Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation

doi: 10.1073/pnas.2321600121

Figure Lengend Snippet: Deletion of PLC components reduces MHC-I surface levels and changes the MHC-I surface composition. MHC-I surface levels of HAP1 cells with single knockouts of PLC components (wt, gray; ΔHLA, red; ΔTAP1, dark blue; ΔTAP2, light blue; ΔTSN, orange; ΔCRT, yellow; ΔERp57, green; ΔERAP1, purple). Flow cytometric analysis of total MHC-I (W6/32), A*02:01 (BB7.2), and B*40:01 (JOAN-1) surface levels was performed by using the respective primary antibody and Nb AF647 . Exemplary histograms and surface quantity of total MHC-I ( A ), A*02:01 ( B ), and B*40:01 ( C ) molecules per cell. The number of MHC-I surface molecules was determined by using Quantum TM AF647 MESF microspheres and normalizing to ΔHLA cells (mean ± SD, n = 4). Proportion of A*02:01 ( D ), B*40:01 ( E ), and the sum of A*02:01 and B*40:01 proportions ( F ) of the total MHC-I molecules (mean ± SD, n = 4). Dark and light colors correspond to A*02:01 and B*40:01 molecules, displayed in ( D ) and ( E ), respectively. The black dashed line represents the value of wt cells. Welch ANOVA comparing ΔTAP1, ΔTAP2, ΔTSN, ΔCRT, ΔERp57, and ΔERAP1 with HAP1 wt cells was performed (ns, nonsignificant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: HAP1 wt cell line (HLA allomorphs A*02:01, B*40:01, C*03:04) and HAP1 knockout cell lines (ΔHLA with knockouts of HLA-A, -B, -C, and -G, ΔTAP1, ΔTSN, ΔCRT, ΔERp57, ΔERAP1) were kindly provided by Robbert Spaapen (Sanquin Research, Netherlands) ( ).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation

doi: 10.1073/pnas.2321600121

Figure Lengend Snippet: Knockout of PLC components increases the ratio of suboptimally loaded A*02:01 surface complexes. Flow cytometric analyses of extracellular peptide exchange on HAP1 wt cells and HAP1 cells with knockouts of individual components of the antigen-processing machinery after pulsing with ELA ( Top ) or TQV ( Bottom ) peptide (1 µM each). ELA and TQV peptide were used crosswise for background correction. ( A ) Activation of the reporter T cells DMF5 NFκB::eGFP ( Top ) and 1G4 NFκB::eGFP ( Bottom ) upon coculture with pulsed HAP1 cells. T cell activation was determined by eGFP median fluorescence intensity (MFI) and normalized to coculture with HAP1 wt cells (mean ± SD, n = 4). ( B ) Number of ELA-A2*02:01 ( Top ) and TQV-A2*02:01 complexes ( Bottom ) per cell was determined by using the enhanced-affinity sTCRs MEL5 Spy-AF647 and 1G4 Spy-AF647 , respectively, and Quantum TM AF647 MESF microspheres (mean ± SD, n = 4). ( C ) Presentation of ELA-A2*02:01 ( Top ) and TQV-A2*02:01 complexes ( Bottom ) in relation to A*02:01 levels and HAP1 wt cells, illustrating the relative peptide exchange (mean ± SD, n = 4). The black dashed line represents the value of HAP1 wt cells. Welch ANOVA comparing ΔTAP1, ΔTAP2, ΔTSN, ΔCRT, ΔERp57, and ΔERAP1 with HAP1 wt cells was performed (ns, nonsignificant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: HAP1 wt cell line (HLA allomorphs A*02:01, B*40:01, C*03:04) and HAP1 knockout cell lines (ΔHLA with knockouts of HLA-A, -B, -C, and -G, ΔTAP1, ΔTSN, ΔCRT, ΔERp57, ΔERAP1) were kindly provided by Robbert Spaapen (Sanquin Research, Netherlands) ( ).

Techniques: Knock-Out, Activation Assay, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation

doi: 10.1073/pnas.2321600121

Figure Lengend Snippet: Deficiencies in the editing module lead to elevated presentation of abundant, high-affinity peptides. Flow cytometric analyses of ELA-A*02:01 and TQV-A*02:01 presentation by HAP1 wt cells and HAP1 cells with knockouts of individual components of the antigen-processing machinery upon transfection with plasmids encoding for peptide expression of either ELA ( Top ) or TQV ( Bottom ). ELA and TQV peptide were used crosswise for background correction. ( A ) Activation of DMF5 NFκB::eGFP ( Top ) and 1G4 NFκB::eGFP ( Bottom ) reporter T cells upon coculture with peptide-expressing HAP1 cells. T cell activation was determined by eGFP MFI and normalized to coculture with HAP1 wt cells (mean ± SD, n = 4). ( B ) Number of ELA-A2*02:01 ( Top ) and TQV-A2*02:01 complexes ( Bottom ) per peptide-expressing cell was determined by using enhanced-affinity sTCRs MEL5 Spy-AF647 and 1G4 Spy-AF647 , respectively, and Quantum TM AF647 MESF microspheres (mean ± SD, n = 4). ( C ) Presentation of ELA-A2*02:01 ( Top ) and TQV-A2*02:01 complexes ( Bottom ) in relation to A*02:01 levels in peptide-expressing HAP1 cells and HAP1 wt cells (mean ± SD, n = 4). The black dashed line represents the value of HAP1 wt cells. Welch ANOVA comparing ΔTAP1, ΔTAP2, ΔTSN, ΔCRT, ΔERp57, and ΔERAP1 with HAP1 wt cells was performed (ns, nonsignificant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: HAP1 wt cell line (HLA allomorphs A*02:01, B*40:01, C*03:04) and HAP1 knockout cell lines (ΔHLA with knockouts of HLA-A, -B, -C, and -G, ΔTAP1, ΔTSN, ΔCRT, ΔERp57, ΔERAP1) were kindly provided by Robbert Spaapen (Sanquin Research, Netherlands) ( ).

Techniques: Transfection, Expressing, Activation Assay

Journal: Cell reports. Medicine

Article Title: Inflammation-induced mitochondrial and metabolic disturbances in sensory neurons control the switch from acute to chronic pain.

doi: 10.1016/j.xcrm.2023.101265

Figure Lengend Snippet: Figure 4. ATPSc-KMT affects cellular metabolism and redox balance (A) Unsupervised principal-component analysis (PCA) of whole metabolomics from DI-HMRS dataset of HAP1 cells expressing ATPSc-KMT (WT), cells deficient of ATPSc-KMT (knockout [KO]), and KO cells reconstituted with WT (KO + WT) or with catalytically inactive ATPSc-KMT mutant (KO + E117A). (B) Heatmap of metabolite levels that were significantly changed between WT and ATPSc-KMT/ cells. Blue represents reduced intensity, and red represents increased intensity. (C and D) Intensity of lactic acid and GA3P in (C) HAP1 cells and (D) N2A cells that are ATPSc-KMT proficient or deficient. (E) Direct ratio of 3-hydroxybutyrate to acetoacetate measured with DI-HMRS as proxy for mitochondrial NAD+/NADH ratio. (F and G) NAD+/NADH ratio calculated from direct measurement of cellular NAD+ and NADH in (F) HAP1 WT and ATPSc-KMT-deficient cells and (G) in N2A cells after overexpression of ATPSc-KMT or control EV.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Capsaicin Sigma-Aldrich M2028 Fura-2, AM Thermo Fisher Scientific F1221 NaCL Sigma-Aldrich 7647-14-5 KCL Merck 4936 HEPES Thermo Fisher Scientific, Gibco 15630–049 D-(+)-Glucose Sigma-Aldrich G8270 NaOH Merck 1310-73-2 Collagenase type XI Sigma-Aldrich C7657 Dispase Thermo Fisher Scientific, Gibco 17105–041 Poly-L-Lysine Sigma-Aldrich P9155 Laminin Sigma-Aldrich L2020 Critical commercial assays SYBR Select Master Mix Applied biosystems 4472908 Seahorse Xfe24 FluxPak Agilent Technologies 102340–100 Experimental models: Cell lines Neuro 2A ATCC ATCC-CLL-131 HAP1 WT (wild-type parental cell line) Horizon Discovery C631 HAP1 ATPSCKMT/FAM173B KO cells Horizon Discovery HZGHC000533c006 HAP1 ATPSCKMT/FAM173B KO cells complemented with human ATPSCKMT-3xFLAG, either WT or E117A-mutated Malecki et al.17 N/A Deposited data Metabolomics data (related to Figures 2 and 4) Dataverse.nl https://doi.org/10.34894/MRMGNW Experimental models: Organisms/strains Mouse: C57Bl/6JRj Janvier https://janvier-labs.com/en/fiche_ produit/2_c57bl-6jrj_mouse/ Mouse: C57BL/6NJ-Atpsckmtem1(IMPC)J/Mmjax Jackson Laboratory 051063-JAX Oligonucleotides For quantitative polymerase chain reaction primers, see text This paper N/A For phosphorothioated antisense oligonucleotides, see text This paper N/A Recombinant DNA HSV plasmid S0109-EV Roy et al.79 N/A HSV plasmid S0109- ATPSc-KMT Willemen et al.16 N/A Software and algorithms ImageJ N/A N/A MetaboAnalyst https://www.metaboanalyst.ca N/A Diva BDbiosciences N/A Graphpad Prism 8.3 Graphpad N/A Qual Browser Thermo Fisher Scientific v2.0.7 Proteome Discoverer Thermo Fisher Scientific N/A Exactive Tune Software (version 2.9.0) Thermo Fisher Scientific N/A Chipsoft (version 8.3.1) Advion Biosciences N/A Xcalibur software (version 3.0) Thermo Fisher Scientific N/A Thermo TraceFinderTM 4.1 Thermo Fisher Scientific N/A HEKA Patchmaster 2x90.2 Multichannel Systems MCS GmbH N/A Other LSRFortessa flow cytometer BDbiosciences https://www.bdbiosciences.com/ en-nl/products/instruments/flowcytometers/research-cellanalyzers/bd-lsrfortessa (Continued on next page) e2 Cell Reports Medicine 4, 101265, November 21, 2023

Techniques: Expressing, Knock-Out, Mutagenesis, Over Expression, Control