standard buffer z 747 jo urn al pr e p oo f 33  (Oroboros Instruments)

Journal: JCI Insight

Article Title: ICOS + CD4 + T cells define a high susceptibility to anti–PD-1 therapy–induced lung pathogenesis

doi: 10.1172/jci.insight.186483

Figure Lengend Snippet: MC38-bearing young or aged mice were treated with anti–PD-L1 Ab. ( A – C ) Lung-infiltrating CD45 + cells were isolated from MC38-bearing young or aged mice that were treated with anti–PD-L1 Ab, and then were analyzed by scRNA-seq. Cluster analysis was performed and plotted by UMAP dimensionality reduction. Subclustering was performed on the data from the Cd4 + T cell populations in young and aged mice, and lower bar plots in A indicate their frequencies. Dot plots shown in B represent the expression of canonical marker genes across the indicated Cd4 + subclusters. Relative expression of indicated genes across subclustered populations from young and aged mice are shown in C . ( D ) Representative images of DAPI staining (left) and in situ RNA hybridization (right; white, ICOS; red, CD19; green, CD4) in lung from aged mice are shown. Scale bars: 100 μm (left) and 50 μm (right). ( E ) Representative dot plots of CD45 iv- cell populations within the lungs are shown. ( F ) Frequencies of indicated lung-infiltrating CD45 – cell populations were assessed in mice treated with indicated Abs. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA followed by Tukey-Kramer post hoc test.

Article Snippet: The target mRNA in the tissues was hybridized by incubating in RNAscope buffered Z probes for Icos (Advanced Cell Diagnostics, 552451), Cd4 (Advanced Cell Diagnostics, 406841-C2), and Cd19 (Advanced Cell Diagnostics, 314711-C3) at 40°C for 2 hours.

Techniques: Isolation, Expressing, Marker, Staining, In Situ, Hybridization

Journal: JCI Insight

Article Title: ICOS + CD4 + T cells define a high susceptibility to anti–PD-1 therapy–induced lung pathogenesis

doi: 10.1172/jci.insight.186483

Figure Lengend Snippet: Tumor-bearing young or aged mice were administered with anti–PD-L1 Ab. Anti-ICOSL Ab was injected to block the ICOS-ICOSL interaction. ( A and B ) Representative dot plots of CD45 iv- lung-infiltrating cell populations from MC38-bearing mice ( A ) and their frequencies ( B ) are shown. ( C and D ) Representative images of CD138 IHC in the lung of tumor-bearing aged mice ( C ), and the percentage area of TLS (left) and CD138 + plasma cells (right) in each lung section ( D ) are shown. Scale bars: 100 μm. ( E ) The levels of SP-D in the serum are shown. ( F ) Lung dysfunction in tumor-bearing mice was assessed by mechanical ventilation system. Data are representative of 3 independent experiments with similar results and presented as mean ± SEM ( n = 4–8). * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA followed by Tukey-Kramer post hoc test.

Article Snippet: The target mRNA in the tissues was hybridized by incubating in RNAscope buffered Z probes for Icos (Advanced Cell Diagnostics, 552451), Cd4 (Advanced Cell Diagnostics, 406841-C2), and Cd19 (Advanced Cell Diagnostics, 314711-C3) at 40°C for 2 hours.

Techniques: Injection, Blocking Assay, Clinical Proteomics

Journal: JCI Insight

Article Title: ICOS + CD4 + T cells define a high susceptibility to anti–PD-1 therapy–induced lung pathogenesis

doi: 10.1172/jci.insight.186483

Figure Lengend Snippet: ( A ) CD4 + T cells were isolated from the lungs of aged mice treated anti–PD-1 therapy and then were stimulated with anti-CD3/anti-CD28 Abs together with anti-ICOS or anti-CD153 agonistic Ab ex vivo. Fold inductions of indicated mRNA expression relative to that in control Ab-stimulated cells are shown. ( B ) Concentration of IL-21 or CXCL13 in BALF isolated from young or aged mice with indicated treatment was measured. Data are mean ± SEM ( n = 3–6). One-way ANOVA followed by Tukey’s post hoc test was used. ( C – F ) WT or IL-21–deficient aged mice were treated with anti–PD-1 and anti-ICOSL Abs, and inoculated intranasally (i.n.) with recombinant IL-21. Representative plots of CD95 + GL7 + cells ( C ) and IgG + cells ( D ) among lung-infiltrating B cells, their frequencies ( E ), and serum concentrations of SP-D ( F ) are shown. Data are mean ± SEM, and the results from 2 independent experiments were combined (n = 4–6 per experiment). Then, 1-way ANOVA followed by Tukey-Kramer post hoc test was conducted for aged WT or IL-21–KO groups separately. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The target mRNA in the tissues was hybridized by incubating in RNAscope buffered Z probes for Icos (Advanced Cell Diagnostics, 552451), Cd4 (Advanced Cell Diagnostics, 406841-C2), and Cd19 (Advanced Cell Diagnostics, 314711-C3) at 40°C for 2 hours.

Techniques: Isolation, Ex Vivo, Expressing, Control, Concentration Assay, Recombinant

Journal: JCI Insight

Article Title: ICOS + CD4 + T cells define a high susceptibility to anti–PD-1 therapy–induced lung pathogenesis

doi: 10.1172/jci.insight.186483

Figure Lengend Snippet: MC38-bearing young or aged mice were treated with control or anti–PD-(L)1 Ab. ( A and B ) The representative dot plots of blood-circulating CD4 + T cells 13 days after tumor inoculation ( A ), and kinetic changes in frequencies of the indicated populations ( B ) are shown ( n = 5–6). ( C ) Frequencies of Foxp3 – or Foxp3 + ICOS + CD4 + T cells in the peripheral blood were analyzed ( n = 8–9). ** P < 0.01, *** P < 0.001 by 1-way ANOVA followed by Tukey’s post hoc test. ( D ) Correlation between the frequencies of indicated populations and the concentration of SP-D is shown. Simple linear regression analysis was conducted. Data are mean ± SEM from more than 2 independent experiments with similar results.

Article Snippet: The target mRNA in the tissues was hybridized by incubating in RNAscope buffered Z probes for Icos (Advanced Cell Diagnostics, 552451), Cd4 (Advanced Cell Diagnostics, 406841-C2), and Cd19 (Advanced Cell Diagnostics, 314711-C3) at 40°C for 2 hours.

Techniques: Control, Concentration Assay

Journal: JCI Insight

Article Title: ICOS + CD4 + T cells define a high susceptibility to anti–PD-1 therapy–induced lung pathogenesis

doi: 10.1172/jci.insight.186483

Figure Lengend Snippet: ( A ) The frequency of ICOS + cells in peripheral CD4 + T cells was analyzed before (Pre) and 4 weeks after initial anti–PD-(L)1 therapy (On) in patients with ( n = 8) or without ( n = 13) the irAE pneumonitis. * P < 0.05 by Wilcoxon’s signed-rank test. ( B ) Changes in the frequencies of indicated CD4 + or CD8 + T cell subsets during 4 weeks of anti–PD-(L)1 therapy in patients with ( n = 34) or without ( n = 13) irAEs, were analyzed. Red dots indicate the values for patients with pneumonitis. ( C ) Changes in the frequency of ICOS + CD4 + T cells in patients with indicated irAEs are shown. “Others” includes symptoms with infusion reaction, fever, adrenal irAEs, carditis, diarrhea, hypophysitis, and neurologic irAEs. * P < 0.05; ** P < 0.01 by Mann-Whitney U test. ( D ) Univariate (ΔICOS, fold change in CXCL13 or IL-6) and multivariate (ΔICOS plus changes in CXCL13) ROC analyses of the predictive values for pneumonitis development. ( E ) Distribution of indicated values in the combined predictive model for pneumonitis incidence in patients with pneumonitis ( n = 8) or without ( n = 12) irAEs. Adonis test based on the Bray-Curtis distance was used. ( F ) Kaplan-Meier plots of progression-free survival of patients with NSCLC stratified according to the median value of the change in peripheral ICOS + CD4 + T cells are shown. Log-rank (Mantel-Cox) test and univariate Cox proportional hazards analyses were performed.

Article Snippet: The target mRNA in the tissues was hybridized by incubating in RNAscope buffered Z probes for Icos (Advanced Cell Diagnostics, 552451), Cd4 (Advanced Cell Diagnostics, 406841-C2), and Cd19 (Advanced Cell Diagnostics, 314711-C3) at 40°C for 2 hours.

Techniques: MANN-WHITNEY