female cd45 2 thy1 2 c57bl 6  (Charles River Laboratories)

Journal: Science (New York, N.Y.)

Article Title: Rewiring STAT signaling from the cell surface with Trikine immunotherapeutics

doi: 10.1126/science.adx9954

Figure Lengend Snippet: ( A-B ) Mouse CD8+ T cells were treated with indicated cytokines for 24 hours prior to RNA isolation for bulk RNAseq analysis; (A) Geneset enrichment analysis (GSEA) for DEGs between IL-2/21-Trikine-treated vs sIL2-treated mouse CD8+ T cells (left) and heatmap of leading-edge genes (right). The DEG ranks were projected onto two gene sets defined based on a previously published ChIP-seq data . pSTAT3-promoted genes, genes with stronger ChIP peaks in STAT3-WT mice. (B) Relative expression of stemness genes Tcf7 , Bach2 , Il7r , Slamf6 , and Sell between mouse CD8+ T cells treated with either sIL-2 or IL-2/21-Trikine. Results from one independent experiment with 3 technical replicates. ( C-F ), C57BL/6 mice bearing established s.c. B16F10 tumors received intravenous (i.v.) adoptive cell transfer (ACT) of pmel CD8+ T cells (1 x 10 7 ) that were preconditioned for 7 days with sIL-2, sIL-2 + IL-21, or IL-2/21-Trikine on day 5, followed by i.p. injections of cytokine (5 μg functional cytokine) every other day until day 12 (n = 5 animals); the experimental timeline (C), average tumor growth curves (D), average weight change curves (E), and survival curves (F). Results from one independent experiment. ( G-I ) Healthy C57BL/6 mice were dosed with 10 μg functional dose of sIL-2, IL-21, sIL-2 + IL-21, IL-2/21-Trikine or PBS every other day for four total doses (n = 10 mice); experimental timeline (G) percent weight change at study endpoint (H), serum amyloid A concentrations at study endpoint (I). Results are combined from 2 independent experiments. ( J-M ), C57BL/6 mice bearing established s.c. B16F10 tumors received intravenous (i.v.) adoptive cell transfer (ACT) of pmel CD8+ T cells (1 x 10 7 ) that were preconditioned for 7 days with sIL-2, sIL-2 + IL-21, or IL-2/21-Trikine on day 5, followed by i.p. injections of cytokine (2.5 μg functional cytokine) every other day until day 12 (n = 14/15 animals); the experimental timeline (J), average tumor growth curves (K), average weight change curves (L), and survival curves (M). Results are combined from 2 independent experiments. ( N ) Proportions of pmel T cell memory populations, TCM and TEM in the draining lymph node 16 days post tumor injection from mice receiving low dose regimen (J-M) (n = 8). Results representative of 2 independent experiments. ( O-P ) MFI of Sca1 (O) and IL7Ra (P) cells in the draining lymph node 12 days post ACT compared to on the day of ACT from mice receiving low dose regimen (J) (n = 8). Results representative of 2 independent experiments. ( Q ) Intratumoral, live Thy1.1+CD8+ cells were sorted from dissociated tumor 12 days after ACT as in (J) and sent for scRNA-seq. 8 transcriptionally distinct clusters are seen by UMAP (left) and cluster composition by treatment is displayed (right). scRNAseq data is from one independent experiment. Schematics in C, G, and J created using BioRender.com .

Article Snippet: For experiments performed at Stanford University, five- to six-week-old Thy1.2 + C57BL/6 (C57BL/6J) mice and B6129SF1/J (F1, Strain #101043) mice were purchased from Jackson Laboratory.

Techniques: Isolation, RNA sequencing, ChIP-sequencing, Expressing, Functional Assay, Injection

Journal: Science (New York, N.Y.)

Article Title: Rewiring STAT signaling from the cell surface with Trikine immunotherapeutics

doi: 10.1126/science.adx9954

Figure Lengend Snippet: ( A-C ), C57BL/6 mice bearing established s.c. B16F10 tumors received intravenous (i.v.) adoptive cell transfer (ACT) of pmel CD8 + T cells (5 × 10 6 ) on day 5, followed by i.p. injections of cytokine (3 μg functional cytokine) or PBS every other day until day 20 (n = 5 animals); the experimental timeline (A), average tumor growth curves (B), and survival curves (C). Results representative of 2 independent experiments. ( D-I ) Experimental setting is described in . C57BL/6 mice bearing established s.c. B16F10 tumors received i.v. ACT of pmel CD8 + T cells (5 × 10 6 ) on day 9, followed by i.p. injections of cytokine (3 μg functional cytokine) or left NT on days 9 and 11 (n = 5 animals). Mice were sacrificed on day 13, tumors were collected for flow cytometry analysis. Frequencies of Thy1.1 + CD8 + tumor-infiltrating lymphocytes (TILs) among single-live cells (D), frequencies of SCA-1 + CD62L + cells among Thy1.1 + CD8 + TILs (E), frequencies of PD-1+CD44+ cells among Thy1.1+CD8+ TILs (F) frequencies of Granzyme B+ cells among PD-1+CD44+ Thy1.1+CD8+ TILs (G), frequencies of IFN-γ+ cells among PD-1+CD44+ Thy1.1+CD8+ TILs (H), frequencies of TCF1+TIM-3- cells among PD-1+CD44+ Thy1.1+CD8+ TILs (I). Results representative of 2 independent experiments. ( J-L ), C57BL/6 mice bearing established s.c. B16F10 tumors (n = 5-10 animals) were administered cytokine (3 μg functional cytokine), PBS, or left NT i.p. on day 5 and every other day until day 19. For combination therapy, anti-PD-1 antibody (200 μg per dose) were i.p. administered on days 7, 11, 15, and 19; the experimental timeline (J), average tumor growth curves (K), survival curves (L). Results representative of 2 independent experiments. ( M ) Schematic of patient-derived organoid experiment. ( N ) Percentages of T133 tumor organoid viability in experiment as in (M). Results representative of 2 independent experiments. All data represent mean ± s.e.m. and are analyzed by one-way (D-I, N) or two-way (B, K) ANOVA with Tukey’s post-test. Schematics in D and M created using BioRender.com .

Article Snippet: For experiments performed at Stanford University, five- to six-week-old Thy1.2 + C57BL/6 (C57BL/6J) mice and B6129SF1/J (F1, Strain #101043) mice were purchased from Jackson Laboratory.

Techniques: Functional Assay, Flow Cytometry, Derivative Assay

Journal: Science (New York, N.Y.)

Article Title: Rewiring STAT signaling from the cell surface with Trikine immunotherapeutics

doi: 10.1126/science.adx9954

Figure Lengend Snippet: ( A-B ), C57BL/6 mice bearing established s.c. B16F10 tumors received i.p. injections of PBS, mono-IL-10, or IL-10/2-trikine (equivalent to 3 μg functional IL-10) starting on day 5, administered every other day until day 19 (n = 9 animals); the experimental timeline (A) and average tumor growth curves (B). Results are combined from 2 independent experiments. ( C-L ), C57BL/6 mice bearing established s.c. B16F10 tumors received i.p. injections of cytokine (3 μg functional cytokine) or left NT on days 9 and 11 (n = 5 animals). Mice were sacrificed on day 13, tumors were collected for flow cytometry analysis. (C), Average frequencies of CD45 + cells among single-live cells. (D- F), Counts of CD8 + T cells (D), CD4 + T cells (E), NK cells (F), myeloid cells (G), macrophages (H), monocytes (I), CD103 + dendritic cells (J), CD11b + dendritic cells (K), and B cells (L) per mg of tumor tissue. Results are representative of 2 independent experiments. ( M-O ), C57BL/6 mice bearing established subcutaneous C2 pancreatic tumors were treated intraperitoneally with cytokine (5.6 μg functional cytokine per dose) or left untreated (NT), administered every other day from day 6 to day 20 (n = 5 mice per group); experimental timeline (M), average tumor growth curves (N) and survival curves (O) of female mice in experiment described in (M). Results are representative of 2 independent experiments. Indicated statistical significance is IL-10/2-Trikine vs. PBS, IL-10/2-Trikine vs. ICB, and IL-10/2-Trikine vs. mono-IL-10. Indicated statistical significance in (O) is mono-IL-10 vs. PBS, is IL-10/2-Trikine vs. PBS, IL-10/2-Trikine vs. ICB, and IL-10/2-Trikine vs. mono-IL-10. Data represent mean ± s.e.m. and are analyzed by one-way (C to L) or two-way (B, N) ANOVA with Tukey’s post-test. Schematics in A and M created using BioRender.com . ( P-T ) C57BL/6 mice were inoculated subcutaneously. with 2.5 × 10 5 6694c2vTRP1 cells and treated with PBS, mono-IL-10 (26 μg/mouse, n = 5), or IL-10/2-Trikine (30.6 μg/mouse, n = 5) intraperitoneally on days 8 and 10 post-inoculation. Tumors were harvested on day 14 and CD45+ cells were isolated by magnetic bead separation, individually labeled with hashtag antibodies, pooled by treatment group, and subjected to Single Cell 5' RNA and cell surface protein sequencing. (P) UMAP of tumor-infiltrating CD3 + T and NK cells colored by unsupervised clusters. (Q) Bar graph shows cell cluster composition across treatment groups. Statistical significance was assessed by unpaired t test. Black asterisks indicate comparisons between PBS and IL-10/2-Trikine; white asterisks indicate comparisons between IL-10 and IL-10/2-Trikine. *P < 0.05, **P < 0.01. (R) Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) between all clusters from IL-10/2-Trikine–treated and mono–IL-10–treated groups using MSigDB Hallmark gene sets. (S) UMAP of tumor-infiltrating T/NK cells colored by a STAT5 target gene expression score ( Gzmb , Prf1 , Nkg7 , Gzma , Klrd1 , Klrb1c , Klra7 , Ccr5 , Ly6a , Thy1 ). (T) Mean STAT5 scores per mouse across all clusters, normalized per mouse. Each dot represents one mouse; error bars indicate standard deviation. Statistical significance was assessed by Wilcoxon test. **P < 0.01. scRNAseq data are the result of one independent experiment.

Article Snippet: For experiments performed at Stanford University, five- to six-week-old Thy1.2 + C57BL/6 (C57BL/6J) mice and B6129SF1/J (F1, Strain #101043) mice were purchased from Jackson Laboratory.

Techniques: Functional Assay, Flow Cytometry, Isolation, Labeling, Single Cell, Sequencing, Targeted Gene Expression, Standard Deviation

Journal: The Journal of experimental medicine

Article Title: Lung tissue-resident memory T cells optimize protection by IL-10 regulation of innate immunity

doi: 10.1084/jem.20242307

Figure Lengend Snippet: (A) Quantification of total CD4 + and CD8 + T cells in the lungs of mice treated with anti-Thy1.2 depleting Abs (orange) or IgG control Abs (gray). Statistical significance was determined by the student’s t-test. Boxes indicate interquartile range with a line drawn at the mean; whiskers indicate the minimum and maximum values. (B) T RM content in the lungs of T cell-depleted or control mice in (A) shown in representative flow cytometry plots (left) and compiled results (right) of CD45 expression from i.v. Ab administration (CD45(IV), left) and CD69 expression (right) by CD4 + (top), and CD8 + (bottom) T cells. (C) Lung macrophage accumulation after influenza infection in T RM -depleted (blue) and control (green) mice undergoing secondary infection at 4dpi compared to uninfected counterparts shown in representative flow cytometry plots (left) and compiled results (right) of F4/80 + macrophages in the lungs. Statistical significance for (A-C) was determined by the student’s t-test. (D-E) Pearson correlation analyses between T RM and CD4 + and CD8 + T RM subsets with (D) macrophages and with (E) Arg1 + macrophages. Data are representative of two independent experiments, with n=7–10 mice per group. ****, p ≤ 0.0001; ***; p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.

Article Snippet: To deplete T RM from mice, WT mice were infected with X31 influenza virus as above and 3 weeks post infection (wkpi), the resultant memory mice were administered 10μg/g of Thy1.2-depleting antibody (BioXcell; BE0066) by intraperitoneal (IP) injection four times over the course of one week.

Techniques: Control, Flow Cytometry, Expressing, Infection