Journal: Science translational medicine

Article Title: The Stoichiometric Production of IL-2 and IFN-γ mRNA Defines Memory T Cells That Can Self-Renew After Adoptive Transfer in Humans

doi: 10.1126/scitranslmed.3004306

Figure Lengend Snippet: Defining the stoichiometric production of IL-2 and IFN-γ mRNA by memory CD8+ T cells. (A) (Left) Representative PBMC sorting for defined memory subsets. (Middle) Expression of IFN-γ (black bars) and IL-2 (gray bars) mRNA from sort-purified memory subsets after 3-hour stimulation with anti-CD3/CD28 beads. (Right) Comparison of the IL-2:IFN index. Data are means ± SEM of three independent patients. *P < 0.05, paired t test. (B) Quantitative comparison of antigen-induced IFN-γ and IL-2 mRNA by gp100-reactive microcultures (n = 9). (C) FACS analysis of the percent of gp100 tetramer+CD8+ cells (black dots) found in microcultures. Numbers in dot plot denote percent of CD8+ cells that are gp100 tetramer+. (D) Correlation between the percent of gp100 tetramer+CD8+ cells in microcultures and IFN-γ mRNA (top) or IL-2 mRNA (bottom). (E) Calculated IL-2:IFN index for microcultures. (F) FACS analysis of the memory phenotype of gp100 tetramer+CD8+ cells found in microcultures. Overlaid dot plots represent all cells within the lymphocyte gate; the gp100 tetramer+CD8+ T cells are shown in black dots and all other lymphocytes are shown in gray dots. Numbers in dot plot denote percent of gp100 tetramer+CD8+ cells that have a TCM phenotype. (G) Correlation between the percent of gp100 tetramer+CD8+ T cells with a TCM phenotype and their IL-2:IFN index.

Article Snippet: One day after completion of their lymphodepleting regimen, patients received expanded T cell clones infused intravenously followed by high-dose IL-2 (720,000 IU/kg) (aldesleukin; Novartis) every 8 hours to tolerance.

Techniques: Expressing, Purification

Journal: Science translational medicine

Article Title: The Stoichiometric Production of IL-2 and IFN-γ mRNA Defines Memory T Cells That Can Self-Renew After Adoptive Transfer in Humans

doi: 10.1126/scitranslmed.3004306

Figure Lengend Snippet: Identification of melanoma-specific TCM and TEM by profiling for the IL-2:IFN index. (A) Comparison of cytokine mRNA production, IL-2:IFN index, and memory phenotype from paired antigen-sensitized microcultures from HLA-A2+ melanoma patients (n = 6). Numbers in FACS dot plots correspond to percent of tetramer+ cells that are CD62L+. (B) Correlation between the percent of tetramer+CD8+ cells with a TCM phenotype and the IL-2:IFN index of the culture (n = 12). (C) Paired comparison of the percent of tetramer+CD8+ cells with a TCM phenotype in cultures with an IL-2:IFN index <10 versus >50. (D) Paired comparison of the percent of total CD8+ T cells that are tetramer+ in cultures with IL-2:IFN index <10 versus >50. Microculture pairs from six individual melanoma patients are denoted by an individual symbol. Statistical comparison was performed with paired t test. Bar represents mean.

Article Snippet: One day after completion of their lymphodepleting regimen, patients received expanded T cell clones infused intravenously followed by high-dose IL-2 (720,000 IU/kg) (aldesleukin; Novartis) every 8 hours to tolerance.

Techniques:

Journal: Science translational medicine

Article Title: The Stoichiometric Production of IL-2 and IFN-γ mRNA Defines Memory T Cells That Can Self-Renew After Adoptive Transfer in Humans

doi: 10.1126/scitranslmed.3004306

Figure Lengend Snippet: Melanoma-specific CD8+ T cells with a high IL-2:IFN index demonstrate superior in vitro proliferation and responsiveness to IL-2 exposure. (A) Representative FACS comparing the percent of gp100 tetramer+CD8+ T cells found in paired microcultures with high IL-2:IFN index (>50) and low IL-2:IFN index (<10) before and after polyclonal expansion with anti-CD3 antibody, IL-2 (300 IU/ml), and irradiated PBMC feeder cells. Numbers in dot plot denote percent of CD8+ cells that are gp100 tetramer+. (B) Fold expansion of tetramer+CD8+ and tetramer−CD8+ cells described in (A) at day 12. (C) Summary of day 12 proliferation cell counts from paired microcultures (high index versus low index) from four melanoma patients. Paired microcultures from individual patients are denoted by an individual symbol. Statistical comparison was performed with paired t test. Bars and numbers represent means. (D) Comparative CFSE dilution in tetramer+CD8+ and tetramer−CD8+ T cells from paired microcultures with high or low IL-2:IFN indices after 5-day exposure to anti-CD3/CD28 stimulation beads. Numbers in dot plots corresponding to percent of total CD8+ T cells that are tetramer+. Tetramer+CD8+ cells are shown in black dots and tetramer−CD8+ cells are in gray dots. Overlaid histograms demonstrate CFSE expression of tetramer+CD8+ cells (shaded black) and tetramer−CD8+ cells (shaded gray). Histogram gating reflects percent of tetramer+CD8+ cells with diluted CFSE compared to baseline. MFI values reflect fluorescence of CFSE in tetramer+CD8+ cells. Analysis is representative of four independent experiments performed. (E) Representative comparison of CFSE dilution in tetramer+CD8+ and tetramer−CD8+ T cells from paired microcultures with high and low IL-2:IFN indices after 7-day exposure to varying concentrations of IL-2. Numbers in dot plots corresponding to percent of tetramer+ (red) and tetramer− cells (black) with diluted CFSE compared to baseline. (F) Dose-response relationship between supplemented exogenous IL-2 and percent of tetramer+CD8+ and teteramer−CD8+ cells with diluted CFSE. Data are means ± SEM of four independent pairs of high-versus low-index cultures from two patients. *P < 0.05, paired t test; ***P < 0.001, two-way ANOVA. EC50 refers to the concentration of IL-2 (IU/ml).

Article Snippet: One day after completion of their lymphodepleting regimen, patients received expanded T cell clones infused intravenously followed by high-dose IL-2 (720,000 IU/kg) (aldesleukin; Novartis) every 8 hours to tolerance.

Techniques: In Vitro, Irradiation, Expressing, Fluorescence, Concentration Assay

Journal: Science translational medicine

Article Title: The Stoichiometric Production of IL-2 and IFN-γ mRNA Defines Memory T Cells That Can Self-Renew After Adoptive Transfer in Humans

doi: 10.1126/scitranslmed.3004306

Figure Lengend Snippet: Melanoma-specific CD8+ TE clones generated from high and low IL-2:IFN index precursors acquire similar cytolytic effector characteristics but differ in the expression of cell survival genes. (A) Representative comparison of parental melanoma-specific CD8+ memory T cells with dichotomous IL-2:IFN index and CD62L expression by FACS. Numbers in dot plots correspond to percent of gp100 tetramer+ cells that are CD62L+. (B) (Left) Phenotype of derived CD8+ T cell clones. Overlaid histograms reflect tetramergated cells; isotype staining is shown in black and specific antibody staining in gray. Values in FACS histograms correspond to percent shift from isotype control. (Right) Reactivity of derived CD8+ T cell clones. Intracellular cytokine FACS for IL-2 and IFN-γ after stimulation with T2 cells pulsed with gp100 peptide (gray dots) versus HIVpol control peptide (black dots). Numbers in dot plots represent percent of gp100-reactive cells found in respective gates. Tumor cytotoxicity was determined by coculture against 526 mel (HLA-A2+/gp100+) (black boxes) and 888 mel (HLA-A2−/gp100+) (white boxes). Plots demonstrate means ± SEM of triplicate samples. Analysis is representative of five independent patient clones that were examined. (C) Heat map of differentially expressed genes (P < 0.01) between resting clone groups (high IL-2:IFN index derived versus low IL-2:IFN index derived). (D) Ingenuity Pathway Analysis of differentially expressed genes; shown are cell functions ranked by the number of identified genes associated with the function. (E) TaqMan analysis for the expression of CD40L and BMF in derived clones. Values are reported as mRNA copies relative to 104 copies of β-actin.

Article Snippet: One day after completion of their lymphodepleting regimen, patients received expanded T cell clones infused intravenously followed by high-dose IL-2 (720,000 IU/kg) (aldesleukin; Novartis) every 8 hours to tolerance.

Techniques: Clone Assay, Generated, Expressing, Derivative Assay, Staining

Journal: Science translational medicine

Article Title: The Stoichiometric Production of IL-2 and IFN-γ mRNA Defines Memory T Cells That Can Self-Renew After Adoptive Transfer in Humans

doi: 10.1126/scitranslmed.3004306

Figure Lengend Snippet: In vitro and in vivo reactivity of adoptively transferred gp100-specific CD8+ T cell clones. (A) Intracellular cytokine FACS for IL-2 and IFN-γ production performed on infused gp100-specific clones. Clones were stimulated with T2 cells pulsed with gp100 peptide (gray dots) versus HIVpol control peptide (black dots). Numbers in dot plots represent percent of gp100-reactive cells in respective gates after CD3 gating. (B) Pie chart showing the percent of reactive cells producing the denoted cytokine (mean values from five patients). (C) (Top left) Representative photograph of skin rash seen on the torso of patient 1 on day 5 after clone infusion. (Top right) Close-up view of rash. (Bottom) Histologic comparison of pre- and post-infusion (day +5) skin biopsies (original magnification, ×20) demonstrating treatment-induced intraepidermal spongiosis (black arrows) seen on hematoxylin and eosin (H&E) staining, CD8+ lymphocyte infiltration (yellow arrows), and loss of melanocytes (red arrows) seen with Melan-A staining. Analysis is representative of biopsies from five independent patients.

Article Snippet: One day after completion of their lymphodepleting regimen, patients received expanded T cell clones infused intravenously followed by high-dose IL-2 (720,000 IU/kg) (aldesleukin; Novartis) every 8 hours to tolerance.

Techniques: In Vitro, In Vivo, Clone Assay, Staining

Journal: Science translational medicine

Article Title: The Stoichiometric Production of IL-2 and IFN-γ mRNA Defines Memory T Cells That Can Self-Renew After Adoptive Transfer in Humans

doi: 10.1126/scitranslmed.3004306

Figure Lengend Snippet: In vivo persistence and self-renewal of gp100-specific CD8+ T cell clones after adoptive transfer. (A) FACS for the percent of gp100 tetramer+CD8+ T cells found in pre-infusion peripheral blood lymphocytes (PBLs), infusion product, and post-infusion (day +30) PBL samples. Numbers in dot plot denote percent of CD8+ cells that are gp100 tetramer+. Infusion product contained >99% gp100 tetramer+CD8+ T cells with clonality verified by TCR gene sequencing. (B) Absolute number of gp100 tetramer+CD8+ T cells per microliter ofblood (top) and FACS analysis for the percent ofgp100tetramer+CD8+ T cells found in PBLs at specified time points for patient 3 (bottom). Arrow denotes day of infusion. (C) Reactivity of gp100+CD8+ T cells persisting in PBLs. Day 30 PBMC samples were thawed, stimulated with T2 cells pulsed with gp100 peptide or HIVpol control peptide, and analyzed by intracellular cytokine FACS for IL-2 and IFN-γ. FACS dot plots represent CD3-gated cells. Color coding of cytokine-producing cells is based on isotype gating. (D) Pie chart showing the percent of gp100-reactive cells producing the denoted cytokines (mean values from four patients). (E) Phenotypic comparison of the gp100+CD8+ T cell clones that were infused (Infu) versus those cells that persisted (Pers). Red squares denote mean values and error bars denote SEM of four independent patients.

Article Snippet: One day after completion of their lymphodepleting regimen, patients received expanded T cell clones infused intravenously followed by high-dose IL-2 (720,000 IU/kg) (aldesleukin; Novartis) every 8 hours to tolerance.

Techniques: In Vivo, Clone Assay, Adoptive Transfer Assay, Sequencing