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IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the <t>GAPDH</t> control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .
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IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the <t>GAPDH</t> control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .
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IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the <t>GAPDH</t> control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .
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IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the <t>GAPDH</t> control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .
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IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the <t>GAPDH</t> control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .
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IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the <t>GAPDH</t> control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .
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IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the GAPDH control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: IFNγ-induced GUCY2C loss is mediated by cellular stress signaling. (A) Gene Set Enrichment Analysis (GSEA) of significantly up- and down-regulated Hallmark pathways in RNAseq data from LS174T cells treated with conditioned media (CM) from T84 co-cultures with control or GucyCART. (B) Significantly upregulated stress pathway-related genes from (A) . (C, D) LS174T cells were treated for 48 hours with CM ± 13 μg/mL anti-IFNγ neutralizing antibody (αIFNγ, C ) or 2.5 μM ruxolitinib (Ruxo, D ) and CHOP protein was quantified. (E) LS174T cells were treated with control or 2 μg/mL tunicamycin for 48 hours, and CHOP and GUCY2C proteins were quantified. Each data point in (C–E) represents the average from biological replicates in separate experiments (N = 3-4). In (E) , CHOP and GUCY2C were examined on the same blot and normalized to the GAPDH control (shown only below GUCY2C). One-way ANOVA was used to compare each condition in (C) and (D) , and a paired T-test was used to compare conditions in (E) ; ns = p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: Membranes were probed using an anti-human GUCY2C antibody (37517, Cell Signaling Technology), anti-human GAPDH (2118S, Cell Signaling Technology), anti-human STAT1 (14994T, Cell Signaling Technology), anti-human phospho-STAT1 (9167S, Cell Signaling Technology), anti-human CHOP (2895S, Cell Signaling Technology), anti-human β-actin (2128S, Cell Signaling Technology), anti-human EpCAM (2929S, Cell Signaling Technology) anti-human CDH17 (88594T, Cell Signaling Technology), and anti-human HER2 (2165T, Cell Signaling Technology).

Techniques: RNA sequencing, Control, Generated