Journal: iScience

Article Title: The dual specificity phosphatase 2 act as distal regulatory node in T cell signaling

doi: 10.1016/j.isci.2026.116690

Figure Lengend Snippet: DUSP2 dephoshorylates the MAP kinases ERK1/2, p38 MAPK, and JNK but not STAT3 in T cells (A)Western blot of Jurkat cells either left untreated or pretreated with CHX (10 μg/mL for 30 min) prior to activation with 5 μg/mL aCD3 and 10 nM PMA. Cell extracts were collected at indicated time points and western blots were probed with the antibodies detecting phosphorylated MAP kinases. (B) Relative mRNA expression levels of the inducible DUSPs (DUSP1, 2, 4, 5) in Jurkat cells unstimulated or stimulated for 2 h or 4 h with 250 ng/mL aCD3/and 250 ng/mL PMA ( n = 3). (C) RT-qPCR analysis was conducted to verify the induction of DUSP2 mRNA expression in Jurkat WT cells within 6 h after stimulation with 5 μg/mL aCD3 and 10 nM PMA or (D) within 4 h after stimulation with aCD3/aCD28 coated beads. Data are presented as DUSP2 mRNA expression normalized to geometric mean of GAPDH and TBP as control ( n = 3). (E) Western blot analysis of stimulated (5 μg/mL aCD3/10 nM PMA) Jurkat cells showed an inverse correlation of DUSP2 protein levels with MAPK phosphorylation levels after initial induction. (F) DUSP2 KO led to sustained MAPK phosphorylation after stimulation. (G) The rescue of Jurkat DUSP2 KO cells with wild-type DUSP2 (DUSP2 resWT ) restored the DUSP2 phosphatase function completely. This effect was abrogated in Jurkat cells (H) rescued with DUSP2 resKIM or (I) with DUSP2 resCS mutants. (J) Stimulation (5 μg/mL aCD3/10 nM PMA) of doxycycline pretreated (2 μg/mL, 3h) DUSP2 resWT (Tet) cells showed a DUSP2 dependent dephosphorlyation of ERK1/2 and p38, (K) while STAT3 was not dephosphorylated in a DUSP2 dependent manner in DUSP2 resWT (Tet) cells after doxycycline pretreatment (2 μg/mL, 3 h) and INFα (100 ng/mL) stimulation. For all experiments cells were starved overnight, and majority of experiments were performed in at least three replicates. Expression data are presented as mean ± SEM. Unpaired student’s t test was calculated compared to unstimulated cells, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; CHX = cycloheximide, PMA = phorbol-12-myristate-13-acetate; INFα = interferon alpha, TPM = transcripts per million, ° unspecific band.

Article Snippet: For ERK inhibitor experiments primary CD4 + T or Jurkat wild-type cells were pretreated with or without a final concentration of 1 μM ERK1/2 inhibitor SCH772984 (MedChemExpress) for 30 min before adding the respective stimulation.

Techniques: Western Blot, Activation Assay, Expressing, Quantitative RT-PCR, Control, Phospho-proteomics

Journal: iScience

Article Title: The dual specificity phosphatase 2 act as distal regulatory node in T cell signaling

doi: 10.1016/j.isci.2026.116690

Figure Lengend Snippet: Mutations outside DUSP2 key residues can impair its phosphatase activity (A) Schematic representation of the amino acid sequence of wild-type DUSP2 compared to mutated allele leading to a C-terminal extended DUSP2 protein (41 kDa) of 380aa. (B–D) Western blot analysis of (B) Jurkat cells, (C) single-allele knock-out Jurkat cells expressing DUSP2 32kDA and (D) single-allele knock-out Jurkat cells expressing DUSP2 41kDA after stimulation with 5 μg/mL aCD3/10 nM PMA with the indicated antibodies. (E) Western blot analysis of extracts derived from stimulated (aCD3/PMA) Jurkat DUSP2 KO cells rescued with a lentiviral expression vector for the 41 kDa allele of DUSP2 and probed with the indicated antibodies. For all western blot experiments cells were starved overnight. (F) Microscopic analysis showed U2OS cells transfected with myc-tagged expression vectors encoding either DUSP2 WT or DUSP2 41kDa and stained with an anti-myc antibody to detect DUSP2. The scale bars indicated a length of 25 μm. (G–I) In vitro phosphatase assays were performed using 300 ng of (G) wild-type human DUSP2 WT protein, (H) catalytically inactive human DUSP2 protein (DUSP2 CS ), or (I) human DUSP2 41kDa protein in combination with the indicated MAPKs (1 μg). Data are presented as mean ± SD of relative increased fluorescence signal (RFU 60 min/RFU 0 min), n = 3. Unpaired student’s t test, ∗∗∗ p < 0.001. DSP, dual specificity phosphatase; PMA, phorbol-12-myristate-13-acetate.

Article Snippet: For ERK inhibitor experiments primary CD4 + T or Jurkat wild-type cells were pretreated with or without a final concentration of 1 μM ERK1/2 inhibitor SCH772984 (MedChemExpress) for 30 min before adding the respective stimulation.

Techniques: Activity Assay, Sequencing, Western Blot, Knock-Out, Expressing, Derivative Assay, Plasmid Preparation, Transfection, Staining, In Vitro, Fluorescence

Journal: iScience

Article Title: The dual specificity phosphatase 2 act as distal regulatory node in T cell signaling

doi: 10.1016/j.isci.2026.116690

Figure Lengend Snippet: Loss of DUSP2 is compensated by DUSP5 (A) RT-qPCR analysis of DUSP5 mRNA expression in Jurkat and DUSP2 KO cells stimulated with aCD3/aCD28 coated beads (bead/cell ration 1:1) for 0–4 h. (B) RT-qPCR analysis of DUSP5 mRNA expression in Jurkat and DUSP2 KO cells stimulated with 5 μg/mL aCD3/10 nM PMA for 0–6 h. (C–E) Western blots analysis of DUSP2, DUSP5, and EGR1 protein levels and phosphorylation of ERK1/2, and p38 in (C) Jurkat, (D) DUSP2 KO , (E) and DUSP2/DUSP5 KO cells after stimulation with 5 μg/mL aCD3/10 nM PMA. The faint DUSP5 band occurred only in this replicate of the DUSP2/DUSP5 KO blot, but was absent in all other replicates (Document S2). (F) RT-qPCR confirmation of EGR1 mRNA expression in Jurkat and DUSP2 KO cells stimulated with aCD3/aCD28 coated beads (bead/cell ration 1:1) for 0–4 h. (G) RT-qPCR confirmation of EGR1 mRNA expression in Jurkat and DUSP2 KO cells stimulated with 5 μg/mL aCD3/10 nM PMA for 0–6 h. (H) Comparison of DUSP2 and DUSP5 mRNA expression levels in Jurkat and EGR1 KO cells stimulated with 5 μg/mL aCD3/10 nM PMA. (I) Western blot analysis of extracts from Jurkat and EGR1 KO cells stimulated with 5 μg/mL aCD3/10 nM PMA and probed with indicated antibodies. (J) Western blot analysis of Jurkat cells either untreated or pretreated with ERK inhibitor (SCH772984, 1 μM, 30 min) prior to stimulation with 5 μg/mL aCD3/10 nM PMA, probed with indicated antibodies. Cells for all experiments were starved overnight prior to stimulation with at least in three replicates. Gene expression data are presented as target gene mRNA expression normalized to geometric mean of GAPDH and TBP as control ( n = 3). Data are presented as mean ± SD, unpaired student’s t test, ∗ p < 0.05, ∗∗∗ p < 0.001. PMA = phorbol-12-myristate-13-acetate.

Article Snippet: For ERK inhibitor experiments primary CD4 + T or Jurkat wild-type cells were pretreated with or without a final concentration of 1 μM ERK1/2 inhibitor SCH772984 (MedChemExpress) for 30 min before adding the respective stimulation.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Phospho-proteomics, Comparison, Gene Expression, Control

Journal: iScience

Article Title: The dual specificity phosphatase 2 act as distal regulatory node in T cell signaling

doi: 10.1016/j.isci.2026.116690

Figure Lengend Snippet: DUSP2 and DUSP5 act in concert to regulate IL2 production in T cells (A–C) RT-qPCR analysis of Jurkat, DUSP2 KO , DUSP2 resWT and DUSP/DUSP5 KO cells stimulated with 250 ng/mL aCD3/250 ng/mL PMA. The analysis determined mRNA expression of (A) the EGR1 (B) DUSP5 and (C) IL2 . Gene expression data are presented as target gene mRNA expression normalized to geometric mean of GAPDH and TBP as control and for statistics compared to 0 h of respective cell line. (D) ELISA analysis of IL2 secretion of Jurkat, DUSP2 KO , DUSP2 resWT , and DUSP/DUSP5 KO cells 24 h after stimulation with 250 ng/mL aCD3/250 ng/mL PMA. (E) ELISA analysis of IL2 secretion of Jurkat, DUSP2 KO , DUSP2 resWT , and DUSP/DUSP5 KO stimulated for 24 h with 250 ng/mL aCD3/250 ng/mL PMA. Cells were either left unstimulated, stimulated, treated with ERK inhibitor (SCH772984, 1 μM) 30 min before stimulation or treated with ERK inhibitor 2 h or 3 h after stimulation. All experiments were performed at least in three replicates ( n ≥ 3), data are presented as mean ± SD, unpaired student’s t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. PMA = phorbol-12-myristate-13-acetate.

Article Snippet: For ERK inhibitor experiments primary CD4 + T or Jurkat wild-type cells were pretreated with or without a final concentration of 1 μM ERK1/2 inhibitor SCH772984 (MedChemExpress) for 30 min before adding the respective stimulation.

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Control, Enzyme-linked Immunosorbent Assay

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: