histological sections  (Novus Biologicals)

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A ) CD8⁺ T cells isolated from either the tumor site or spleen of C57BL/6 mice ( n = 4) bearing YUMM1.7 melanoma were assessed for p-p38 expression. The adjacent bar plot summarizes pooled data from four tumor-bearing mice. ( B ) Intratumoral CD8⁺ T cells from C57BL/6 mice ( n = 4/group) with subcutaneous YUMM1.7 melanoma, treated with vehicle control or p38i, were evaluated for the frequency of terminally exhausted CD8⁺ T cells (PD1⁺Tim3⁺). The adjacent bar plot summarizes pooled data from four mice per group. ( C ) Adoptively transferred Pmel-1 T cells transduced with either control shRNA or shRNA targeting PERK, isolated from tumors of C57BL/6 mice ( n = 4/group) bearing subcutaneous B16-F10 melanoma and treated with or without anti-PD1 antibody, were evaluated for the frequency of terminally exhausted CD8⁺ T cells (PD1⁺Tim3⁺). The adjacent bar plot summarizes pooled data from four mice per group. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001 ns, nonsignificant ( P > 0.05). The error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( A , B ) and one-way ANOVA. .

Article Snippet: C57BL/6 mice (JAX stock #000664) and Pmel-1 mice (JAX stock #005023) were procured from Jackson Laboratories, USA, and were housed under specific pathogen-free (SPF) conditions in the CSIR-IICB animal facility with controlled temperature, humidity, and a 12-h light/dark cycle, and were provided autoclaved food and water ad libitum under the supervision of trained veterinary staff.

Techniques: Isolation, Expressing, Control, Transduction, shRNA, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A – D ) C57BL/6 mice ( n = 4 mice/group) with subcutaneously established YUMM1.7 melanoma tumor treated either with vehicle control or p38i, as ( A ) represented schematically, were evaluated for: ( B ) tumor growth, ( C ) frequency of CD8 + T cells at the tumor site, ( D ) the ability of CD8 + T cells from the tumor site to produce different effector cytokines. ( E ) Schematic representation of the ACT protocol where C57BL/6 mice ( n = 4 mice/group) bearing subcutaneous B16-F10 tumors were adoptively transferred with 0.75 × 10⁶ Pmel-1 T cells, followed by treatment with or without anti-PD1 antibody (Clone# RMP1-14; 200 µg/mouse twice weekly), combined with p38i or vehicle control. Mice were subsequently evaluated for: ( F ) tumor growth ( n = 4), ( G ) frequency of Vβ13 + CD8 + T cells at the tumor site ( n = 4), and ( H ) Intracellular expression of effector cytokines in intratumoral Pmel-1 T cells following in vitro restimulation ( n = 4). ( I ) Schematic representation of the ACT protocol where C57BL/6 mice ( n = 4 mice/group) bearing subcutaneous B16-F10 tumors were adoptively transferred with 0.75 × 10⁶ Pmel-1 T cells transduced with either control shRNA (Pmel WT ) or shRNA targeting PERK (Pmel PERK ), followed by treatment with or without anti-PD1 antibody (Clone# RMP1-14; 200 µg/mouse twice weekly). Mice were evaluated for: ( J ) tumor growth ( n = 4), ( K ) frequency of Vβ13 + CD8 + T cells at the tumor site ( n = 4), and ( L ) Intracellular expression of effector cytokines in intratumoral Pmel-1 T cells following in vitro restimulation ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( C , D ), one-way ANOVA ( G , H , K , L ), and two-way ANOVA test ( B , F , J ). .

Article Snippet: C57BL/6 mice (JAX stock #000664) and Pmel-1 mice (JAX stock #005023) were procured from Jackson Laboratories, USA, and were housed under specific pathogen-free (SPF) conditions in the CSIR-IICB animal facility with controlled temperature, humidity, and a 12-h light/dark cycle, and were provided autoclaved food and water ad libitum under the supervision of trained veterinary staff.

Techniques: Control, Expressing, In Vitro, Transduction, shRNA, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A ) CD8⁺ T cells isolated from either the tumor site or spleen of C57BL/6 mice ( n = 4) bearing YUMM1.7 melanoma were assessed for p-p38 expression. The adjacent bar plot summarizes pooled data from four tumor-bearing mice. ( B ) Intratumoral CD8⁺ T cells from C57BL/6 mice ( n = 4/group) with subcutaneous YUMM1.7 melanoma, treated with vehicle control or p38i, were evaluated for the frequency of terminally exhausted CD8⁺ T cells (PD1⁺Tim3⁺). The adjacent bar plot summarizes pooled data from four mice per group. ( C ) Adoptively transferred Pmel-1 T cells transduced with either control shRNA or shRNA targeting PERK, isolated from tumors of C57BL/6 mice ( n = 4/group) bearing subcutaneous B16-F10 melanoma and treated with or without anti-PD1 antibody, were evaluated for the frequency of terminally exhausted CD8⁺ T cells (PD1⁺Tim3⁺). The adjacent bar plot summarizes pooled data from four mice per group. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001 ns, nonsignificant ( P > 0.05). The error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( A , B ) and one-way ANOVA. .

Article Snippet: Mouse: pmel-1 TCR transgenic mouse , Jackson Laboratory , NA.

Techniques: Isolation, Expressing, Control, Transduction, shRNA, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A – D ) C57BL/6 mice ( n = 4 mice/group) with subcutaneously established YUMM1.7 melanoma tumor treated either with vehicle control or p38i, as ( A ) represented schematically, were evaluated for: ( B ) tumor growth, ( C ) frequency of CD8 + T cells at the tumor site, ( D ) the ability of CD8 + T cells from the tumor site to produce different effector cytokines. ( E ) Schematic representation of the ACT protocol where C57BL/6 mice ( n = 4 mice/group) bearing subcutaneous B16-F10 tumors were adoptively transferred with 0.75 × 10⁶ Pmel-1 T cells, followed by treatment with or without anti-PD1 antibody (Clone# RMP1-14; 200 µg/mouse twice weekly), combined with p38i or vehicle control. Mice were subsequently evaluated for: ( F ) tumor growth ( n = 4), ( G ) frequency of Vβ13 + CD8 + T cells at the tumor site ( n = 4), and ( H ) Intracellular expression of effector cytokines in intratumoral Pmel-1 T cells following in vitro restimulation ( n = 4). ( I ) Schematic representation of the ACT protocol where C57BL/6 mice ( n = 4 mice/group) bearing subcutaneous B16-F10 tumors were adoptively transferred with 0.75 × 10⁶ Pmel-1 T cells transduced with either control shRNA (Pmel WT ) or shRNA targeting PERK (Pmel PERK ), followed by treatment with or without anti-PD1 antibody (Clone# RMP1-14; 200 µg/mouse twice weekly). Mice were evaluated for: ( J ) tumor growth ( n = 4), ( K ) frequency of Vβ13 + CD8 + T cells at the tumor site ( n = 4), and ( L ) Intracellular expression of effector cytokines in intratumoral Pmel-1 T cells following in vitro restimulation ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( C , D ), one-way ANOVA ( G , H , K , L ), and two-way ANOVA test ( B , F , J ). .

Article Snippet: Mouse: pmel-1 TCR transgenic mouse , Jackson Laboratory , NA.

Techniques: Control, Expressing, In Vitro, Transduction, shRNA, Standard Deviation, Derivative Assay, Two Tailed Test

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

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: 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: Functional Assay, Flow Cytometry, Derivative Assay