Journal: Cell Reports Methods

Article Title: Generation of T cell responses against broad KRAS hotspot neoantigens for cell therapy or TCR discovery

doi: 10.1016/j.crmeth.2025.101049

Figure Lengend Snippet: Enrichment of antigen-specific T cells (A) Representative flow-cytometry plots demonstrating the upregulation of activation markers on antigen-specific CD8 + cells (red) compared to bystander cells (gray). Red numbers: % of CD8 + cells. (B) Aggregate data from six independent experiments using either 4-1BB (left) or CD69 (right) as enrichment marker. Note increase in frequency or de novo detection of antigen-specific cells. Colors denote different KRAS neoantigens. (C and D) Enrichment of antigen-specific CD4 + cells with either 4-1BB- or CD69-based enrichment compared to a non-enriched condition. (C) Representative flow-cytometry plots from one healthy donor identifying mKRAS-specific CD4 + T cells via the delta IFNγ or TNF-α expression with or without an overnight peptide stimulation. (D) Responses across three donors (4-1BB in dark green, CD69 in green). Horizontal bars indicate the mean. p values per one-way ANOVA with Dunnett’s correction for multiple comparisons: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (E) First four runs of the large-scale enrichment process. Cells were expanded post enrichment and antigen-specific CD8 + T cell frequency was determined pre-enrichment (day 13), post enrichment (day 14 or day 16), and post expansion (day 26). Dotted line indicates target frequency. (F and G) In vitro cytotoxicity using a representative DP. DP was co-cultured with GFP-labeled target cells. (F) A375 melanoma cells transduced to express HLA-A∗11:01 at an approximate antigen-specific effector to target (E:T) ratio of 5:1. Target cells were either loaded with WT KRAS peptide (purple) or G12V neoantigen peptide (blue). (G) SW620 colorectal adenocarcinoma cells, which endogenously express KRAS G12V, transduced to express HLA-A∗11:01 at an approximate antigen-specific E:T ratio of 2.5:1 (blue). Data points represent the mean of GFP-labeled target cell area from technical duplicates (F) or triplicates (G) ± SD. p values per two-way ANOVA: ∗∗∗∗ p < 0.0001. See also .

Article Snippet: CD69 + Microbeads , Miltenyi , Cat#130-092-355.

Techniques: Flow Cytometry, Activation Assay, Marker, Immunopeptidomics, Expressing, In Vitro, Cell Culture, Labeling

Journal: Nature immunology

Article Title: Lymph Node Conduits Transport Virions for Rapid T Cell Activation

doi: 10.1038/s41590-019-0342-0

Figure Lengend Snippet: a ) Blended projections of T cells (red) clustered around a conduit (white)-associated VACV-infected cell (green) near a high endothelial venule (HEV, indicated in picture). Arrows point to conduits. Scalebar = 20 μm. Area in circle is magnified on the right. Scalebar = 5 μm. ERTR7= white, VACV-infected cells = green, OT-I CD8 + T cells = red, B220 = blue. Results are representative of 30 LN sections taken from 5 experiments. b ) OT-I CD8 + T cell activation as determined by flow cytometry of single-cell suspensions of popliteal LNs harvested 8 h p.i. 10 6 OT-I cells were transferred 12–24 h prior to infection. NP-eGFP (no SIINFEKL) was given at 10 7 pfu; all other infections used NP-S-eGFP (containing SIINFEKL) at the indicated dose. Activation was determined by CD69 expression. MFI is shown on the right. Dots = individual LNs. n = 6. Results were repeated 3 times with 3–6 mice/group. Bars = mean. Error bars = SEM. c ) OT-I CD8 + T cell activation in (b) but 24 h p.i. Dots = individual LNs. n = 6. Results were repeated 3 times with 3–6 mice/group. Bars = mean. Error bars = SEM. d ) Quantitation of the percentage of VACV- or MVA-infected cells in either the SCS and IFA region or T cell zone contacted by OT-I CD8 + T cells (n = 37 VACV or 32 MVA; dots indicate individual sections). Bars = mean. Error bars = SEM. e ) Percentage of activated OT-I CD8 + T cells (those with CD69 MFI > 50) in each region of 10 LN sections harvested 8 h after infection with 10 8 pfu VACV. Percentages shown are activated cells/total cells in each region. Dots = individual LN sections. Bars = mean. Error bars = SEM. f ) Percentage of only the activated OT-I CD8 + T cells (as opposed to total cells in (e)) found in each LN region. g ) MIP LN section 8 h p.i. showing OT-I CD8 + T cell activation. B = B cell follicle, SCS = subcapsular sinus. Arrow indicates activated T cell cluster in the T cell zone, magnified on the right. Scalebars = 50 μm, left and 5 μm, right. OT-I CD8 + T cells = red, CD69 = white, VACV-infected cells = green, B220 = blue. h ) MFI of CD69 on all activated cells in each region of the popliteal LN shown in (g). n =197 T cells. Bars = mean. Error bars = SEM. i ) MIP of popliteal LN harvested 8 h post infection with 10 8 pfu VACV (green) without OT-I transfer. Endogenous, polyclonal CD8 + T cells = red. Colocalization of polyclonal CD8 + T cell signal (red) and CD69 (white) signal is shown in purple. B220 = blue, VACV-infected cells = green. The boxed area is magnified in (j). scalebars = 100 μm j ) Higher magnification MIP image of (i) showing both the SCS&IFA (labeled SCS) and T cell zone (T). Colocalization of polyclonal CD8 + T cell signal (red) and CD69 (white) signal is shown in purple. scalebars = 50 μm. f,h,i,j) 6 LNs from 3 experiments were analyzed. g) 10 LNs from 5 experiments were analyzed. Statistics = unpaired two-tailed t test.

Article Snippet: Sections were blocked with 5% goat, donkey, bovine, rat or rabbit serum and then stained with one or more of the following Abs: CD11b (clone M1/70, eBioscience, Lot#E022424, E15525–01, 4319572, or B259438); CD11c (clone N418, eBioscience, Lot#4288340, 4339477, or E029528); CD205 (clone NLDC-145, Biolegend, Lot#B251007); B220 (clone RA3–6B2, eBioscience, Lot#4288340, 4339477, or 4306068); CD8α (clone 53–6.7, eBioscience, many different lots); Lyve-1 (clone ALY7, eBioscience, Lot#4291625 or 4311240); ERTR7 (staining a fibroblastic reticular cell antigen and used to identify the LN stromal network including conduits, blood vessels and lymphatic sinuses; rat monoclonal, Abcam (cat#51824); CD69 (polyclonal goat, R&D Systems, Cat#AF2386, Lot 0117021 or 0107081); anti-GFP (polyclonal, eBioscience, Lot#E051981634); anti-vaccinia virus (Accurate Chemical Group, Lot#J2368); anti-NS2B (Genetex, Lot#42508); and/or Lyve-1 (clone ALY7, eBioscience, Lot#4291625, 4311240 or 1978147).

Techniques: Infection, Activation Assay, Flow Cytometry, Expressing, Quantitation Assay, Labeling, Two Tailed Test

Journal: Nature immunology

Article Title: Lymph Node Conduits Transport Virions for Rapid T Cell Activation

doi: 10.1038/s41590-019-0342-0

Figure Lengend Snippet: a ) Maximum intensity projection (MIPs) of sections from two different popliteal LNs (left and right) harvested 8 h after footpad injection of 10 6 pfu of VACV. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green, B220 = blue. Arrows indicate the location of VACV-infected paracortical cell. scalebars = 100 μm. Higher magnification images are shown on the right; lower panels lack the blue channel for clarity. Scalebars = 50 μm. Results are representative of 10 LNs from 3 experiments. b ) MIP of a section of a popliteal LN harvested 8 h after footpad injection of 10 6 pfu of VACV. Prior to infection, 10 6 OT-I CD8 + T cells were transferred (pink). ERTR7 = red, B220 = blue, CD69 = white, VACV-infected cells = green. Arrows indicate two areas of T cell activation that are shown in higher magnification insets on the left. Scalebar = 200 μm. Top panels of insets show merge; bottom panels do not show OT-I signal (pink) in order to reveal CD69 staining more clearly. Scalebars = 20 μm. Results are representative of 10 LNs from 3 experiments. c ) MIP of a section of a popliteal LN harvested 8 h after footpad infection with 10 6 pfu of VACV showing an infected paracortical CD205 + DC. Scalebar = 200 μm. Higher magnification images are shown in the insets showing: top left) merge; top right) conduits + VACV-infected cells; lower left) conduits + VACV-infected cells + CD11c + CD11b; lower right) conduits + VACV-infected cells + CD205. Scalebar = 20 μm. ERTR7 = red, Lyve-1 = white, VACV-infected cells = green, B220 = blue, CD11c = yellow, CD205 = purple, and CD11b = brown. Results are representative of 10 LNs from 3 experiments.

Article Snippet: Sections were blocked with 5% goat, donkey, bovine, rat or rabbit serum and then stained with one or more of the following Abs: CD11b (clone M1/70, eBioscience, Lot#E022424, E15525–01, 4319572, or B259438); CD11c (clone N418, eBioscience, Lot#4288340, 4339477, or E029528); CD205 (clone NLDC-145, Biolegend, Lot#B251007); B220 (clone RA3–6B2, eBioscience, Lot#4288340, 4339477, or 4306068); CD8α (clone 53–6.7, eBioscience, many different lots); Lyve-1 (clone ALY7, eBioscience, Lot#4291625 or 4311240); ERTR7 (staining a fibroblastic reticular cell antigen and used to identify the LN stromal network including conduits, blood vessels and lymphatic sinuses; rat monoclonal, Abcam (cat#51824); CD69 (polyclonal goat, R&D Systems, Cat#AF2386, Lot 0117021 or 0107081); anti-GFP (polyclonal, eBioscience, Lot#E051981634); anti-vaccinia virus (Accurate Chemical Group, Lot#J2368); anti-NS2B (Genetex, Lot#42508); and/or Lyve-1 (clone ALY7, eBioscience, Lot#4291625, 4311240 or 1978147).

Techniques: Injection, Infection, Activation Assay, Staining

Journal: Frontiers in Immunology

Article Title: The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial

doi: 10.3389/fimmu.2019.01206

Figure Lengend Snippet: Tumors from patients treated with Tadalafil and MUC1/polyICLC vaccine show a lower infiltration of MDSCs and Treg and a higher infiltration of activated CD8 in the tumor bed. Computer based image cytometry was performed to enumerate the number of (A) MDSC, (B) IL4Rα − myeloid cells, (C) CD4 + T cell subsets, or (D) CD8 + T cells. (E) CD69 expression within the CD8 is reported normalized on the CD69 expression on all the cells evaluated. Depending on the region of interest evaluated, at least 10 5 -10 6 cells were analyzed. (F) The expression of CD69 in CD8 + T cells was plotted against MUC1 IHC score of the corresponding tumor. Two ways T -test and relevant pearson correlation parameters are reported.

Article Snippet: Rabbit polyclonal anti-human CD8 antibody (dilution 1/30, Abcam) and goat polyclonal anti-human CD69 antibody (dilution 1/25, R&D System).

Techniques: Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial

doi: 10.3389/fimmu.2019.01206

Figure Lengend Snippet: Tadalafil and MUC1/polyICLC vaccine treatments modulate the expression of PDL1 in the tumor microenvironment. The expression of PDL1 within the CD163+ (A) or the CD163 − cells (B) was quantified by image cytometry in the tumor, at the tumor edge, or in “normal” surrounding tissue in the tumor specimen from the control (black filled circle) or Tadalafil and MUC1/polyICLC vaccine treated (gray filled circle) patients. Two way T -test p -value are reported. (C) Correlation between the expression of CD69 in the CD8 + T cells and PDL1 expression on the CD163 − cells. (D) Summary of the one way RM ANOVA analysis.

Article Snippet: Rabbit polyclonal anti-human CD8 antibody (dilution 1/30, Abcam) and goat polyclonal anti-human CD69 antibody (dilution 1/25, R&D System).

Techniques: Expressing, Cytometry, Control

Journal: Frontiers in Immunology

Article Title: The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial

doi: 10.3389/fimmu.2019.01206

Figure Lengend Snippet: PDL1 expression limits the beneficial prognostic value of CD8a and CD69 in the tumor. KM plotter analysis (kmplot.com) was performed on tumor RNAseq data from patients with HNSCC ( n = 499, all tumor stages) using the mean expression of the indicated genes with weight =1 and auto select best cutoff selected.

Article Snippet: Rabbit polyclonal anti-human CD8 antibody (dilution 1/30, Abcam) and goat polyclonal anti-human CD69 antibody (dilution 1/25, R&D System).

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 . 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