αil 6  (Bio X Cell)

Journal: Immunity

Article Title: Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4 + T Cells

doi: 10.1016/j.immuni.2019.12.007

Figure Lengend Snippet: Increased IL-2 Availability after Treg Cell Depletion Contributes to Shaping the Th Cell Phenotype within Tumors (A and B) MCA205-tumor-bearing mice were treated with αCTLA-4 on days 6, 9, and 12 alone or with αIL-2, αCD8, and αMHC class II on days 6, 9, 12, and 15 after tumor implantation. (A) Individual tumor growth curves and (B) cumulative survival are shown. (C–G) MCA205-tumor-bearing mice were treated with αCTLA-4, αIL-2, or combination as in (A). TILs and dLNs were isolated 13 days post-tumor inoculation. (C) CD4eff/Treg cells in tumors (N = 10/group in two independent experiments). (D) TILs and dLNs from MCA205-bearing mice were re-stimulated with IL-2. Representative plots of pSTAT5 expression in CD4 + T cells and quantification of pSTAT5-expressing CD4eff T cells are shown (N = 5/group in two independent experiments). (E) Expression of GITR, CD69, and PD-1 by CD4eff TILs. Representative plots are shown with mean percentage of expression or mean fluorescence intensity (N = 5–10/group; two independent experiments). (F) Quantification of GzmB-expressing cells within CD4eff TILs and Treg TILs (N = 10/group in two independent experiments. (G) Quantification of GzmB-expressing cells within CD8 TILs and expression of Eomes and T-bet by CD4eff and CD8 TILs. Representative plots are shown with mean percentage of expression or mean fluorescent intensity (N = 10/group; two independent experiments). (H) dLN-infiltrating CD4 + T cells were cultured unstimulated or stimulated with MCA205-pulsed dendritic cells (DCs) or empty (np) DCs on αGzmB-coated ELISPOT plate for 24 h. Numbers represent GzmB spots per 10,000 responding CD4 + T cells. Graphical representation and quantification are shown. (I) Immunohistochemical analysis of GzmB expression by CD4 + T cells in human melanoma. Representative plots pre- and post-therapy are shown, with CD4 staining in brown, FOXP3 in green, and GZMB in blue. Quantification of CD4 + GZMB + cells within tumor pre- and post-treatment and ratio of CD4eff to CD4 + FOXP3 + cells are shown (n = 10 patients, Wilcoxon matched-pairs signed rank test, one tailed). All other quantification plots show mean ± SEM (one-way ANOVA).

Article Snippet: Therapeutic antibodies: αCTLA-4 (9H10), αCD4 (GK1.5) and αCD8 (2.43), αIL-2 (JES-6-1A12), αMHC-II (M5/114), αIL-15 (AIO.3) and αIL-7 (M25) were purchased from BioXcell.

Techniques: Tumor Implantation, Isolation, Expressing, Fluorescence, Cell Culture, Enzyme-linked Immunospot, Immunohistochemical staining, Staining, One-tailed Test

Journal: Immunity

Article Title: Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4 + T Cells

doi: 10.1016/j.immuni.2019.12.007

Figure Lengend Snippet: T-bet Is Not Required for CTLA-4-Mediated Rejection of MCA205 Sarcoma (A–C) WT and Tbx21 −/− MCA205-tumor-bearing mice were treated with αCTLA-4, αIL-2, or their combination. TILs and dLNs were isolated 13 days post-tumor inoculation for analysis of (A) Treg cells (N = 7–9/group in two independent experiments) and IFN-γ (N = 4–5/group) within CD4 + TILs and (B) GzmB-expressing cells within CD4eff and CD8 + TILs (N = 7–9/group in two independent experiments). (C) T-bet and Eomes expression by GzmB + CD4eff and CD8 + TILs (N = 7–9/group in two experiments). (D) Tumor growth and survival in WT and Tbx21 −/− mice bearing MCA205 tumors and treated with αCTLA-4 alone or combined with depleting αCD8 or αCD4 antibodies on days 1, 3, 8, and 17 post-tumor implantation. (E) qRT-PCR for transcription factors in purified MCA205 CD4 + Fopx3 − TILs and LNs at day 12 post-tumor inoculation (untreated versus αCTLA-4 treated mice). Results shown are expression relative to Hprt1 expression using the 2 −ΔΔC(t) method (N = 6/condition). All quantification plots show mean ± SEM (one-way ANOVA).

Article Snippet: Therapeutic antibodies: αCTLA-4 (9H10), αCD4 (GK1.5) and αCD8 (2.43), αIL-2 (JES-6-1A12), αMHC-II (M5/114), αIL-15 (AIO.3) and αIL-7 (M25) were purchased from BioXcell.

Techniques: Isolation, Expressing, Tumor Implantation, Quantitative RT-PCR, Purification

Journal: Immunity

Article Title: Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4 + T Cells

doi: 10.1016/j.immuni.2019.12.007

Figure Lengend Snippet: IL-2 Controls Cytotoxic CD4 + T Cell Differentiation in a Blimp-1-Dependent Manner (A) Prdm1 fl/fl and Prdm1 −/− Cd4 cre mice bearing MCA205 tumors were treated or not with αCTLA-4 and monitored for tumor growth and survival. (B and C) TILs and dLNs were isolated at day 12 post-tumor inoculation for quantification of (B) GzmB-expressing cells within CD4eff and CD8 TILs and (C) T-bet-expressing cells within CD4eff and CD8 TILs (N = 9–11/group in two experiments). (D) Purified CD4 + T cells from dLNs and tumors from MCA205-bearing Prdm1 fl/fl and Prdm1 −/− Cd4 cre mice were transferred to MCA205-bearing Rag1 −/− mice at day 3 post-inoculation followed by αCTLA-4. Overall survival is shown (N = 5/group). (E) Quantification of CD25- and IL-2-stimulated pSTAT5-expressing CD4eff TILs (N = 5–11 group in two experiments). (F) Prdm1 fl/fl and Prdm1 −/− Cd4 cre MCA205 tumor-bearing mice were treated with αCTLA-4 alone or in combination with high-dose intratumoral IL-2. TILs and dLNs were isolated at day 12 post-tumor inoculation for quantification of GzmB- and Prf-1-expressing cells within CD4 + TILs (N = 5/group). (G) Purified CD4 + T cells from WT ( Prdm1 fl/fl ) and Prdm1 −/− Cd4 cre mice were transduced with Trp1 TCR-expressing vector and transferred to B16-bearing mice alone or in combination with αCTLA-4 + RT. (H) Representative plots and quantification of GzmB- and IFN-γ-expressing cells within Trp1 effector TILs (N = 6/group in two independent experiments). (I) Transduced WT and Blimp-1-deficient Trp1 cells co-transferred 1:1 to the same host. Representative plots and quantification of GzmB-and Prf-1-expressing cells within Trp1 TILs are shown (N = 5/group). (J) Transduced cells as in (H) were transferred at day 8 to B16-bearing WT or Ifngr −/− mice alone or in combination with αCTLA-4 + RT. Cumulative tumor growth and survival are shown (N = 5/group). All quantification plots show mean ± SEM (one-way ANOVA) (J, two-way ANOVA).

Article Snippet: Therapeutic antibodies: αCTLA-4 (9H10), αCD4 (GK1.5) and αCD8 (2.43), αIL-2 (JES-6-1A12), αMHC-II (M5/114), αIL-15 (AIO.3) and αIL-7 (M25) were purchased from BioXcell.

Techniques: Cell Differentiation, Isolation, Expressing, Purification, Transduction, Plasmid Preparation