Journal: The Journal of Experimental Medicine

Article Title: Multi-transcription factor reporter mice delineate early precursors to the ILC and LTi lineages

doi: 10.1084/jem.20200487

Figure Lengend Snippet: Clonal potential of FL ILC progenitors. Single-cell potential of the indicated ILC progenitors sorted from Zbtb16 EGFPCre Tcf7 mCherry Rorc Thy1.1 E14-E16 FLs onto OP9 stromal cells. Pie charts represent the frequency of single ILC1 (NK1.1 + ICOS − α4β7 − ), ILC2 (NK1.1 − ICOS + α4β7 − ), and ILC3/LTi 0 (NK1.1 − ICOS +/− α4β7 + CD4 − ); two or more ILC subsets (multi); LTi 4 (NK1.1 − ICOS +/− α4β7 + CD4 + ); and mixed ILC1/2/3/LTi 0 and LTi 4 (mixILC/LTi). Data are pooled from >20 independent experiments. P values for pie charts were calculated using Chi-Square Test for Independence followed by post-hoc analysis with Bonferroni correction; comparisons were made against the Flt3 + αLP (black), ILCP (blue), and LTiP (orange).

Article Snippet: The following fluorochrome- or biotin-conjugated antibodies were used: α4β7 (DATK32), CCR6 (29-2L17), CD11c (N418), CD19 (6D5), CD90.2 (Thy-1.2; 53–2.1), CD127 (IL-7Rα; A7R34), CXCR5 (L138D7), GR-1 (RB6-8C5), ICOS (C398.4A), PD-1 (29F.1A12), Ter119 (TER-119), mouse IgG1 κ-chain (MG1-45), mouse IgG2a κ-chain (MG2a-53), and rat IgG2b κ-chain (RTK45-30; all from BioLegend); B220 (RA3-6B2), CD3ε (145-2C11), CD4 (GK1.5), CD8α (53–6.7), CD11b (M1/70), Flt3 (A2F10.1), NK1.1 (PK136), TCRβ (H57-597), Thy1.1 (OX-7), RORγt (Q31-378), and PLZF (R17-809; all from BD Biosciences); TCF1 (Cell Signaling Technologies); and polyclonal goat α-Rb IgG (Abcam).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Multi-transcription factor reporter mice delineate early precursors to the ILC and LTi lineages

doi: 10.1084/jem.20200487

Figure Lengend Snippet: Identification of Rorc expression in the FL αLP. (A) Representative flow cytometry plot of αLP from Zbtb16 EGFPCre Tcf7 mCherry Rorc Thy1.1 E15 FL ( n = 7). Adjacent plot reflects population frequencies. (B) Representative histogram plot of ID2-EYFP expression in the indicated ILC progenitors from Zbtb16 EGFPCre Tcf7 mCherry Rorc Thy1.1 ID2 EYFP E15 FL ( n = 6). Associated plots reflect population MFI. (C) MFI of Rorc -Thy1.1, CCR6, IL-7Rα, and PD-1 on Rorc + αLP, iLTiP, and LTiP from Zbtb16 EGFPCre Tcf7 mCherry Rorc Thy1.1 E15 FL ( n = 8). (D) Quantification of Flt3 − Rorc − αLP, Rorc + αLP, iLTiP, and LTiP from Zbtb16 EGFPCre Tcf7 mCherry Rorc Thy1.1 FL at E11, E13, and E15. (E) Representative flow plots and histograms of bulk cultured Flt3 − Rorc − αLP, Rorc + αLP, iLTiP, and LTiP from Zbtb16 EGFPCre Tcf7 mCherry Rorc Thy1.1 E15 FL cultured for 2 d on OP9 stromal cells ( n = 3). Adjacent plot reflects CD4 + frequency among Rorc + Tcf7 + cells. (F) Single-cell potential of the indicated αLP precursors sorted from Zbtb16 EGFPCre Tcf7 mCherry Rorc Thy1.1 E15 FL onto OP9 stromal cells. (G) Hierarchical clustering dendrogram of ILC progenitor clonal potential on OP9 stromal cells calculated from Pearson correlation. For plots, each symbol represents an individual FL; data are presented as mean ± SEM; and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. P values for pie charts were calculated using Chi-Square Test for Independence followed by post-hoc analysis with Bonferroni correction; comparisons were made against the Flt3 + Rorc − αLP (black). Data are pooled from at least two independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The following fluorochrome- or biotin-conjugated antibodies were used: α4β7 (DATK32), CCR6 (29-2L17), CD11c (N418), CD19 (6D5), CD90.2 (Thy-1.2; 53–2.1), CD127 (IL-7Rα; A7R34), CXCR5 (L138D7), GR-1 (RB6-8C5), ICOS (C398.4A), PD-1 (29F.1A12), Ter119 (TER-119), mouse IgG1 κ-chain (MG1-45), mouse IgG2a κ-chain (MG2a-53), and rat IgG2b κ-chain (RTK45-30; all from BioLegend); B220 (RA3-6B2), CD3ε (145-2C11), CD4 (GK1.5), CD8α (53–6.7), CD11b (M1/70), Flt3 (A2F10.1), NK1.1 (PK136), TCRβ (H57-597), Thy1.1 (OX-7), RORγt (Q31-378), and PLZF (R17-809; all from BD Biosciences); TCF1 (Cell Signaling Technologies); and polyclonal goat α-Rb IgG (Abcam).

Techniques: Expressing, Flow Cytometry, Cell Culture

Journal: Nutrients

Article Title: Calorie Restriction Impairs Anti-Tumor Immune Responses in an Immunogenic Preclinical Cancer Model

doi: 10.3390/nu15163638

Figure Lengend Snippet: Effect of CR on the population of tumor-infiltrating lymphocytes (TILs). ( A ) Representative dot plots depicting (upper panels) or percentages (lower panel) of TILs expressing CD3 and CD45. B16-OVA-inoculated mice were treated with/without anti-PD-1 antibody (0.2 mg/mouse i.p.) at days 3, 6, and 9 from day 0. ( B ) Representative dot plots depicting (upper panels) or percentages (lower panel) of cells expressing CD4 and CD8 in the gated CD3 + CD45 + TILs. ( C ) Representative histograms representing surface expression (upper panels) or median fluorescence intensity (MFI) values (lower panel) of CD44 on CD3 + CD8 + CD45 + TILs derived from B16-OVA-inoculated mice. Each plot represents the mean ± SEM ( n = 4); indicated p values were obtained from a statistical comparison; one-way ANOVA with Bonferroni’s multiple comparisons correction.

Article Snippet: The cells were then incubated with a saturating amount of fluorophore-conjugated antibodies against PE-Cy/7 CD45 (30-F11), FITC CD3ε (145-2C11), PerCP-Cy5.5 CD4 (GK1.5), APC CD8 (53-6.7), PE CD44 (IM7), APC-Cy7 CD62L (MEL-14) were purchased from Biolegend (San Diego, CA, USA).

Techniques: Expressing, Fluorescence, Derivative Assay, Comparison

Journal: Stem Cells International

Article Title: Immune Suppressive Effects of Tonsil-Derived Mesenchymal Stem Cells on Mouse Bone-Marrow-Derived Dendritic Cells

doi: 10.1155/2015/106540

Figure Lengend Snippet: T-MSCs inhibit DC-mediated T cell proliferation and differentiation of the CD4 + T cell subset. (a) T cells were isolated from mouse spleens (BALB/c, 8-week-old, female) using MACS. (b) T cells were cocultured with mature DCs in the presence or absence of T-MSCs, and differentiation into CD4 + T cell subsets was induced using anti-CD3 and anti-CD28 antibodies. T cell numbers were increased upon coculturing with mature DCs. In the presence of T-MSCs, however, T cell proliferation was inhibited by 80%. (c) Differentiation into Th1, Th2, and Th17 cells also was decreased upon addition of T-MSCs.

Article Snippet: Separated cells were stained with PE-conjugated anti-mouse CD4 antibody (GK 1.5, Rat IgG 2b , BioLegend) and analyzed by flow cytometry to determine the purity of the cell population.

Techniques: Isolation

Journal: Journal for Immunotherapy of Cancer

Article Title: Combination of radiation therapy, bempegaldesleukin, and checkpoint blockade eradicates advanced solid tumors and metastases in mice

doi: 10.1136/jitc-2021-002715

Figure Lengend Snippet: External beam radiation primes B78 tumor-bearing mice for BEMPEG treatment by increasing CD122 expression on CD8+ and CD4+ T-helper cells in the spleen; RT+BEMPEG increases the number of TILs in the B78 melanoma model. (A) Immune cell profile in the spleen 5 days after RT showing CD4+ T helpers/CD45+, CD4+ CD122+ T helpers/CD4+ T helpers, and CD122 MFI on CD4+ T helpers. (B) Immune cell profile in the spleen 5 days after RT showing CD8+/CD45+, CD8+ CD122+/CD8+, and CD122 MFI on CD8+T cells. A separate independent experiment (with a smaller sample size) demonstrated similar trends as the data presented in (A, B) (analyses here done by unpaired t-test). (C) The combined results from two independent flow experiments (n=5 per group, per experiment) showing the number of TILs as a ratio of live cells (or as a ratio to T regulatory cells for the last two panels) in the tumor following treatment with buffer (black), RT alone (blue), BEMPEG alone (pink), or RT+BEMPEG (purple) on day 14 after treatment began. Each symbol represents the TIL or splenic immune cell profile from one mouse (analyses done by one-way ANOVA). P values calculated using one-way ANOVA with Tukey’s multiple comparison tests. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. For comparisons where there is no p value shown, the p value was not significant. ANOVA, analysis of variance; BEMPEG, bempegaldesleukin; RT, radiation therapy; TIL, tumor-infiltrating lymphocyte.

Article Snippet: Cells were incubated with anti-CD16/32 (Biolegend, Clone 93) for 10 min at room temperature and then stained for 30 min at 4°C with the following antibodies: CD45 BV510 (30-F11), CD4 BV785 (GK1.5), CD8 APC-R700 (53–6.7), and NK1.1 PE-CF594 (PK136) all from BD Biosciences.

Techniques: Expressing