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Jackson Laboratory thy1 1 mice

( A ) Recipient mice (Thy1.2 + ) were transferred with mixed OVA-specific Cd69 +/+ <t>(Thy1.1</t> + Thy1.2 + ) and Cd69 −/− (Thy1.1 + Thy1.2 − ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.n.: intranasally; i.v.: intravenously. ( B ) Plots of Cd69 +/+ and Cd69 −/− CD45(iv) − CD4 + T cells in the lung (left) and absolute numbers ( n = 5) (right). ( C ) Confocal micrograph of the lung at day 35 (left). V: vessels. Frequency of transferred Cd69 +/+ (yellow) and Cd69 −/− (red) CD4 + T cells in iBALT ( n = 5) (right). DAPI: 4′,6-diamidino-2-phenylindole. ( D ) Recipient mice (Ly5.2 + ) were transferred with the mixed OVA-specific creER T2 Cd69 +/+ (Ly5.1 + Ly5.2 − ) and Cd69 fl/fl (Ly5.1 + Ly5.2 + ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.p.: intraperitoneally. ( E ) Plots of creER T2 Cd69 +/+ and Cd69 fl/fl cells in Ly5.1 + CD45(iv) − CD4 + T cells in the lung on day 49 and ( F ) absolute numbers (PBS, n = 8; TAM, n = 13). ( G ) Experimental protocol to investigate the functional impact of CD4 + T RM cell egress from the lungs on airway inflammation. ( H ) Absolute numbers of eosinophils (Eos.), neutrophils (Neut.), lymphocytes (Lymph.), and macrophages (Mac.) in the bronchoalveolar lavage fluid (BALF). ( I ) Representative H&E-stained lung sections. ( J ) Experimental protocol to assess the impact of CD69 ablation in CD4 + T RM cells under BCG infection. ( K ) Representative plots of CD45(iv) − CD44 hi CD69 + CD4 + T cells in the lung at day 49. ( L ) Absolute numbers of CD45(iv) − CD4 + T cells in PBS-treated ( n = 10) or TAM-treated ( n = 10) mice. Pooled data from two independent experiments. [(B), (C), (F), and (H)] Data of more than two independent experiments. Significance was determined by an unpaired U test in (B) and (C) and a one-way ANOVA in (F), (H), and (L). Data represent the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

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1) Product Images from "CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells"

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

Journal: Science Advances

doi: 10.1126/sciadv.adw1038

( A ) Recipient mice (Thy1.2 + ) were transferred with mixed OVA-specific Cd69 +/+ (Thy1.1 + Thy1.2 + ) and Cd69 −/− (Thy1.1 + Thy1.2 − ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.n.: intranasally; i.v.: intravenously. ( B ) Plots of Cd69 +/+ and Cd69 −/− CD45(iv) − CD4 + T cells in the lung (left) and absolute numbers ( n = 5) (right). ( C ) Confocal micrograph of the lung at day 35 (left). V: vessels. Frequency of transferred Cd69 +/+ (yellow) and Cd69 −/− (red) CD4 + T cells in iBALT ( n = 5) (right). DAPI: 4′,6-diamidino-2-phenylindole. ( D ) Recipient mice (Ly5.2 + ) were transferred with the mixed OVA-specific creER T2 Cd69 +/+ (Ly5.1 + Ly5.2 − ) and Cd69 fl/fl (Ly5.1 + Ly5.2 + ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.p.: intraperitoneally. ( E ) Plots of creER T2 Cd69 +/+ and Cd69 fl/fl cells in Ly5.1 + CD45(iv) − CD4 + T cells in the lung on day 49 and ( F ) absolute numbers (PBS, n = 8; TAM, n = 13). ( G ) Experimental protocol to investigate the functional impact of CD4 + T RM cell egress from the lungs on airway inflammation. ( H ) Absolute numbers of eosinophils (Eos.), neutrophils (Neut.), lymphocytes (Lymph.), and macrophages (Mac.) in the bronchoalveolar lavage fluid (BALF). ( I ) Representative H&E-stained lung sections. ( J ) Experimental protocol to assess the impact of CD69 ablation in CD4 + T RM cells under BCG infection. ( K ) Representative plots of CD45(iv) − CD44 hi CD69 + CD4 + T cells in the lung at day 49. ( L ) Absolute numbers of CD45(iv) − CD4 + T cells in PBS-treated ( n = 10) or TAM-treated ( n = 10) mice. Pooled data from two independent experiments. [(B), (C), (F), and (H)] Data of more than two independent experiments. Significance was determined by an unpaired U test in (B) and (C) and a one-way ANOVA in (F), (H), and (L). Data represent the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Figure Legend Snippet: ( A ) Recipient mice (Thy1.2 + ) were transferred with mixed OVA-specific Cd69 +/+ (Thy1.1 + Thy1.2 + ) and Cd69 −/− (Thy1.1 + Thy1.2 − ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.n.: intranasally; i.v.: intravenously. ( B ) Plots of Cd69 +/+ and Cd69 −/− CD45(iv) − CD4 + T cells in the lung (left) and absolute numbers ( n = 5) (right). ( C ) Confocal micrograph of the lung at day 35 (left). V: vessels. Frequency of transferred Cd69 +/+ (yellow) and Cd69 −/− (red) CD4 + T cells in iBALT ( n = 5) (right). DAPI: 4′,6-diamidino-2-phenylindole. ( D ) Recipient mice (Ly5.2 + ) were transferred with the mixed OVA-specific creER T2 Cd69 +/+ (Ly5.1 + Ly5.2 − ) and Cd69 fl/fl (Ly5.1 + Ly5.2 + ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.p.: intraperitoneally. ( E ) Plots of creER T2 Cd69 +/+ and Cd69 fl/fl cells in Ly5.1 + CD45(iv) − CD4 + T cells in the lung on day 49 and ( F ) absolute numbers (PBS, n = 8; TAM, n = 13). ( G ) Experimental protocol to investigate the functional impact of CD4 + T RM cell egress from the lungs on airway inflammation. ( H ) Absolute numbers of eosinophils (Eos.), neutrophils (Neut.), lymphocytes (Lymph.), and macrophages (Mac.) in the bronchoalveolar lavage fluid (BALF). ( I ) Representative H&E-stained lung sections. ( J ) Experimental protocol to assess the impact of CD69 ablation in CD4 + T RM cells under BCG infection. ( K ) Representative plots of CD45(iv) − CD44 hi CD69 + CD4 + T cells in the lung at day 49. ( L ) Absolute numbers of CD45(iv) − CD4 + T cells in PBS-treated ( n = 10) or TAM-treated ( n = 10) mice. Pooled data from two independent experiments. [(B), (C), (F), and (H)] Data of more than two independent experiments. Significance was determined by an unpaired U test in (B) and (C) and a one-way ANOVA in (F), (H), and (L). Data represent the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Techniques Used: Functional Assay, Staining, Infection

( A ) Volcano plot of the differential gene expression between CD4 + T RM cells and CD4 + T CIRC cells identified in fig. S5 (A to E). ( B ) Plots of CD69 and S1PR1 in Ly5.1 + CD45(iv) − CD4 + T cells in the lung of the OVA-treated mice at day 49 shown in fig. S6A. ( C ) Absolute numbers of CD45(iv) − S1PR1 + CD4 + T cells in the mice transferred with creER T2 Cd69 +/+ T H 2 cells ( n = 9) or creER T2 Cd69 fl/fl T H 2 cells ( n = 10). Pooled data from two independent experiments. ( D ) Absolute numbers of CD45(iv) − CD4 + T cells in the three groups ( n = 4 or 5). Data of two independent experiments. ( E ) Confocal micrograph of the lungs at day 49. B: bronchiole. ( F ) Absolute numbers of Ly5.1 + memory CD4 + T cells in iBALT. Data of two independent experiments. ( G ) H&E-stained lung section (top). Lungs were collected from the mice treated as shown in fig. S2A. The vessel wall, iBALT, and a parenchyma area located 200 μm from blood vessels were chosen to measure S1P levels by mass spectroscopy. The S1P intensity in these regions is shown (bottom). ( H ) Average peak intensities of S1P in each region. ( I ) Confocal micrograph of the lungs at day 35. Lungs were collected from the mice treated as shown in fig. S6H. ( J ) Absolute numbers of CD69 + Thy1.1 + cells (left) and CD69 − Thy1.1 + cells (right) in concentric distance zones from Lyve-1 + lymphatic vessels. Data represent the means ± SEM. Significance was determined by a one-way ANOVA in (C), (D), and (F), by paired one-way ANOVA in (H), and by an unpaired U test in (J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure Legend Snippet: ( A ) Volcano plot of the differential gene expression between CD4 + T RM cells and CD4 + T CIRC cells identified in fig. S5 (A to E). ( B ) Plots of CD69 and S1PR1 in Ly5.1 + CD45(iv) − CD4 + T cells in the lung of the OVA-treated mice at day 49 shown in fig. S6A. ( C ) Absolute numbers of CD45(iv) − S1PR1 + CD4 + T cells in the mice transferred with creER T2 Cd69 +/+ T H 2 cells ( n = 9) or creER T2 Cd69 fl/fl T H 2 cells ( n = 10). Pooled data from two independent experiments. ( D ) Absolute numbers of CD45(iv) − CD4 + T cells in the three groups ( n = 4 or 5). Data of two independent experiments. ( E ) Confocal micrograph of the lungs at day 49. B: bronchiole. ( F ) Absolute numbers of Ly5.1 + memory CD4 + T cells in iBALT. Data of two independent experiments. ( G ) H&E-stained lung section (top). Lungs were collected from the mice treated as shown in fig. S2A. The vessel wall, iBALT, and a parenchyma area located 200 μm from blood vessels were chosen to measure S1P levels by mass spectroscopy. The S1P intensity in these regions is shown (bottom). ( H ) Average peak intensities of S1P in each region. ( I ) Confocal micrograph of the lungs at day 35. Lungs were collected from the mice treated as shown in fig. S6H. ( J ) Absolute numbers of CD69 + Thy1.1 + cells (left) and CD69 − Thy1.1 + cells (right) in concentric distance zones from Lyve-1 + lymphatic vessels. Data represent the means ± SEM. Significance was determined by a one-way ANOVA in (C), (D), and (F), by paired one-way ANOVA in (H), and by an unpaired U test in (J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Techniques Used: Gene Expression, Staining, Mass Spectrometry

Image Search Results

Journal: Science Advances

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

doi: 10.1126/sciadv.adw1038

Figure Lengend Snippet: ( A ) Recipient mice (Thy1.2 + ) were transferred with mixed OVA-specific Cd69 +/+ (Thy1.1 + Thy1.2 + ) and Cd69 −/− (Thy1.1 + Thy1.2 − ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.n.: intranasally; i.v.: intravenously. ( B ) Plots of Cd69 +/+ and Cd69 −/− CD45(iv) − CD4 + T cells in the lung (left) and absolute numbers ( n = 5) (right). ( C ) Confocal micrograph of the lung at day 35 (left). V: vessels. Frequency of transferred Cd69 +/+ (yellow) and Cd69 −/− (red) CD4 + T cells in iBALT ( n = 5) (right). DAPI: 4′,6-diamidino-2-phenylindole. ( D ) Recipient mice (Ly5.2 + ) were transferred with the mixed OVA-specific creER T2 Cd69 +/+ (Ly5.1 + Ly5.2 − ) and Cd69 fl/fl (Ly5.1 + Ly5.2 + ) T H 2 cells (1 × 10 7 cells) followed by intranasal OVA exposure. i.p.: intraperitoneally. ( E ) Plots of creER T2 Cd69 +/+ and Cd69 fl/fl cells in Ly5.1 + CD45(iv) − CD4 + T cells in the lung on day 49 and ( F ) absolute numbers (PBS, n = 8; TAM, n = 13). ( G ) Experimental protocol to investigate the functional impact of CD4 + T RM cell egress from the lungs on airway inflammation. ( H ) Absolute numbers of eosinophils (Eos.), neutrophils (Neut.), lymphocytes (Lymph.), and macrophages (Mac.) in the bronchoalveolar lavage fluid (BALF). ( I ) Representative H&E-stained lung sections. ( J ) Experimental protocol to assess the impact of CD69 ablation in CD4 + T RM cells under BCG infection. ( K ) Representative plots of CD45(iv) − CD44 hi CD69 + CD4 + T cells in the lung at day 49. ( L ) Absolute numbers of CD45(iv) − CD4 + T cells in PBS-treated ( n = 10) or TAM-treated ( n = 10) mice. Pooled data from two independent experiments. [(B), (C), (F), and (H)] Data of more than two independent experiments. Significance was determined by an unpaired U test in (B) and (C) and a one-way ANOVA in (F), (H), and (L). Data represent the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Thy1.1 + mice were purchased from the Jackson Laboratory.

Techniques: Functional Assay, Staining, Infection

Journal: Science Advances

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

doi: 10.1126/sciadv.adw1038

Figure Lengend Snippet: ( A ) Volcano plot of the differential gene expression between CD4 + T RM cells and CD4 + T CIRC cells identified in fig. S5 (A to E). ( B ) Plots of CD69 and S1PR1 in Ly5.1 + CD45(iv) − CD4 + T cells in the lung of the OVA-treated mice at day 49 shown in fig. S6A. ( C ) Absolute numbers of CD45(iv) − S1PR1 + CD4 + T cells in the mice transferred with creER T2 Cd69 +/+ T H 2 cells ( n = 9) or creER T2 Cd69 fl/fl T H 2 cells ( n = 10). Pooled data from two independent experiments. ( D ) Absolute numbers of CD45(iv) − CD4 + T cells in the three groups ( n = 4 or 5). Data of two independent experiments. ( E ) Confocal micrograph of the lungs at day 49. B: bronchiole. ( F ) Absolute numbers of Ly5.1 + memory CD4 + T cells in iBALT. Data of two independent experiments. ( G ) H&E-stained lung section (top). Lungs were collected from the mice treated as shown in fig. S2A. The vessel wall, iBALT, and a parenchyma area located 200 μm from blood vessels were chosen to measure S1P levels by mass spectroscopy. The S1P intensity in these regions is shown (bottom). ( H ) Average peak intensities of S1P in each region. ( I ) Confocal micrograph of the lungs at day 35. Lungs were collected from the mice treated as shown in fig. S6H. ( J ) Absolute numbers of CD69 + Thy1.1 + cells (left) and CD69 − Thy1.1 + cells (right) in concentric distance zones from Lyve-1 + lymphatic vessels. Data represent the means ± SEM. Significance was determined by a one-way ANOVA in (C), (D), and (F), by paired one-way ANOVA in (H), and by an unpaired U test in (J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Thy1.1 + mice were purchased from the Jackson Laboratory.

Techniques: Gene Expression, Staining, Mass Spectrometry

a–d , Therapeutic effect of TGM1–IL-2 in DSS-induced colitis (PBS, n = 9 mice; TGM1–IL-2, n = 10 mice). a , Individual weight changes of mice in the indicated groups. b , Representative flow cytometry plots and quantification of IFN-γ + and IL-17a + CD4 + T-cell frequencies in the colon. c , Representative flow cytometry plots and quantification of Foxp3 + and Foxp3 - Thy1.1 + OT-II cell frequencies among total CD4 + T cells in the indicated tissues. d , Representative flow cytometry plots and quantification of Rorγt + and Gata3 + cell frequencies among Foxp3 + OT-II cells in the indicated tissues. e , UMAP plots showing the indicated transcription factor activity scores across clusters. f , Venn diagram illustrating the overlap of up- and down-regulated DEGs between the indicated groups. DEGs were filtered using an adjusted p value < 0.0001 and log 2 fold change > 1. g , Gene regulatory networks and signature genes identified by scRNA-seq analysis. The two numbers in parentheses represent the specific values from the Venn diagram shown in Extended Data Fig. 12f (right). h , Schematic illustration of antigen-specific pTreg differentiation in vivo driven by TCR stimulation, TGM1–IL-2, and key transcription factors. Created in BioRender; Sun, Q. https://BioRender.com/2rbbpii (2026). Data are presented as mean ± s.e.m. The data in a–d are representative of two independent experiments. The statistics were obtained by unpaired Welch’s t-test (two-tailed) ( b and c ).

Journal: Nature

Article Title: Facile induction of immune tolerance by an interleukin-2–TGFβ surrogate agonist

doi: 10.1038/s41586-026-10208-0

Figure Lengend Snippet: a–d , Therapeutic effect of TGM1–IL-2 in DSS-induced colitis (PBS, n = 9 mice; TGM1–IL-2, n = 10 mice). a , Individual weight changes of mice in the indicated groups. b , Representative flow cytometry plots and quantification of IFN-γ + and IL-17a + CD4 + T-cell frequencies in the colon. c , Representative flow cytometry plots and quantification of Foxp3 + and Foxp3 - Thy1.1 + OT-II cell frequencies among total CD4 + T cells in the indicated tissues. d , Representative flow cytometry plots and quantification of Rorγt + and Gata3 + cell frequencies among Foxp3 + OT-II cells in the indicated tissues. e , UMAP plots showing the indicated transcription factor activity scores across clusters. f , Venn diagram illustrating the overlap of up- and down-regulated DEGs between the indicated groups. DEGs were filtered using an adjusted p value < 0.0001 and log 2 fold change > 1. g , Gene regulatory networks and signature genes identified by scRNA-seq analysis. The two numbers in parentheses represent the specific values from the Venn diagram shown in Extended Data Fig. 12f (right). h , Schematic illustration of antigen-specific pTreg differentiation in vivo driven by TCR stimulation, TGM1–IL-2, and key transcription factors. Created in BioRender; Sun, Q. https://BioRender.com/2rbbpii (2026). Data are presented as mean ± s.e.m. The data in a–d are representative of two independent experiments. The statistics were obtained by unpaired Welch’s t-test (two-tailed) ( b and c ).

Article Snippet: Foxp3 -GFP mice (IMSR_JAX:006772) were crossed with OT-II Thy1.1 mice.

Techniques: Flow Cytometry, Activity Assay, In Vivo, Two Tailed Test

( 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 .

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: TCR-transgenic Thy1.1 + pmel-1 (pmel) mice (B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J) were originally purchased from the Jackson Laboratory and maintained in the Stanford University-Lorry Lokey (SIM1) Facility.

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

( 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 .

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 .

Techniques: Functional Assay, Flow Cytometry, Derivative Assay

A. Experimental scheme: naive TCR TAG (Thy1.1) were adoptively transferred into LM TAG -infected C57BL/6 (B6; Thy1.2) or tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice. TCR TAG were re-isolated from spleens and livers of infected mice (green) and tumor mice (blue) for flow cytometric analysis between 2.5-70+ days post-transfer. B. TCR TAG cell numbers in the spleens (left) and livers (right) of LM TAG -infected mice and tumor-bearing mice. Dots represent mean and error bars represent SEM. For B-D and F n = 2-3 mice per group (infection); n = 3 mice per group (tumor). Data is representative of two independent experiments. C. Histograms (left) showing TCR TAG PD1 expression from infection shown in comparison to naive (N; gray). For C-D and F , each symbol represents an individual mouse, and one-sample Student’s t -test with Bonferroni correction was used to determine significance: *, P <0.008 (infection); *, P <0.007 (tumor); ns, not statistically significant. D. Histograms and summary plots of CD38 expression and CD101 expression in spleens and livers from tumor mice shown in comparison to N. E. TCR TAG IFNγ and TNFα production after 4-hour ex vivo TAG peptide stimulation, with inset numbers indicating the percentage of cells in each gate. Gates were set based on no peptide stimulation controls. Right, summary plots of % IFNγ + TCR TAG after peptide stimulation; each symbol represents an individual mouse; n = 3-4 mice per group. *, P <0.05; **, P <0.01; ****, P <0.0001 (one-way ANOVA with post hoc Tukey test). F. Histograms and summary plots of CD122 expression and BCL2 expression from tumor mice shown in comparison to N. All flow plots are gated on live CD8 + Thy1.1 + cells, and flow data for each timepoint is concatenated from all biological replicates. Summary plots (right) show MFI (geometric m ean fluorescence intensity).

Journal: bioRxiv

Article Title: TCF1 lo CD8 T cells proliferate and persist autonomously in tumors

doi: 10.64898/2026.01.17.700120

Figure Lengend Snippet: A. Experimental scheme: naive TCR TAG (Thy1.1) were adoptively transferred into LM TAG -infected C57BL/6 (B6; Thy1.2) or tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice. TCR TAG were re-isolated from spleens and livers of infected mice (green) and tumor mice (blue) for flow cytometric analysis between 2.5-70+ days post-transfer. B. TCR TAG cell numbers in the spleens (left) and livers (right) of LM TAG -infected mice and tumor-bearing mice. Dots represent mean and error bars represent SEM. For B-D and F n = 2-3 mice per group (infection); n = 3 mice per group (tumor). Data is representative of two independent experiments. C. Histograms (left) showing TCR TAG PD1 expression from infection shown in comparison to naive (N; gray). For C-D and F , each symbol represents an individual mouse, and one-sample Student’s t -test with Bonferroni correction was used to determine significance: *, P <0.008 (infection); *, P <0.007 (tumor); ns, not statistically significant. D. Histograms and summary plots of CD38 expression and CD101 expression in spleens and livers from tumor mice shown in comparison to N. E. TCR TAG IFNγ and TNFα production after 4-hour ex vivo TAG peptide stimulation, with inset numbers indicating the percentage of cells in each gate. Gates were set based on no peptide stimulation controls. Right, summary plots of % IFNγ + TCR TAG after peptide stimulation; each symbol represents an individual mouse; n = 3-4 mice per group. *, P <0.05; **, P <0.01; ****, P <0.0001 (one-way ANOVA with post hoc Tukey test). F. Histograms and summary plots of CD122 expression and BCL2 expression from tumor mice shown in comparison to N. All flow plots are gated on live CD8 + Thy1.1 + cells, and flow data for each timepoint is concatenated from all biological replicates. Summary plots (right) show MFI (geometric m ean fluorescence intensity).

Article Snippet: TCR TAG transgenic mice (B6.Cg-Tg(TcraY1,TcrbY1)416Tev/J, RRID:IMSR_JAX:005236) , Cre-ER T2 mice (B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J, RRID:IMSR_JAX:008463), Rosa26-Cas9 mice (Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J, RRID:IMSR_JAX:026179), B6 Thy1.1 mice (B6.PL-Thy1a/CyJ, RRID:IMSR_JAX:000406), and B6 mice (C57BL/6J, RRID:IMSR_JAX:000664) were purchased from the Jackson Laboratory.

Techniques: Infection, Isolation, Expressing, Comparison, Ex Vivo, Fluorescence

A. Experimental setup as shown in . Histograms and summary plots of CD44 expression from infection (green) and tumor (blue) in spleen and liver shown in comparison to naive (N; gray). Each symbol represents an individual mouse, and one-sample Student’s t -test with Bonferroni correction was used to determine significance: *, P <0.008 (infection); *, P <0.007 (tumor); ns, not statistically significant. n = 2-3 mice per group (infection); n = 3 mice per group (tumor). Data is representative of two independent experiments. B. TCR TAG IFNγ and TNFα production after 4-hour ex vivo TAG peptide stimulation, with inset numbers indicating the percentage of cells in each gate. Gates were set based on no peptide stimulation controls. Summary plots show % IFNγ + TCR TAG after peptide stimulation; each symbol represents an individual mouse; n = 3-4 mice per group. ****, P <0.0001 (two-way ANOVA followed by post hoc Šídák’s multiple comparisons test). All flow plots are gated on live CD8 + Thy1.1 + cells and flow data for each timepoint is concatenated from all biological replicates. Data is representative of two independent experiments.

Journal: bioRxiv

Article Title: TCF1 lo CD8 T cells proliferate and persist autonomously in tumors

doi: 10.64898/2026.01.17.700120

Figure Lengend Snippet: A. Experimental setup as shown in . Histograms and summary plots of CD44 expression from infection (green) and tumor (blue) in spleen and liver shown in comparison to naive (N; gray). Each symbol represents an individual mouse, and one-sample Student’s t -test with Bonferroni correction was used to determine significance: *, P <0.008 (infection); *, P <0.007 (tumor); ns, not statistically significant. n = 2-3 mice per group (infection); n = 3 mice per group (tumor). Data is representative of two independent experiments. B. TCR TAG IFNγ and TNFα production after 4-hour ex vivo TAG peptide stimulation, with inset numbers indicating the percentage of cells in each gate. Gates were set based on no peptide stimulation controls. Summary plots show % IFNγ + TCR TAG after peptide stimulation; each symbol represents an individual mouse; n = 3-4 mice per group. ****, P <0.0001 (two-way ANOVA followed by post hoc Šídák’s multiple comparisons test). All flow plots are gated on live CD8 + Thy1.1 + cells and flow data for each timepoint is concatenated from all biological replicates. Data is representative of two independent experiments.

Techniques: Expressing, Infection, Comparison, Ex Vivo

A. Experimental setup as in . Heatmap summarizing data for flow cytometric assessment of activation and homing proteins, inhibitory receptors, cytokine receptors, proliferation and survival markers, and transcription factors. Blue-red color scale shows row normalized log 2 fold change (log2FC) of MFI in comparison to N. Each column represents individual biologic replicates for each condition and time point (the same single naive replicate is shown next to both infection and tumor groups). Data is representative of two independent experiments . B. Corresponding histograms showing expression of activation and homing markers (LY108, CD62L, CD44, CD69) andinhibitory receptors (PD1, CD38, CD101, CD101, and TIM3). C. Histograms showing expression of cytokine receptors (CD132, CD25, CD127, CD122), proliferation and survival markers (KI67, BCL2, BIM), and transcription factors (TOX, LEF1, and TCF1). All flow plots are gated on live CD8 + Thy1.1 + cells and flow data for each timepoint is concatenated from all biological replicates. Data is representative of two independent experiments.

Journal: bioRxiv

Article Title: TCF1 lo CD8 T cells proliferate and persist autonomously in tumors

doi: 10.64898/2026.01.17.700120

Figure Lengend Snippet: A. Experimental setup as in . Heatmap summarizing data for flow cytometric assessment of activation and homing proteins, inhibitory receptors, cytokine receptors, proliferation and survival markers, and transcription factors. Blue-red color scale shows row normalized log 2 fold change (log2FC) of MFI in comparison to N. Each column represents individual biologic replicates for each condition and time point (the same single naive replicate is shown next to both infection and tumor groups). Data is representative of two independent experiments . B. Corresponding histograms showing expression of activation and homing markers (LY108, CD62L, CD44, CD69) andinhibitory receptors (PD1, CD38, CD101, CD101, and TIM3). C. Histograms showing expression of cytokine receptors (CD132, CD25, CD127, CD122), proliferation and survival markers (KI67, BCL2, BIM), and transcription factors (TOX, LEF1, and TCF1). All flow plots are gated on live CD8 + Thy1.1 + cells and flow data for each timepoint is concatenated from all biological replicates. Data is representative of two independent experiments.

Techniques: Activation Assay, Comparison, Infection, Expressing

A. Experimental scheme: naive TCR TAG (Thy1.1) were adoptively transferred into tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice. TCR TAG were re-isolated from spleens and livers for flow cytometric analysis at 2.5, 5, 10, 14, 28, 35, and 70+ days post-transfer. B. Histograms and summary plots of KI67 expression of TCR TAG in spleens and livers from tumor-bearing mice shown in comparison to N, with % KI67 + gate set to exclude N. Flow plots for each time point were concatenated from all biological replicates; each symbol represents an individual mouse. *, P <0.007 determined by one-sample Student’s t -test with Bonferroni correction. C. Left, representative dot plots of KI67 expression and DNA content staining in TCR TAG in spleens and livers of AST;Cre-ER T2 mice, with inset numbers indicating the percentage of cells in G 0 , G 1 , and S-G 2 M phases of cell cycle. Middle, summary plots of the percentage of TCR TAG in G 0 , G 1 , and S-G 2 M, shown as mean ± SEM. Right, summary plots of the percentage of TCR TAG in S-G 2 M. Each symbol represents an individual mouse. *, P <0.007 determined by one-sample Student’s t -test with Bonferroni correction. D. Histograms and summary plots of TCF1 expression in spleens and livers, with TCF1 + gate shown. Flow plots for each time point were concatenated from all biological replicates; each symbol represents an individual mouse. *, P <0.007 determined by one-sample Student’s t -test with Bonferroni correction. E. Longitudinal analysis of the absolute cell numbers of TCR TAG cells (blue) in the spleens and livers of tumor-bearing mice and the % TCF1 + TCR TAG (yellow), shown as mean ± SEM. F. Summary plots of the % TCF1 + (filled bars) and TCF1 − (open bars) TCR TAG within the S-G 2 /M + subset, shown as mean ± SEM. Data is representative of two independent experiments with n = 3 mice per group

Journal: bioRxiv

Article Title: TCF1 lo CD8 T cells proliferate and persist autonomously in tumors

doi: 10.64898/2026.01.17.700120

Figure Lengend Snippet: A. Experimental scheme: naive TCR TAG (Thy1.1) were adoptively transferred into tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice. TCR TAG were re-isolated from spleens and livers for flow cytometric analysis at 2.5, 5, 10, 14, 28, 35, and 70+ days post-transfer. B. Histograms and summary plots of KI67 expression of TCR TAG in spleens and livers from tumor-bearing mice shown in comparison to N, with % KI67 + gate set to exclude N. Flow plots for each time point were concatenated from all biological replicates; each symbol represents an individual mouse. *, P <0.007 determined by one-sample Student’s t -test with Bonferroni correction. C. Left, representative dot plots of KI67 expression and DNA content staining in TCR TAG in spleens and livers of AST;Cre-ER T2 mice, with inset numbers indicating the percentage of cells in G 0 , G 1 , and S-G 2 M phases of cell cycle. Middle, summary plots of the percentage of TCR TAG in G 0 , G 1 , and S-G 2 M, shown as mean ± SEM. Right, summary plots of the percentage of TCR TAG in S-G 2 M. Each symbol represents an individual mouse. *, P <0.007 determined by one-sample Student’s t -test with Bonferroni correction. D. Histograms and summary plots of TCF1 expression in spleens and livers, with TCF1 + gate shown. Flow plots for each time point were concatenated from all biological replicates; each symbol represents an individual mouse. *, P <0.007 determined by one-sample Student’s t -test with Bonferroni correction. E. Longitudinal analysis of the absolute cell numbers of TCR TAG cells (blue) in the spleens and livers of tumor-bearing mice and the % TCF1 + TCR TAG (yellow), shown as mean ± SEM. F. Summary plots of the % TCF1 + (filled bars) and TCF1 − (open bars) TCR TAG within the S-G 2 /M + subset, shown as mean ± SEM. Data is representative of two independent experiments with n = 3 mice per group

Techniques: Isolation, Expressing, Comparison, Staining

A. Experimental scheme: naive TCR TAG (Thy1.1) were adoptively transferred into LM TAG -infected B6 (Thy1.2) or tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice and 5-ethynyl-2’-deoxyurdine (EdU) and 5-bromo-2’-deoxyuridine (BrdU) were administered on days 35 and 42, respectively. TCR TAG were re-isolated on day 43 from spleens of infected mice (green) and spleens and livers of tumor-bearing mice (blue) for flow cytometric analysis. B. Schematic representation of the expected distributions of EdU and BrdU incorporation for T cells proliferating in a progenitor-progeny hierarchy (left) or stochastically (right). C. Top, dot plots of TCR TAG EdU and BrdU incorporation in infected mice and tumor-bearing mice. Unlabeled cells shown in gray with inset numbers showing the percentage of nucleoside labeled cells in each gate. Bottom, summary plots of % EdU + (red), EdU + ,BrdU + (purple), and BrdU + (cyan) TCR TAG within each nucleoside labeled subset. Each symbol represents an individual mouse. **, P <0.01 determined by repeated measures, one-way ANOVA with post hoc Tukey test. D. Concatenated histogram of TCF1 expression in nucleoside-labeled TCR TAG and summary plot of the percentage of nucleoside-labeled TCR TAG expressing TCF1. Each symbol represents an individual mouse. *, P <0.025 determined by one-sample Student’s t -test with Bonferroni correction with n=4-5 mice per group and representative of two independent experiments. Flow plots show data concatenated from all biological replicates.

Journal: bioRxiv

Article Title: TCF1 lo CD8 T cells proliferate and persist autonomously in tumors

doi: 10.64898/2026.01.17.700120

Figure Lengend Snippet: A. Experimental scheme: naive TCR TAG (Thy1.1) were adoptively transferred into LM TAG -infected B6 (Thy1.2) or tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice and 5-ethynyl-2’-deoxyurdine (EdU) and 5-bromo-2’-deoxyuridine (BrdU) were administered on days 35 and 42, respectively. TCR TAG were re-isolated on day 43 from spleens of infected mice (green) and spleens and livers of tumor-bearing mice (blue) for flow cytometric analysis. B. Schematic representation of the expected distributions of EdU and BrdU incorporation for T cells proliferating in a progenitor-progeny hierarchy (left) or stochastically (right). C. Top, dot plots of TCR TAG EdU and BrdU incorporation in infected mice and tumor-bearing mice. Unlabeled cells shown in gray with inset numbers showing the percentage of nucleoside labeled cells in each gate. Bottom, summary plots of % EdU + (red), EdU + ,BrdU + (purple), and BrdU + (cyan) TCR TAG within each nucleoside labeled subset. Each symbol represents an individual mouse. **, P <0.01 determined by repeated measures, one-way ANOVA with post hoc Tukey test. D. Concatenated histogram of TCF1 expression in nucleoside-labeled TCR TAG and summary plot of the percentage of nucleoside-labeled TCR TAG expressing TCF1. Each symbol represents an individual mouse. *, P <0.025 determined by one-sample Student’s t -test with Bonferroni correction with n=4-5 mice per group and representative of two independent experiments. Flow plots show data concatenated from all biological replicates.

Techniques: Infection, Isolation, BrdU Incorporation Assay, Labeling, Expressing

A. Histogram of TCF1 expression 48 hours post transfection of naive Cas9;TCR TAG with sgRNA transfected with non-targeting control sgRNA (NTC; light green) or TCF7 sgRNA (TCF1KO; dark teal), data representative of two independent experiments. B. Experimental scheme: NTC or TCF1KO TCR TAG (Thy1.1) were adoptively transferred into tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice. EdU was administered in the final 3 days. TCR TAG were re-isolated on day 35 from the spleens and livers for flow cytometric analysis. C. NTC and TCF1KO TCR TAG cell numbers recovered from tumor-bearing mice. D. Representative dot plots of KI67 expression and EdU incorporation of NTC and TCF1KO TCR TAG cells, with gates set based on KI67 − /EdU − cells. E. Top, summary plots of the percentage (left) and absolute number (right) of EdU + NTC or TCF1KO TCR TAG . Bottom, summary plots of the percentage (left) and absolute number (right) of KI67 + or TCF1KO TCR TAG . For C and E , each symbol represents an individual mouse, with two-way ANOVA followed by post hoc Šídák’s multiple comparisons test with n=4-5 mice per group and representative of two independent experiments.

Journal: bioRxiv

Article Title: TCF1 lo CD8 T cells proliferate and persist autonomously in tumors

doi: 10.64898/2026.01.17.700120

Figure Lengend Snippet: A. Histogram of TCF1 expression 48 hours post transfection of naive Cas9;TCR TAG with sgRNA transfected with non-targeting control sgRNA (NTC; light green) or TCF7 sgRNA (TCF1KO; dark teal), data representative of two independent experiments. B. Experimental scheme: NTC or TCF1KO TCR TAG (Thy1.1) were adoptively transferred into tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice. EdU was administered in the final 3 days. TCR TAG were re-isolated on day 35 from the spleens and livers for flow cytometric analysis. C. NTC and TCF1KO TCR TAG cell numbers recovered from tumor-bearing mice. D. Representative dot plots of KI67 expression and EdU incorporation of NTC and TCF1KO TCR TAG cells, with gates set based on KI67 − /EdU − cells. E. Top, summary plots of the percentage (left) and absolute number (right) of EdU + NTC or TCF1KO TCR TAG . Bottom, summary plots of the percentage (left) and absolute number (right) of KI67 + or TCF1KO TCR TAG . For C and E , each symbol represents an individual mouse, with two-way ANOVA followed by post hoc Šídák’s multiple comparisons test with n=4-5 mice per group and representative of two independent experiments.

Techniques: Expressing, Transfection, Control, Isolation

A. Experimental scheme: naive TCR TAG (Thy1.1) were adoptively transferred into tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice. FTY720 treatment or vehicle was administered every other day beginning on day −1 for 6 days or day 28 for 3 weeks. EdU was administered for the final 3 days prior to harvest. TCR TAG were re-isolated on day 5 and day 49 from the livers for flow cytometric analysis. B. Representative histogram and summary plot of % CD3ε + cells in peripheral blood 24 hours after initiation of FTY720 (purple) or vehicle (black) treatment. ****, P <000.1 determined by unpaired, two-tailed Student’s t -test. C. TCR TAG cell numbers in the livers of vehicle- (open black bars) and FTY720 (purple bars)-treated mice. Each symbol represents an individual mouse, with ****, P <0.0001 determined by two-way ANOVA followed by post hoc Šídák’s multiple comparisons test. D. Representative histogram of EdU+ incorporation in TCR TAG T cells and summary plot of % EdU + from the livers of vehicle- and FTY720-treated AST;Cre-ER T2 mice. Each symbol represents an individual mouse, two-way ANOVA followed by post hoc Šídák’s multiple comparisons test. E. Left, representative dot plots of TCF1 and KI67 expression in EdU + TCR TAG from the livers of AST;CreER T2 mice at day 5 (top) and day 49 (bottom) following vehicle and FTY720 treatment. Right, summary plots of % TCF1 hi (top) and KI67 + (bottom) of EdU + TCR TAG . Each symbol represents an individual mouse, two-way ANOVA followed by post hoc Šídák’s multiple comparisons test. For B-E , n=4-5 mice per group and representative of two independent experiments.

Journal: bioRxiv

Article Title: TCF1 lo CD8 T cells proliferate and persist autonomously in tumors

doi: 10.64898/2026.01.17.700120

Figure Lengend Snippet: A. Experimental scheme: naive TCR TAG (Thy1.1) were adoptively transferred into tamoxifen-treated AST;Cre-ER T2 (Thy1.2) mice. FTY720 treatment or vehicle was administered every other day beginning on day −1 for 6 days or day 28 for 3 weeks. EdU was administered for the final 3 days prior to harvest. TCR TAG were re-isolated on day 5 and day 49 from the livers for flow cytometric analysis. B. Representative histogram and summary plot of % CD3ε + cells in peripheral blood 24 hours after initiation of FTY720 (purple) or vehicle (black) treatment. ****, P <000.1 determined by unpaired, two-tailed Student’s t -test. C. TCR TAG cell numbers in the livers of vehicle- (open black bars) and FTY720 (purple bars)-treated mice. Each symbol represents an individual mouse, with ****, P <0.0001 determined by two-way ANOVA followed by post hoc Šídák’s multiple comparisons test. D. Representative histogram of EdU+ incorporation in TCR TAG T cells and summary plot of % EdU + from the livers of vehicle- and FTY720-treated AST;Cre-ER T2 mice. Each symbol represents an individual mouse, two-way ANOVA followed by post hoc Šídák’s multiple comparisons test. E. Left, representative dot plots of TCF1 and KI67 expression in EdU + TCR TAG from the livers of AST;CreER T2 mice at day 5 (top) and day 49 (bottom) following vehicle and FTY720 treatment. Right, summary plots of % TCF1 hi (top) and KI67 + (bottom) of EdU + TCR TAG . Each symbol represents an individual mouse, two-way ANOVA followed by post hoc Šídák’s multiple comparisons test. For B-E , n=4-5 mice per group and representative of two independent experiments.

Techniques: Isolation, Two Tailed Test, Expressing

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