Journal: Nature Communications

Article Title: Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation

doi: 10.1038/s41467-024-47574-0

Figure Lengend Snippet: a NK cell viability after thawing assessed via flow cytometry ( n = 5 healthy donors). b NK cell recovery 24, 48, and 72 h after thawing in comparison to starting NK cell number at time 0 h after thawing ( n = 10 healthy donors). c CD107A degranulation assay before and after thawing. NK cells were cultured with K562 cells at 1:1 effector:target ratio for 3 h ( n = 4 healthy donors). d NK cytotoxicity assay before and after cryopreservation with B2M KO Raji cells ( n = 4 healthy donors). e NK cytotoxicity assay before and after cryopreservation with K562 cells ( n = 4 healthy donors). f ADCC assay using anti-CD20 antibody and Jeko-1 cells ( n = 3 healthy donors). g Single-cell secretome profile of cryopreserved and non-cryopreserved NK cells treated with R848 for 24 h). Heatmap showing individual cytokines that NK cell secreted ( n = 3 healthy donors). h Bulk RNA volcano plot comparing before, 24 and 72 h after cryopreservation ( n = 5 healthy donors). i GSEA of IL-2-STAT5 pathway. Normality test was used to determine the distribution of the data then parametric test t test was used for ( a , c ). Two-way RM ANOVA was used for ( d – f ). The Benjamini–Hochberg method was used to obtain P value shown in ( h ). A two-tailed test was used for ( a , c , h ). g Error bars are shown as mean SD. All other graphs are shown as mean ± SEM.

Article Snippet: NK cells were thawed and rested in NK media for 24 h prior to stimulation with 1 μg/ml R848 (Invivogen # tlrl-r848) for 16 h. NK cells were then stained with cell trace violet membrane dye and loaded into IsoPlexis single-cell human NK cell secretome chips, which were loaded into the IsoSpark Duo instrument and analyzed with IsoSpeak software according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Comparison, Degranulation Assay, Cell Culture, Cytotoxicity Assay, ADCC Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation

doi: 10.1038/s41467-024-47574-0

Figure Lengend Snippet: a NK cell recovery immediately before and after cryopreservation ( n = 4 healthy donors). b Incucyte live imagining using caspase-3/7 cleavable dye to indicate early apoptosis ( n = 4 healthy donors). c Incucyte live imagining using Cytox NIR dye to indicate loss of plasma membrane integrity ( n = 4 healthy donors. d granzyme B levels at time 0 and 24 h after thawing ( n = 5 healthy donors). e GZMB knockout NK cells recovery after cryopreservation ( n = 4 healthy donors). f Perforin knockout NK cells recovery after cryopreservation ( n = 4 healthy donors). g , h GZMB and mock KO NK cells were cocultured at 1 ratio of 1:10 in both directions to determine the role of fratricide in NK cell death ( n = 4 healthy donors). i , j Confocal microscopy imaging showing the extent of colocalization of GZMB and LAMP-3, a marker for cytotoxic granules in fresh versus cryopreserved NK cells (fresh n = 29, cryopreserved n = 42). Normality test was used to determine the distribution of the data then parametric test t test was used for ( a , d , e , f , h ). nonparametric Mann–Whitney test was used for ( i ). A two-tailed test was used for ( a , d , e , f , h , i ). All graphs are shown as mean ± SEM.

Article Snippet: NK cells were thawed and rested in NK media for 24 h prior to stimulation with 1 μg/ml R848 (Invivogen # tlrl-r848) for 16 h. NK cells were then stained with cell trace violet membrane dye and loaded into IsoPlexis single-cell human NK cell secretome chips, which were loaded into the IsoSpark Duo instrument and analyzed with IsoSpeak software according to the manufacturer’s instructions.

Techniques: Membrane, Knock-Out, Confocal Microscopy, Imaging, Marker, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation

doi: 10.1038/s41467-024-47574-0

Figure Lengend Snippet: a , b Pretreating NK cells with cytokines 24 h before cryopreservation ( n = 10 healthy donors ( a ) and n = 6 healthy donors for ( b ). c GZMB MFI 24 h after vehicle or IL-12 treatment ( n = 7 healthy donors). d NK cell-mediated killing assay using IL−15 + IL−18-pretreated NK cell before and after cryopreservation ( n = 4 healthy donors). e Single-cell secretome profile of IL-15 + IL-18-treated cryopreserved and fresh NK cells treated with R848 for 24 h ( n = 3 healthy donors). f CD107A degranulation after IL-15 + IL-18 treatment ( n = 6 healthy donors). g Granzyme B ELISA 24 h after IL−15 + IL-18 treatment ( n = 6 healthy donors). h Comparing MFI of IL-15 + IL-18-treated NK cells at 24- and 72-h post-treatment ( n = 7 healthy donors). i Volcano plot comparing IL-15 + IL-18-treated vs untreated NK cells ( n = 3 healthy donors). j Heatmap showing RNA levels using NanoString assay ( n = 3 healthy donors). k MFI quantification of BCL2L1 after IL-15 + IL-18 treatment ( n = 3 healthy donors). l Recovery of perforin KO and BCL2L1 KO NK cells with and without IL-15 + IL-18 pretreatment ( n = 3 healthy donors). m IL-15 + IL-18-treated NK cell cryopreservation using GMP control rate freezer ( n = 4 healthy donors). Normality test was used to determine the distribution of the data then parametric test t test was used for ( c , g , k , l , m ), nonparametric Wilcoxon matched pairs t test was used for ( a , b , f , h ). Two-way RM ANOVA was used for ( d ). The Benjamini–Hochberg method was used to obtain P value shown in ( i , j ). Two-tailed test was used for ( a – c , f – m ). e Error bars are shown as mean SD. All other graphs are shown as mean ± SEM.

Article Snippet: NK cells were thawed and rested in NK media for 24 h prior to stimulation with 1 μg/ml R848 (Invivogen # tlrl-r848) for 16 h. NK cells were then stained with cell trace violet membrane dye and loaded into IsoPlexis single-cell human NK cell secretome chips, which were loaded into the IsoSpark Duo instrument and analyzed with IsoSpeak software according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation

doi: 10.1038/s41467-024-47574-0

Figure Lengend Snippet: a FlowSOM cluster map ( n = 3 healthy donors). b individual marker expression in UMAP-1 ( n = 3 healthy donors). c heatmap showing markers and expression levels in different clusters ( n = 3 healthy donors). d UMAP showing NK cells distribution at different time points after thawing ( n = 3 healthy donors).

Article Snippet: NK cells were thawed and rested in NK media for 24 h prior to stimulation with 1 μg/ml R848 (Invivogen # tlrl-r848) for 16 h. NK cells were then stained with cell trace violet membrane dye and loaded into IsoPlexis single-cell human NK cell secretome chips, which were loaded into the IsoSpark Duo instrument and analyzed with IsoSpeak software according to the manufacturer’s instructions.

Techniques: Marker, Expressing

Journal: Nature Communications

Article Title: Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation

doi: 10.1038/s41467-024-47574-0

Figure Lengend Snippet: a Schematic diagram for in vivo experiment. b Luminescent images of treated mice ( n = 9 mice/group). c quantification of tumor burden (flux) ( n = 9 mice/group). d NK cell number in the blood of treated mice ( n = 9 mice/group) . e Survival curve of NK treated mice ( n = 9 mice/group) ( e ). Two-way ANOVA was used for ( c ). Normality test was used to determine the distribution of the data then parametric test t test was used for d. Simple survival analysis (Kaplan–Meier) with no data adjustment were made for ( e ). A two-tailed test was used for ( d ). All graphs are shown as mean ± SEM.

Article Snippet: NK cells were thawed and rested in NK media for 24 h prior to stimulation with 1 μg/ml R848 (Invivogen # tlrl-r848) for 16 h. NK cells were then stained with cell trace violet membrane dye and loaded into IsoPlexis single-cell human NK cell secretome chips, which were loaded into the IsoSpark Duo instrument and analyzed with IsoSpeak software according to the manufacturer’s instructions.

Techniques: In Vivo, Two Tailed Test

Journal: bioRxiv

Article Title: BMP antagonist CHRDL2 enhances the cancer stem cell phenotype and increases chemotherapy resistance in Colorectal Cancer

doi: 10.1101/2024.01.23.576664

Figure Lengend Snippet: Inducible CHRDL2 overexpression in CRC cell lines alters proliferation in a glucose dependent manner and cell cycle status A) Table of doxycycline treatment used. B) qPCR of mRNA levels of CHRDL2 expressed as fold change in 4 experimental cell lines. Cell lines were grown with doxycycline at: 0.1 μg/ml, 1 μg/ml or 10 μg/ml to induce expression. RKO DMSO-10μg/ml p<0.01, COLO320 DMSO-10μg/ml p<0.05, CACO2 DMSO-10μg/ml p<0.01, LS180 DMSO-10μg/ml p<0.01. N=3, T-test. C) Western blotting of corresponding protein levels of CHRDL2 in cell lines with lentiviral overexpression, and secreted CHRDL2 present in cell culture media. D) Quantification of CHRDL2 protein levels as measure by western blot using Image J software. E) MTT assay of cellular proliferation of CHRDL2 cell lines. In, COLO320 and RKO cells lines small but significant decreases in CHRDL2 expression were shown. Two-way RM ANOVA P<0.01, P<0.036 respectively F) Cellular proliferation analysis on cells grown in low glucose conditions, given 10 μg/ml doxycycline overexpression of CHRDL2. In CACO2, COLO320, LS180 and RKO cells lines, proliferation was significantly decreased with CHRDL2 expression. T-test; P<0.01, P<0.01, P<0.001 and P<0.01 respectively. G) Western blotting of SMAD1/5 phosphorylation in cell lines overexpressing CHRDL2. H) Flow cytometry analysis of COLO320 cells given CHRDL2++ overexpression. Cell number increased in S phase by CHRDL2 overexpression, T-test P<0.05. I) Crystal violet staining of colonies of RKO cells treated with 1 μg/ml and 10 μg/ml doxycycline to induce CHRDL2 expression. J) Quantification of colonies established in our 4 experimental cell lines with CHRDL2 overexpression. CACO2 and RKO cell lines both showed reduced colony formation in the low and high CHRDL2 treated groups, p<0.01, p<0.001, p<0.05, P<0.001, T-test. COLO320 and LS180 both showed a reduction in colony formation in the high CHRDL2 group only, p<0.01. N=3. Error bars given as ± SEM.

Article Snippet: To confirm overexpression, doxycycline was added at (0.1 μg/ml, 1 μg/ml (CHRDL2 +) or 10 μg/ml (CHRDL2 ++) RNA was extracted (RNeasy, QIAGEN) and quantified by real-time reverse transcriptase polymerase chain reaction (qPCR) using TaqMan technology (Hs00248808_m1) according to the manufacturers protocol (Applied biosystems).

Techniques: Over Expression, Expressing, Western Blot, Cell Culture, Software, MTT Assay, Phospho-proteomics, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: BMP antagonist CHRDL2 enhances the cancer stem cell phenotype and increases chemotherapy resistance in Colorectal Cancer

doi: 10.1101/2024.01.23.576664

Figure Lengend Snippet: CHRDL2 overexpression increases resistance to common CRC chemotherapies. A) Drug dose response curves using CACO2 cells and 5FU, COLO320 cells and Irinotecan, and LS180 and RKO cells and Oxaliplatin N=3. Two-way ANOVA was used to find differences between curves, P<0.0068, P<0.0001, P<0.0006, P<0.005. B) Average difference in IC50 valued across all cell lines and 3 chemotherapy drugs. P< 0.005. C) Table of ratio differences in IC50 values between CHRDL2++ cells and control for each chemotherapy drug and cell-line. N=3. D) Flow cytometry analysis of COLO320 cells treated with Oxaliplatin and CHRDL2 Overexpression. CHRDL2 increased the number of cells in G1 phase. (students T-test P<0.003). E) Flow cytometry analysis of COLO320 cells treated with Oxaliplatin and CHRDL2 overexpression. F) Quantification of cell percentages of live, apoptotic and dead cells in COLO320 cells treated with oxaliplatin and given CHRDL2 overexpression. CHRDL2 increased the % of live cells (students T-test P<0.03) and decreased the % of early apoptotic cells (P<0.023). N=3. G) Drug dose response curves of CACO2 and RKO cells with CHRDL2 conditioned media and Oxaliplatin N=3. Two-way ANOVA was used to find differences between curves, P<0.005, P<0.0001 H) Average IC50 values for chemotherapy drug Oxaliplatin, on cell lines CACO2 and RKO with CHRDL2 conditioned media. CHRDL2 against control p< 0.0305. N=3. I) Cell count after irradiation of RKO cells overexpressing CHRDL2++ N=3. T-test 4GY: P<0.038, 6GY: P<0.0241. Error bars given as ± SEM.

Article Snippet: To confirm overexpression, doxycycline was added at (0.1 μg/ml, 1 μg/ml (CHRDL2 +) or 10 μg/ml (CHRDL2 ++) RNA was extracted (RNeasy, QIAGEN) and quantified by real-time reverse transcriptase polymerase chain reaction (qPCR) using TaqMan technology (Hs00248808_m1) according to the manufacturers protocol (Applied biosystems).

Techniques: Over Expression, Control, Flow Cytometry, Cell Counting, Irradiation

Journal: bioRxiv

Article Title: BMP antagonist CHRDL2 enhances the cancer stem cell phenotype and increases chemotherapy resistance in Colorectal Cancer

doi: 10.1101/2024.01.23.576664

Figure Lengend Snippet: CHRDL2 overexpression decreases DNA damage marked by γH2AX during chemotherapy treatment and enhances expression of ATM. A) Representative immunofluorescence of γH2AX on COLO320 cells treated with 5 μM oxaliplatin at 24, 48 and 72 hrs. Foci indicated by red arrows. B) Quantification of γH2AX foci in COLO320 cells overexpressing CHRDL2 treated with 5 μM oxaliplatin at 24, 48, and 72 hrs. Cells were treated with DMSO control reagent, or Doxycycline to induce CHRDL2++ overexpression. 24hs P< 0.0001, 48hrs P<0.01, 72hrs P<0.0001. N=3. T-test C) Immunofluorescence staining of ATM on COLO320 cells treated with 5 μM oxaliplatin. D) Quantification of ATM staining on COLO320 cells. Immunofluorescence given as Corrected Total Cell Fluorescence (CTCF). Cells were treated with DMSO control reagent, or Doxycycline to induce CHRDL2++ overexpression. P<0.0001 N=3 T-test. E) Comet assay of RKO cells treated with IC50 Oxaliplatin. Cells were then treated with CHRDL2 ++ overexpression or a control. F) Quantification of Comet assay, T-test P<0.0001. N=3.Error bars given as ± SEM. Quantification carried out using Image J.

Article Snippet: To confirm overexpression, doxycycline was added at (0.1 μg/ml, 1 μg/ml (CHRDL2 +) or 10 μg/ml (CHRDL2 ++) RNA was extracted (RNeasy, QIAGEN) and quantified by real-time reverse transcriptase polymerase chain reaction (qPCR) using TaqMan technology (Hs00248808_m1) according to the manufacturers protocol (Applied biosystems).

Techniques: Over Expression, Expressing, Immunofluorescence, Control, Staining, Fluorescence, Single Cell Gel Electrophoresis

Journal: bioRxiv

Article Title: BMP antagonist CHRDL2 enhances the cancer stem cell phenotype and increases chemotherapy resistance in Colorectal Cancer

doi: 10.1101/2024.01.23.576664

Figure Lengend Snippet: Secreted CHRDL2 decreases murine small intestinal organoid budding and increases stem cell marker expression. A) Image of intestinal organoid diagram depicting organoid structure. B) Image of murine-derived organoids treated with conditioned media containing secreted forms of CHRDL2 compared to conditioned media from control cells with no CHRDL2 overexpression. C) Quantification of buds per organoid in CHRDL2 treated murine organoids compared to a control. T-test P<0.0001. D) Quantification of average organoid diameter in CHRDL2 treated murine organoids compared to a control. T-test P<0.001. E) Immunofluorescence staining of OLFM4 on murine organoids treated with secreted CHRDL2 compared to a control after 1 week. F) Quantification of immunofluorescence scoring of OLFM4 on murine organoids treated with secreted CHRDL2 compared to a control. T-test P<0.0001. G) qPCR of stem cell markers from CHRDL2 treated murine organoids compared to a control. T-test SOX9. Students T-test P<0.0014, LGR5 P<0.04, P<0.043, BMI1 P<0.0113, MSI1 P<0.0067, P<0.009. Error bars given as ± SEM.

Article Snippet: To confirm overexpression, doxycycline was added at (0.1 μg/ml, 1 μg/ml (CHRDL2 +) or 10 μg/ml (CHRDL2 ++) RNA was extracted (RNeasy, QIAGEN) and quantified by real-time reverse transcriptase polymerase chain reaction (qPCR) using TaqMan technology (Hs00248808_m1) according to the manufacturers protocol (Applied biosystems).

Techniques: Marker, Expressing, Derivative Assay, Control, Over Expression, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: BMP antagonist CHRDL2 enhances the cancer stem cell phenotype and increases chemotherapy resistance in Colorectal Cancer

doi: 10.1101/2024.01.23.576664

Figure Lengend Snippet: RNAseq analysis demonstrates that CHRDL2 expression enhances cancer stem-cell and other cancer hallmark pathways. RNAseq analysis of CACO2 cells treated with CHRDL2 + or CHRDL2 ++ compared to a control. N=3 A) Volcano plot of differentially expressed genes from cells with CHRDL2 overexpression (CHRDL2 + and CHRDL2 ++) from RNAseq analysis. Expressed as Log2 Fold change against control. B) Bar-plot of significantly differentially expressed genes by CHRDL2+ and CHRDL2 ++. Genes included pass the threshold of P<0.001 for CHRDL2+ and P<0.001 from CHRDL2 ++. C) Intersect of highly differentially expressed genes in both the CHRDL2 + and CHRDL2++ treated groups P<0.001. 21 genes were differentially expressed in both groups. D) qPCR validation of differentially expressed gene from RNAseq data in CACO2 and COLO320 cells N=3.E) GSEA plots of differentially expressed pathways in CACO2 cells treated with CHRDL2++. F) qPCR validation of stem cell markers in CACO2 and COLO320 cells N=3. CACO2 BMI1 P<0.05, CACO2 LGR6 P<0.05, COLO320 LGR5 P<0.01. N=3. Error bars given as ± SEM.

Article Snippet: To confirm overexpression, doxycycline was added at (0.1 μg/ml, 1 μg/ml (CHRDL2 +) or 10 μg/ml (CHRDL2 ++) RNA was extracted (RNeasy, QIAGEN) and quantified by real-time reverse transcriptase polymerase chain reaction (qPCR) using TaqMan technology (Hs00248808_m1) according to the manufacturers protocol (Applied biosystems).

Techniques: Expressing, Control, Over Expression, Biomarker Discovery

Journal: Frontiers in Medicine

Article Title: Single Cell RNA Sequencing in Autoimmune Inflammatory Rheumatic Diseases: Current Applications, Challenges and a Step Toward Precision Medicine

doi: 10.3389/fmed.2021.822804

Figure Lengend Snippet: Single cell isolation, capture and library generation using droplet-based microfluidic system. Tissue biopsies are usually dissociated mechanically and/or enzymatically. Single cells can be subsequently isolated and captured into individual reaction units using various methods/platforms. The traditional methods include limiting dilution, micromanipulation, laser capture microdissection (LCM), and flow-activated cell sorting (FACS). Although these methods can be used to separate individual cells into compartments, the downstream analytical processes (cell lysis, reverse transcription, and library construction) cannot be performed directly in these compartments. In contrast, microdroplet technology can be used for both: to capture each individual cell into one compartment/reaction unit, as well as to perform downstream reactions directly in every unit. In the droplet-based microfluidic system (i.e., 10x Genomics Chromium), aqueous droplets are formed in a continuous oil phase. Each droplet contains individual cell mixed with gel beads containing oligo sequences for a bead-specific barcode, unique molecular identifier (UMI) and poly-dT sequence which hybridizes with poly(A) tails of each mRNA. With reverse transcription, the bead-specific barcode integrates into the cDNA, allowing subsequent identification of the cell origin. The figure was created with BioRender.com .

Article Snippet: Human synovial fluid , PsA patients ( n = 3) , Synovial fluid cells (ND) , 10X Genomics Chromium , Reduced CD14+CD6- classical and increased CD14+CD16+ intermediate monocytes/macrophages in PsA, Monocyte/macrophage PAR2 activation by tryptase-6 resulted in increased secretion of MCP-1. , Abji et al. ( ) .

Techniques: Single-cell Isolation, Isolation, Micromanipulation, Laser Capture Microdissection, FACS, Lysis, Reverse Transcription, Sequencing

Journal: Frontiers in Medicine

Article Title: Single Cell RNA Sequencing in Autoimmune Inflammatory Rheumatic Diseases: Current Applications, Challenges and a Step Toward Precision Medicine

doi: 10.3389/fmed.2021.822804

Figure Lengend Snippet: Application of scRNA-seq technology in rheumatoid arthritis.

Article Snippet: Human synovial fluid , PsA patients ( n = 3) , Synovial fluid cells (ND) , 10X Genomics Chromium , Reduced CD14+CD6- classical and increased CD14+CD16+ intermediate monocytes/macrophages in PsA, Monocyte/macrophage PAR2 activation by tryptase-6 resulted in increased secretion of MCP-1. , Abji et al. ( ) .

Techniques: Activity Assay, Mouse Assay, Blocking Assay, Gene Expression, Produced, In Vitro, Modification, Mutagenesis, Expressing

Journal: Frontiers in Medicine

Article Title: Single Cell RNA Sequencing in Autoimmune Inflammatory Rheumatic Diseases: Current Applications, Challenges and a Step Toward Precision Medicine

doi: 10.3389/fmed.2021.822804

Figure Lengend Snippet: Application of scRNA-seq technology in systemic lupus erythematosus.

Article Snippet: Human synovial fluid , PsA patients ( n = 3) , Synovial fluid cells (ND) , 10X Genomics Chromium , Reduced CD14+CD6- classical and increased CD14+CD16+ intermediate monocytes/macrophages in PsA, Monocyte/macrophage PAR2 activation by tryptase-6 resulted in increased secretion of MCP-1. , Abji et al. ( ) .

Techniques: Expressing, Gene Expression, Derivative Assay, Clinical Proteomics, Activity Assay, Isolation, Activation Assay

Journal: Frontiers in Medicine

Article Title: Single Cell RNA Sequencing in Autoimmune Inflammatory Rheumatic Diseases: Current Applications, Challenges and a Step Toward Precision Medicine

doi: 10.3389/fmed.2021.822804

Figure Lengend Snippet: Application of scRNA-seq technology in systemic sclerosis.

Article Snippet: Human synovial fluid , PsA patients ( n = 3) , Synovial fluid cells (ND) , 10X Genomics Chromium , Reduced CD14+CD6- classical and increased CD14+CD16+ intermediate monocytes/macrophages in PsA, Monocyte/macrophage PAR2 activation by tryptase-6 resulted in increased secretion of MCP-1. , Abji et al. ( ) .

Techniques: Expressing, Mouse Assay, Control, Migration, Amplification, Gene Expression

Journal: Frontiers in Medicine

Article Title: Single Cell RNA Sequencing in Autoimmune Inflammatory Rheumatic Diseases: Current Applications, Challenges and a Step Toward Precision Medicine

doi: 10.3389/fmed.2021.822804

Figure Lengend Snippet: Application of scRNA-seq technology in PsA, AxSpA, SjS and KD.

Article Snippet: Human synovial fluid , PsA patients ( n = 3) , Synovial fluid cells (ND) , 10X Genomics Chromium , Reduced CD14+CD6- classical and increased CD14+CD16+ intermediate monocytes/macrophages in PsA, Monocyte/macrophage PAR2 activation by tryptase-6 resulted in increased secretion of MCP-1. , Abji et al. ( ) .

Techniques: Activation Assay, Expressing, Clinical Proteomics, Isolation, Biomarker Discovery

Journal: Nature Communications

Article Title: CAR T-cell-mediated delivery of bispecific innate immune cell engagers for neuroblastoma

doi: 10.1038/s41467-024-51337-2

Figure Lengend Snippet: a CD16a IHC of representative high-risk ( MYCN amplified; left ) and low-risk ( right ) neuroblastoma tumors, respectively. Scales bars are indicated. b CD3 IHC of the same tumors shown in ( a ). Scales bars are indicated. c Percentage of CD16a + cells in tumors stratified by International Neuroblastoma Risk Group (INRG) (low/intermediate; n = 22 vs. high; n = 23), previous chemotherapy treatment [diagnosis; n = 25 vs. post-chemo (only high-risk); n = 10] and MYCN amplification (non-amplified; n = 14 vs. amplified; n = 8) included in the TMA. * P = 0.038 and ** P = 0.004 (Unpaired t -test; two-tailed). Means and SDs are shown. d Percentage of CD3 + cells in tumors stratified by INRG (low/intermediate; n = 22 vs. high; n = 24), previous chemotherapy treatment [diagnosis; n = 26 vs. post-chemo (only high-risk); n = 9] and MYCN amplification (non-amplified; n = 15 vs. amplified; n = 9) included in the TMA. * P = 0.034 and ** P = 0.027 (Unpaired t -test; two-tailed). MS neuroblastomas were excluded from the analyses in ( c and d ). Means and SDs are shown. e Expression of PTPRC (encoding CD45), FCGR3A (encoding CD16a), CD68 (defining macrophages) and NKG7 (defining NK-cells) in 2 neuroblastoma single cell datasets (6442 and 13,281 cells, respectively). SingleR analysis was also used to label different cell types [NK-cells and T-cells (helper and cytotoxic); right ]. f Heatmap showing single-cell expression profiles ( n = 16 NK-related genes) in the subset of NK-cells identified in dataset in ( e ) (top). Red box indicates the subpopulation of NK-cells with potential dysfunctional properties. FCGR3A is highlighted in bold. g Percentage of CD16a-positive cells in immune cell subsets present in neuroblastoma-infiltrated bone marrows. P values are shown in graph (One-way ANOVA plus Dunn’s multiple comparison test). Means and SDs are shown ( n = 9). h Immune cell types present in neuroblastoma-infiltrated bone marrows. Percentage of total CD45 cells is indicated. P values are shown in graph (One-way ANOVA plus Dunn’s multiple comparison test). Means and SDs are shown ( n = 9). Gating strategies are shown in Supplementary Fig. . i ( left ) Dot plots showing co-expression of CD56 and CD16a on NK-cells isolated from a neuroblastoma-infiltrating patient BM specimen. ( right ) Flow cytometry histograms showing TIGIT and LAG3 levels on BM-derived NK-cells. For further clinical information see Supplementary Table . US unstained, inter intermediate, chemo chemotherapy. Source data are provided as a Source Data file.

Article Snippet: After 24 hours of co-culture, NK-cells were separated from tumor cells by positive isolation using CD45 magnetic beads (130-045-801; Miltenyi Biotec), NK-cells were loaded on a Single-Cell Secretome Adaptive Immune: Human NK-cell chip (Isoplexis; ISOCODE-3L03-4) and run on an IsoSpark System (IsoPlexis) using the manufacturer’s protocols.

Techniques: Amplification, Biomarker Discovery, Two Tailed Test, Expressing, Comparison, Isolation, Flow Cytometry, Derivative Assay

Journal: Nature Communications

Article Title: CAR T-cell-mediated delivery of bispecific innate immune cell engagers for neuroblastoma

doi: 10.1038/s41467-024-51337-2

Figure Lengend Snippet: a Graphical illustration of T-cell GPC2.CAR expression and GD2.BiCE secretion. b Schematic representation of transgenes for two BiCE-secreting GPC2.CAR constructs targeting GD2 and CD19 (GPC2.CAR-GD2.BiCE and GPC2.CAR-CD19.BiCE, respectively), as well as single GPC2.CAR, CD19.CAR and GD2.BiCE constructs. c Detection of GD2 BiCE by western blot (His-tag) in cSN from HEK293T cells transfected with GPC2.CAR-GD2.BiCE vector. d Binding assays detecting secondary His-tag expression on tumor cells incubated with cSN from HEK293T cells transfected with different constructs as indicated. e Correlation between GD2 BiCE binding and GD2 cell surface expression in NB cell lines. Spearman correlation (two-tailed). f Illustration ( left ) and histograms ( right ) of binding assay evaluating human recombinant CD16a protein binding to tumor cells incubated with different CAR construct cSNs. g Illustration ( left ) and histograms ( right ) of binding assay evaluating 1A7 binding to human primary NK-cells or T-cells incubated with different CAR construct cSNs. 1, control medium; 2, GPC2.CAR; 3, GPC2.CAR-CD19.BiCE; and 4, GPC2.CAR-GD2.BiCE in ( d , f , and g ). h NK-cell-mediated specific lysis of NB-EbC1 cells in the presence of cSNs (5 ng/mL of His-tagged BiCE), or dinutuximab (10 µg/mL) as a positive control. * P = 0.0009 and ** P = 0.0028 (One-way ANOVA plus Tukey’s multiple comparison test). Means and SDs are shown ( n = 3 independent donors). i Co-expression of CD107a and CD69 by flow cytometry in NK-cells from the cytotoxicity experiment in ( h ). * P = 0.0003 (One-way ANOVA plus Tukey’s multiple comparison test). Means and SDs are shown ( n = 3 independent donors). j IFN-γ levels measured by ELISA in NK-cell supernatants from the cytotoxicity experiment in ( h ). * P = 0.0028 (One-way ANOVA plus Tukey’s multiple comparison test). Means and SDs are shown ( n = 3 independent donors). k Polyfunctionality pie charts indicating the percentage of single NK-cells secreting 1 or more cytokine when cultured alone or in the presence of NB-EbC1 cells together with GD2 BiCE, CD19 BiCE or dinutuximab as in ( h ). l NK92 WT vs. NK92-CD16a cell cytotoxicity against NB-EbC1 cells co-incubated with different cSNs or dinutuximab (10 µg/mL) as a positive control after 24 hours at a 10:1 E:T ratio. Means and SDs are shown (n = 3 technical replicates). m ( left ) Percentage of CD107a + NK-cells isolated from either neuroblastoma-infiltrating BM aspirates or peripheral blood of healthy donors after 24 hours of co-incubation with NB-EbC1 cells and cSNs (from GPC2.CAR-GD2.BiCE or GPC2.CAR-CD19.BiCE; 5 ng/mL). * P = 0.0139 and ** P < 0.0001 (Paired, two-way ANOVA plus Šídák’s multiple comparisons test). ( right ) Quantification of residual tumor cells (CD45 - /GD2 + ) in the same samples. A total of 4 BMs were utilized and shown with different symbols. The degree of opacity indicates the E:T ratio utilized for each BM sample (10:1, 5:1 and 2.5:1; lower to higher opacity). The same E:T ratios were utilized for healthy donor blood NK-cells to facilitate comparison between groups. n ( left ) Representative contour plots of phagocytosis assay with GFP-labeled NB-EbC1 cells exposed to different cSNs or dinutuximab (10 µg/mL) together with macrophages. Macrophages alone are shown to define GFP + /CD11b + cells. ( right ) Quantification of NB-EbC1 phagocytosis. Means and SDs are shown ( n = 3 technical replicates). TM transmembrane, neg negative, cSN concentrated supernatant, BM bone marrow, HD healthy donor. Represented data has been validated with at least 2 independent experiments. Figure 3 a , f and g created with BioRender.com, and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Article Snippet: After 24 hours of co-culture, NK-cells were separated from tumor cells by positive isolation using CD45 magnetic beads (130-045-801; Miltenyi Biotec), NK-cells were loaded on a Single-Cell Secretome Adaptive Immune: Human NK-cell chip (Isoplexis; ISOCODE-3L03-4) and run on an IsoSpark System (IsoPlexis) using the manufacturer’s protocols.

Techniques: Expressing, Construct, Western Blot, Transfection, Plasmid Preparation, Binding Assay, Incubation, Two Tailed Test, Recombinant, Protein Binding, Control, Lysis, Positive Control, Comparison, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Isolation, Phagocytosis Assay, Labeling

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) NetMHCPan 4.1 summary of the top 10 peptide sequences that are most likely to be presented by HLA-A2 from the first 30 amino acids of KRAS G12V . Peptides containing the G12V mutation (including mutations in red text) are in black text; others are in gray. (b) Log 10 (EC 50 (M)) comparison of recognition of synthetic KRAS G12V 5-14 peptide (KLVVVGAVGV) presented by HLA-A2 by an initially generated set of MHC class I-restricted TCRs isolated to be specific for this epitope. Lower values indicate higher functional avidity. (c) % IFNγ + T cells, transduced with TCR 2 , after exposure to B-LCL cell lines expressing different Class I HLA alleles found on CFPAC1 cells without KRAS G12V 5-14 peptide loaded. Cell lines are color coded by allele. (d) MS results of RAS family peptide fragments detected after tryptic digestion of immunoprecipitated whole KRAS protein from cell lysate of DAN-G and CFPAC1 pancreatic adenocarcinoma cell lines. Lysine side chain trimethylation is indicated in red with an asterisk. (e) Reconstitution of properly folded HLA-A2 surface expression on T2 cells after exogeneous loading by wild type or G12V/D mutant KRAS 5-14 peptides with or without lysine-5 side chain methylations. (f) Additional views of structural models of HLA-A2-presented KRAS G12V 5-14 peptide including possible lysine-5 side chain methylation, supplementing by highlighting the protrusion of the lysine-5 side chain (circled in red) out of the HLA-A2 groove. (g) Overview of structural prediction of a TCR-pMHC complex using a combined Alphafold-Rosetta pipeline. Alphafold allows for deep learning-based prediction of proteins and protein complexes, which we applied to predict TCR-pMHC complex structure. However, this does not yet contain PTMs, which Alphafold cannot incorporate reliably. Thus, we used the resulting structure as input into Rosetta, which used physics-based energy functions to introduce PTMs and reoptimize the structure to accommodate them.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Mutagenesis, Comparison, Generated, Isolation, Functional Assay, Transduction, Expressing, Immunoprecipitation, Methylation, Structural Proteomics, Introduce

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Log 10 (EC 50 (M)) comparison of recognition of different concentrations of a synthetic KRAS G12V 5-14 peptide (KLVVVGAVGV) presented by HLA-A2 by different MHC class I-restricted TCRs specific for the epitope. Lower values indicate higher functional avidity. (b) Killing of live CFPAC1 and DAN-G HLA-A2 + KRAS G12V+ pancreatic adenocarcinoma cells cocultured with CD8 + T cells expressing the TCRs shown in (a) using a 4:1 T cell:tumor cell ratio. Cocultures were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR: red fluorescent protein; NG: green fluorescent protein. (c) ARTEMIS MS data of peptides eluted from HLA-A2. Data is from the 293F cell line, as commonly used for proteomics assays requiring high protein expression. All cell lines were transduced with an HLA-A2 single chain secreted dimer as well as a KRAS G12V constitutive expression construct to increase presentation and the likelihood of detecting a presented peptide by MS, of KRAS G12V -related epitopes. The first 100 amino acids of KRAS G12V are shown, with the G12V mutation highlighted in yellow. Epitopes predicted by NetMHCPan 4.1 are shown in purple; epitopes detected from ARTEMIS are shown in blue. ARTEMIS detection of KRAS G12V 5-14 is outlined in red. (d) Heatmap of – log 10 (EC 50 (μg/ml)) of CD8 + T cells expressing TCR 2 or TCR 19 against candidate KRAS G12V epitopes, with and without methylation of the lysine-5 side chain of the epitope, presented by HLA-A2. Higher values indicate higher functional avidity. EC 50 values were calculated from T cell exposure to peptide concentrations ranging from 1 µg/ml to 10 - µg/ml. Gray squares indicate EC 50 calculations that lacked a stable fit, e.g., due to lack of response to peptide even at high doses. (e) Rosetta structural modeling of HLA-A2 presenting KRAS G12V 5-14 and its methylated variants. Red circles indicate the amine group of the lysine-5 side chain; arrows indicate its movement with different methylation states. A reference point is drawn at the same position in each image to help illustrate positional changes. (f) Alanine scan of the KRAS G12V epitope and resulting response by CD8 + T cells expressing TCR 2 and TCR 19 . The epitope with each individual residue substituted with alanine (or, for alanine-11, substituted with either glycine or threonine) was presented by antigen-presenting cells to stimulate primary human CD8 + T cells expressing a TCR, and subsequent IFNγ expression was measured. Higher IFNγ induction indicates greater tolerance for amino acid substitution at that position. X-axis numbers indicate the residue number on the KRAS protein, and letters indicate the amino acid substitution. A: alanine; G: glycine; T: threonine. Last column is unmodified KRAS G12V 5-14.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Comparison, Functional Assay, Expressing, Imaging, Transduction, Construct, Mutagenesis, Methylation, Residue

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Structural model, as predicted by a combined Alphafold-Rosetta pipeline, of a ‘core’ CDR3α sequence (sequence EDNT) of an existing TCR recognizing KRAS G12V 5-14 and its relation to the lysine-5 side chain (circled in red) of the epitope as presented by HLA-A2. (b) Generation of TCR mutagenesis libraries by mutagenesis of the core CDR3α sequence followed by transduction of the TCRs into Nur77-GFP reporter Jurkat cells and subsequent clonal expansions. Tetramer binding patterns of HLA-A2-KRAS G12V 5-14 peptide with unmodified or methylated lysine-5 side chain at the end of cell stimulation are shown. Each circle indicates a population with a specific pattern of unmethylated vs. methylated peptide tetramer binding that was sorted and sequenced. (c) Examples of mutated CDR3α ‘core’ sequences resulting from sorted clones of the mutagenesis library that exhibit the noted different patterns of PTM peptide recognition. (d) Heatmap of – log 10 (EC 50 (μg/ml)) of TCRs derived from the mutagenesis library and the original TCR 2 responding to different degrees of lysine-5 methylation of KRAS G12V 5-14 presented by HLA-A2, as calculated from dose response assays. Gray squares indicate EC 50 values that lacked a stable fit, e.g., due to limited response to peptide even at high doses. TCR 4UM is the 4 th TCR clonotype identified from an initial sequencing dataset from sorted Jurkat cells binding to both unmethylated (“U”) and methylated (“M”) tetramers; other TCRs are labeled by changes in the ‘core’ CDR3α sequence (e.g., EDST). (e) Killing of HLA-A2 + KRAS + CFPAC1 and DAN-G pancreatic adenocarcinoma cells by CD8 + T cells expressing selected mutagenized TCRs. Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Sequencing, Mutagenesis, Transduction, Binding Assay, Methylation, Cell Stimulation, Clone Assay, Derivative Assay, Labeling, Expressing, Imaging

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: Structural predictions of unmethylated and methylated variants of KRAS G12V 5-14, as well as unmethylated wild type or G12D peptide, presented by HLA-A2 to interface with (a) TCR 2 and (b) mutagenized TCR EDST TCRs. The CDR3α ‘core’ sequence is shown as individual amino acids within the TCRs. In (b), the TCR 2 -pMHC interfaces from (a) are overlaid with transparency to facilitate visual comparison. With TCR EDST , residues in the core sequence of CDR3α and the lysine-5 side chain of each variant of KRAS G12V epitope appear closer to each other than with TCR 2 .

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Methylation, Sequencing, Comparison, Variant Assay

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Dose response curves of CD8 + T cells expressing selected TCRs generated from the HLA-A2 KRAS G12V TCR mutagenesis library after exposure to wild type or G12D KRAS 5-14 peptides presented by HLA-A2. (b) Failure of CD8 + T cell killing and progressive growth of human HLA-A2 + tumor cell lines that express wild type KRAS (HeLa cervical carcinoma cell line; left) or KRAS G12D (Panc1 pancreatic adenocarcinoma; right) and no KRAS G12V . Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein. (c) Alanine scan of KRAS G12V 5-14 epitope tested against TCRs selected from the mutagenesis library. Each cell indicates the resulting expression of Nur77-GFP upon replacement of a KRAS G12V residue with alanine (or in the case of alanine-11, replacement with either glycine or threonine) and with or without lysine-5 side chain dimethylation. (d) IFNγ secretion by CD8 + T cells expressing mutagenized TCRs after exposure to synthetic peptides from the human proteome fitting the K-x-x-V-V-x-A-x-x-x tolerance pattern identified from the alanine scan shown in (c), with a concentration of 0.1 μg/ml for each peptide. (e) Dose response curves of CD8 + T cells expressing mutagenized TCRs after exposure to the potential human off-target peptides demonstrated above to generate the three largest responses at 0.1 μg/ml: CFA61 23-32/607-616/667-676 , RSLBB 35-44 , and TRXR1 342-351 , compared to the KRAS G12V epitope.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Expressing, Generated, Mutagenesis, Imaging, Residue, Concentration Assay

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Experimental workflow to enrich and characterize T cell clones with TCRs recognizing a defined peptide epitope. (b) Tetramer binding patterns of primary human T cells stimulated with unmethylated and methylated KRAS G12V peptide presented by HLA-A2. Plots also indicate gates used to sort cells with different affinities for unmethylated and methylated epitopes. (c) Dose response curves of TCRs against wild type and KRAS G12D 5-14 peptides presented by HLA-A2. (d) Tumor cell growth during coculture of CD8 + T cells expressing selected TCRs with HLA-A2 + , wild type KRAS HeLa and KRAS G12D Panc1 pancreatic adenocarcinoma cell lines. Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein. NR: red fluorescent protein; NG: green fluorescent protein. (e) Alanine scan of KRAS G12V 5-14 epitope tested against TCR A2UoM1-1 . Each box indicates the resulting expression of Nur77-GFP upon replacement of a KRAS G12V residue with alanine (or in the case of alanine-11, replacement with either glycine or threonine). (f) Dose response curve of CD8 + Nur77-GFP Jurkat cells expressing TCR A2UoM1-1 after exposure to EPIPL 1948-1957 , the potential human off-target peptide as determined from alanine scan results.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Clone Assay, Binding Assay, Methylation, Expressing, Imaging, Residue

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Heatmap of –log 10 (EC 50 (μg/ml)) of selected TCRs responding to the unmethylated and methylated epitopes presented by HLA-A2, as calculated from dose response assays. Higher values indicate higher functional avidity. Gray squares indicate EC 50 values that lacked a stable fit, e.g., due to limited response to peptide even at high doses. (b) Killing of HLA-A2 + KRAS G12V+ CFPAC1 and DAN-G pancreatic adenocarcinoma cells by CD8 + T cells expressing selected TCRs. Cocultures of fluorescently labeled tumor cells and TCR-T cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR: red fluorescent protein; NG: green fluorescent protein. (c) Alphafold-Rosetta visualizations of TCR:pMHC complexes for TCR A2UoM1-1 , isolated from expansion of responding primary human CD8 + T cells from normal repertoires. Structures were generated for unmethylated and mono/di/trimethylated versions of the epitope, presented by HLA-A2. The four CDR3α amino acids in closest proximity to the lysine-5 side chain of KRAS G12V are shown with individual molecular bonds.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Methylation, Functional Assay, Expressing, Labeling, Imaging, Isolation, Generated

Journal: bioRxiv

Article Title: Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity

doi: 10.1101/2024.09.18.612965

Figure Lengend Snippet: (a) Overall workflow: DAN-G pancreatic adenocarcinoma cells were transduced with a CRISPR knockout library targeting methylation-related genes, and then cocultured with CD8 + T cells expressing either TCR 2 or mutagenized TCR EDST , which respectively have weak or strong recognition of methylated KRAS G12V epitopes. Tumor cells surviving at the end of coculture were sequenced to identify gRNA enrichment or depletion associated with immune evasion. (b) Volcano plot summarizing log 2 (fold change) of gRNA read frequencies targeting individual genes recovered after coculture of DAN-G cells with T cells expressing TCR EDST versus TCR 2 . The threshold of significance was set at log 10 ( p ) < 1.2 ( p < 0.05) and is denoted by the horizontal dashed line. Mean counts of all gRNAs per gene were used to calculate fold change values. The data point for gRNAs targeting SUPT6H is specifically labeled. The knockouts depleted or enriched after cocultures using TCR EDST versus TCR 2 are colored blue and red, respectively. (c) Killing of DAN-G cells with or without SUPT6H knockout in coculture with selected TCR-T cells (untransduced or expressing TCR 2 , TCR EDST , or TCR A2UoM1-1 ). Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed green nuclear fluorescent protein (“NG”). (d) Kaplan-Meier survival plots for individuals who have tumors with KRAS mutations, using public TCGA data. Left: only tumors with the KRAS G12V mutant, with or without concurrent SUPT6H mutation. Right: tumors with any KRAS mutant, with or without a concurrent SUPT6H mutation.

Article Snippet: Briefly, we transduced these cell lines with a construct expressing secreted, His-tagged HLA-A2 single chain dimer (SCD) with mCherry and a construct for constitutive expression of the first 100 amino acids of human KRAS G12V , based on Lenti-mCherry-G12V-KRAS-IRES-Blast (Addgene #153336).

Techniques: Transduction, CRISPR, Knock-Out, Methylation, Expressing, Labeling, Imaging, Mutagenesis

Journal: British Journal of Pharmacology

Article Title: Alzheimer's disease, ?-amyloid, glutamate, NMDA receptors and memantine - searching for the connections

doi: 10.1111/j.1476-5381.2012.02057.x

Figure Lengend Snippet: Effect of Aβ on glutamate homeostasis (studies showing effects leading to decrease in synaptic glutamate are in bold text)

Article Snippet: LTP hippocampal slices DG , Aβ naturally secreted (CHO cells expressing human APP751) and synthetic Aβ 1–42 (Bachem) , Inhibited the induction of LTP (natural at 1 μM and synthetic at 100–200 nM). mGluR5 antagonists blocked this effect of Aβ , Wang et al . (2004b ).

Techniques: Expressing, Incubation, Cell Culture, Concentration Assay, Activity Assay, Microscopy, Conjugation Assay, Inhibition, In Vitro, In Vivo

Journal: British Journal of Pharmacology

Article Title: Alzheimer's disease, ?-amyloid, glutamate, NMDA receptors and memantine - searching for the connections

doi: 10.1111/j.1476-5381.2012.02057.x

Figure Lengend Snippet: Effect of Aβ on NMDA receptor function (studies showing a decrease of NMDA function after Aβ are indicated by bold text)

Article Snippet: LTP hippocampal slices DG , Aβ naturally secreted (CHO cells expressing human APP751) and synthetic Aβ 1–42 (Bachem) , Inhibited the induction of LTP (natural at 1 μM and synthetic at 100–200 nM). mGluR5 antagonists blocked this effect of Aβ , Wang et al . (2004b ).

Techniques: Patch Clamp, Transferring, In Vivo, Phospho-proteomics, Fluorescence, Imaging, Incubation, Expressing, Produced, Centrifugation, Cell Culture, Binding Assay, Functional Assay, Activation Assay, Concentration Assay, Activity Assay, Injection, Single-unit Recording, Sonication

Journal: British Journal of Pharmacology

Article Title: Alzheimer's disease, ?-amyloid, glutamate, NMDA receptors and memantine - searching for the connections

doi: 10.1111/j.1476-5381.2012.02057.x

Figure Lengend Snippet: Effect of Aβ on LTP. Majority of studies show disruption of LTP, few studies show an enhancement of LTP by Aβ (indicated by bold text)

Article Snippet: LTP hippocampal slices DG , Aβ naturally secreted (CHO cells expressing human APP751) and synthetic Aβ 1–42 (Bachem) , Inhibited the induction of LTP (natural at 1 μM and synthetic at 100–200 nM). mGluR5 antagonists blocked this effect of Aβ , Wang et al . (2004b ).

Techniques: Disruption, Incubation, In Vivo, In Vitro, Transgenic Assay, Concentration Assay, Binding Assay, Functional Assay, Derivative Assay, Inhibition, Transmission Assay, Isolation, Expressing