Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Neutralization of the adaptor protein PAG by monoclonal antibody limits murine tumor growth

doi: 10.1016/j.omtm.2022.10.012

Figure Lengend Snippet: Clone 7M16A binds to the extracellular domain of PAG (A) ELISA with immobilized PAG peptide (human, mouse, or cynomolgus) using hybridoma supernatants. (B) 7M16A staining of non-permeabilized wild-type C57BL6 or PAG knockout (KO) murine splenocytes shows specificity of binding. (C) Western blot of A549 cells made to stably overexpress PAG-GFP. (D) Western blot of fractionated cell lysates showing that PAG-GFP is expressed at high levels at the cell membrane. Confocal imaging showing the localization of PAG-GFP at the plasma membrane. (E) 7M16A staining of non-permeabilized A549 cells shows binding to PAG-GFP on the surface. (F) ELISA with immobilized human PAG peptide using purified monoclonal antibodies. (G) ELISA of IL-2 secretion from primary human CD3 + T cells stimulated as indicated for 48 h. n = 3; ∗p ≤ 0.05 and ∗∗p ≤ 0.01. (H and I) Jurkat T cells stably express PAG-GFP with or without PD-1-SNAP incubated with Raji B cells in the presence of SEE. Cells were imaged using confocal microscopy to assess the location of PAG and PD-1 relative to the immune synapse. White arrow indicates the center of the immune synapse where PAG was excluded with 7M16A pretreatment. White star indicates the point in the cell with enrichment of PD-1 either at or away from the immune synapse. Quantification of the number of cells per phenotype. ∗p ≤ 0.05.

Article Snippet: Primary human CD3 + T cells were purified from peripheral blood using RosetteSep CD3 + Negative Selection (catalog no. 15021; Stemcell) followed by Lymphoprep separation.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Knock-Out, Binding Assay, Western Blot, Stable Transfection, Membrane, Imaging, Clinical Proteomics, Purification, Bioprocessing, Incubation, Confocal Microscopy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Neutralization of the adaptor protein PAG by monoclonal antibody limits murine tumor growth

doi: 10.1016/j.omtm.2022.10.012

Figure Lengend Snippet: PAG antibody and PD-1 antibody in combination enhance T cell infiltration into MC38 tumors Immunohistochemistry of dissected tumors at the study endpoint. Sections were stained with anti-CD3 from 2 tumors per condition; representative images shown (40×) (A). (B) Quantification of CD3 + cells per high-power field (HPF; 20×) from 2 tumors per condition. ∗p ≤ 0.05.

Article Snippet: Primary human CD3 + T cells were purified from peripheral blood using RosetteSep CD3 + Negative Selection (catalog no. 15021; Stemcell) followed by Lymphoprep separation.

Techniques: Immunohistochemistry, Staining

Journal: Nature biomedical engineering

Article Title: Classification of T-cell activation via autofluorescence lifetime imaging

doi: 10.1038/s41551-020-0592-z

Figure Lengend Snippet: ( a ) Representative optical redox ratio, NAD(P)H τ m , and FAD τ m images (4 images selected out of 202 images acquired from 6 different donors with similar results) of quiescent (columns 1, 3) and activated (columns 2, 4) CD3 + (rows 1–3) and CD3 + CD8 + (row 4–6) T cells from two different donors. Scale bar is 20 μm. Cell size ( b ), optical redox ratio ( c ), NAD(P)H τ m ( d ), FAD τ m ( e ), NAD(P)H α 1 ( f ), and FAD α 1 ( g ) of quiescent and activated CD3 + and CD3 + CD8 + T cells. Black circles represent mean of all data (6 donors), triangles (donors A [dark red], B [medium red], and F [light red]) represent data from female donors, squares (donors C [dark blue], D [medium blue], and E [light blue]) represent data from male donors. Each colour shade represents data from an individual donor. Data are mean +/− 99% CI. Horizontal lines indicate statistical comparisons, Stars (*** p<0.001) indicate statistical comparisons at the cellular level (n=4,877 biologically independent CD3 + T cells from 6 donors and n=3,478 biologically independent CD3 + CD8 + T cells from 6 donors) from a two-sided logistic regression, generalized linear model using an α significance level of 0.05. Carets (B: ˆˆ p=0.001, ˆ=0.016; C: ˆ p=0.011, ˆˆ p=0.002; D: ˆ p=0.015, ˆˆ p=0.002; E: ˆ p=0.022; F: ˆˆ p=0.008, ˆˆˆ p<0.001; G: ˆˆ p=0.002, ˆ p=0.021) indicate significance at the donor level, n=6 biologically independent donors. Aggregated cellular data was compared using a double-sided paired t-test. ( h-j ) Cellular respiration increases in activated T cells. The oxygen consumption rate (OCR; panel H) and extracellular acidification rate (ECAR, panel I) are increased in activated bulk CD3 + and isolated CD3 + CD8 + T cells. The ratio of OCR to ECAR (J) is significantly decreased in activated bulk CD3 + and isolated CD3 + CD8 + T cells as compared with that of quiescent T cells. *** p<1*10 −5 , horizontal lines indicate statistical comparisons, double-sided, Student’s t-test, n=6 wells/group CD3 + CD8 + isolation, n=12 wells/group CD3 + isolation (1 donor). Mean +/− 95% CI. Error bars smaller than the symbol for the mean are not shown.

Article Snippet: Following the RosetteSep Protocol, the CD3 + or CD8 + RosetteSep Cocktail (StemCell Technologies) was added to the blood (50 μl per ml of blood), mixed, and incubated at room temperature for 10 minutes.

Techniques: Isolation

Journal: Nature biomedical engineering

Article Title: Classification of T-cell activation via autofluorescence lifetime imaging

doi: 10.1038/s41551-020-0592-z

Figure Lengend Snippet: ( A-B ) UMAP data reduction technique allows visual representation of the separation between quiescent (“Q”) and activated (“Act”) bulk CD3 + (A) and isolated CD3 + CD8 + (B) T cells. Each colour corresponds to a different donor, greys correspond to quiescent cells and green or purple to activated CD3 + or CD3 + CD8 + T cells, respectively. Data are from 6 donors. Each dot represents a single cell, n=4,877 CD3 + T cells and n=3,478 CD3 + CD8 + T cells. ( C ) Feature weights for classification of quiescent versus activated T cells by the gain ratio method. Analysis was performed at the cellular level with data from 6 donors. ( D ) ROC curves of the test data for logistic regression models for classification of activation state within bulk CD3 + T cells, bulk CD3 + T cells normalized within each donor (CD3 + Norm), isolated CD3 + CD8 + T cells, and isolated CD3 + CD8 + T cells normalized within each donor (CD3 + CD8 + Norm). ( E-F ) ROC curves of the test data for logistic regression classification models computed using different features for the classification of (E) quiescent or activated bulk CD3 + or (F) isolated CD3 + CD8 + T cells. Models for subfigures D-F were trained on cells that lacked same cell validation data from donors A, B, C, and D but were known to be quiescent or activated by culture conditions (n = 4,131 biologically independent CD3 + cells, n=2,655 biological independent CD3 + CD8 + cells), and cells from donors B, E, and F with CD69 validation of activation state were used to test the models (n = 696 biologically independent CD3 + cells, n=595 biologically independent CD3 + CD8 + cells). Redox Ratio = NAD(P)H/(NAD(P)H+FAD).

Article Snippet: Following the RosetteSep Protocol, the CD3 + or CD8 + RosetteSep Cocktail (StemCell Technologies) was added to the blood (50 μl per ml of blood), mixed, and incubated at room temperature for 10 minutes.

Techniques: Isolation, Activation Assay, Biomarker Discovery

Journal: Nature biomedical engineering

Article Title: Classification of T-cell activation via autofluorescence lifetime imaging

doi: 10.1038/s41551-020-0592-z

Figure Lengend Snippet: (A) Heatmap of z-scores of NAD(P)H and FAD autofluorescence imaging endpoints where each row is the mean data aggregating all cells from a single donor, subtype (CD3 + or CD3 + CD8 + ), and activation, n=6 biologically independent donors. Data clusters by activation state and isolation (bulk CD3 + or isolated CD3 + CD8 + ). (B) Heatmap of z-scores of NAD(P)H and FAD autofluorescence imaging endpoints of CD3 + CD8 + T cells from a single donor, each row is a single cell (n=635 cells). Distinct clusters are identified within the quiescent and activated CD3 + CD8 + T cells. (C) Histogram analysis of NAD(P)H τ m reveals two populations in quiescent CD3 + CD8 + T cells across all 6 donors (n=2126 quiescent cells, 1352 activated cells, - Act = quiescent cells, + Act = cells exposed to anti-CD3/CD2/CD28 for 48hr). (D) NAD(P)H τ m is decreased in CD45RO + CD3 + CD8 + T cells compared to NAD(P)H τ m of CD45RA + CD3 + CD8 + T cells (CD45RA + n=265 cells, CD45RO + n=33 cells from 3 donors, *** p=0.00058, two-sided logistic regression, generalized linear model. p>0.05 for data aggregated to the donor level, two-sided paired t-test.) Mean +/− 95% confidence interval. Redox Ratio = NAD(P)H/(NAD(P)H+FAD).

Article Snippet: Following the RosetteSep Protocol, the CD3 + or CD8 + RosetteSep Cocktail (StemCell Technologies) was added to the blood (50 μl per ml of blood), mixed, and incubated at room temperature for 10 minutes.

Techniques: Imaging, Activation Assay, Isolation

Journal: Nature biomedical engineering

Article Title: Classification of T-cell activation via autofluorescence lifetime imaging

doi: 10.1038/s41551-020-0592-z

Figure Lengend Snippet: (A) UMAP of NAD(P)H and FAD autofluorescence endpoints of quiescent and activated (“Act”) CD3 + CD8 + T cells identified within bulk CD3 + and specific CD3 + CD8 + isolations, n=477 biologically independent cells from 3 donors. (B) Optical redox ratio and (C) NAD(P)H α 1 of CD3 + CD8 + T cells cultured as an isolated population (CD3 + CD8 + specific isolation, n=39 quiescent cells, n=174 activated cells, from 3 donors) and with CD3 + CD4 + T cells (bulk CD3 + isolation, n=83 quiescent cells, n=170 activated cells, from 3 donors). Mean +/− 95% confidence interval. Horizontal lines indicate statistical comparisons, ** p=0.002, *** p<0.001 for cell level comparisons using a two sided logistic regression, linear generalized model. Donor level p-values provided in – . (D) Accuracy of random forest classification of quiescent versus activated CD3 + CD8 + T cells from CD3 + CD8 + specific isolation (n=213 cells, 3 donors) and bulk CD3 + isolation (n=253 cells, 3 donors). Mean +/− 95% confidence interval for 50 iterations. (E) UMAP of NAD(P)H and FAD autofluorescence imaging endpoints of quiescent and activated CD3 + CD4 + and CD3 + CD8 + cells identified within bulk CD3 + populations, n=583 biologically independent cells from 3 donors. (F) NAD(P)H τ 2 of quiescent CD3 + CD4 + and CD3 + CD8 + cells (bulk CD3 + isolation, n=66 quiescent CD3 + CD4 + T cells, n=83 quiescent CD3 + CD8 + T cells from 3 donors, * p=0.04, two-sided logistic regression, generalized linear model; p>0.05 for two sided paired t-test at the donor level, mean +/− 95% confidence interval). (G) NAD(P)H α 1 of activated CD3 + CD4 + and CD3 + CD8 + cells (bulk CD3 + isolation, n=264 activated CD3 + CD4 + T cells, n=170 activated CD3 + CD8 + T cells from 3 donors, *** p=0.0004, two-sided logistic regression, generalized linear model; p>0.05 for two sided paired t-test at the donor level, mean +/− 95% confidence interval. (H) Accuracy of random forest classification of CD3 + CD4 + and CD3 + CD8 + T cells from quiescent (2 group classification, “CD3 + Q”), activated (2 group classification, “CD3 + Act”), or both quiescent and activated T cells (4 group classification, “CD3 + All”) within bulk CD3 + isolations, total observations include 66 quiescent CD3 + CD4 + T cells, 83 quiescent CD3 + CD8 + T cells, 264 activated CD3 + CD4 + T cells, and 170 activated CD3 + CD8 + T cells from 3 donors. Mean +/− 95% confidence interval for 50 iterations.

Article Snippet: Following the RosetteSep Protocol, the CD3 + or CD8 + RosetteSep Cocktail (StemCell Technologies) was added to the blood (50 μl per ml of blood), mixed, and incubated at room temperature for 10 minutes.

Techniques: Cell Culture, Isolation, Imaging

Journal: Nature biomedical engineering

Article Title: Classification of T-cell activation via autofluorescence lifetime imaging

doi: 10.1038/s41551-020-0592-z

Figure Lengend Snippet: (A) Representative NAD(P)H α 1 image of 4 images acquired with similar results of combined quiescent (CD69 − ) and activated (CD69 + ) T cells with CD69 immunofluorescence overlaid in pink. Scale bar is 30 μm. CD69 image is shifted to account for cell movement between frames. (B) UMAP representation of NAD(P)H and FAD imaging endpoints of CD69 − and CD69 + CD3 + T cells from a combined population of quiescent and activated T cells, n=265 biologically independent cells from 1 donor. (C) Optical redox ratio and (D) NAD(P)H α 1 of isolated (“Iso.”) and combined quiescent (CD69 − ) and activated (CD69 + ) CD3 + T cells. Mean +/− 95% confidence interval. Horizontal lines indicate statistical comparisons, *** p<1*10 −9 for cell level analysis using a two sided logistic regression, generalized linear model, n=733 biologically independent cells, single donor. (E) ROC curves of logistic regression classification of quiescent and activated CD3 + T cells from a combined population of CD69 − and CD69 + T cells from a single donor, n=250 biologically independent cells. (F) Percent difference of NAD(P)H α 1 and fluorescence intensity in CD3 + T cell nuclei and cytoplasms over time. Anti-CD2/CD3/CD28 added at t=0 m. Mean +/− SE of n=94 biologically independent cells from 3 different donors.

Article Snippet: Following the RosetteSep Protocol, the CD3 + or CD8 + RosetteSep Cocktail (StemCell Technologies) was added to the blood (50 μl per ml of blood), mixed, and incubated at room temperature for 10 minutes.

Techniques: Immunofluorescence, Imaging, Isolation, Fluorescence