Journal: Indian Journal of Pharmacology

Article Title: Evaluation of peripheral lymphocyte subsets in acne vulgaris patients before and after systemic isotretinoin treatment

doi: 10.4103/ijp.ijp_695_21

Figure Lengend Snippet: Comparison of lymphocyte subsets by acne severity

Article Snippet: CD3/CD16/CD56 monoclonal antibody (M2AB, C5.9, J5511), fluorescein isothiocyanate, PE (catalog number: MA1–12207), MS anti-HU CD45 PERCP (catalog number: MHCD4531), anti-human CD19 APC SJ25C1 (catalog number: 17-0198-41), human CD3/CD4/CD8 mixture CD3/CD4/CD8 (catalog number: CD348A), anti-human CD38 APC-eFluor® 780 (catalog number: 47-0389-41), anti-human HLA-DR APC (LN3) (catalog number: 17-9956-41), anti-human CD45RA APC HI100 (catalog number: 17-0458-41), and anti-human CD45RO APC-eFluor® 780 (catalog number: 47-0457-41) were obtained from Thermo Fisher Scientific (Meridian Road Rockford, IL 61105 USA).

Techniques:

Journal: Indian Journal of Pharmacology

Article Title: Evaluation of peripheral lymphocyte subsets in acne vulgaris patients before and after systemic isotretinoin treatment

doi: 10.4103/ijp.ijp_695_21

Figure Lengend Snippet: Comparison of lymphocyte subsets in patients with acne vulgaris before and after systemic isotretinoin treatment

Article Snippet: CD3/CD16/CD56 monoclonal antibody (M2AB, C5.9, J5511), fluorescein isothiocyanate, PE (catalog number: MA1–12207), MS anti-HU CD45 PERCP (catalog number: MHCD4531), anti-human CD19 APC SJ25C1 (catalog number: 17-0198-41), human CD3/CD4/CD8 mixture CD3/CD4/CD8 (catalog number: CD348A), anti-human CD38 APC-eFluor® 780 (catalog number: 47-0389-41), anti-human HLA-DR APC (LN3) (catalog number: 17-9956-41), anti-human CD45RA APC HI100 (catalog number: 17-0458-41), and anti-human CD45RO APC-eFluor® 780 (catalog number: 47-0457-41) were obtained from Thermo Fisher Scientific (Meridian Road Rockford, IL 61105 USA).

Techniques:

Journal: Nature Communications

Article Title: IFNγ signaling in cytotoxic T cells restricts anti-tumor responses by inhibiting the maintenance and diversity of intra-tumoral stem-like T cells

doi: 10.1038/s41467-023-35948-9

Figure Lengend Snippet: Control ( n = 3) and CD8-IFNγRKO ( n = 3) mice were engrafted with B16-OVA tumors. CD45 + CD3 + CD8 + Tomato + cells were sorted from tumors and subjected to scRNA-seq analysis. a - Graph-based clustering ( n = 15706 total) identified 5 clusters. b - The violin plots show the expression levels (y axes) of selected exhaustion markers (Pdcd1 (encoding PD-1), Ctla4, Lag3, Tigit, Havcr2 (encoding Tim-3), and Tox) in each of the identified clusters (x axes). c - UMAP visualization of CD8 Stem-like signature. d - UMAP visualization of the distribution of selected transcripts defining the Stem-like gene signature. Genes include Tcf7 (encoding Tcf1), Il7r, Ccr7, IFNγ, Gzmb (encoding granzyme B) and Pdcd1 (encoding PD-1). e - Pseudotime trajectory across the 5 TIL clusters. f - The violin plots show the expression score (y axes) of the exhaustion gene signature in each of the identified clusters (x axes). Clusters have been re-ordered to follow the exhaustion score and pseudo-time. Box plots indicate median (middle line), 25th, 75th percentile (box). ( n = 15706 total). g - UMAP visualization of CD8 T cell terminal exhaustion signature. h - The violin plots show the expression score (y axes) of the TCR signaling gene signature in each of the identified clusters ( x axes). Box plots indicate median (middle line), 25th, 75th percentile (box). Pairwise group comparisons with two-sided Wilcoxon signed-rank test. ( n = 15706 total). i - The dot plot shows the relative expression and percent of cells expressing the transcription factor Tox and the receptor Entpd1 (encoding CD39) within the different clusters.

Article Snippet: TILs from tumor-bearing Tcf1/7-GFP mice were enriched using a CD45 (TIL) Microbeads enrichment kit (Miltenyi Biotech) and stained for cell sorting.

Techniques: Control, Expressing

Journal: Nature Communications

Article Title: IFNγ signaling in cytotoxic T cells restricts anti-tumor responses by inhibiting the maintenance and diversity of intra-tumoral stem-like T cells

doi: 10.1038/s41467-023-35948-9

Figure Lengend Snippet: Control and CD8-IFNγRKO mice were engrafted with B16-OVA tumors. a–b and d–e CD45 + CD3 + CD8 + Tomato + cells were sorted from tumors and subjected to scRNA-seq analysis. a - Graph-based clustering ( n = 15,706 total) of the assembled cell states according to the signatures identified in Fig. 4. b - The bar plots show the percentages of CD8 T cells from Control (red) and CD8-IFNγRKO (grey) mice that were found in each cell state. Permutation test. c - Tumors were harvested 10-12 days post-tumor engraftment and cell suspensions were stained for CD45, CD3, PD-1, and TCF1/7 and subjected to flow cytometry. Percentage of TCF1/7 + PD-1 + CD8 T cells in tumors from Control (red) and CD8-IFNγRKO (grey) mice Each point represents one individual mouse ( n = 27 for Ctrl; n = 18 for CD8-IFNγRKO), from 4 independent experiments. Data shows Mean +/− SEM. Unpaired t test. d - Heatmap of control TILs shows the relative average expression of IFNγR1 and IFNγR2 in the different cell states in control mice. The average expression of IFNγR1 and IFNγR2 in CD8-IFNγRKO cells from the exhausted state was used as a control for negative IFNγR1 expression. e - The dot plot shows the relative average expression and percent of cells expressing transcripts implicated in IFNγ signaling and response within the different cell states. f–h CD45 + CD3 + CD8 + Tomato + cells were sorted from tumors and subjected to scRNA-seq and scTCR-seq analysis. f - The relative frequency of the top 20 clones and their distribution throughout the cell states was analyzed within Control and CD8-IFNγRKO mice. Each color corresponds to a different clone. g - Relative TCR clonal abundance by cell state and genotype. Clonotype abundance (as frequency of total repertoire per cell state per genotype) is defined as: Rare = 0 < X < = 0.01%; Small = 0.01 < X < = 0.05%; Medium = 0.005 < X < = 0.1%; Large 0.1 < X < = 0.25%; Hyperexpanded 0.25 < X < = 1%. h - Graph-based clustering of TILs ( n = 7000 for each genotype) from control (left) and CD8-IFNγRKO (right) mice overlaid with the frequency of clonotypes. The black line corresponds to the pseudotime trajectory, as in Fig. . Clonotype abundance is defined as in ( g ).

Article Snippet: TILs from tumor-bearing Tcf1/7-GFP mice were enriched using a CD45 (TIL) Microbeads enrichment kit (Miltenyi Biotech) and stained for cell sorting.

Techniques: Control, Staining, Flow Cytometry, Expressing, Clone Assay

Journal: iScience

Article Title: Tocilizumab-treated convalescent COVID-19 patients retain the cross-neutralization potential against SARS-CoV-2 variants

doi: 10.1016/j.isci.2023.106124

Figure Lengend Snippet:

Article Snippet: APC-eFluorTM 780 Mouse anti-human CD8a (Clone OKT8) , Thermo Fisher Scientific , Cat# 47-0086-42; RRID: AB_2573945.

Techniques: Virus, Variant Assay, Clinical Proteomics, Recombinant, Viscosity, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: Nature Communications

Article Title: High p16 expression and heterozygous RB1 loss are biomarkers for CDK4/6 inhibitor resistance in ER + breast cancer

doi: 10.1038/s41467-022-32828-6

Figure Lengend Snippet: A Half-maximal inhibitory concentration (IC 50 ) and IC 50 fold-change (fc) values for ribociclib, fulvestrant and the combination of drugs in T47D and MCF7 cells overexpressing p16 compared to control cells (MOCK), treated for 6 days and evaluated as shown underneath. Data are presented as mean values ± SEM of at least three independent experiments. B Immunoblot of the indicated proteins in MOCK and p16 overexpressing T47D and MCF7 cells untreated or treated with ribociclib for 5 days at the indicated concentrations. C Immunoblot of the indicated proteins in a cell competition assay. Cells were treated with vehicle or ribociclib 1 µM for the indicated days. D Comparison of structural models for the complexes of p16 bound to P18IN003 and p16 bound to CDK4. Gray cartoon is p16, orange spheres represent P18IN003, cyan cartoon is CDK4; zoomed view are the binding pocket with p16 shown as gray surface and highlighting the residues in the binding pocket of p16 as sticks and the hydrogen bonds made between P18IN003 and p16 shown as black dashed lines. E Relative spheroid area of PDC191 and PDC313 after treatment with 1 µM ribociclib, 20 nM P18IN003 and the combination for 7 days. Data are presented as mean values of three independent experiments ± SEM. Two-tailed p -values are based on the one-way ANOVA test with Tukey’s method correction compared with the vehicle (black line). Dashed line indicates the optimal cut-off established in Fig. . p16 and pRb scores of each PDC are indicated. F IC 50 and IC 50 fc values for ribociclib, fulvestrant and the combination of drugs in T47D and MCF7 cells overexpressing cyclin D1, compared to control cells (MOCK), treated for 6 days and evaluated as shown underneath. Data are presented as mean values ± SEM from at least three independent experiments. G Immunoblot of indicated proteins in control (MOCK) and cyclin D1-overexpressing T47D and MCF7 cells untreated or treated with 0.5 µM ribociclib for 48 h. H Immunoblot as indicated for panel ( C ). Source data are provided as a Source Data file.

Article Snippet: Fulvestrant and letrozole were purchased from Selleckchem.

Techniques: Concentration Assay, Control, Western Blot, Competitive Binding Assay, Comparison, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: High p16 expression and heterozygous RB1 loss are biomarkers for CDK4/6 inhibitor resistance in ER + breast cancer

doi: 10.1038/s41467-022-32828-6

Figure Lengend Snippet: A Waterfall plot showing the growth of n = 23 PDX treated with ribociclib 75 mg/kg plus alpelisib 35 mg/kg (bars and black dots) and vehicle (white circles). The percentage change from the initial volume is shown at day 35 of treatment. Dashed lines indicate the range of PD (>20%), SD (20% to −30%) and PR/CR (<−30%). The number of tumors treated per model is indicated in the brackets (n). Hashtags (#) indicate models harboring mutations in PIK3CA . Data represent mean values and error bars ± SEM. Boxes underneath show the molecular and intrinsic tumor subtype as well as their responses to the indicated treatments. The preclinical benefit to each drug is indicated as percentage in brackets. n.d, not determined. B IHC analysis of KI67 (left graph ; p -value < 0.001) and phospho-pRb S807/811 (right graph; p -value < 0.01) in vehicle and alpelisib plus ribociclib-treated PDXs according to the PDX response to alpelisib plus ribociclib. Data are presented as mean values of each PDX. Two-tailed p- values are based on Mann-Whitney U test for all biological replicates. Different colors represent the tumor’s intrinsic subtype. R, resistant; S, sensitive; Alp, alpelisib; Rib, ribociclib. C Relative tumor growth of PDX039 and PDX191 after treatment with the indicated drugs and time. Dashed lines indicate the range of PD (>1.2), SD (1.2 to −0.7) and PR/CR (<−0.7). Two-tailed p -values are based on the two-way ANOVA test with Bonferroni’s method correction. V, vehicle; R, ribociclib; A, alpelisib. D , E Relative tumor growth of the ribociclib-resistant PDX244LR#18R and PDX450 after treatment with the indicated drug(s) for the indicated period of time, respectively. Dashed lines indicate the range of PD (>1.2), SD (1.2 to −0.7) and PR/CR (<−0.7). F Immunoblot of the indicated proteins in PDC476.2 treated with vehicle or 500 nM palbociclib as single-agent or combined with 100 nM fulvestrant and/or 2.5 µM alpelisib in ex vivo cultures for 48 h. At least two independent experiments were conducted. Source data are provided as a Source Data file.

Article Snippet: Fulvestrant and letrozole were purchased from Selleckchem.

Techniques: Two Tailed Test, MANN-WHITNEY, Western Blot, Ex Vivo

Journal: Med (New York, N.y.)

Article Title: Alterations in T and B cell function persist in convalescent COVID-19 patients

doi: 10.1016/j.medj.2021.03.013

Figure Lengend Snippet: Alterations in B cell subsets during acute COVID-19 are recovered upon convalescence (A) Cumulative data show ex vivo frequency of CD19 + B cells in healthy individuals (n = 38) and COVID-19 patients with mild (n = 24), moderate (n = 26), and severe (n = 12) disease and at convalescence (n = 83). (B) Cumulative data show Ki-67 expression by B cells in healthy individuals (n = 28) and COVID-19 patients with mild (n = 13), moderate (n = 15), and severe (n = 9) disease and at convalescence (n = 75). (C and D) Representative flow cytometry plots and cumulative data show frequencies of naive (CD27 − IgD + ), unswitched memory (CD27 + IgD + ), switched memory (CD27 + IgD − ), and double-negative (CD27 − IgD − ) B cells in healthy individuals (n = 38–40) and COVID-19 with mild (n = 22–24), moderate (n = 25–26), and severe (n = 12–13) disease and at convalescence (n = 78–80). (E and F) Representative flow cytometry plots and cumulative data show ex vivo frequency of CD24 hi CD38 hi transitional B cells and CD24 int CD38 int mature B cells in healthy individuals (n = 37) and COVID-19 patients with mild (n = 24), moderate (n = 23), and severe (n = 11) disease and at convalescence (n = 80). (G) tSNE projection of flow cytometry panel visualizing B cell subsets in PBMCs. Representative images for healthy individuals, severe COVID-19 patients, and convalescent patients. Key indicates cell subsets identified on the image. (H) Cumulative data show frequency of CD27 hi CD38 hi plasmablasts in healthy controls (n = 38) and COVID-19 patients with mild (n = 23), moderate (n = 23), and severe (n = 12) disease and at convalescence (n = 81). (I) Graph showing correlation between plasmablasts and IgG + (left), IgA + (center), or IgM + (right) B cell frequencies in acute COVID-19 patients. Graphs show individual patient data, with the bar representing median values. In all graphs, open triangles represent SARS-CoV-2 PCR − patients. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, 1-way ANOVA with Kruskal-Wallis test with Dunn’s post hoc testing for multiple comparisons or Spearman ranked coefficient correlation test. See also and .

Article Snippet: APC-eFluor 780 anti-human CD24 (clone: eBioSN3, 1 in 50) , eBioscience , Cat# 47-0247-42; RRID: AB_10735091.

Techniques: Ex Vivo, Expressing, Flow Cytometry

Journal: Med (New York, N.y.)

Article Title: Alterations in T and B cell function persist in convalescent COVID-19 patients

doi: 10.1016/j.medj.2021.03.013

Figure Lengend Snippet:

Article Snippet: APC-eFluor 780 anti-human CD24 (clone: eBioSN3, 1 in 50) , eBioscience , Cat# 47-0247-42; RRID: AB_10735091.

Techniques: Blocking Assay, Recombinant, Staining, Software