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cd69 fitc (Miltenyi Biotec)

Name: cd69 fitc
Brand: Miltenyi Biotec
Rating: 95 (71 reviews)

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Miltenyi Biotec cd69 fitc

( A ) Flow cytometric analysis (FCA) of CD25 (left) and <t>CD69</t> (right) staining and quantification in CTL, n = 6/11 individual patients. ( B ) FCA of lymphocyte-activation gene 3 (LAG3), programmed cell death protein 1 (PD1), and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) staining and quantification as indicated, n = 6/4 (not activated/+CD3/CD28) individual patients. P values (left to right): 0.2842, 0.2188, 0.6875, 0.5494, 0.1250, and >0.9999. ( C ) Quantification of relative cytotoxicity using a flow cytometric killing assay, n = 5 individual patients. ( D ) Quantification of caspase-3 activity reporter using a CTL-K562 killing assay, n = 4 individual patients. Data were represented as mean ± SEM with dots indicating individual values ( A , B ) or as individual data points ( C , D ). P values: as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test, as appropriate.

Cd69 Fitc, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 71 article reviews

cd69 fitc - by Bioz Stars, 2026-02

95/100 stars

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1) Product Images from "Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery"

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

Journal: EMBO Molecular Medicine

doi: 10.1038/s44321-025-00324-1

( A ) Flow cytometric analysis (FCA) of CD25 (left) and CD69 (right) staining and quantification in CTL, n = 6/11 individual patients. ( B ) FCA of lymphocyte-activation gene 3 (LAG3), programmed cell death protein 1 (PD1), and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) staining and quantification as indicated, n = 6/4 (not activated/+CD3/CD28) individual patients. P values (left to right): 0.2842, 0.2188, 0.6875, 0.5494, 0.1250, and >0.9999. ( C ) Quantification of relative cytotoxicity using a flow cytometric killing assay, n = 5 individual patients. ( D ) Quantification of caspase-3 activity reporter using a CTL-K562 killing assay, n = 4 individual patients. Data were represented as mean ± SEM with dots indicating individual values ( A , B ) or as individual data points ( C , D ). P values: as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test, as appropriate.

Figure Legend Snippet: ( A ) Flow cytometric analysis (FCA) of CD25 (left) and CD69 (right) staining and quantification in CTL, n = 6/11 individual patients. ( B ) FCA of lymphocyte-activation gene 3 (LAG3), programmed cell death protein 1 (PD1), and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) staining and quantification as indicated, n = 6/4 (not activated/+CD3/CD28) individual patients. P values (left to right): 0.2842, 0.2188, 0.6875, 0.5494, 0.1250, and >0.9999. ( C ) Quantification of relative cytotoxicity using a flow cytometric killing assay, n = 5 individual patients. ( D ) Quantification of caspase-3 activity reporter using a CTL-K562 killing assay, n = 4 individual patients. Data were represented as mean ± SEM with dots indicating individual values ( A , B ) or as individual data points ( C , D ). P values: as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test, as appropriate.

Techniques Used: Staining, Activation Assay, Activity Assay

( A ) FCA of MitoTracker green and quantification in isolated mitochondria from CTL, n = 7 individual patients. ( B ) FCA of JC1 green and quantification of red/green fluorescence in isolated mitochondria from CTL, n = 4 individual patients. ( C ) Immunoblot of OXPHOS complexes using isolated mitochondria of CTL and normalized densitometric quantification of complex V, n = 5 individual patients. I–V complex I–V; H heat shock protein 60 (HSP60). Subunits detected by the antibody cocktail: complex I: subunit NDUFB8; complex II: subunit 30 kDa (SDHB); complex III: subunit Core 2 (UQCRC2); complex IV: subunit II (COXII); complex V: ATP synthase subunit alpha (ATP5). ( D , E ) Representative immunoblot of mitochondrial fusion and fission proteins of isolated mitochondria of CTL ( D ) and normalized densitometric quantification ( E ) of the respective proteins, n = 5/4/3 individual patients. ( F ) Representative projections of 3D image stacks of CTL stained with MitoTracker (MT, green, left) and the respective skeleton images (white, right), generated using mitochondrial analyzer, time points as indicated, representative of six individual patients. ( G ) Quantification of mitochondrial count per cell, n = 29 cells from three individual patients. ( H ) Quantification of mitochondrial length (left) and mean mitochondrial area using mitochondrial analyzer (right), n = 180/170 mitochondria from 14/22 cells from three individual patients (length) and n = 50 cells from three individual patients (area). ( I ) Representative projection of 3D image stacks (two per time point) of translocated mitochondria to the IS in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, and quantification of mitochondrial translocation to the CTL IS, time points as indicated, n = 14 from 5 individual patients. If not stated otherwise, data were represented as mean ± SEM. P values as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test ( A – E ), and unpaired t -test or Mann–Whitney test ( G – I ), as appropriate. .

Figure Legend Snippet: ( A ) FCA of MitoTracker green and quantification in isolated mitochondria from CTL, n = 7 individual patients. ( B ) FCA of JC1 green and quantification of red/green fluorescence in isolated mitochondria from CTL, n = 4 individual patients. ( C ) Immunoblot of OXPHOS complexes using isolated mitochondria of CTL and normalized densitometric quantification of complex V, n = 5 individual patients. I–V complex I–V; H heat shock protein 60 (HSP60). Subunits detected by the antibody cocktail: complex I: subunit NDUFB8; complex II: subunit 30 kDa (SDHB); complex III: subunit Core 2 (UQCRC2); complex IV: subunit II (COXII); complex V: ATP synthase subunit alpha (ATP5). ( D , E ) Representative immunoblot of mitochondrial fusion and fission proteins of isolated mitochondria of CTL ( D ) and normalized densitometric quantification ( E ) of the respective proteins, n = 5/4/3 individual patients. ( F ) Representative projections of 3D image stacks of CTL stained with MitoTracker (MT, green, left) and the respective skeleton images (white, right), generated using mitochondrial analyzer, time points as indicated, representative of six individual patients. ( G ) Quantification of mitochondrial count per cell, n = 29 cells from three individual patients. ( H ) Quantification of mitochondrial length (left) and mean mitochondrial area using mitochondrial analyzer (right), n = 180/170 mitochondria from 14/22 cells from three individual patients (length) and n = 50 cells from three individual patients (area). ( I ) Representative projection of 3D image stacks (two per time point) of translocated mitochondria to the IS in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, and quantification of mitochondrial translocation to the CTL IS, time points as indicated, n = 14 from 5 individual patients. If not stated otherwise, data were represented as mean ± SEM. P values as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test ( A – E ), and unpaired t -test or Mann–Whitney test ( G – I ), as appropriate. .

Techniques Used: Isolation, Fluorescence, Western Blot, Staining, Generated, Translocation Assay, MANN-WHITNEY

( A ) FCA of MitoTracker green and quantification in CD8 + CTL, n = 7 individual patients. ( B ) Representative Seahorse OCR plot. ( C ) Fold change of basal OCR, maximum OCR, spare respiratory capacity (SRC) and ATP production, n = 6 individual patients. ( D ) Quantification of mitochondrial length, n = 92 mitochondria from 28 cells from three individual patients. ( E ) Quantification of mitochondrial count per cell, n = 30/26 cells from three individual patients. ( F ) Representative confocal microscopy image of mitochondria translocated to the TCR in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, T2 image as depicted in Fig. , upper right image. ( G ) Quantification of mitochondrial translocation in proximity to the CTL IS, n = 12 from four individual patients. ( H ) Representative impedance plot and ( I ) quantification of relative CTL cytotoxicity using ECIS, n = 6 individual patients. If not stated otherwise, data are represented as mean ± SEM. P values as indicated, one-sample t -test ( C ), paired t -test or Wilcoxon matched-pairs signed-rank test ( A – C, H , I ), and unpaired t -test or Mann–Whitney test ( D – G ), as appropriate. .

Figure Legend Snippet: ( A ) FCA of MitoTracker green and quantification in CD8 + CTL, n = 7 individual patients. ( B ) Representative Seahorse OCR plot. ( C ) Fold change of basal OCR, maximum OCR, spare respiratory capacity (SRC) and ATP production, n = 6 individual patients. ( D ) Quantification of mitochondrial length, n = 92 mitochondria from 28 cells from three individual patients. ( E ) Quantification of mitochondrial count per cell, n = 30/26 cells from three individual patients. ( F ) Representative confocal microscopy image of mitochondria translocated to the TCR in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, T2 image as depicted in Fig. , upper right image. ( G ) Quantification of mitochondrial translocation in proximity to the CTL IS, n = 12 from four individual patients. ( H ) Representative impedance plot and ( I ) quantification of relative CTL cytotoxicity using ECIS, n = 6 individual patients. If not stated otherwise, data are represented as mean ± SEM. P values as indicated, one-sample t -test ( C ), paired t -test or Wilcoxon matched-pairs signed-rank test ( A – C, H , I ), and unpaired t -test or Mann–Whitney test ( D – G ), as appropriate. .

Techniques Used: Confocal Microscopy, Staining, Translocation Assay, MANN-WHITNEY

Image Search Results

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) Flow cytometric analysis (FCA) of CD25 (left) and CD69 (right) staining and quantification in CTL, n = 6/11 individual patients. ( B ) FCA of lymphocyte-activation gene 3 (LAG3), programmed cell death protein 1 (PD1), and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) staining and quantification as indicated, n = 6/4 (not activated/+CD3/CD28) individual patients. P values (left to right): 0.2842, 0.2188, 0.6875, 0.5494, 0.1250, and >0.9999. ( C ) Quantification of relative cytotoxicity using a flow cytometric killing assay, n = 5 individual patients. ( D ) Quantification of caspase-3 activity reporter using a CTL-K562 killing assay, n = 4 individual patients. Data were represented as mean ± SEM with dots indicating individual values ( A , B ) or as individual data points ( C , D ). P values: as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test, as appropriate.

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Staining, Activation Assay, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in isolated mitochondria from CTL, n = 7 individual patients. ( B ) FCA of JC1 green and quantification of red/green fluorescence in isolated mitochondria from CTL, n = 4 individual patients. ( C ) Immunoblot of OXPHOS complexes using isolated mitochondria of CTL and normalized densitometric quantification of complex V, n = 5 individual patients. I–V complex I–V; H heat shock protein 60 (HSP60). Subunits detected by the antibody cocktail: complex I: subunit NDUFB8; complex II: subunit 30 kDa (SDHB); complex III: subunit Core 2 (UQCRC2); complex IV: subunit II (COXII); complex V: ATP synthase subunit alpha (ATP5). ( D , E ) Representative immunoblot of mitochondrial fusion and fission proteins of isolated mitochondria of CTL ( D ) and normalized densitometric quantification ( E ) of the respective proteins, n = 5/4/3 individual patients. ( F ) Representative projections of 3D image stacks of CTL stained with MitoTracker (MT, green, left) and the respective skeleton images (white, right), generated using mitochondrial analyzer, time points as indicated, representative of six individual patients. ( G ) Quantification of mitochondrial count per cell, n = 29 cells from three individual patients. ( H ) Quantification of mitochondrial length (left) and mean mitochondrial area using mitochondrial analyzer (right), n = 180/170 mitochondria from 14/22 cells from three individual patients (length) and n = 50 cells from three individual patients (area). ( I ) Representative projection of 3D image stacks (two per time point) of translocated mitochondria to the IS in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, and quantification of mitochondrial translocation to the CTL IS, time points as indicated, n = 14 from 5 individual patients. If not stated otherwise, data were represented as mean ± SEM. P values as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test ( A – E ), and unpaired t -test or Mann–Whitney test ( G – I ), as appropriate. .

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Isolation, Fluorescence, Western Blot, Staining, Generated, Translocation Assay, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in CD8 + CTL, n = 7 individual patients. ( B ) Representative Seahorse OCR plot. ( C ) Fold change of basal OCR, maximum OCR, spare respiratory capacity (SRC) and ATP production, n = 6 individual patients. ( D ) Quantification of mitochondrial length, n = 92 mitochondria from 28 cells from three individual patients. ( E ) Quantification of mitochondrial count per cell, n = 30/26 cells from three individual patients. ( F ) Representative confocal microscopy image of mitochondria translocated to the TCR in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, T2 image as depicted in Fig. , upper right image. ( G ) Quantification of mitochondrial translocation in proximity to the CTL IS, n = 12 from four individual patients. ( H ) Representative impedance plot and ( I ) quantification of relative CTL cytotoxicity using ECIS, n = 6 individual patients. If not stated otherwise, data are represented as mean ± SEM. P values as indicated, one-sample t -test ( C ), paired t -test or Wilcoxon matched-pairs signed-rank test ( A – C, H , I ), and unpaired t -test or Mann–Whitney test ( D – G ), as appropriate. .

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Confocal Microscopy, Staining, Translocation Assay, MANN-WHITNEY

CAR-T cell cytotoxicity in short term cultures is comparable between NFC, Matrigel, and BME CD20 CAR-T cells were co-cultured with Daudi (Burkitt lymphoma cell line) cells labeled with CellTrace Violet (CTV) for 24 h at the different effector to target (E:T) ratios specified, in standard 2D suspension (control) or embedded in the specified hydrogels. (A) Representative gating strategy for analyzing the viability of Daudi and CD20 CAR-T cells by flow cytometry. (B) Number of viable CAR-T cells recovered after culture in the 1:1 E:T condition (50,000 Daudi +50,000 CAR-T cells/well) as measured by flow cytometry using counting beads (Flow-Count fluorospheres). Data were analyzed using a one-way ANOVA with Tukey’s multiple comparison test. (C) Specific lysis (%) of Daudi cells induced by CD20 CAR-T cells at the specified E:T ratios. Specific apoptosis was calculated by applying the following formula: [(%viable untreated − %viable treated)/%viable untreated] × 100. (D and E) Bar plot showing median fluorescence intensity (MFI) of (D) IFNγ and (E) CD69 in CAR-T cells cultured for 24 h in the described conditions. Data from two or three independent experiments are presented as means ± SD. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparison test (∗ p < 0.05, ∗∗ p < 0.005). NFC, nanofibrillar cellulose; MG, Matrigel; BME, basement membrane extract.

Journal: iScience

Article Title: Impact of 3D cell culture hydrogels derived from basement membrane extracts or nanofibrillar cellulose on CAR-T cell activation

doi: 10.1016/j.isci.2025.113234

Figure Lengend Snippet: CAR-T cell cytotoxicity in short term cultures is comparable between NFC, Matrigel, and BME CD20 CAR-T cells were co-cultured with Daudi (Burkitt lymphoma cell line) cells labeled with CellTrace Violet (CTV) for 24 h at the different effector to target (E:T) ratios specified, in standard 2D suspension (control) or embedded in the specified hydrogels. (A) Representative gating strategy for analyzing the viability of Daudi and CD20 CAR-T cells by flow cytometry. (B) Number of viable CAR-T cells recovered after culture in the 1:1 E:T condition (50,000 Daudi +50,000 CAR-T cells/well) as measured by flow cytometry using counting beads (Flow-Count fluorospheres). Data were analyzed using a one-way ANOVA with Tukey’s multiple comparison test. (C) Specific lysis (%) of Daudi cells induced by CD20 CAR-T cells at the specified E:T ratios. Specific apoptosis was calculated by applying the following formula: [(%viable untreated − %viable treated)/%viable untreated] × 100. (D and E) Bar plot showing median fluorescence intensity (MFI) of (D) IFNγ and (E) CD69 in CAR-T cells cultured for 24 h in the described conditions. Data from two or three independent experiments are presented as means ± SD. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparison test (∗ p < 0.05, ∗∗ p < 0.005). NFC, nanofibrillar cellulose; MG, Matrigel; BME, basement membrane extract.

Article Snippet: Anti-Human CD69 FITC , Miltenyi , Cat# 130-113-523; RRID: AB_2733656.

Techniques: Cell Culture, Labeling, Suspension, Control, Flow Cytometry, Comparison, Lysis, Fluorescence, Membrane

A549 lung metastases was developed in 6–8 wk post-humanized mice and lung met-bearing humanized mice were then treated with NPRL2 , pembrolizumab, and its combination. 3-to 5 d after the treatment, lung metastases tissues were analyzed for infiltrating human immune cells. Single-cell suspensions were prepared from fresh lung metastases and in-depth immune analyses were performed using multicolor flow cytometry for determining human (A) CD45 +leukocytes, ( B ) CD3 + T, ( C ) CD8 + T, ( D ) regulatory T cells, and ( E ) natural killer (NK), cells. The level of human immune cells is shown for different treatment groups. ( F ) The level of activating CD8 + T cells was determined by the expression of CD69 expressing markers on infiltrating T cells among different treatment groups. ( G ) Percentage of PD1 expressing CD8 + T cells and its alterations after treatment are shown. ( H–I ) NPRL2 mediated alteration on the level of effector memory and central memory of CD8 + T cells in the tumor microenvironment. ( J ) Percentages of tissue-resident T cells (T RM ) in tumors and their alteration by NPRL2 treatment. CD103 + expressing T cells were considered as T RM . ( K ) The effect of NPRL2 treatment on the myeloid populations was also investigated. The level of HLA-DR + DC cells was determined among the lineage-negative population. ( L ) Level of human myeloid-derived suppressor cells (MDSC) based on the expression of CD33 + HLA-DR-ve population and the changes of MDSC in different treatment groups shown in lung met and (M) the percentage of M1 macrophages and its alteration upon NPRL2 treatment in lung metastases. These populations were gated as Lin-ve >CD11b+ve > HLA-DR+ve > CD163-ve. Statistics are shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5.p<0.05; **p<0.005; ***p<0.0005. Figure 3—source data 1. NPRL2 induces antitumor immune responses in anti-PD1 resistant KRAS/LKB1 mutant A549-Lung Met in humanized mice: Tumor microenvironment analysis in humanized mice.

Journal: eLife

Article Title: NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

doi: 10.7554/eLife.98258

Figure Lengend Snippet: A549 lung metastases was developed in 6–8 wk post-humanized mice and lung met-bearing humanized mice were then treated with NPRL2 , pembrolizumab, and its combination. 3-to 5 d after the treatment, lung metastases tissues were analyzed for infiltrating human immune cells. Single-cell suspensions were prepared from fresh lung metastases and in-depth immune analyses were performed using multicolor flow cytometry for determining human (A) CD45 +leukocytes, ( B ) CD3 + T, ( C ) CD8 + T, ( D ) regulatory T cells, and ( E ) natural killer (NK), cells. The level of human immune cells is shown for different treatment groups. ( F ) The level of activating CD8 + T cells was determined by the expression of CD69 expressing markers on infiltrating T cells among different treatment groups. ( G ) Percentage of PD1 expressing CD8 + T cells and its alterations after treatment are shown. ( H–I ) NPRL2 mediated alteration on the level of effector memory and central memory of CD8 + T cells in the tumor microenvironment. ( J ) Percentages of tissue-resident T cells (T RM ) in tumors and their alteration by NPRL2 treatment. CD103 + expressing T cells were considered as T RM . ( K ) The effect of NPRL2 treatment on the myeloid populations was also investigated. The level of HLA-DR + DC cells was determined among the lineage-negative population. ( L ) Level of human myeloid-derived suppressor cells (MDSC) based on the expression of CD33 + HLA-DR-ve population and the changes of MDSC in different treatment groups shown in lung met and (M) the percentage of M1 macrophages and its alteration upon NPRL2 treatment in lung metastases. These populations were gated as Lin-ve >CD11b+ve > HLA-DR+ve > CD163-ve. Statistics are shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5.p<0.05; **p<0.005; ***p<0.0005. Figure 3—source data 1. NPRL2 induces antitumor immune responses in anti-PD1 resistant KRAS/LKB1 mutant A549-Lung Met in humanized mice: Tumor microenvironment analysis in humanized mice.

Article Snippet: Fluorochrome–conjugated monoclonal antibodies to the following human antigens were used: CD45-Alexa Fluor 700 (clone 2D1, HI30), CD45-phycoerythrin (PE; clone 2D1, HI30), CD3-PerCp/cy5.5 (clone HIT3a), CD19-PE-cyanine 7 (clone HIB19), CD8-allophycocyanin-cyanine 7 (clone RPA-T8, HIT8a), CD4-Pacific blue (clone OKT4), CD56-PE/BV510 (clone HCD56), CD69-FITC/APC/PE-Alexa Flour 610 (clone FN50; Thermo fisher), HLA-DR-PerCp/cy5.5 (clone LN3), CD33-PE (clone WM-53) (Thermo Fisher), CD11b-PE-Cy7 (clone 1CRF-44) (Thermo Fisher), Granzyme B-FITC (clone GB11), and IFN-γ-APC (clone 4 S.B3), CD103-Super bright 600 (Colne B-LY7; Thermo fisher), CD279 (PD-1)-Super Bright 702 (Clone J105; Thermo Fisher), CCR7-FITC (Clone G043H7), CD45RA-PE (Clone HI100), CD25-APC (clone CD25-4E3), Lin-FITC (Biolegend), CD163-APC (clone ebioGH1/61; Thermo fisher), CD11c-Pacific blue (clone Bu15; Thermo Fisher).

Techniques: Flow Cytometry, Expressing, Derivative Assay, Mutagenesis

( A ) Experimental strategy of H1299 subcutaneous tumor development in humanized mice. NSG mice were humanized for 7–8 wk and humanization was verified by blood screening followed by tumor cell injections. Subcutaneous tumors were developed for another 6 wk to obtain a tumor size of about 200 mm 3 . Tumors were treated with NPRL2 (i.v) and pembrolizumab (i.p.) treatment for 2 wk. ( B ) Humanization status was checked by detecting the human CD45 cells in the blood before tumor cell implantation. ( C ) At the end of the experiment, the humanization level was checked based on the number of human CD45 cells in the mouse. ( D ) H1299 tumors were treated with NPRL2 and pembrolizumab and tumor volume was measured twice a week. The tumor growth curve was generated based on tumor volumes and the antitumor effect was evaluated. ( E ) Growth curves showed the individual mouse response to treatment. ( F–K ) tumor microenvironment analysis was performed to evaluate the immune cell infiltration into tumors. Fresh tumors were harvested within 3–5 d after treatment and single-cell suspensions were prepared for in-depth immune analyses by using multicolor flow cytometry for determining the level of human (F) cytotoxic CD8 + T, ( G ) effector natural killer (NK) cells, and (H) antigen-presenting HLA-DR + DC. ( I ) Level of activating CD8 + T cells was determined by the expression of CD69 expressing markers on infiltrating T cells among different treatment groups. ( J ) Percentage of PD1 expressing CD8 + T cells and its alterations after treatment are shown. ( K ) Percentages of tissue-resident T cells (T RM ) in tumors and their alteration by NPRL2 treatment. CD103 +expressing T cells were considered as T RM . In-vivo experiment was repeated three times with at least 5 mice/group used in each experiment. Statistics are shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5. *p<0.05; **p<0.005; ***p<0.0005. Figure 4—source data 1. NPRL2 induced synergistic antitumor immune response with pembrolizumab on anti-PD1 responsive H1299 tumors in humanized mice: NPRL2 antitumor effect on H1299 tumors and tumor microenvironment analysis.

Journal: eLife

Article Title: NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

doi: 10.7554/eLife.98258

Figure Lengend Snippet: ( A ) Experimental strategy of H1299 subcutaneous tumor development in humanized mice. NSG mice were humanized for 7–8 wk and humanization was verified by blood screening followed by tumor cell injections. Subcutaneous tumors were developed for another 6 wk to obtain a tumor size of about 200 mm 3 . Tumors were treated with NPRL2 (i.v) and pembrolizumab (i.p.) treatment for 2 wk. ( B ) Humanization status was checked by detecting the human CD45 cells in the blood before tumor cell implantation. ( C ) At the end of the experiment, the humanization level was checked based on the number of human CD45 cells in the mouse. ( D ) H1299 tumors were treated with NPRL2 and pembrolizumab and tumor volume was measured twice a week. The tumor growth curve was generated based on tumor volumes and the antitumor effect was evaluated. ( E ) Growth curves showed the individual mouse response to treatment. ( F–K ) tumor microenvironment analysis was performed to evaluate the immune cell infiltration into tumors. Fresh tumors were harvested within 3–5 d after treatment and single-cell suspensions were prepared for in-depth immune analyses by using multicolor flow cytometry for determining the level of human (F) cytotoxic CD8 + T, ( G ) effector natural killer (NK) cells, and (H) antigen-presenting HLA-DR + DC. ( I ) Level of activating CD8 + T cells was determined by the expression of CD69 expressing markers on infiltrating T cells among different treatment groups. ( J ) Percentage of PD1 expressing CD8 + T cells and its alterations after treatment are shown. ( K ) Percentages of tissue-resident T cells (T RM ) in tumors and their alteration by NPRL2 treatment. CD103 +expressing T cells were considered as T RM . In-vivo experiment was repeated three times with at least 5 mice/group used in each experiment. Statistics are shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5. *p<0.05; **p<0.005; ***p<0.0005. Figure 4—source data 1. NPRL2 induced synergistic antitumor immune response with pembrolizumab on anti-PD1 responsive H1299 tumors in humanized mice: NPRL2 antitumor effect on H1299 tumors and tumor microenvironment analysis.

Techniques: Generated, Flow Cytometry, Expressing, In Vivo

NPRL2 stable clones in A549 and H1299 NSCLC cells were generated and developed tumors in humanized mice for tumor microenvironment analysis. The in-vitro assays were performed using these stable clones to elucidate cell death and molecular signaling. ( A ) Scheme showing the experimental strategy where NSG mice were humanized for 6–8 wk followed by H1299- NPRL2 ++/++ tumor cell implantation to develop the tumors for TME analysis. ( B ) Humanization status was checked before tumor implantation based on the level of human CD45 cells. Mice containing 25% or more human CD45 + cells were considered as humanized mice and only the verified mice were used for the tumor implantation. The status of CD3 + T, NK, and B cells was evaluated prior to tumor implantation. ( C ) Tumor growth curves showed the rate of tumor growth and differences in tumor growth between parental H1299 tumors and H1299- NPRL2 ++/++ tumors in humanized mice. ( D–N ) At day 43, fresh tumors were harvested, single cells were prepared, and multicolor flow cytometry was run for multiple innate and adaptive panels to identify the human immune cell populations in the tumor microenvironment. ( D–H ) The percentage of major human immune cells including (D) human CD45 + , ( E ) human CD19 + B, ( F ) human CD3 + T, ( G ) CD4 + T, and CD8 + T, and ( H ) natural killer (NK) cells in parental tumors and their alterations in NPRL2 -stably expressing tumors in humanized mice. ( I–K ) The status of ( I ) regulatory T cells, ( J ) PD1 expressing T cells (CD274 +CD8 + T), and ( K ) activating T cells (CD69 +CD8 + T) in both parental and NPRL2 -stably expressing tumors. ( L–N ) The presence of innate cells was also investigated and analyzed after gating out the lineage-positive population to identify the level of ( L ) MDSC (CD33 +HLA-DR- MDSC), ( M ) TAM (CD11b+HLA-DR-CD163+TAM), and (N) HLA-DR +DC in tumors in humanized mice. ( O–S ) A549- NPRL2 ++/++ and H1299- NPRL2 ++/++ cells were used for in-vitro assays compared with their respective parental counterparts. ( O ) Stable expression level of NPRL2 was verified by western blot, ( P ) Colony forming assay in H1299 and H1299- NPRL2 ++/++ cells showed the differences in colony formation inhibition in the absence or presence of different concentrations of carboplatin, ( Q ) Apoptosis assay was also performed in both pairs of cell lines to detect the annexin V positive apoptotic cells by flow cytometry following carboplatin treatment. The difference in the level of apoptosis was estimated and compared between parental and NPRL2 stably expressing counterparts. ( R ) The level of apoptosis was verified by PARP cleavage by detecting the cleavage of PARP by western blots in NRPL2 stable cells after carboplatin treatment. ( S ) Western blots were performed to detect a list of signaling molecules involved in downstream and upstream of the PI3K/AKT/mTOR signaling pathway, which included p-AKT, p-mTOR, p-S6, p-4E-BP, p-PRAS40, p-GSK-3b. The MAPK pathway was investigated based on the level of expression of pERK1/2. The in-vivo experiment was repeated three times with at least N=5 mice/group used in each experiment. Statistics were shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5. *p<0.05; **p<0.005; ***p<0.0005. Figure 8—source data 1. Restoration of NPRL2 expression altered tumor microenvironment (TME), induced apoptosis, inhibited cell growth and signaling: Tumor microenvironment analysis in NPRL2 stable expressing tumors. Figure 8—source data 2. PDF file containing original western blots for , indicating relevant bands, treatments and samples. Figure 8—source data 3. Original files for western blot images displayed in .

Journal: eLife

Article Title: NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

doi: 10.7554/eLife.98258

Figure Lengend Snippet: NPRL2 stable clones in A549 and H1299 NSCLC cells were generated and developed tumors in humanized mice for tumor microenvironment analysis. The in-vitro assays were performed using these stable clones to elucidate cell death and molecular signaling. ( A ) Scheme showing the experimental strategy where NSG mice were humanized for 6–8 wk followed by H1299- NPRL2 ++/++ tumor cell implantation to develop the tumors for TME analysis. ( B ) Humanization status was checked before tumor implantation based on the level of human CD45 cells. Mice containing 25% or more human CD45 + cells were considered as humanized mice and only the verified mice were used for the tumor implantation. The status of CD3 + T, NK, and B cells was evaluated prior to tumor implantation. ( C ) Tumor growth curves showed the rate of tumor growth and differences in tumor growth between parental H1299 tumors and H1299- NPRL2 ++/++ tumors in humanized mice. ( D–N ) At day 43, fresh tumors were harvested, single cells were prepared, and multicolor flow cytometry was run for multiple innate and adaptive panels to identify the human immune cell populations in the tumor microenvironment. ( D–H ) The percentage of major human immune cells including (D) human CD45 + , ( E ) human CD19 + B, ( F ) human CD3 + T, ( G ) CD4 + T, and CD8 + T, and ( H ) natural killer (NK) cells in parental tumors and their alterations in NPRL2 -stably expressing tumors in humanized mice. ( I–K ) The status of ( I ) regulatory T cells, ( J ) PD1 expressing T cells (CD274 +CD8 + T), and ( K ) activating T cells (CD69 +CD8 + T) in both parental and NPRL2 -stably expressing tumors. ( L–N ) The presence of innate cells was also investigated and analyzed after gating out the lineage-positive population to identify the level of ( L ) MDSC (CD33 +HLA-DR- MDSC), ( M ) TAM (CD11b+HLA-DR-CD163+TAM), and (N) HLA-DR +DC in tumors in humanized mice. ( O–S ) A549- NPRL2 ++/++ and H1299- NPRL2 ++/++ cells were used for in-vitro assays compared with their respective parental counterparts. ( O ) Stable expression level of NPRL2 was verified by western blot, ( P ) Colony forming assay in H1299 and H1299- NPRL2 ++/++ cells showed the differences in colony formation inhibition in the absence or presence of different concentrations of carboplatin, ( Q ) Apoptosis assay was also performed in both pairs of cell lines to detect the annexin V positive apoptotic cells by flow cytometry following carboplatin treatment. The difference in the level of apoptosis was estimated and compared between parental and NPRL2 stably expressing counterparts. ( R ) The level of apoptosis was verified by PARP cleavage by detecting the cleavage of PARP by western blots in NRPL2 stable cells after carboplatin treatment. ( S ) Western blots were performed to detect a list of signaling molecules involved in downstream and upstream of the PI3K/AKT/mTOR signaling pathway, which included p-AKT, p-mTOR, p-S6, p-4E-BP, p-PRAS40, p-GSK-3b. The MAPK pathway was investigated based on the level of expression of pERK1/2. The in-vivo experiment was repeated three times with at least N=5 mice/group used in each experiment. Statistics were shown at a significance level of p<0.05 unless otherwise noted. Data is shown as mean percentage ± SD, n=5. *p<0.05; **p<0.005; ***p<0.0005. Figure 8—source data 1. Restoration of NPRL2 expression altered tumor microenvironment (TME), induced apoptosis, inhibited cell growth and signaling: Tumor microenvironment analysis in NPRL2 stable expressing tumors. Figure 8—source data 2. PDF file containing original western blots for , indicating relevant bands, treatments and samples. Figure 8—source data 3. Original files for western blot images displayed in .

Techniques: Clone Assay, Generated, In Vitro, Tumor Implantation, Flow Cytometry, Stable Transfection, Expressing, Western Blot, Inhibition, Apoptosis Assay, In Vivo

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