Journal: JID Innovations

Article Title: CIT tumor lines: A series of immunogenic murine cutaneous squamous cell carcinoma cell lines derived from chemical carcinogenesis

doi: 10.1016/j.xjidi.2026.100477

Figure Lengend Snippet: CIT lines express identifiable neoantigens whose cognate CD8+ T cells are responsive to immunotherapy. ( a ) Selection criteria for CIT6 mutations predicted to generate neoantigens. (b) Predicted peptide log affinity ratio (affinity ratio is defined as the ratio between predicted binding affinity of mutant versus wild-type sequence) and gene expression level of mutations predicted to be top CIT6 candidate neoantigens, shown for H2-Dq and H2-Kq alleles. Affinity predictions were generated using the NetH2Pan algorithm. (c) Schematic of IFNγ ELISpot assay. For screening of CD8+ T cell and CD4+ T cell responses, CD8+ or CD4+ T cells were isolated from the spleens of tumor-bearing mice and co-cultured with total splenocytes from a healthy tumor-naïve mouse. (d) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Brinp3 peptide CPAFLPCTV (top row) and negative control (no peptide, bottom row). (e) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Brinp3 peptide CPAFLPCTV. Each colored line represents data points from a different mouse (n = 4 mice). (f) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Ttll4 peptide VPPSSLLPL (top row) and negative control (no peptide, bottom row). (g) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Ttll4 peptide VPPSSLLPL. Each colored line represents data points from a different mouse (n = 4 mice). (h) Spot counts from ELISpot assays performed with CD4+ T cells isolated from the spleens of CIT6-bearing mice and cultured with splenocytes from tumor-naïve mice loaded with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 4 mice). (i) Spot counts from ELISpot assays performed with total splenocytes from CIT6-bearing mice cultured with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 10 – 11 mice). (j) Schematic of ICI treatment regimen and ELISpot analysis. (k) Average spot counts of ICI-treated splenocytes cultured with 80 ug/ml of Ttll4 peptide vs negative control. Each line represents data points from a different mouse (n = 6 mice). Statistical analysis in panels e, g, h, i, and k was done by a paired t- test. ∗ P < .05, ∗∗ P < .01. Error bars in panels e, g, h and i represent mean and SD. CIT, carcinogen-induced tumor; ICI, Immune checkpoint inhibitor.

Article Snippet: Eight-week-old wild-type FVB/N mice were purchased from Jackson Laboratories.

Techniques: Immunopeptidomics, Selection, Binding Assay, Mutagenesis, Sequencing, Gene Expression, Generated, Enzyme-linked Immunospot, Isolation, Cell Culture, Negative Control

Journal: iScience

Article Title: MAP3K1 integrates RhoA/ROCK signaling to regulate epithelial morphogenesis

doi: 10.1016/j.isci.2026.115853

Figure Lengend Snippet: S1P activates the MAP3K1-RhoA signaling network (A) Western blot and (B) densitometric analysis of phosphoproteins in wild type and Map3k1 −/− keratinocytes treated with S1P (10 μM, 1h). (C) Western blot and (D) quantification of phosphoproteins (p) in Map3k1 TG keratinocytes infected with Ad-GFP or Ad-Cre, with or without MAP2K4/7 inhibitor (BSJ-04-122, BSJ) or JNK inhibitor (SP600125, SP). (E) Quantification of p -PXN (Y) in S1P-treated HEK293 cells (Control, Ctl), cells expressing kinase dead MAP3K1 (KM), or cells with siRNA-mediated MAP3K1 knockdown (siRNA), and in Ad-Cre infected Map3k1TG keratinocytes treated with MAP2K4/7, JNK, or ROCK (Y27632, Y) inhibitors. (F) p -PXN (Y) in S1P-treated wild-type cells with or without ROCK inhibitor (Y) and in Rhoa Δ/Δ ;Rock1 Δ/Δ cells ( R Δ/Δ ;R Δ/Δ ) keratinocytes. (G) Quantification of CN03 (RhoA activator)-induced phosphoproteins in HEK293 cells expressing MAP3K1-KM. (H) S1P-stimulated AP-1 reporter activity and (I) Wound-closure assays in keratinocytes under the indicated genetic perturbations and inhibitor treatments. (J) Schematic model of MAP3K1 signaling network. Data represent mean ± SEM. from ≥3 independent biological replicates. ∗∗∗ p < 0.001 in (B and G) (unpaired two-tailed t test); ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 in (D-F and H-I) (one-way ANOVA followed by Dunnett’s post hoc test).

Article Snippet: Wild type C57BL/6J strain , The Jackson Laboratory , RRID:IMSR_JAX:000664.

Techniques: Western Blot, Infection, Control, Expressing, Knockdown, Activity Assay, Two Tailed Test

Journal: iScience

Article Title: MAP3K1 integrates RhoA/ROCK signaling to regulate epithelial morphogenesis

doi: 10.1016/j.isci.2026.115853

Figure Lengend Snippet: G protein subunits and RhoGEFs connect S1P signaling to RhoA and MAP3K1 activation (A) Schematic model depicting candidate molecular links between S1P receptors, MAP3K1, and RhoA. (B and D) Western blot analyses and (C and E) quantification of phosphoproteins in S1P-treated HEK293 cells in the presence or absence of Gβ inhibitor (Gallein) or siRNAs targeting ARHGEF1 and ARHGEF5 (SC, scrambled control). (F) AP-1 reporter activity in S1P-treated HEK293 cells or MAP3K1-wild type (MAP3K1-WT) adenovirus infected cells with or without pathway inhibitors or ARHGEF knockdown. Data represent mean ± SEM. from ≥3 independent experiments. ∗∗∗ p < 0.001 in (C) (unpaired two-tailed t test); ∗∗ p < 0.01 and ∗∗∗ p < 0.001 in (E and F) (one-way ANOVA followed by Dunnett’s post hoc test).

Article Snippet: Wild type C57BL/6J strain , The Jackson Laboratory , RRID:IMSR_JAX:000664.

Techniques: Activation Assay, Western Blot, Control, Activity Assay, Infection, Knockdown, Two Tailed Test

Journal: iScience

Article Title: MAP3K1 integrates RhoA/ROCK signaling to regulate epithelial morphogenesis

doi: 10.1016/j.isci.2026.115853

Figure Lengend Snippet: G×G and G×E interactions converge on cytoskeleton reorganization (A) Gross eye images at birth (P0) and H&E-stained sections of wild type and Rhoa Δ/Δ ;Rock1 Δ/Δ embryonic eyes at E14.5-E16.5. Red arrows indicate open eye at birth (EOB) defects and the eyelid leading edge. (B) Phalloidin staining and (C) quantification of actin filaments (green) in wild type and Rhoa Δ/Δ ;Rock1 Δ/Δ ( R Δ/Δ ;R Δ/Δ ) eyelid epithelium at E15.5. Left: low magnification; right: enlargements of boxed areas. White arrowheads in (B) mark enriched F-actin at the epithelial leading edge. Dashed lines denote the basement membrane. (D) Phalloidin staining (red) and (E) quantification of F-actin in Rhoa flox/flox ;Rock1 flox/flox keratinocytes infected with Ad-GFP or Ad-GFP-Cre; only GFP positive cells were analyzed. Nuclei are counterstained with Hoechst (blue). Quantification of F-actin intensity in (F) TCDD-treated wild type and Rock1 Δ/Δ pups and (G) Ad-GFP (wild type) and Ad-GFP-Cre infected Rock1 flox/flox ( Rock1 Δ/Δ ) keratinocytes with or without TCDD treatment. Images are representative of at least three mice/genotypes or experimental replicates. CO, cornea; LE, lens; EL, eyelid; RE, retina. Data represent mean ± SEM. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 in (C, E-F) (unpaired two-tailed t test), ∗∗ p < 0.01 in (G) (one-way ANOVA followed by Dunnett’s post hoc test). Scale bars, 500 μm and 200 μm (A) and 50 μm (B and D).

Article Snippet: Wild type C57BL/6J strain , The Jackson Laboratory , RRID:IMSR_JAX:000664.

Techniques: Staining, Membrane, Infection, Two Tailed Test

Journal: iScience

Article Title: MAP3K1 integrates RhoA/ROCK signaling to regulate epithelial morphogenesis

doi: 10.1016/j.isci.2026.115853

Figure Lengend Snippet: Genetic and environmental interactions regulate epithelial differentiation (A) Cell proliferation in wild type and Rhoa Δ/Δ ;Rock1 Δ/Δ ( R Δ/Δ ;R Δ/Δ ) eyelid at E15.5 assessed by EdU labeling. (B) Immunostaining and (C) quantification of Krt1 (green) as a marker of terminal epidermal differentiation in E15.5 eyelids of the indicated genotypes with or without TCDD exposure. Left: low magnification, right: enlarged boxed areas. Dashed lines mark the basement membrane. Nuclei are counterstained with Hoechst (blue). Images represent at least three embryos/genotypes or independent experiments. EL, eyelid. (D and E) RT-qPCR quantification of Krt1 (D) and Krt10 (E) mRNA in Ad-GFP-infected (wild type), Ad-Cre-infected Rock1 flox/flox ( Rock1 Δ/Δ ) and Rhoa flox/flox ; Rock1 flox/flox ( Rhoa Δ/Δ ; Rock1 Δ/Δ ) keratinocytes with or without TCDD treatment. Data represent mean ± SEM. ∗∗ p < 0.01 in (A) (unpaired two-tailed t test) and ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 in (C-E) (one-way ANOVA followed by Dunnett’s post hoc test). Scale bars, 50 μm.

Article Snippet: Wild type C57BL/6J strain , The Jackson Laboratory , RRID:IMSR_JAX:000664.

Techniques: Labeling, Immunostaining, Marker, Membrane, Quantitative RT-PCR, Infection, Two Tailed Test

Journal: iScience

Article Title: Regulation of antiviral immunity by PD-1 during respiratory syncitial virus infection and upon vaccination

doi: 10.1016/j.isci.2026.116157

Figure Lengend Snippet: PD-1/PD-L1 axis is the predominant inhibitory pathway during RSV infection (A) Male C57BL/6J mice, aged 6 to 8 weeks, were challenged with 1 × 10 6 PFU of RSV strain A2 intranasal instillation. Daily monitoring of behavior, weight, and non-invasive airway function (Penh) measurements was performed to track the progression of infection. Euthanasia was carried out at 5- and 9-dpi. Several infection parameters were measured in the C57BL/6J mice infected with RSV to corroborate that viral infections promote the disease. Days post-infection (dpi). (B) Neutrophil gating strategy (C), Graph of body weight changes, and (D) differences in airway obstruction were evaluated by measuring Penh via whole-body plethysmography between 0 and 5 dpi. (E) N-RSV gene expression was quantified by RT-qPCR in the lungs and normalized by 5,000 copies of β-actin. (F) Neutrophil infiltration in the lungs was determined by flow cytometry. (G–I) PD-1 expression on lung CD8 + cells and expression of its ligand PD-L1 on APCs (J–L) and (M–O) epithelial cells in lung homogenates were determined by flow cytometry. Data represent the mean ± SEM. For A and B, n = 12 for each group. For C and D, n = 6 per group. A multiple Mann-Whitney test. p < 0.05 was considered statistically significant.

Article Snippet: Initial colonies of wild-type C57BL/6J (B6) (cat#000664) and Pdcd1 −/− (cat#028276) mice were purchased from The Jackson Laboratory, and mice were subsequently bred at the Pontificia Universidad Católica de Chile facility.

Techniques: Infection, Gene Expression, Quantitative RT-PCR, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: iScience

Article Title: Regulation of antiviral immunity by PD-1 during respiratory syncitial virus infection and upon vaccination

doi: 10.1016/j.isci.2026.116157

Figure Lengend Snippet: PD-1 expression on CD8 + T cells in mice treated with anti-F-RSV antibody or immunized with rBCG-N-RSV vaccine (A) Male C57BL/6J mice (5–8 weeks old) received an intraperitoneal injection of 500 μg of anti-F-RSV antibody one day before the RSV infection. (B) Weight change and (C) differences in airway obstruction by measuring Penh via whole-body plethysmography between 0 and 7 dpi. The euthanasia was performed at 7 dpi (Red arrow). (D) Viral loads (N-RSV expression) were quantified by RT-qPCR in the lungs and normalized to 5,000 copies of β-actin. (E) Neutrophil infiltration into the lung homogenates was determined by flow cytometry at 7 dpi. (F) PD-1 expression on CD8 + T cells was determined in lung homogenates by flow cytometry at 7 dpi and (G) PD-1 MFI. (H) In a separate group, mice were immunized subcutaneously with 1–5×10 5 CFU of rBCG-N-RSV in two doses. Twenty-one days later, the mice receiving rBCG-N-RSV were infected intranasally with 1 × 10 6 PFU of RSV strain A2. (I) Weight change and (J) differences in airway obstruction by measuring Penh via whole-body plethysmography between 0 and 7 dpi. The euthanasia was performed at 7 dpi (Red arrow). (K) Viral loads (N-RSV expression) were quantified by quantitative PCR (qPCR) in the lungs and normalized to 5,000 copies of β-actin. (L) Infiltration of neutrophils into the lung homogenates was determined by flow cytometry at 7 dpi. PD-1 expression on CD8 + T cells was determined in lung homogenates by flow cytometry at 7 dpi. (M) PD-1 count and (N) PD-1 MFI. Data represent the mean ± SEM of two independent experiments with three mice per group ( n = 6). (B-D and I-K) A mixed-effects model was used for multiple comparisons, followed by Dunnett’s post hoc test. (E–H and L-N) Kruskal-Wallis’s test was applied for multiple group comparisons, followed by Dunn’s post hoc test. p < 0.05 was considered statistically significant for both statistical analyses used for this figure.

Article Snippet: Initial colonies of wild-type C57BL/6J (B6) (cat#000664) and Pdcd1 −/− (cat#028276) mice were purchased from The Jackson Laboratory, and mice were subsequently bred at the Pontificia Universidad Católica de Chile facility.

Techniques: Expressing, Injection, Infection, Quantitative RT-PCR, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: Regulation of antiviral immunity by PD-1 during respiratory syncitial virus infection and upon vaccination

doi: 10.1016/j.isci.2026.116157

Figure Lengend Snippet: Infection parameters in C57BL/6J and Pdcd1 −/− mice immunized with rBCG-N-RSV (A) C57BL/6J mice were immunized intradermally with a 2-dose schedule (0 and 14 days) of rBCG-N-RSV and treated intraperitoneally with 100 μg of nivolumab twice a week throughout the protocol. (B) Simultaneously, another group of Pdcd1 −/− mice received the same immunization schedule. Euthanasia was carried out 14 days after the booster. Both groups were challenged with RSV 7 days after the booster, and euthanasia was performed at 7 dpi. (C–F) body weight changes relative to day 0 for (C) C57BL/6J mice, (D) C57BL/6J mice treated with Nivolumab, (E) Pdcd1 −/− mice, and (F) combined comparison of RSV-infected groups from different mice genotypes and treatments. (G-H) Viral load, represented as the copy number of N-RSV per 5000 copies of β-actin, was quantified by RT-qPCR in lung homogenates of each group. Data are presented as mean ± SEM from two independent experiments with three mice per group ( n = 6). (C-F) Multiple comparisons were performed using a mixed-effects model followed by Dunnett’s post hoc test. (G and H) two-way ANOVA followed by Bonferroni’s multiple comparisons test was applied. p < 0.05 was considered statistically significant for both statistical analyses used for this figure. p values shown in blue indicate comparisons made exclusively against the mock group.

Article Snippet: Initial colonies of wild-type C57BL/6J (B6) (cat#000664) and Pdcd1 −/− (cat#028276) mice were purchased from The Jackson Laboratory, and mice were subsequently bred at the Pontificia Universidad Católica de Chile facility.

Techniques: Infection, Comparison, Quantitative RT-PCR

Journal: iScience

Article Title: Regulation of antiviral immunity by PD-1 during respiratory syncitial virus infection and upon vaccination

doi: 10.1016/j.isci.2026.116157

Figure Lengend Snippet: Effect of PD-1 blockade and ablation on follicular CD4 + T cells and T cell memory differentiation in vaccine mice C57BL/6J mice were immunized intradermally with a 2-dose schedule (0–14 days) of rBCG-N-RSV and treated intraperitoneally with 100 μg of nivolumab twice a week throughout the protocol. Simultaneously, another group of Pdcd1 −/− mice received the same immunization schedule. Euthanasia was carried out 14 days after the booster. Memory compartment cells and CD4 + follicular T cells were also analyzed by flow cytometry in splenocytes extracted from rBCG-N-RSV-immunized mice after 72 h of stimulation with N-RSV protein (10 μg/mL) (A) Gating strategy (B) Change in the number of follicular CD4 + T cells. (C) Percent of Memory CD4 + T cells. (D) Percent of Memory CD8 + T cells. Naive: CD62L + CD44 − , TCM: CD62L + CD44 + , and TEM: CD62L − CD44 + . Data represent the mean ± SEM of two independent experiments with three mice per group ( n = 6). (B) Kruskal-Wallis’s test was applied for multiple group comparisons, followed by Dunn’s post hoc test. (C and D) Mixed-effect analysis followed by a Bonferroni test was used for multiple comparisons. p < 0.05 was considered statistically significant for both statistical analyses used for this figure.

Article Snippet: Initial colonies of wild-type C57BL/6J (B6) (cat#000664) and Pdcd1 −/− (cat#028276) mice were purchased from The Jackson Laboratory, and mice were subsequently bred at the Pontificia Universidad Católica de Chile facility.

Techniques: Flow Cytometry

Journal: iScience

Article Title: Regulation of antiviral immunity by PD-1 during respiratory syncitial virus infection and upon vaccination

doi: 10.1016/j.isci.2026.116157

Figure Lengend Snippet: IFN-γ and IL-17 responses in rBCG-N-RSV–immunized mice under PD-1 modulation C57BL/6J mice were immunized intradermally with a 2-dose schedule (0–14 days) of rBCG-N-RSV and treated intraperitoneally with 100 μg of nivolumab twice a week throughout the protocol. Simultaneously, another group of Pdcd1 −/− mice received the same immunization schedule. Euthanasia was carried out 14 days after the booster. The splenocytes (3 × 10 5 cells/well) from mice were stimulated with N-RSV protein (10 μg/mL) for 72 h, and (A) IFN-γ- and (B) IL-17-producing SFCs were quantified by ELISPOT. (C) Representative ELISPOT images are shown in the last panel. Data represent the mean ± SEM of two independent experiments with three mice per group ( n = 6). Kruskal-Wallis’s test was applied for multiple group comparisons, followed by Dunn’s post hoc test. p < 0.05 was considered statistically significant.

Article Snippet: Initial colonies of wild-type C57BL/6J (B6) (cat#000664) and Pdcd1 −/− (cat#028276) mice were purchased from The Jackson Laboratory, and mice were subsequently bred at the Pontificia Universidad Católica de Chile facility.

Techniques: Enzyme-linked Immunospot

Journal: iScience

Article Title: Regulation of antiviral immunity by PD-1 during respiratory syncitial virus infection and upon vaccination

doi: 10.1016/j.isci.2026.116157

Figure Lengend Snippet: Changes in humoral immune response induced by the vaccine C57BL/6J mice were immunized intradermally with a 2-dose schedule (0–14 days) of rBCG-N-RSV and treated intraperitoneally with 100 μg of nivolumab twice a week throughout the protocol. Simultaneously, another group of Pdcd1 −/− mice received the same immunization schedule. Euthanasia was carried out 14 days after the booster. Both groups were challenged with RSV 7 days after the booster, and euthanasia was performed at 7 dpi. (A) Neutralizing antibodies and (B) total anti-N-RSV IgG levels and (C) avidity percentage in serum were determined. Data represent the mean ± SEM of two independent experiments with three mice per group ( n = 6). two-way ANOVA followed by a Bonferroni test was used for multiple comparisons. p < 0.05 was considered statistically significant.

Article Snippet: Initial colonies of wild-type C57BL/6J (B6) (cat#000664) and Pdcd1 −/− (cat#028276) mice were purchased from The Jackson Laboratory, and mice were subsequently bred at the Pontificia Universidad Católica de Chile facility.

Techniques: