Journal:

Article Title: Expansion of activated human na?ve T-cells precedes effector function

doi: 10.1046/j.1365-2249.2002.02015.x

Figure Lengend Snippet: Surface markers and cytokines expressed by naïve and memory T-cells

Article Snippet: Antigen-experienced cells were purified by incubating cells with a FITC-conjugated antibody to CD45RA, then depleted by MACS sorting using anti-FITC magnetic microbeads (Miltenyi, Auburn, CA, USA).

Techniques: Expressing

Journal:

Article Title: Expansion of activated human na?ve T-cells precedes effector function

doi: 10.1046/j.1365-2249.2002.02015.x

Figure Lengend Snippet: .Stimulated antigen-experienced T-cells gain effector function without cell division. PBMC from healthy adult donors depleted of CD45RA+ T-cells, were stained with 0·25 µm CFSE and were then stimulated for 4 days with PHA or SEB, or for 6 days by MLR. Shown is a dot plot panel of surface markers and cytokine production for antigen-experienced CD4+ or CD8+ gated T-cells.

Article Snippet: Antigen-experienced cells were purified by incubating cells with a FITC-conjugated antibody to CD45RA, then depleted by MACS sorting using anti-FITC magnetic microbeads (Miltenyi, Auburn, CA, USA).

Techniques: Staining

Journal:

Article Title: Expansion of activated human na?ve T-cells precedes effector function

doi: 10.1046/j.1365-2249.2002.02015.x

Figure Lengend Snippet: IL-7 drives proliferation of naïve T-cells in MHC independent fashion. Cord blood PBMC were stained with 0·25 µm CFSE and were then cultured for 9 days in 10 ng/ml IL-7 with isotypic control antibodies (a) or 10 µg/ml blocking antibodies recognizing HLA A, B, C, DR, DP, and DQ (b) Dot plots of CD4+ or CD8+ gated T-cells show expression patterns of CD45RA, and CD45RO with respect to cell division. Boxed areas of CD4 and CD8 plots represent the percentage of cultured cells that had proliferated more than three times for IL-7 alone (a) and IL-7+ anti-MHC antibodies (b).

Article Snippet: Antigen-experienced cells were purified by incubating cells with a FITC-conjugated antibody to CD45RA, then depleted by MACS sorting using anti-FITC magnetic microbeads (Miltenyi, Auburn, CA, USA).

Techniques: Staining, Cell Culture, Control, Blocking Assay, Expressing

Journal:

Article Title: Expansion of activated human na?ve T-cells precedes effector function

doi: 10.1046/j.1365-2249.2002.02015.x

Figure Lengend Snippet: IL-2 is Required for IL-7 induced expansion of memory T-Cells present in umbilical cord blood. Cord blood PBMC were stained with 0·25 µm CFSE and were then cultured for 9 days in 10 ng/ml IL-7 with or without 10 µg/ml depleting antibodies to IL-2. After culturing, PBMC were stained with CD4 and CD45RO or CD45RA, and CD8 and CD45RA or CD45RO. Dot plots of CD4+ or CD8+ T-cells show expression patterns of CD45RA, CD45RO, CD25, and IFN-γ with respect to cell division.

Article Snippet: Antigen-experienced cells were purified by incubating cells with a FITC-conjugated antibody to CD45RA, then depleted by MACS sorting using anti-FITC magnetic microbeads (Miltenyi, Auburn, CA, USA).

Techniques: Staining, Cell Culture, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Interleukin-12 encoded by the oncolytic virus VSV-GP enhances therapeutic antitumor efficacy by inducing CD8+ T-cell responses with a long-lived effector cell phenotype

doi: 10.1136/jitc-2024-010675

Figure Lengend Snippet: Phenotypic analysis of total and N-specific blood CD8+ T cells. Mixed TC-1:TC-1 ifnar1 −/− (80:20) tumor-bearing mice were treated with phosphate-buffered saline (control; n=5), VSV-GP (n=5) or VSV-GP-IL12 (n=5) and peripheral blood was analyzed 14 days post treatment for CD44, CD62L, KLRG1, CD43, CD27 and CD127 expression by flow cytometry. N-specific CD8+ T cells (Ntet+) were determined by tetramer staining. ( A ) The gating strategy is shown for one representative mouse out of five treated with VSV-GP-IL12. CD8+ T cells (CD3×CD8 plot) were derived from gating on lymphocytes>singlets>living CD45 positive cells. Using CD44 and CD62L (CD44×CD62L plot) as markers, total CD8+ T and N-tetramer positive (Ntet+) virus-specific cells were further divided into CD44 low CD62L + naïve, CD44 high CD62L + central memory (TCM), and CD44 + CD62L − effector/effector-memory (TE/TEM) populations. Within total naïve, TCM and TE/TEM populations, as well as in N-specific TE/TEMs, CD43 and KLRG1 (CD43×KLRG1 dot plots) expression patterns defined subpopulations of CD43 neg KLRG1 neg (black), CD43 pos KLRG1 neg (blue), CD43 pos KLRG1 pos (purple) and CD43 pos KLRG1 pos (red) cells. ( B ) There was no significant difference in the frequencies of Ntet+ virus-specific CD8+ T cells induced by VSV-GP and VSV-GP-IL12 treatments. Statistical analysis was performed using the non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons (*p<0.05; ns, not significant). ( C ) The gating on naïve, TCM, and TE/TEM populations revealed that the vast majority of N-specific cells displayed a TE/TEM phenotype. The numbers represent the mean percentage of the corresponding population within Ntet+ CD8+ T cells (n=5 per treatment groups). ( D ) 5000 CD8+T cells per treated animal were analyzed and the mean percentage of CD43 pos KLRG1 neg (blue), CD43 pos KLRG1 pos (purple), CD43 pos KLRG1 pos (red) and CD43 neg KLRG1 neg (black) subpopulations within total naïve, TCM and TE/TEM as well as in N-specific (Ntet+) TE/TEMs after VSV-GP and VSV-GP-IL12 treatments are shown. ( E ) Ntet+ cells from 5000 CD8+ T cells per treated animal were concatenated and are presented in color-coded CD43×KLRG1dot plots. The expression of CD27 and CD127 in these CD43/KLRG1 subpopulations is shown in color-coded histograms. Numbers within the histograms represent mean fluorescence intensities to the respective peak (n.a.; not applicable due to low cell numbers). Color-coded CD43/KLRG1 subpopulations were also divided into KLRG1 neg CD127 low early effector cell (EEC), KLRG1 pos CD127 low short-lived effector cell (SLEC), KLRG1 pos CD127 pos double positive (DP), and KLRG1 neg CD127 pos memory precursor cell (MPEC) populations based on the expression of KLRG1 and CD127. GP, glycoprotein; IL, interleukin; VSV, vesicular stomatitis virus.

Article Snippet: Subsequently, surface staining was carried out for 20 min at 4°C with the following antibodies: CD43-FITC (clone 1B11, BD Biosciences), CD3e-PE (clone 145–2 C11, BD Biosciences), CD3-BUV395 (clone 145–2 C11, BD Bioscience), CD127-BV711 (clone SB/199, BD Bioscience), CD62L-APC-Cy7 (clone MEL-14, BD Bioscience), CD45-PerCP (clone 30-F11, Miltenyi), CD27-PerCP-Cy5.5 (clone LG.3A10, BD Biosciences), KLRG1-PE-Cy7 (clone 2F1, BioLegend), CD8-BV421 (clone 53–6.7, BD Biosciences), CX3CR1-BV510 (clone Z8-50, BD Biosciences), CD44-AF700 (clone IM7, BD Bioscience), NK1.1-APC (clone PK136, BD Biosciences) and NKp46-FITC (CD335, clone REA815, Miltenyi Biotec).

Techniques: Saline, Control, Expressing, Flow Cytometry, Staining, Derivative Assay, Virus, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Interleukin-12 encoded by the oncolytic virus VSV-GP enhances therapeutic antitumor efficacy by inducing CD8+ T-cell responses with a long-lived effector cell phenotype

doi: 10.1136/jitc-2024-010675

Figure Lengend Snippet: Functional analysis of tumor CD8+ T cells. Mixed TC-1:TC-1 ifnar1 −/− (80:20) tumor-bearing mice were treated with VSV-GP or VSV-GP-IL12 and tumor tissues were harvested at day 7 and 14 post treatment (n=4–5 mice). Tumors were enzymatically digested to generate single-cell suspensions. 10 6 tumor cells were stimulated with 2 µg/mL N or E7 peptides for 3 hours. As a control, tumor cells were cultivated without peptides (no stimulation). To determine degranulation, BV786-labeled CD107a antibody was present during the 3 hours incubation. Following incubation, cells were stained for cell surface markers and after permeabilization stained with a PE-labeled IFN-γ antibody. ( A ) The gating strategy is shown for one representative mouse out of five treated with VSV-GP or VSV-GP-IL12. CD8+ T cells (CD3×CD8) were derived from gating on lymphocytes>singlets>living CD45 positive cells. Representative color-coded dot plots show the distribution of CD43 pos KLRG1 neg (blue), CD43 pos KLRG1 pos (purple), CD43 neg KLRG1 pos (red) and CD43 neg KLRG1 neg (black) subpopulations within total CD8+ T cells after 7 and 14 dpt. ( B ) Percentage of CD43 pos KLRG1 neg (blue), CD43 pos KLRG1 pos (purple), CD43 neg KLRG1 pos (red) and CD43 neg KLRG1 neg (black) subpopulations within total CD8+ T cells after 7 and 14 dpt derived from n=4–5 mice per group. Percentage and mean fluorescence intensities (MFI) of IFN-γ positive tumor CD8+ T cells derived 7 ( C ) and 14 dpt ( D ) with VSV-GP or VSV-GP-IL12 after 3 hours ex vivo stimulation with N or E7 peptides. ( E ) Relative percentage of CD43 pos KLRG1 neg (blue), CD43 pos KLRG1 pos (purple), CD43 neg KLRG1 pos (red) and CD43 neg KLRG1 neg (black) subpopulations is shown within IFN-γ positive tumor CD8+ T cells. Data in E were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons test. Asterisks indicate significant differences between the corresponding colored populations in the VSV-GP and VSV-GP-IL12 groups (**p<0.01; ***p<0.001; ****p<0.0001; ns, not significant). Representative color-coded dot-plots and histograms from one representative mouse out of five treated with VSV-GP or VSV-GP-IL12 reflecting different CD43/KLRG1 subpopulations ( F and H ) and percentage of CD107a single, CD107a/IFN-γ double and IFN-γ single positive tumor CD8+ T cells ( G and I ) after 3 hours ex vivo peptide stimulation derived from mice at 7 ( F and G ) and 14 days ( H and I ) post VSV-GP or VSV-GP-IL12 treatments. Data were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons for B , C , D , G and I (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). ANOVA, analysis of variance; GP, glycoprotein; IFN, interferon; IL, interleukin; VSV, vesicular stomatitis virus.

Article Snippet: Subsequently, surface staining was carried out for 20 min at 4°C with the following antibodies: CD43-FITC (clone 1B11, BD Biosciences), CD3e-PE (clone 145–2 C11, BD Biosciences), CD3-BUV395 (clone 145–2 C11, BD Bioscience), CD127-BV711 (clone SB/199, BD Bioscience), CD62L-APC-Cy7 (clone MEL-14, BD Bioscience), CD45-PerCP (clone 30-F11, Miltenyi), CD27-PerCP-Cy5.5 (clone LG.3A10, BD Biosciences), KLRG1-PE-Cy7 (clone 2F1, BioLegend), CD8-BV421 (clone 53–6.7, BD Biosciences), CX3CR1-BV510 (clone Z8-50, BD Biosciences), CD44-AF700 (clone IM7, BD Bioscience), NK1.1-APC (clone PK136, BD Biosciences) and NKp46-FITC (CD335, clone REA815, Miltenyi Biotec).

Techniques: Functional Assay, Control, Labeling, Incubation, Staining, Derivative Assay, Fluorescence, Ex Vivo, Virus