cd30 elisa  (R&D Systems)

Journal: Clinical & Translational Immunology

Article Title: Memory stem T cells modified with a redesigned CD30‐chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma

doi: 10.1002/cti2.1268

Figure Lengend Snippet: T SCM‐like are highly enriched, CD30‐CAR‐transduced and expanded in vitro despite CD30 expression. Naïve T cells from healthy donors ( n = 7) were cultured with CD3/CD28 costimulation in the presence of IL‐7, IL‐15 and IL‐21 during 10 days. (a) Frequencies of CD4 + and CD8 + T‐cell subpopulations at the end of culture. T SCM‐like cells were the most prevalent T‐cell population (mean ± SD). (b) Representative plot of T cells transduced at day 2 with CD30‐CAR‐encoding lentivirus and CD30‐CAR expression and analysed by flow cytometry using an anti‐CD3 and anti‐EGFRt antibodies. (c) Expression of CD30 receptor on CD4 + and CD8 + T‐cell subpopulations in untransduced cells (white bars) and transduced cells (black bars) during the culture (days 4, 7 and 10; mean ± SD). (d) Percentage of T‐cell viability in untransduced cells (grey line) and transduced cells (black line) at days 4, 7 and 10 of culture (mean ± SD). (e) Fold expansion of CD4 + and CD8 + T cells during 10‐day culture in untransduced cells (grey line) and transduced cells (black line; mean ± SD). Data are representative of seven independently repeated experiments.

Article Snippet: Soluble CD30 protein was measured in supernatants of L540 and L428 cell cultures by enzyme‐linked immunoabsorbent assay (Human CD30 PicoKine ELISA Kit; Boster Biological Technology, Pleasanton, California, USA).

Techniques: In Vitro, Expressing, Cell Culture, Flow Cytometry

Journal: Clinical & Translational Immunology

Article Title: Memory stem T cells modified with a redesigned CD30‐chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma

doi: 10.1002/cti2.1268

Figure Lengend Snippet: CD30‐CAR T SCM‐like enriched culture efficiently eradicates HL in vitro . (a) T SCM‐like enriched cells expressing CD30‐CAR ( n = 7) were exposed to CD30 + target cells (L540 or L428 tumor cells) and control CD30 − cell line (Raji) at different effector:target (E:T) ratios. Specific cytolytic activity was measured at 24 h by bioluminescence assay. Untransduced T SCM‐like cells were used as negative control (mean ± SD). (b) Cytolytic capacity of untransduced and CD30‐CAR T SCM‐like cells ( n = 3) was measured against CD30 + target cells (L540 and L428) at 24 h, in the presence or absence of saturated concentration of soluble CD30 protein (sCD30; 20 μg), at different E:T ratios. Raji cell line (CD30 − ) was used as negative target control (mean ± SD). (c) Cytokine secretion of CD30‐CAR T SCM‐like enriched culture ( n = 3) 24 h after L540, L428 and Raji co‐culture. Untransduced T SCM‐like cells were used as negative control. Cytokine levels of IL‐2, IL‐6, IL‐10, IFN‐γ and TNF‐α were measured by cytometry‐based multiplex analysis (mean ± SD). Data are representative of seven (a) or three (b, c) independently repeated experiments. For each donor, technical duplicate was used (c) .

Article Snippet: Soluble CD30 protein was measured in supernatants of L540 and L428 cell cultures by enzyme‐linked immunoabsorbent assay (Human CD30 PicoKine ELISA Kit; Boster Biological Technology, Pleasanton, California, USA).

Techniques: In Vitro, Expressing, Control, Activity Assay, ATP Bioluminescent Assay, Negative Control, Concentration Assay, Co-Culture Assay, Cytometry, Multiplex Assay

Journal: Clinical & Translational Immunology

Article Title: Memory stem T cells modified with a redesigned CD30‐chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma

doi: 10.1002/cti2.1268

Figure Lengend Snippet: CD30‐CAR T SCM‐like function persists after CD30 + antigen re‐exposition. (a) Representative graphic of untransduced and CD30‐CAR T SCM‐like enriched cells exposed to CD30 + target cells (L540) at different effector:target (E:T) ratios every 72 h after each antigen re‐exposition (total stimulations: 3). Specific cytolytic activity was measured after 24 h of each antigen stimulation by bioluminescence. (b) Mean fluorescence intensity (MFI) of CD30‐CAR expression and (c) percentage of PD‐1‐TIM‐3 + T cells during antigen re‐exposures (mean ± SD). * P < 0.05; ** P < 0.01. Data are representative of three independently repeated experiments.

Article Snippet: Soluble CD30 protein was measured in supernatants of L540 and L428 cell cultures by enzyme‐linked immunoabsorbent assay (Human CD30 PicoKine ELISA Kit; Boster Biological Technology, Pleasanton, California, USA).

Techniques: Activity Assay, Fluorescence, Expressing

Journal: Clinical & Translational Immunology

Article Title: Memory stem T cells modified with a redesigned CD30‐chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma

doi: 10.1002/cti2.1268

Figure Lengend Snippet: Therapeutic treatment with CD30‐CAR T SCM‐like enriched cell products induces a potent antitumor response against HL in vivo . NSG mice ( n = 4 for both experimental and control groups) were injected with (a) 2.5 × 10 6 L540 tumor cells (sc) on day 0 and treated 3 days later with 5 or 10 × 10 6 CD30‐CAR T SCM‐like enriched cells (iv), or (b) 2 × 10 6 L428 tumor cells (iv) on day 0 and treated with 5 or 10 × 10 6 CD30‐CAR T SCM‐like enriched cells (iv) 22 days after tumor challenge. In both models, control mice received 10 × 10 6 untransduced (UN) T SCM‐like enriched cells. Mice were monitored every other day for survival, and tumor growth was measured by in vivo bioluminescence. (c) Expression of exhaustion markers (PD1 and TIM3) in CD4 + and CD8 + tumor‐infiltrating T cells found in L540‐bearing mice treated with 5 × 10 6 CD30‐CAR T SCM‐like enriched cells, 48 days after tumor challenge, compared with CD4 + and CD8 + pre‐infused T cells (mean ± SD). * P < 0.05; ** P < 0.01. Data are representative of two independent experiments.

Article Snippet: Soluble CD30 protein was measured in supernatants of L540 and L428 cell cultures by enzyme‐linked immunoabsorbent assay (Human CD30 PicoKine ELISA Kit; Boster Biological Technology, Pleasanton, California, USA).

Techniques: In Vivo, Control, Injection, Expressing

Journal: Clinical & Translational Immunology

Article Title: Memory stem T cells modified with a redesigned CD30‐chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma

doi: 10.1002/cti2.1268

Figure Lengend Snippet: T SCM‐like enriched cells expressing CD30‐CAR have high persistence in vivo and confer long‐lasting immunity against HL. (a) NSG mice surviving from L428 tumor administration ( n = 4) were rechallenged again with the same tumor dose (2 × 10 6 cells per mouse, iv), 79 days (arrow) after the first tumor challenge (grey discontinued line). An age‐matched mouse group ( n = 4) was injected with 2 × 10 6 L428 tumor cells (iv) as tumor control (pointed line). Black line represents control mice receiving 10 × 10 6 untransduced (UN) T SCM‐like enriched cells. Mice were followed every other day for survival, and tumor growth control was done by in vivo bioluminescence. (b) Bone marrow and lymph nodes from surviving CD30‐CAR‐treated mice were analysed for CAR T cell by flow cytometry (mean ± SD). (c) T‐cell subpopulations were analysed in bone marrow and lymph nodes by flow cytometry (mean ± SD). (d) Expression of exhaustion markers (PD1, TIM3 and LAG‐3) was analysed by flow cytometry in T cells found in lymph nodes and bone marrow (mean ± SD). Data are representative of two independent experiments.

Article Snippet: Soluble CD30 protein was measured in supernatants of L540 and L428 cell cultures by enzyme‐linked immunoabsorbent assay (Human CD30 PicoKine ELISA Kit; Boster Biological Technology, Pleasanton, California, USA).

Techniques: Expressing, In Vivo, Injection, Control, Flow Cytometry

Journal: Clinical & Translational Immunology

Article Title: Memory stem T cells modified with a redesigned CD30‐chimeric antigen receptor show an enhanced antitumor effect in Hodgkin lymphoma

doi: 10.1002/cti2.1268

Figure Lengend Snippet: CD30‐CAR T SCM‐like H products have higher antitumor efficacy and T‐cell persistence than CD30‐CAR T SCM‐like L products. (a) Composition of T SCM‐like H and T SCM‐like L cultures at the day of treatment (mean ± SEM). (b, c) NSG mice ( N = 4/group) were challenged with 2.5 × 10 6 L540 tumor cells (sc) on day 0 and treated when the tumor was well established (day 5) with 5 × 10 6 CD30‐CAR T SCM‐like H (pointed line and ▲) or T SCM‐like L (discontinued line and ■) cells (iv). A group of mice were injected with 2.5 × 10 6 L540 tumor cells (sc) as tumor control (black line and ●). Mice were monitored every other day for (B) tumor growth and (c) survival, measured by in vivo bioluminescence (mean ± SD). (d) Percentage of CD30‐CAR expression in tumor‐infiltrating T cells was analysed in T SCM‐like H and T SCM‐like L treated mice by flow cytometry (e) CD30‐CAR T‐cell detection in blood from mice treated with T SCM‐like H or T SCM‐like L cultures. (mean ± SD). * P < 0.05; ** P < 0.01; *** P < 0.001. Data are representative of two independent experiments.

Article Snippet: Soluble CD30 protein was measured in supernatants of L540 and L428 cell cultures by enzyme‐linked immunoabsorbent assay (Human CD30 PicoKine ELISA Kit; Boster Biological Technology, Pleasanton, California, USA).

Techniques: Injection, Control, In Vivo, Expressing, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice

doi: 10.1371/journal.ppat.1008477

Figure Lengend Snippet: A) RNA expression profiling of the human B cell transcriptome of mice from three independent experiments with EBV − FK506 – (n = 5, clear), EBV − FK506 + (n = 6, orange), EBV + FK506 – (n = 7, purple) and EBV + FK506 + (n = 8, magenta). Principal component plot: Sample similarity in a 2D projection by multi-dimensional scaling. Spurious batch effects of unknown origin and HFL donor-specific effects were corrected by surrogate variable analysis. The two principal components represent 60% of the total variance in the data set. B) Volcano plots: The horizontal line corresponds to FDR = 0.01 and genes below this line are labeled ‘not significant’. The two vertical lines correspond to a 2-fold change in expression and genes outside this range are labelled ‘strong’. The legend refers to ‘not’—not significant and weak effect; ‘signif & strong’—significant and strong effect; ‘signif’—significant but weak effect; ‘strong’—not significant but strong effect. C) Normalized AmpliSeq expression levels of CXCL10 , CD28 , IL6 and TNFRSF8 (CD30) are depicted for individual mice with mean ± SEM and FDR-corrected p-value summaries (*: FDR-p<0.05, **: FDR-p<0.01, ***: FDR-p<0.001.) D) Upset plot identifies private, shared and promiscuous DEGs among groups. The vertical bar chart indicates the intersection size of transcripts shared between certain groups defined by solid points below the chart. The total number of DEGs for each condition is depicted in the horizontal bar chart. See also – Figs and and Tables.

Article Snippet: Commercially available ELISA kits (CD30 Human Instant ELISA Kit, ThermoFisher Scientific; lens epithelial protein (LENEP) ELISA Kit, MyBiosource; PRR4 ELISA Kit (Proline Rich 4, Lacrimal), Cloud-Clone Corp.) were used to determine the concentration of sCD30, PRR4 and LENEP in the serum of individual mice or patients and in the supernatants of cultured LCLs according to the manufacturer’s instructions.

Techniques: RNA Expression, Labeling, Expressing

Journal: PLoS Pathogens

Article Title: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice

doi: 10.1371/journal.ppat.1008477

Figure Lengend Snippet: A-C) Protein concentration of sCD30 was measured in serum samples with ELISA. A) Serum was obtained at the day of sacrifice from animals from two independent experiments. B) Post hoc stratification of FK506-treated mice with or without macroscopically visible tumors with EBV-infection status indicated by black (EBV + ) or clear (EBV – ) symbols. A-B) Mean ± SEM, MWT. Dashed lines indicate quantification thresholds. C) sCD30 was measured in the sera of 31 healthy children undergoing elective tonsillectomy, 21 children that presented with non-EBV associated fever (Febrile non-EBV, EBV VCA-IgM negative) and 13 pediatric PTLD patients around diagnosis (PTLD Acute) and again at least 36 months later (PTLD Recovered) (See and Tables). Dashed line represents the median of the healthy controls. MWT and Wilcoxon matched-pairs test. D) Representative immunohistochemistry staining of CD30 (red) and PAX5 (brown), or CD30 (red) and EBNA2 (brown), in splenic sections or tumor tissue of huNSG-A2 mice. Scale bars: 50μm. Quantification of CD30 + (n = 7) and CD30 + EBNA2 + cells (n = 6) per mm 2 in tumor sections and non-tumorous spleen tissue of EBV-infected mice is depicted for two independent experiments. E) Cytokine concentration was measured in the serum of EBV-PTLD patients (n = 8) at diagnosis (acute) and again at recovery time point at least 36m post-diagnosis and in FK506-treated EBV − (n = 8) and EBV + (n = 10) experimental mice, composite data from two independent experiments. Upper panel: Heatmaps and hierarchical clustering of pediatric patients or huNSG-A2 mice based on serum cytokine levels (IFNγ, TNFα, IL-6, IL-8 and IL-10) at diagnosis (red) and recovery (blue) for PTLD (left) and on the day of sacrifice for huNSG-A2 mice (right) treated with FK506 (blue) and additionally EBV-infected (red). Tumor presence versus absence in huNSG-A2 mice is indicated in black and grey respectively. Lower panel: Cytokine concentration (median (IQR) with min and max range as whiskers) in the serum of EBV-PTLD patients and in FK506-treated EBV − and EBV + experimental mice. Wilcoxon matched-pairs test and MWT respectively. *P < 0.05, **P < 0.01, ***P < 0.001. See also and and Tables.

Article Snippet: Commercially available ELISA kits (CD30 Human Instant ELISA Kit, ThermoFisher Scientific; lens epithelial protein (LENEP) ELISA Kit, MyBiosource; PRR4 ELISA Kit (Proline Rich 4, Lacrimal), Cloud-Clone Corp.) were used to determine the concentration of sCD30, PRR4 and LENEP in the serum of individual mice or patients and in the supernatants of cultured LCLs according to the manufacturer’s instructions.

Techniques: Protein Concentration, Enzyme-linked Immunosorbent Assay, Infection, Biomarker Discovery, Immunohistochemistry, Staining, Concentration Assay