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: Stem Cells International

Article Title: Induced Pluripotent Stem Cells in Dental and Nondental Tissue Regeneration: A Review of an Unexploited Potential

doi: 10.1155/2020/1941629

Figure Lengend Snippet: Studies investigating the regenerative potential of iPSCs.

Article Snippet: Xie et al., 2016 [ ] , Mouse MiPS-01 cell line , In vitro In vivo , Biomimetic nanofiber HA/Col/CTS , Biomimetic nanofiber HA/Col/CTS was associated with upregulation of osteogenic genes..

Techniques: In Vitro, In Vivo, Functional Assay, Irradiation, Transplantation Assay, Cell Culture, Clinical Proteomics, Transduction, Activity Assay, Comparison, Derivative Assay, Over Expression, Expressing, Marker, Modification, Suspension, Mutagenesis, Transfection