissue. P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated above.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Workflow of single-cell RNA sequencing from HIV infected tonsil tissue isolated CD8 + T-cells pre-sorted on HIV-, CMV- and ‘non-specific’ CD8 + T-cells from HIV infected participants indicated in Table S2. B . Dimensionality reduction using tSNE on scRNA-Seq cells coloured by Louvain cluster (top), participant ID (middle), and tetramer specificity (bottom). C . Heatmap of z-scored gene expression of top differentially expressed genes (t-test) between Louvain clusters from scRNA-seq data with cells grouped by Louvain cluster, genes grouped by hierarchical clustering (full gene lists in supplemental Table S3). D . Gene set enrichment scores for each of the 4 Louvain clusters (0-3) shown for ‘ T RM ’ , ‘Exhaustion’ , ‘proliferation’ and ‘activation’ [ , ] published gene lists. E . Heatmaps of z-scored gene expression of top differentially expressed genes (t-test) between single cells with high and low normalized MFI values of CD69, CD103, PD-1 and CD127. Selected genes labelled in plot, full gene lists in Table S4-S5. F . Gene lists from (E) scored against the published gene lists as indicated in D.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: RNA Sequencing, Infection, Isolation, Gene Expression, Activation Assay