Journal: European journal of immunology

Article Title: Guidelines for the use of flow cytometry and cell sorting in immunological studies

doi: 10.1002/eji.201646632

Figure Lengend Snippet: Uni-, bi- and multi-parameter presentation of flow data. Comparison of two gender- and age-matched patients: a healthy one (67 years) and a patient with B-CLL (64 years) from 294. (A) 1D-histogram presentation of CD3 expression on lymphocytes (red: B-CLL, grey: healthy), (B) 2D-dot-plot presentation of CD3 expression on x-axis vs. CD16/56 expression on y-axis, (C) multivariate presentation of expression of 12 different antibodies on 9 colors (OMIP-023, exclusion of low CD25 expression) for 9 different leukocyte subsets in a radar-plot. Abbreviations used: B-CLL (B-cell chronic lymphocytic leukemia), Th (CD4+ T-helper cell), Tc (CD8+ cytotoxic T cell), NK (natural killer cell).

Article Snippet: 6 Monoclonal antibodies 6.1 Surface staining: BD Biosciences : CD4 BUV 395 (SK3), CD45RA BV421 (HI100), CCR7 BUV395 (150503), CD45RA BV650 (HI100), CXCR5 Alexa Fluor® 488 (clone RF8B2), CD25 APC (clone 2A3) CD161 FITC (DX12). eBioscience : CD3 PE (UCHT1), KLRG1 AF488 (clone 13F12F2), CD4 PerCP-eFluor 710 (clone SK3), CD127 PE-Cy7 (clone eBioRDR5), CD27 APC-eFluor 780 (clone O323), CD107a FITC (clone H4A3) Biolegend : CD27 APC-Fire 750 (O323), CCR6 Alexa Fluor® 647 (clone G034E3), CCR7 BV421 (clone G043H7), CX3CR1 FITC (clone 2A9–1), CCR4 BV421 (L291H4), CD28 Alexa Fluor 700 (CD28.2), CD127 BV650 (A019D5).

Techniques: Expressing

Journal: European journal of immunology

Article Title: Guidelines for the use of flow cytometry and cell sorting in immunological studies

doi: 10.1002/eji.201646632

Figure Lengend Snippet: Most common transcription factors measured by flow cytometry. For more information about them http://www.uniprot.org/uniprot/

Article Snippet: 6 Monoclonal antibodies 6.1 Surface staining: BD Biosciences : CD4 BUV 395 (SK3), CD45RA BV421 (HI100), CCR7 BUV395 (150503), CD45RA BV650 (HI100), CXCR5 Alexa Fluor® 488 (clone RF8B2), CD25 APC (clone 2A3) CD161 FITC (DX12). eBioscience : CD3 PE (UCHT1), KLRG1 AF488 (clone 13F12F2), CD4 PerCP-eFluor 710 (clone SK3), CD127 PE-Cy7 (clone eBioRDR5), CD27 APC-eFluor 780 (clone O323), CD107a FITC (clone H4A3) Biolegend : CD27 APC-Fire 750 (O323), CCR6 Alexa Fluor® 647 (clone G034E3), CCR7 BV421 (clone G043H7), CX3CR1 FITC (clone 2A9–1), CCR4 BV421 (L291H4), CD28 Alexa Fluor 700 (CD28.2), CD127 BV650 (A019D5).

Techniques: Flow Cytometry

Journal: Nature

Article Title: Engineered CD47 protects T cells for enhanced antitumour immunity

doi: 10.1038/s41586-024-07443-8

Figure Lengend Snippet: (a) Her2.BBζ-CAR ± B6H12 treated 143B tumour survival. n = 5 mice/arm. (b) B7H3.BBζ- or GD2.BBζ-CAR ± B6H12 treated MG63.3 tumour survival. n = 5 mice/arm. (c) B7H3.BBζ-CAR ± B6H12 treated D425 tumour growth by BLI. CD19.BBζ-CAR is included as a non-tumour targeting control. Mean ± SEM of n = 5 (B6H12) or n = 6 (all others) mice/arm. Representative of two independent experiments. (d) B7H3.BBζ-CAR ± B6H12 treated D425 tumour survival. CD19.BBζ-CAR is included as a non-tumour targeting control. n = 5 (B6H12) or n = 6 (all others) mice per treatment arm. Representative of two independent experiments. (e) Representative flow cytometry plots of hCD45 + T cells identified in the blood and tumour in the B7H3.BBζ-CAR ± B6H12 treated MG63.3 model on day 30 post tumour engraftment. (f) Representative flow cytometry plots of hCD45 + T cells identified in the blood of non-tumour bearing mice co-treated with CD19.28ζ-CAR T cells and either PBS, B6H12, or mIgG1 isotype control. Representative of two independent experiments. (g) hCD8 + (top) and hCD4 + (bottom) T cells in the blood of mice on day 5 in the isotype control model, treated as in (f). Mean ± SD of n = 5 mice. Representative of two independent experiments. (h) T cells (hCD4 + and hCD8 + ) in the blood of mice on day 12 in Her2.BBζ ± B6H12 treated mice in the 143B model. Mean ± SD of n = 5 mice. (i) Low-dose NY-ESO-1-TCR ± B6H12 treated A375 tumour survival. n = 5 mice/arm. (j) High-dose NY-ESO-1-TCR ± B6H12 treated A375 tumour growth. Mean ± SEM of n = 5 mice/arm. (k) T cells (hCD4 + and hCD8 + ) in the blood by flow cytometry on day 16 of mice treated with high-dose NY-ESO-1-TCR ± B6H12 in the A375 model. Mean ± SD of n = 5 mice. (l) High-dose CD19.28ζ-CAR ± B6H12 treated Nalm6 tumour growth by BLI. Mean ± SEM of n = 5 mice/arm. (m) Low-dose CD19.28ζ-CAR ± B6H12 treated Nalm6 tumour growth by BLI. Mean ± SEM of n = 5 mice/arm. (n) Images of Nalm6 tumour BLI in the high-dose (left) and low-dose (right) CAR-T model on day 17. (o) Low-dose CD19.28ζ-CAR ± B6H12 treated Nalm6 survival. n = 5 mice/arm. (p) T cell BLI in the high-dose CAR-T – Nalm6 model, treated as in (l). Mean ± SEM of n = 5 mice/arm. (q) T cell BLI in the low-dose CAR-T – Nalm6 model, treated as in (m). Mean ± SEM of n = 5 mice/arm. (r) Images of CD19.28ζ-nLuc-CAR T cell BLI in the low-dose CAR T – Nalm6 model, treated as in (m), on day 11. (s) Quantification of T cells by flow cytometry from the spleen in the high-dose CAR T – Nalm6 model, treated as in (l). Mean ± SD of n = 4 mice. [(a), (b), (d), (i), (o)] Log-rank Mantel-Cox test. [(c), (j), (l), (m), (p), (q)] Two-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test. [(g), (h), (k), (s)] Unpaired two-tailed Student’s t test. For all: ns = not significant.

Article Snippet: T cells were stained with anti-hCD4 (BUV 395, SK3, BD, 1:200), anti-hCD8 (BUV 805, SK1, BD, 1:400), anti-hCD47 or mIgG1 isotype control (PE, B11/6, Abcam, 1:20), anti-hCD45RA (BV785, HI100, BioLegend, 1:100) and anti-hCD62L (BV605, DREG-56, BD, 1:100) antibodies.

Techniques: Flow Cytometry, Comparison, Two Tailed Test

Journal: Nature

Article Title: Engineered CD47 protects T cells for enhanced antitumour immunity

doi: 10.1038/s41586-024-07443-8

Figure Lengend Snippet: (a) CD19.BBζ-CAR ± CV-1 (Fc-dead) treated Nalm6 tumour survival. n = 5 mice/arm. (b) Images of Nalm6 BLI on day 11 of high-dose CD19.BBζ-CAR ± CV-1 treated mice, treated as in (a). (c) Low-dose CD19.28ζ-CAR ± CV-1 (Fc-dead) treated Nalm6 tumour growth. Mean ± SEM of n = 5 mice/arm. (d) Images of Nalm6 BLI of low-dose CD19.28ζ-CAR ± CV-1 treated mice, treated as in (c). (e - f) Images (e) and quantification (f) of T cell BLI post CV-1 treatment of Nalm6 tumour-bearing mice. Top: high-dose CD19.BBζ-CAR ± CV-1, treated as in (a), on day 9 (four days post CV-1 treatment). Bottom: low-dose CD19.28ζ-CAR ± CV-1, treated as in (c), day 11 (six days post CV-1 treatment). For (f): mean ± SD of n = 5 mice for each dose condition. (g) CRISPR/Cas9 mediated CD47 knock-out (CD47 KO ) efficiency in primary human T cells by flow cytometry. CD47 WT cells are CD47 KO with exogenous expression of wild-type CD47. Representative of n > 3 donors. (h) Images of Nalm6 tumour progression (top five rows) and T cells on day 11 (bottom row) by BLI in the Nalm6 – 47 KO -CD19.28ζ-CAR T model. (i) Survival of Nalm6 bearing mice shown in (h). n = 5 mice per treatment arm. (j) Representative flow cytometry plots of hCD45 + T cells identified in the blood of 47 WT - or 47 KO -CD19.28ζ-nLuc-CAR ± B6H12 treated, non-tumour bearing mice. (k) Images of T cell BLI following treatment with 47 WT - or 47 KO -CD19.28ζ-nLuc-CAR T cells before and after B6H12 treatment. (l) T cell BLI of mice shown in (k) treated with 47 WT - or 47 KO -CD19.28ζ-nLuc-CAR T, before and after B6H12 treatment. Dashed line indicates limit of detection. Mean ± SD of n = 5 mice. (m) hCD8 + and hCD4 + T cells in the blood on day 6 or 7 by flow cytometry following treatment 47 WT - or 47 KO -CD19.28ζ-nLuc-CAR T ± B6H12 in separate experiments, treated as in (k). Mean ± SD of n = 5 mice. [(c), (l), (m)] Two-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test. ns = not significant. [(f - top)] Two-tailed Mann-Whitney test. [(f - bottom)] Unpaired two-tailed Student’s t test.

Article Snippet: T cells were stained with anti-hCD4 (BUV 395, SK3, BD, 1:200), anti-hCD8 (BUV 805, SK1, BD, 1:400), anti-hCD47 or mIgG1 isotype control (PE, B11/6, Abcam, 1:20), anti-hCD45RA (BV785, HI100, BioLegend, 1:100) and anti-hCD62L (BV605, DREG-56, BD, 1:100) antibodies.

Techniques: CRISPR, Knock-Out, Flow Cytometry, Expressing, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Engineered CD47 protects T cells for enhanced antitumour immunity

doi: 10.1038/s41586-024-07443-8

Figure Lengend Snippet: (a) Representative flow cytometry histograms of 50 nM hSIRPα, 50 nM mSIRPα, 500 nM B6H12, 500 nM TJC4, and 50 nM Hu5F9 binding to 47 WT , 47 A30P , 47 Q31P , and 47 A30P-Q31A over-expressed on primary human T cells. Data are representative of n = 3 different T cell donors in independent experiments. (b) Left: Representative flow cytometry histograms of 100 nM hSIRPα, 100 nM mSIRPα, and 500 nM B6H12 binding to 47 WT , 47 A30P , and 47 Q31P expressed on Jurkats with endogenous 47 KO . Right: Binding of B6H12, hSIRPα, and mSIRPα to full-length 47 WT , 47 A30P , and 47 Q31P expressed on Jurkat cells with endogenous 47 KO . Mean ± SD of n = 3 independent experiments, normalized to fraction binding to 47 WT . (c) Phagocytosis by primary human macrophages from multiple donors of CFSE labelled Jurkats with endogenous 47 KO , expressing 47 WT , 47 A30P , or 47 Q31P variants after one hour of co-culture ± B6H12. Mean ± SD of n = 3 triplicate wells. (d) Phagocytosis by primary human macrophages from two donors of pHrodo Red labelled primary human CD19.28ζ-CAR-T cells from three donors with endogenous 47 KO , expressing 47 WT or 47 E (47 Q31P ) after three hours of co-culture ± B6H12. Mean ± SD of n = 3 triplicate wells. (e) Images of T cell BLI following treatment with 47 WT - or 47 E -CD19.28ζ-nLuc-CAR-T cells ± B6H12 in non-tumour bearing mice. (f) T cell BLI before and after αCD47 treatment, of mice treated as in (e). Mean ± SD of n = 5 mice. (g) hCD8 + and hCD4 + T cells in the blood on day 6 by flow cytometry of mice treated as in (e). Mean ± SD of n = 5 mice. (h) Representative flow plots and gating strategy for detection of CFSE + macrophages and T cells in dissociated 143B tumour samples in the in vivo phagocytosis model treated with Her2.BBζ-CAR T cells ± B6H12. Representative of n = 5 mice/arm. (i) Representative flow plot and gating strategy for detection of CFSE + cells of a dissociated 143B tumour from a mouse that did not receive CAR T cells. (j) mF4/80 + /CD11b + macrophages identified by flow cytometry of dissociated tumours treated as in (h). Mean ± SD of n = 5 mice. (k) hCD3 + /hCD45 + human T cells identified by flow cytometry of dissociated tumours treated as in (h). Mean ± SD of n = 5 mice. (l) Percentage of phagocytosed T cells identified in each dissociated tumour, treated as in (h), calculated as the number of CFSE + macrophages per 100,000 lives cells per sample divided by the total number of CFSE + macrophages and T cells per 100,000 live cells per sample, identified by flow cytometry. Mean ± SD of n = 5 mice. [(b), (c), (d), (f), (g)] Two-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test. ns = not significant. (b) comparison is between indicated group and binding to 47 WT expressing cells. [(j), (k)] Unpaired two-tailed Student’s t test. [(l)] Two-tailed Mann-Whitney Test.

Article Snippet: T cells were stained with anti-hCD4 (BUV 395, SK3, BD, 1:200), anti-hCD8 (BUV 805, SK1, BD, 1:400), anti-hCD47 or mIgG1 isotype control (PE, B11/6, Abcam, 1:20), anti-hCD45RA (BV785, HI100, BioLegend, 1:100) and anti-hCD62L (BV605, DREG-56, BD, 1:100) antibodies.

Techniques: Flow Cytometry, Binding Assay, Expressing, Co-Culture Assay, In Vivo, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Engineered CD47 protects T cells for enhanced antitumour immunity

doi: 10.1038/s41586-024-07443-8

Figure Lengend Snippet: (a – b) hCD8 + (left, a) and hCD4 + (right, a & b) T cells derived from two independent donors (represented by panels a & b) in the blood on day 14 by flow cytometry after B6H12 treatment in 143B tumour bearing mice, treated with 47 WT - or 47 E -Her2.BBζ-Antares-CAR-T ± B6H12. Mean ± SD of n = 5 mice. (c) hCD4 + and hCD8 + T cells in the blood on day 14 and day 27 of tumour growth in 143B tumour bearing mice, treated with 47 E -Her2.BBζ-Antares-CAR-T + B6H12. Datapoints represent individual mice, with values from the same mouse connected by lines between time points. (d) T cell BLI prior to B6H12 treatment in mice treated as in (a). Mean ± SD of n = 5 mice. (e) T cell BLI after B6H12 treatment on day 13 in mice treated as in (a). Mean ± SD of n = 5 mice. (f) 47 WT - or 47 E -Her2.BBζ-Antares-CAR-T ± B6H12 treated 143B tumour survival. n = 5 mice/arm. (g and h) 47 WT - or 47 E -Her2.BBζ-Antares-CAR-T ± B6H12 treated 143B tumour (g) growth and (h) survival, using T cells derived from a different donor than (f). Mean ± SEM (g) or representative (h) of n = 5 mice. (i) hCD8 + (left) and hCD4 + (right) T cells derived from in the blood on day 12 by flow cytometry after treatment with 47 WT - or 47 E -Her2.BBζ-CAR-T ± low-doses of 75 µg or 25 µg B6H12 treatment in 143B tumour bearing mice. Mean ± SD of n = 5 mice. (j) 47 E -Her2.BBζ-CAR-T ± low-dose B6H12 (75 µg or 25 µg) treated 143B tumour growth, using T cells derived from two different donors (left and right panels, respectively). Mice treated with mock T cells were co-treated ± 250 µg (~10 mg/kg) B6H12 (left panel) or 75 µg (~3 mg/kg) B6H12 (right panel). Mean ± SEM of n = 5 mice. [(a), (b), (d), (e), (g), (i), (j)] Two-way ANOVA test with Tukey’s multiple comparison test. ns = not significant. [(f), (h)] Log-rank Mantel-Cox test. ns = not significant.

Article Snippet: T cells were stained with anti-hCD4 (BUV 395, SK3, BD, 1:200), anti-hCD8 (BUV 805, SK1, BD, 1:400), anti-hCD47 or mIgG1 isotype control (PE, B11/6, Abcam, 1:20), anti-hCD45RA (BV785, HI100, BioLegend, 1:100) and anti-hCD62L (BV605, DREG-56, BD, 1:100) antibodies.

Techniques: Derivative Assay, Flow Cytometry, Comparison

Journal: Nature

Article Title: Engineered CD47 protects T cells for enhanced antitumour immunity

doi: 10.1038/s41586-024-07443-8

Figure Lengend Snippet: (a) hCD8 + (left) and hCD4 + (right) T cells by flow cytometry from the blood of CAR-T treated mice on day 15 in CHLA-255 tumour bearing mice, treated with 47 WT - or 47 E -B7H3.BBζ-nLuc-CAR-T ± B6H12. Mean ± SD of n = 5 mice. (b) T cell BLI on day 14 in CHLA-255 tumour bearing mice, treated as in (a). Mean ± SD of n = 5 mice. (c) Images of CHLA-255 tumour progression (top four rows) and T cells on day 14 (bottom) by BLI, treated as in (a). (d) 47 WT - or 47 E -B7H3.BBζ-nLuc-CAR-T ± B6H12 treated CHLA-255 tumour growth by BLI. Mean ± SEM of n = 5 mice/arm. (e) 47 WT - or 47 E -CD19.28ζ-CAR-T ± B6H12 treated Nalm6 tumour growth by BLI. Mean ± SEM of n = 5 mice/arm. (f) 47 WT - or 47 E -CD19.28ζ-CAR-T ± B6H12 treated Nalm6 survival. n = 5 mice/arm. (g) T cell by BLI in A375 tumour bearing mice, treated with 47 E -NY-ESO-1-Antares-TCR-T cells ± B6H12, before (day 9, left) and after (day 14, right) αCD47 treatment. Mean ± SD of n = 5 mice. (h) hCD4 + (left) and hCD8 + (right) T cells by flow cytometry from the blood in the A375 tumour bearing mice, treated as in (g). Mean ± SD of n = 5 mice. (i) 47 E -NY-ESO-1-TCR-T ± B6H12 treated A375 tumour growth. Data are individual tumour growth traces of n = 5 mice/arm. (j) 47 E -NY-ESO-1-TCR-T ± B6H12 treated A375 tumour growth, with T cells derived from a different donor than shown in (i). Mean ± SEM of n = 5 mice/arm. [(a), (b), (d), (e), (g), (h), (j)] Two-way ANOVA test with Tukey’s multiple comparison test. ns = not significant. [(f)] Log-rank Mantel-Cox test. ns = not significant.

Article Snippet: T cells were stained with anti-hCD4 (BUV 395, SK3, BD, 1:200), anti-hCD8 (BUV 805, SK1, BD, 1:400), anti-hCD47 or mIgG1 isotype control (PE, B11/6, Abcam, 1:20), anti-hCD45RA (BV785, HI100, BioLegend, 1:100) and anti-hCD62L (BV605, DREG-56, BD, 1:100) antibodies.

Techniques: Flow Cytometry, Derivative Assay, Comparison