human gp100 peptide  (AnaSpec)

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Body weight (percentage change from baseline) of mice (n=8 mice/group) treated with PBS, IL-9 (50 µg or 100 µg), or IL-2 (50 µg) i.p. every other day. (B) Survival of mice from (A). (C) Sum of distance traveled over 30 second period in mice from (A), plotted relative to the average distance traveled by mice in the PBS group. (D) Graphical representation of mouse movement (n=8 mice/group) over a period of 30 seconds at baseline (top panel) and six days after starting treatment (bottom panel). (E) Serum IFNγ levels measured by ELISA six days after initiating treatment (n=3 mice/group) as described in A-C. (F) B16-F10 tumor growth (mean ± SEM, n=5 mice/group) after ACT with pmel T cells engineered with o9R or IL-9R (0.4×10 6 transduced cells, i.v.), and cytokine treatment with oIL-2 or IL-9, respectively (5×10 4 IU i.p., daily for 5 days starting with ACT). Data is representative of three independent experiments. (G) Survival of mice from (F). (H) Quantification of transduced pmel T cells in the blood five days after ACT (n=5 mice/group). (I) Quantification of transduced pmel T cells (Thy1.1 + YFP + ) across different tissues five days after ACT (n=5 mice/group). (J) Transduced (YFP + ) pmel T cells as percentage of all pmel T cells within the tumor 14 days after ACT (left panel) and total number of CD8+ T cells per gram of tumor 14 days after ACT (right panel) (n=5 mice per group). (K) o9R or IL9R transduced C57BL/6 T cells stimulated for 24h with IL-2 (10nM), IL-9 (10nM), oIL-2 (10µM), or a combination as noted. The proportion of naïve (CD62L+CD44-), central memory (CD62L+CD44+), effector (CD44+CD62L-) or double negative (CD62L-CD44-) T cells. Data from two independent experiments, with 4 technical replicates per condition. (L) The proportion of T SCM cells (CD44-, CD62L+, Sca-1+) from (K). For in vivo experiments, cytokines were tagged with mouse serum albumin (MSA) for half-life extension. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 (one-way ANOVA for A-C and E-F; Mantel-Cox for G; unpaired t test for H and J, two-way ANOVA for K-L).

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: Enzyme-linked Immunosorbent Assay, In Vivo

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Tumor growth (from ) with data shown for individual mice. (B) B16-F10 tumor growth (mean ± SEM, n=5 mice/group) in mice treated with either PBS or IL-9 (5×10 4 IU, i.p/dose) starting five days after tumor inoculation and continuing every other day for five doses. (C) KP-gp100 tumor growth (mean ± SEM and individual mice, n=6 mice / group) after ACT with pmel T cells engineered with IL-9R (1.6×10 6 transduced cells, i.v.), and cytokine treatment with PBS or IL-9, respectively (5×10 4 IU i.p., every other day for 5 days starting with ACT). Data is representative of two independent experiments. (D) Quantification of transduced pmel T cells (Thy1.1 + YFP + ) across different tissues 14 days after ACT and five doses of MSA-oIL2 or MSA-mIL9 (n=5 mice/group). (E) Representative gating strategy of CD44 and CD62L phenotyping for IL-9R or o9R-engineered T cells 24h after stimulation with IL-2 (10nM), IL-9 (10nM), or MSA-oIL2 (10µM). See . (F) Related to . The percentage of naïve (CD62L+CD44-), central memory (CD62L+CD44+), effector (CD44+CD62L-) or double negative (CD62L-CD44-) T cells as bar plots. (G) Proportion of T SCM cells (CD44-, CD62L+, Sca-1+) from (E) presented as bar plots. For all in vivo experiments, cytokines were tagged with mouse serum albumin (MSA) for half-life extension. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 (two-way ANOVA for B; Welch’s T-test for C; one-way ANOVA for D; two-way ANOVA for F-G).

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: In Vivo

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Dose-response curves of STAT1, STAT3, and STAT5 phosphorylation in IL-9R or o9R transduced (YFP + ) pmel T cells stimulated with either oIL-2 or IL-9 for 20 minutes (shown are technical duplicates; representative of at least three independent experiments). (B) Volcano plots depicting differential gene expression based on RNA-sequencing of IL-9R or o9R transduced C57BL/6 T cells and treated with IL-9 (10 nM), IL-2 (10 nM) or oIL2 (10 µM) for 48 hours. Comparisons for each volcano plot are shown below the x-axis. Significance (red) indicates adjusted p < 10 -5 and absolute fold change ≥2. (C) Differentially phosphorylated proteins between IL-9R transduced C57BL/6 T cells stimulated for 20’ with either IL-2 (10nM) or no cytokine (left) or IL-9 (10nM) versus no cytokine (right). Significance (red) indicates adjusted p<0.05 and log 2 (fold change)≥ 0.5. (D) Dose-response curve for STAT4 phosphorylation among IL-9R transduced (YFP + ) pmel T cells treated for 20 minutes with IL-9 or IL-12, or o9R transduced T cells treated with oIL-2 (shown are technical duplicates; representative of two independent experiments). (E) Waterfall plot summarizing transcription factor enrichment scores based on RNA-seq data of IL-9R T cells treated with IL-9 or IL-2 (top 15 for each). Enrichment score is inferred by fitting a linear model that predicts observed gene expression based on prior knowledge of a curated set of transcription factors and their target genes. (F) Heat map of the expression of the Biocarta IL-12 Pathway gene set based on RNA-sequencing from (B). Samples and genes clustered hierarchically without supervision. (G) In vitro expansion of IL-9R (YFP + ) transduced pmel T cells and treated with 10nM cytokine starting on day 3 after activation (n=3 technical replicates/group). Data is representative of three biological replicates and two independent experiments. (H) Representative contour plots of CD44 and CD62L expression of T cells from (G) after 24h treatment with cytokine. (I) Quantification of naïve CD62L + CD44 - (left) and stem-like CD62L + CD44 - Sca-1 + (right) T cells from (H). Data from two independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (one-way ANOVA for G; two-way ANOVA for G, I).

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: Expressing, RNA Sequencing Assay, In Vitro, Activation Assay

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Principal component analysis (PC1 vs PC2) of RNA sequencing of C57BL/6 T cells transduced for 48h with IL-9R and treated with IL-9 (10 nM) or IL-2 (10 nM), or with o9R and treated with oIL2 (10 µM). Samples cluster by treatment. (B) Volcano plots depicting differential gene expression based on RNA-sequencing of C57BL/6 T cells transduced with IL-9R or o9R and treated with IL-9 (10 nM), IL-2 (10 nM) or oIL2 (10 µM) for 48 hours. Shown here is the comparisons between o9R T cells treated with oIL-2 and IL-9R T cells treated with IL-2. Significance (red) indicates adjusted p-value < 1 x 10 -5 . (C) Heatmap of top 100 differentially upregulated (left) and downregulated (right) transcripts in T cell expressing IL-9R treated with IL-9 versus IL-2 for 48h. Shown also are the o9R samples treated with oIL-2, which mimic the expression of the IL-9R + IL-9 samples. (D) Differentially phosphorylated proteins between C57BL/6 T cells transduced with IL-9R and stimulated with IL-2 (10nM) or IL-9 for 20’. Significance (red) indicates adjusted p < 0.05 and log 2 (fold change) ≥ 0.5. (E) Representative gating strategy for flow cytometry data for intracellular staining of phosphoproteins in engineered (YFP + ) T cells. (F) Dose-response curves of ERK or AKT phosphorylation in IL-9R or o9R transduced pmel T cells (YFP + ) and stimulated with either oIL-2, IL-2, or IL-9 for 20 minutes. Error bars represent min/max of technical duplicates. Data representative of two independent experiments.

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: RNA Sequencing Assay, Expressing, Transduction, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Sequence alignment between mouse and human IL-2, IL-9, IL-15, and IL-21 around the conserved glutamine in Helix D. (B) Waterfall plot of maximal tumor size reduction for experiment from , in which B16-F10 tumors in mice (n=7-9 mice/group) treated with IL-9R transduced pmel T cells and either IL-9 WT or IL-9 Q115T . (C) Peripheral blood enrichment of IL-9R transduced pmel T cells over time in mice treated with ACT and either IL-9 WT or IL-9 Q115T cytokine (each cytokine dose indicated by a vertical dashed line). Enrichment was calculated as percentage of transduced (YFP+) pmel T cells as a proportion of all pmel T cells. See also . (D) Dose-response curves of phosphorylation of indicated STAT proteins in pmel T cells transduced with IL-9R WT , IL-9R 3x , and IL-9R 5x and stimulated with IL-2 or IL-9 for 20 minutes. Error bars represent min/max of three biological replicates. For in vivo experiments, cytokines were tagged with mouse serum albumin (MSA) for half-life extension. ***P < 0.001 (Welch’s t-test for C, two-way ANOVA for D).

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: Sequencing, Transduction, In Vivo

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Structural prediction of the interleukin-9 (IL-9) receptor complex based on AlphaFold2. The complex consists of the IL-9 receptor (IL-9R, green), IL-9 (blue), and the γ c (pink). The inset demonstrates the interaction of the glutamine at amino acid position 115 of IL-9 (Q115) with the γ c , which we subsequently mutated (IL-9 Q115T ) to generate an attenuated cytokine. (B) Dose-response curves of STAT1, STAT3, STAT4, and STAT5 phosphorylation in IL-9R transduced pmel T cells stimulated with IL-9 WT (blue) or IL-9 Q115T (light gray) for 20 minutes. Error bars represent SEM of technical duplicates; Data is representative of two independent experiments. (C) Tumor growth after ACT with IL-9R pmel T cells seven days after B16-F10 tumor inoculation. Mice were treated with either IL-9 WT (n=7 mice) or IL-9 Q115T (n=9 mice) (10 doses, every other day). Data is representative of two independent experiments. (D) Peripheral blood quantification of IL-9R transduced pmel T cells over time in mice treated with ACT and either IL-9 WT or IL-9 Q115T cytokine (each cytokine dose indicated by a vertical dashed line). In the IL-9 Q115T group, IL-9R pmel T cells were not observed in the blood at day 41 or after three additional cytokine doses on days 44-48, and thus only the IL-9 WT group received doses beyond day 48. Data is representative of two independent experiments. (E) Schematic of IL-9R variants with either three (IL-9R 3x ) or five (IL-9R 5x ) repeated phospho-tyrosine (pY) elements within the intracellular domain of the IL-9 receptor (created with Biorender.com ). Sequence of the phosphotyrosine element shown in the legend. (F) Phosphorylation of indicated STATs at E max (100nM) for IL-9R WT , IL-9R 3x , and IL-9R 5x transduced pmel T cells stimulated with IL-2 or IL-9 for 20 minutes. Data is representative of three biological experiments (mean ± SEM). (G) B16-F10 tumor growth after ACT with either IL-9R WT (n=7 mice) or IL-9R 3x transduced pmel T cells (n=8 mice). Mice were also treated with IL-9 (5×10 4 IU i.p., every other day for 5 doses starting with ACT). Shown are individual tumor growth curves (left and middle) and mean ± SEM (right). Data is representative of two independent experiments. For in vivo experiments, cytokines were tagged with mouse serum albumin (MSA) for half-life extension. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001. (two-way ANOVA for C; Welch’s t-test for D, G; one-way ANOVA for F). See also .

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: Sequencing, In Vivo

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Schematic of the IL-9 receptor complex, highlighting the phosphotyrosine residue within the IL-9R intracellular domain (ICD) and three adjacent amino acids. A panel of ten single amino acid mutations were generated within the ICD at the proline or glutamine residues (created with Biorender.com ). (B) Heat map of MFI (log-scaled and row-scaled) at E max for phosphorylation of STAT1, STAT3, STAT4, and STAT5 for C57BL/6 T cells transduced with wildtype IL-9R or one of ten IL-9R mutants. Transduced pmel T cells (technical duplicates) were stimulated with IL-9 for 20 minutes. Data is representative of two independent experiments. (C) Dose-response curves of STAT1, STAT3, STAT4, and STAT5 phosphorylation among transduced IL-9R WT , IL-9R AQ , and IL-9R PR pmel T cells (YFP+) stimulated with recombinant IL-9 for 20 minutes. Error bars represent SEM of technical duplicates. Data is representative of three biological replicates. (D) Relative in vitro expansion of YFP + IL-9R WT , IL-9R AQ , IL-9R PR pmel T cells cultured with IL-9 on day 3 post-activation (10 nM; n=3 technical replicates/group). Data is representative of 3 biological replicates. (E) In vitro proliferation index of C57BL/6 T cells engineered with IL-9R WT , IL-9R AQ , and IL-9R PR over the course of nine days post-activation (transduced on day 1), as measured by dilution of CellTrace Violet dye (n=6 replicates/group). Cells were treated with either recombinant IL-2 or IL-9 (10 nM) on day 3 after T cell activation. Proliferation index was quantified using FlowJo software based on values of histogram peaks (see ). (F) Peripheral blood quantification of IL-9R WT , IL-9R AQ , or IL-9R PR transduced (Thy1.1 + YFP + ) pmel T cells in B16-F10 tumor-bearing mice (n=6-7 mice/group) on the indicated days after ACT. IL-9 treatment (5×10 4 IU i.p., every other day) was started with ACT and continued for 5 doses. Data is representative of at least three independent experiments. (G) Quantification of IL-9R WT , IL-9R AQ , or IL-9R PR transduced (YFP+) pmel T cells within the tumors (B16-F10) of mice (n=6-7 mice/group) seven days post adoptive cell transfer (ACT). Data is representative of two independent experiments. (H) B16-F10 tumor growth in mice treated with IL-9R WT (n=7 mice) or IL-9R PR (n=6 mice) pmel T cells. Data is representative of at least three independent experiments. (I) B16-F10 tumor growth in mice treated with IL-9R WT or IL-9R AQ pmel T cells (n=10 mice/group). IL-9 treatment as per (F). Data is representative of at least 4 independent experiments. For in vivo experiments, cytokines were tagged with mouse serum albumin (MSA) for half-life extension. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (two-way ANOVA for D-F; Welch’s t-test for G-I).

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: Residue, Generated, Transduction, Recombinant, In Vitro, Cell Culture, Activation Assay, Software, In Vivo

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Schematic for in vivo experiment to generate single cell RNAseq data. IL-9R WT , IL-9R AQ , or IL-9R PR engineered pmel T cells are adoptively transferred into B16-F10 tumor-bearing mice seven days after tumor inoculation. Mice (n=7-8 mice/group) are treated with IL-9 (5×10 4 I.U. every other day) starting on the day of ACT (day 7) until tumors are harvested on day 15. Transduced pmel T cells (Thy1.1 + YFP + ) are sorted by FACS prior to library preparation and single cell RNAseq. See related – . (B) UMAP plots based on scRNAseq of n=6,706 cells pmel T cells from (A). Ten major clusters colored according to annotation. See related . (C) Propeller plots demonstrating the relative proportion of each cluster from (A), split by treatment group (IL-9R WT , IL-9R AQ , or IL-9R PR ). (D) Violin plots summarizing single cell expression of a gene set differentially expressed between KLRG1 hi effector and naïve mouse T cells (GSE10239). Gene sets are summarized as a module score in Seurat and plotted by treatment group. (E) Violin plots summarizing single cell expression of a gene set differentially expressed between mouse T cells eight days (D8) after encounter with acute infection versus malignancy (GSE60501). Gene sets are summarized as a module score in Seurat and plotted by treatment group. (F) Ridgeplot of pseudotime scores for single cell data from (A) organized by treatment group. (G) UMAP plot of scRNA-sequencing data from (A) annotated with pseudotime trajectories. Cells within the Tscm-like (Tcf7) cluster were selected as the root for the pseudotime analysis (white circles with black outlines). Black circles with white outlines represent nodes of the differentiation trajectory. (H) Schematic for linking phosphoflow data with RNA-sequencing data. o9R, IL-9R WT , IL-9R AQ , IL-9R PR , or IL-9R 5x pmel T cells were treated with cytokines for either 20 mins (phosphoflow) or 48h (RNA-sequencing). MFIs at E max for pSTAT1, pSTAT3, pSTAT4 and pSTAT5 were merged with RNA-sequencing data to identify genes highly correlated with phosphorylation of each STAT protein. (I) Histograms of phosphorylation of STAT1, STAT3, STAT4 and STAT5 for transduced (YFP+) T cells for each condition after 20 minutes of cytokine stimulation at Emax: IL-2 (10nM), IL-9 (10nM) and oIL-2 (10μM). (J) Venn diagram of the top 100 genes most correlated with the phosphorylation of each STAT. (K) Scatterplot depicting the relationship between the pSTAT1 module score (y-axis) and pSTAT1 phosphorylation levels (x-axis) in vitro. Module score was calculated from the expression of 100 genes most strongly correlated with STAT1 phosphorylation in vitro. Shown are biological triplicates for the gene expression data, colored by sample condition. For STAT phosphorylation data, shown are the average of technical replicates. The gray line indicates the linear regression fit. (L) Violin plot depicting projection of pSTAT1 module score from (J)-(K) onto scRNA-seq data from (A), organized by treatment group. (M) Projection of pSTAT1 module score from (J)-(K) onto UMAP of scRNA-seq data from (A). (N) Waterfall plot summarizing transcription factor (TF) enrichment scores when comparing in vitro RNAseq data of IL-9R WT vs IL-9R AQ groups (left) and IL-9R PR vs IL-9R WT groups (right). Gene expression changes across IL-9R AQ ➔ IL-9R WT ➔ IL-9R PR are captured by increasing activity of STAT1 and related TFs (shown in red). (O) Ridgeplots summarizing STAT1, STAT3, STAT4, and STAT5a regulon activity as an AUC score calculated using SCENIC. Results are presented by treatment group, with pairwise statistical comparisons (Wilcoxon Rank-Sum Test).

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: In Vivo, Expressing, Infection, Sequencing, RNA Sequencing Assay, In Vitro, Activity Assay

Journal: bioRxiv

Article Title: IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

doi: 10.1101/2025.01.15.633105

Figure Lengend Snippet: (A) Tumor growth (mean ± SEM) for the experiment described in (A). Data is representative of at least two independent experiments. (B) Transduced (Thy1.1 + YFP + ) pmel T cells as a percentage of total CD8 T cells in the tumor by treatment group for scRNAseq experiment from (A)-(B). (C) Merged UMAP plot based on scRNAseq of of n=6,706 cells pmel T cells transduced with IL-9R WT , IL-9R AQ , or IL-9R PR . Ten major clusters colored according to annotation. See related . (D) Violin plots summarizing single cell expression of stem and memory markers, Tcf7 (top) and Il7r (bottom), by cluster. (E) Feature plot of module score defined by the Gene Ontology Myeloid Differentiation gene set demonstrating high expression exclusively in the Tmyeloid-like cluster. (F) Violin plots summarizing single cell expression of effector molecules, Prf1 (top) and Gzmb (bottom), by treatment group. (G) Ridgeplot of pseudotime scores for single cell data from (A) organized by annotated cluster. (H) Scatterplot depicting the relationship between the pSTAT module scores (y-axis) and pSTAT phosphorylation levels (x-axis) in vitro. Module scores were calculated from the expression of 100 genes most strongly correlated with STAT phosphorylation in vitro. Shown are biological triplicates for the gene expression data, colored by sample condition. For STAT phosphorylation data, shown are the averages of technical duplicates. See also . (I) Projection of pSTAT module scores from (H) onto scRNA-seq data from . (J) Hallmark gene set enrichment analysis based on genes differentially expressed among tumor infiltrating IL-9R AQ pmel T cells from .

Article Snippet: Isolated pmel T cells were activated with 100 U ml -1 recombinant mouse IL-2 (rmIL-2) (Peprotech) and 1 µg ml -1 human gp100 peptide (Anaspec) the day before transduction.

Techniques: Transduction, Expressing, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: Pharmacologic LDH inhibition redirects intratumoral glucose uptake and improves antitumor immunity in solid tumor models

doi: 10.1172/JCI177606

Figure Lengend Snippet: ( A ) Schematic depicting tumor-killing assay with LDHi in which B16-YFP cells were treated with 20 μM LDHi or vehicle 24 hours apart and T cells were added 24 hours after the first LDHi treatment. ( B ) Quantified media glucose from killing assay coculture. ( C ) Flow cytometry quantification of 2-NBDG (MFI) in B16-YFP and CD8 + Pmel-1 T cells from killing assay cocultures 48 hours after last treatment. ( D – F ) ( D ) Quantified YFP + tumor cells and ( E ) representative in vitro killing assay images of YFP + tumor cells after 48 hours of coincubation with Pmel-1 CD8 + T cells as in A . ( F ) Corresponding quantified YFP + tumor cells and percentages of tumor killing in the same conditions as above alongside vehicle supplemented with 10 mM glucose. ( G ) Quantification of killing of OVA 257-264 –pulsed live B16-YFP tumor cells by OVA-primed CD8 + T cells from OT1 transgenic mice upon 48 hours of coculture in the presence of LDHi (as indicated in A ). E:T = 2:1, cocultured over 48 hours. ( H ) Schematic depicting in vitro Treg suppression assay with MACS column–sorted Tregs (CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse) cocultured with αCD3/αCD28-activated CTV-labeled syngeneic CD8 + T cells for 48 hours with the addition of conditioned media from B16 cells treated with 20 μM LDHi or vehicle or fresh media containing 10 mM glucose. ( I ) Percentage of suppression was calculated as percentage reduction in CD8 + T cell proliferation with respect to CD8 + T cells cultured alone in the same treatment and glucose conditions. Data show 1 representative experiment of 3 independent experiments ( n = 3–4 technical replicates). All statistics produced by 2-way ANOVA with Bonferroni’s multiple-comparisons test implemented in GraphPad Prism. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Data are represented as mean ± SEM.

Article Snippet: Splenocytes were primed with OVA (SIINFEKL 257-264, AnaSpec, AS-60193-1) or human gp100 peptide (25-33, AnaSpec, AS-62589) in RPMI media supplemented with 10% FCS and 50 mM BME as previously described ( ).

Techniques: Flow Cytometry, In Vitro, Transgenic Assay, Suppression Assay, Cell Isolation, Labeling, Cell Culture, Produced

Journal: Nature Communications

Article Title: Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin

doi: 10.1038/s41467-023-42714-4

Figure Lengend Snippet: a Single-dose subcutaneous (S.C.) injection of si3033 at 20 mg/kg supports Jak1 silencing in tail skin for over 5 weeks ( n = 5 animals, mean ± s.d.; two-sided unpaired t test, * P < 0.05, ** P < 0.01, *** P < 0.001); QuantiGene 2.0 assay and presented as percentage of siNTC control. b Biodistribution of si3033 at skin local to the injection site and in systemic tissues. ( n = 5 animals, mean ± s.d.). c Blood chemistry diagnostics at 24 h and 72 h post S.C. injection of 20 mg/kg si3033 ( n = 8 animals, mean ± s.d. the current plotting is to support visual clarity, raw data values can be found in the supplied source data file). ALB albumin, ALP alkaline phosphatase, ALT alanine transaminase, AMY amylase, Ca 2+ calcium, CRE creatinine, GLU glucose, Na + sodium, K + potassium, TP total protein, GLOB globulin, WBC white blood cell, LYM lymphocyte, MON monocyte, NEU neutrophil, RBC red blood cell, PLT platelet, HGB hemoglobin, HCT hematocrit, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, RDWc red blood cell distribution width coefficient of variation, RDWs red blood cell distribution width standard deviation, MPV mean platelet volume, PCT procalcitonin, PDWc platelet distribution width coefficient of variation, PDWs platelet distribution width standard deviation. d 0.08 mg of si3033 provides Jak1 silencing in the footpad skin (right vs. left pad) for 2 weeks. Mouse Jak1 mRNA were measured over 5 weeks using the QuantiGene 2.0 assay ( n = 16 animals, mean ± s.d.; two-sided paired t test, * P < 0.05, ** P < 0.01, *** P < 0.001). e si3033 significantly reduces the expression of IFN-γ-inducible chemokines CXCL9 and 10 in an ex vivo skin model of IFN-γ signaling at week 2. IFN-γ signaling was induced using recombinant mouse IFN-γ in a 3-mm skin punch collected at the injection site of footpad. Mouse CXCL9 and CXCL10 were quantified in the ex vivo culture media using ELISA assays ( n = 10 animals, mean ± s.d.; lines represent two-sided paired t test in the same mouse, * P < 0.05). f Schematic of a mouse model of vitiligo that mimics human skin depigmentation. g Skin infiltration of autoreactive PMEL CD8 + T cells ( n = 12 animals, mean ± s.d.; two-sided paired t test, ** P < 0.01). h Representative image reveals that si3033 prevents skin depigmentation in footpads. The locally injected area of left footpad exhibited less severity of depigmentation compared to right footpad treated with siNTC control compound. Source data are provided as a Source Data file.

Article Snippet: For dendritic cell vaccination, the BMDCs were primed with 10 μM human gp10025–33 (gp100) peptide (GenScript; #RP20344) in Opti-MEM media (Gibco; #11058021) at 37 °C for 3 h and washed with PBS for three times.

Techniques: Injection, Control, Concentration Assay, Standard Deviation, Expressing, Ex Vivo, Recombinant, Enzyme-linked Immunosorbent Assay