Journal: International Journal of Molecular Sciences

Article Title: HLA-E-Directed Accumulation of KIR + NKG2C + NK Cells upon HCMV Peptide Presentation In Vitro: Association with the Ex Vivo Phenotype

doi: 10.3390/ijms27136087

Figure Lengend Snippet: NK cell phenotype ex vivo: ( a ) the proportion of KIR + cells in total NK cell population of HCMV − and HCMV + individuals (left), correlation between the proportion of KIR + ex vivo and the titer of antibodies to HCMV (right); ( b ) the proportion of NKG2C + cells in the NK cell population of HCMV − and HCMV + individuals (left), correlation between the proportion of NKG2C + ex vivo and the titer of antibodies to HCMV (right); ( c ) the proportion of KIR + NKG2C + cells in the NK cell population of HCMV − and HCMV + individuals (left), correlation between the proportion of KIR + NKG2C + ex vivo and the titer of antibodies to HCMV (right); ( d ) the proportions of NKG2A + , HLA-DR + , and HLA-DR + CD56 bright cells in the NK cell population of HCMV − and HCMV + individuals; ( e ) hierarchical tree clustering of HCMV + donors based on HLA-DR + , HLA-DR + CD56 bright , NKG2C + , NKG2A + , KIR2DL2/3 + , CD57 + NKG2C + , and KIR2DL2/3 + NKG2C + proportions in NK cell population, Ward’s method, standardized data; ( f ) the proportions of HLA-DR + CD56 bright , KIR2DL2/3 + , HLA-DR + , CD56 bright , NKG2A + , NKG2C + , CD57 + NKG2C + , and KIR2DL2/3 + NKG2C + cells in the NK cell population of different clusters’ donors. Number of donors: N = 91 (HCMV + -67, HCMV − -24). Statistical analysis was performed using nonparametric Mann–Whitney U test ( a – d ) or nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison post hoc test ( f ) (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ns—not significant); means ± SD are shown. Correlation analysis was done using Pearson’s correlation; p < 0.05 was considered statistically significant.

Article Snippet: Mouse anti-human fluorescent-labeled antibodies that were used for PBMC/NK cell surface staining were as follows: CD3-APC-Vio 770 (Clone REA613), CD56-FITC, CD56-PE-Vio 615, CD56-PE-Vio 770 (Clone REA196), CD57-VioBlue, CD57-APC-Vio 770 (Clone TB03), KIR2DL2/3-FITC, KIR2DL2/3-PE-Vio 615 (Clone REA 1006), KIR2DL2/3-APC (Clone DX27), NKG2A-PE, NKG2A-PE-Vio 770 (Clone REA110), NKG2C-FITC, NKG2C-PE (Clone REA205), HLA-DR-PE-Vio 770 (Clone REA805) (Miltenyi Biotech); CD3-PerCP (Clone HIT3a), CD56-Brilliant Violet 421 (Clone 5.1H11) (Sony, San Jose, CA, USA) (also shown in ).

Techniques: Ex Vivo, MANN-WHITNEY, Comparison

Journal: International Journal of Molecular Sciences

Article Title: HLA-E-Directed Accumulation of KIR + NKG2C + NK Cells upon HCMV Peptide Presentation In Vitro: Association with the Ex Vivo Phenotype

doi: 10.3390/ijms27136087

Figure Lengend Snippet: Proliferative activity and phenotype of bulk NK cell cultures obtained with and without LFL presentation: ( a ) expansion coefficient of bulk NK cell cultures after cultivation with and without HCMV peptide presentation, number of donors: N = 18 (HCMV + -13, HCMV − -5); ( b ) proportions of KIR + and KIR + NKG2C + cells in bulk NK cell cultures obtained with and without peptide presentation, with representative cytometric data, N = 18 (HCMV + -13, HCMV − -5); ( c ) correlation between the relative expansion coefficient K = K(LFL)/K (no peptide) and the proportion of KIR + NK cells ex vivo, N = 14 (HCMV + -9, HCMV − -5); ( d ) correlation between the expansion coefficient of NK cell cultures obtained without (left) and with (right) LFL presentation and the proportion of HLA-DR + NK cells ex vivo, N = 13 (HCMV + -8, HCMV − -5); ( e ) correlation between the relative proportion of NKG2C + (NKG2C ratio = NKG2C + ,% (LFL)/NKG2C + ,% (no peptide)) cells in cultures obtained with LFL presentation and the proportion of HLA-DR + ex vivo, N = 11 (HCMV + -6, HCMV − -5); ( f ) correlation between the relative proportion of KIR + cells (KIR ratio = KIR + ,% (LFL)/KIR + ,% (no peptide)) in cultures presented with the LFL peptide and the proportion of CD57 + NKG2C + ex vivo, N = 11 (HCMV + -6, HCMV − -5); ( g ) proportion of non-viable cells in spheroids of HLA-E-expressing tumor lines MCF7 and SKOV3 after co-incubation with bulk cultures of NK cells activated with and without peptide presentation, with representative cytometric data; 6 to 8 replicates are presented. Cultivation time T = 14 days, total number of donors: N = 18 (HCMV + -13, HCMV − -5). Statistical analysis was performed using a nonparametric Wilcoxon’s test ( b ) or nonparametric Mann–Whitney test ( a , g ) (* p < 0.05, ** p <0.01, ns—not significant); means ± SD are shown. Correlation analysis was done using Spearman’s correlation for non-normally distributed data; p < 0.05 was considered statistically significant.

Article Snippet: Mouse anti-human fluorescent-labeled antibodies that were used for PBMC/NK cell surface staining were as follows: CD3-APC-Vio 770 (Clone REA613), CD56-FITC, CD56-PE-Vio 615, CD56-PE-Vio 770 (Clone REA196), CD57-VioBlue, CD57-APC-Vio 770 (Clone TB03), KIR2DL2/3-FITC, KIR2DL2/3-PE-Vio 615 (Clone REA 1006), KIR2DL2/3-APC (Clone DX27), NKG2A-PE, NKG2A-PE-Vio 770 (Clone REA110), NKG2C-FITC, NKG2C-PE (Clone REA205), HLA-DR-PE-Vio 770 (Clone REA805) (Miltenyi Biotech); CD3-PerCP (Clone HIT3a), CD56-Brilliant Violet 421 (Clone 5.1H11) (Sony, San Jose, CA, USA) (also shown in ).

Techniques: Activity Assay, Ex Vivo, Expressing, Incubation, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: HLA-E-Directed Accumulation of KIR + NKG2C + NK Cells upon HCMV Peptide Presentation In Vitro: Association with the Ex Vivo Phenotype

doi: 10.3390/ijms27136087

Figure Lengend Snippet: Phenotype of NK cell subset cultures with different KIR, NKG2C, and CD57 expression profiles, activated in the presence or absence of LFL peptide: ( a ) De novo NKG2C expression in cultures of NKG2C-negative subsets with different expression of KIR and CD57, activated in the presence or absence of LFL peptide; ( b ) stability of NKG2C expression in cultures of NKG2C-positive subsets with different expression of KIR and CD57, activated in the presence or absence of LFL peptide; ( c ) de novo KIR expression in cultures of CD57 − KIR − NKG2C + subset, activated in the presence or absence of LFL; ( d ) stability of NKG2C expression in cultures of CD57 − KIR − NKG2C + , activated in the presence or absence of LFL; ( e , f ) correlation between the proportion of HLA-DR + NK cells ex vivo and the stability of KIR expression ( e ) and NKG2C de novo expression ( f ) in CD57 − KIR + NKG2C − subset cultures that were and were not presented with LFL; ( g ) correlation between the proportion of KIR + NKG2C + NK cells ex vivo and KIR de novo expression in CD57 − KIR − NKG2C + subset cultures that were and were not presented with LFL. Duration of cultivation: T = 12 days, number of donors: N = 17 (HCMV + -11, HCMV − -6). Statistical analysis was performed using a nonparametric Friedman’s test followed by Dunn’s multiple comparison post hoc test ( a , b ) or Wilcoxon’s test ( c , d ) (* p < 0.05, ** p < 0.01, ns—not significant). Correlation analysis was done using Spearman’s correlation; p < 0.05 was considered statistically significant.

Article Snippet: Mouse anti-human fluorescent-labeled antibodies that were used for PBMC/NK cell surface staining were as follows: CD3-APC-Vio 770 (Clone REA613), CD56-FITC, CD56-PE-Vio 615, CD56-PE-Vio 770 (Clone REA196), CD57-VioBlue, CD57-APC-Vio 770 (Clone TB03), KIR2DL2/3-FITC, KIR2DL2/3-PE-Vio 615 (Clone REA 1006), KIR2DL2/3-APC (Clone DX27), NKG2A-PE, NKG2A-PE-Vio 770 (Clone REA110), NKG2C-FITC, NKG2C-PE (Clone REA205), HLA-DR-PE-Vio 770 (Clone REA805) (Miltenyi Biotech); CD3-PerCP (Clone HIT3a), CD56-Brilliant Violet 421 (Clone 5.1H11) (Sony, San Jose, CA, USA) (also shown in ).

Techniques: Expressing, Ex Vivo, Comparison

Journal: International Journal of Molecular Sciences

Article Title: HLA-E-Directed Accumulation of KIR + NKG2C + NK Cells upon HCMV Peptide Presentation In Vitro: Association with the Ex Vivo Phenotype

doi: 10.3390/ijms27136087

Figure Lengend Snippet: Functional activity of NK cell subset cultures with different expression of KIR, NKG2C, and CD57, activated in the presence or absence of LFL peptide: ( a ) proportion of IFNγ-producing NK cells in cultures of NKG2C-positive subsets with different expression of KIR and CD57; ( b ) proportion of IFNγ-producing NK cells in cultures of the CD57 − KIR + NKG2C − subset, to which LFL peptide was and was not presented; ( c ) proportion of degranulating CD107a + NK cells in cultures of NKG2C-positive subsets with different expression of KIR and CD57 upon co-incubation with K562 targets; ( d ) correlation between the proportion of IFNγ-producing cells in CD57 − KIR + NKG2C − subset cultures presented with LFL peptide and the proportion of NK cells NKG2C + ex vivo; ( e ) correlation between the proportion of IFNγ-producing cells in CD57 − KIR − NKG2C + subset cultures presented with LFL peptide and the proportion of NK cells KIR + NKG2C + ex vivo; ( f ) correlation between the proportion of IFNγ-producing cells in CD57 − KIR + NKG2C + subset cultures presented with LFL peptide and the proportion of NK cells KIR + NKG2C + ex vivo; ( g ) correlation between the proportion of CD107a + cells in CD57 − KIR + NKG2C + subset cultures presented with LFL peptide and the proportion of HLA-DR + NK cells in the CD56 bright fraction ex vivo; ( h ) correlation between the relative proportion of CD107a + cells in CD57 + KIR + NKG2C + subset cultures presented with LFL peptide and the proportion of HLA-DR + CD56 bright NK cells ex vivo; ( i ) correlation between the proportion of IFNγ-producing cells in CD57 − KIR + NKG2C + subset cultures presented with LFL peptide and the proportion of NKG2A + NK cells ex vivo; ( j ) correlation between the proportion of CD107a + cells in CD57 + KIR + NKG2C + subset cultures presented with the LFL peptide and the proportion of NKG2A + NK cells ex vivo. Duration of cultivation: T = 17 days, number of donors: N = 17 (HCMV + -11, HCMV − -6). Statistical analysis was performed using a nonparametric Wilcoxon’s test ( a – c ) (* p < 0.05, ** p < 0.01, ns—not significant). Correlation analysis was done using Spearman’s correlation; p < 0.05 was considered statistically significant.

Article Snippet: Mouse anti-human fluorescent-labeled antibodies that were used for PBMC/NK cell surface staining were as follows: CD3-APC-Vio 770 (Clone REA613), CD56-FITC, CD56-PE-Vio 615, CD56-PE-Vio 770 (Clone REA196), CD57-VioBlue, CD57-APC-Vio 770 (Clone TB03), KIR2DL2/3-FITC, KIR2DL2/3-PE-Vio 615 (Clone REA 1006), KIR2DL2/3-APC (Clone DX27), NKG2A-PE, NKG2A-PE-Vio 770 (Clone REA110), NKG2C-FITC, NKG2C-PE (Clone REA205), HLA-DR-PE-Vio 770 (Clone REA805) (Miltenyi Biotech); CD3-PerCP (Clone HIT3a), CD56-Brilliant Violet 421 (Clone 5.1H11) (Sony, San Jose, CA, USA) (also shown in ).

Techniques: Functional Assay, Activity Assay, Expressing, Incubation, Ex Vivo

Journal: Frontiers in Immunology

Article Title: Clinical MAPPs: a personalized healthcare-driven assay for the direct identification of potential T cell epitopes in patients

doi: 10.3389/fimmu.2026.1879253

Figure Lengend Snippet: Heat map of HLA-DR-associated adalimumab peptides presented by moDCs from 17 healthy buffy-coat donors. HLA-DR-associated peptides were immunoprecipitated from 5.4x10 6 adalimumab-loaded moDCs and eluted peptides were identified by LC-MS/MS using the QE-HFX mass spectrometer. The adalimumab sequence regions are separated by the variable domain of the heavy chain (VH), constant domain of the heavy chain (CH1), variable domain of the light chain (VL), constant region of the kappa type light chain (Ck), and the fragment crystallizable (Fc) region. Identified clusters are signified by the colored regions spanning from dark red to yellow, which represent the antibody peptide abundance (as a log2 score) per position from low to high, respectively (indicated in the legend). Complementarity-determining regions (CDRs) 1 to 3 are indicated as pink, green, and blue vertical lines, respectively, along the sequence of the VH and VL domains. Identified peptide clusters are indicated as C1 to C11 below the horizontal axis of the relative adalimumab amino acid sequence position (see Supplementary Table 1 ). Donor number (D1-D17) is denoted on the vertical axis. Vertical pink lines denote a modified amino acid (here oxidized VH Met 86 ). The data was generated using a Q-Exactive™ HF-X Orbitrap mass spectrometer.

Article Snippet: Lysates were incubated either with 10 μg (unless otherwise specified) anti-human HLA-DR biotin (L243 clone, RayBiotech; Cat: #150-10306) or with a mixed preparation of 10 μg anti-human HLA-DR biotin (L243 clone, RayBiotech), 15 μg anti-human HLA-DQ biotin (clone SPV-L3, Biotium; Cat: #BNCB0200-500) and 15 μg anti-human MHC-II pan biotin (clone WR18; Bio-Techne, Cat: #NB100-64358B).

Techniques: Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Modification, Generated

Journal: Frontiers in Immunology

Article Title: Clinical MAPPs: a personalized healthcare-driven assay for the direct identification of potential T cell epitopes in patients

doi: 10.3389/fimmu.2026.1879253

Figure Lengend Snippet: An automated sample preparation protocol leads to the presentation of additional adalimumab-derived peptide clusters overlapping with CDRs in the MAPPs assay. Comparative heat map of HLA-DR-associated adalimumab-derived peptides presented by differing numbers (3.5x10 5 , 7.0x10 5 , 1.35x10 6 , 2.7x10 6 , 5.4x10 6 ) moDCs from the same buffy-coat donor. Eluted peptides from both preparation types were identified by LC-MS/MS using the Bruker TIMS ToF mass spectrometer. The adalimumab sequence regions are separated by the variable domain of the heavy chain (VH) and light chain (VL), constant domain of the heavy chain (CH1), constant region of the kappa type light chain (Ck), and the fragment crystallizable (Fc) region. Clusters are signified by the colored regions spanning from dark red to yellow, which represent the antibody peptide abundance (as a log2 score) per position from low to high, respectively (indicated in the legend). Complementarity-determining regions (CDRs) 1 to 3 are indicated as pink, green, and blue vertical lines, respectively, along the sequence of the VH and VL domains. Identified clusters are indicated as C1 to C16 below the horizontal axis of the relative amino acid sequence corresponding to each domain (see Supplementary Table 1 ). Donor number, protocol used (Bravo vs manual), and cell number of each sample is denoted on the vertical axis. Identified peptide clusters in relation to the clusters identified in Figure 1 are indicated below the horizontal axis of the relative adalimumab amino acid sequence position. Data obtained from 3 buffy-coat donors. Vertical pink lines denote a modified amino acid. The data was generated using a TIMS ToF Pro2 mass spectrometer.

Article Snippet: Lysates were incubated either with 10 μg (unless otherwise specified) anti-human HLA-DR biotin (L243 clone, RayBiotech; Cat: #150-10306) or with a mixed preparation of 10 μg anti-human HLA-DR biotin (L243 clone, RayBiotech), 15 μg anti-human HLA-DQ biotin (clone SPV-L3, Biotium; Cat: #BNCB0200-500) and 15 μg anti-human MHC-II pan biotin (clone WR18; Bio-Techne, Cat: #NB100-64358B).

Techniques: Automated Sample Preparation, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Modification, Generated

Journal: Frontiers in Immunology

Article Title: Clinical MAPPs: a personalized healthcare-driven assay for the direct identification of potential T cell epitopes in patients

doi: 10.3389/fimmu.2026.1879253

Figure Lengend Snippet: Applying the clinical MAPPs assay protocol to be set forth using cryopreserved PBMCs in comparison to fresh PBMCs. Cryopreserved and fresh PBMCs each obtained from 10 mL whole blood per donor were subjected to the MAPPs assay. moDCs were challenged with KLH and HLA-DR-associated peptides were immunoprecipitated and identified by LC-MS/MS using the TIMS ToF mass spectrometer. Identified clusters are signified by the colored regions spanning from dark red to yellow, which represent the abundance of KLH-derived peptides from low to high, respectively. Donor number and starting material (fresh or frozen PBMCs) is denoted on the vertical axis. Data obtained from 10 whole blood donors. Starting number of CD14 + monocytes: D43, 2.63x10 5 cells; D44, 1.97x10 5 cells; D45, 2.40x10 5 cells; D46, 2.08x10 5 cells; D47, 1.26x10 5 cells; D48, 4.48x10 5 cells; D49, 1.86x10 5 cells; D50, 2.13x10 5 cells; D51, 1.09x10 5 cells; D52, 3.01x10 5 cells. All data were generated using a TIMS-ToF Pro2 mass spectrometer.

Article Snippet: Lysates were incubated either with 10 μg (unless otherwise specified) anti-human HLA-DR biotin (L243 clone, RayBiotech; Cat: #150-10306) or with a mixed preparation of 10 μg anti-human HLA-DR biotin (L243 clone, RayBiotech), 15 μg anti-human HLA-DQ biotin (clone SPV-L3, Biotium; Cat: #BNCB0200-500) and 15 μg anti-human MHC-II pan biotin (clone WR18; Bio-Techne, Cat: #NB100-64358B).

Techniques: Comparison, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Derivative Assay, Generated

Journal: Frontiers in Immunology

Article Title: Clinical MAPPs: a personalized healthcare-driven assay for the direct identification of potential T cell epitopes in patients

doi: 10.3389/fimmu.2026.1879253

Figure Lengend Snippet: moDCs and DCs present similar phenotypic characteristics when used in the MAPPs assay (A) Flow cytometry analysis comparing monocytes with immature and stimulated moDC. Shown are the CD83 and CD14 surface marker expression identified from the parent CD11c + CD14 population. Data is representative of 2 buffy-coat donors. (B) Flow cytometry analysis demonstrative of highly enriched, sorted DCs. Left panel : the low signals in the gated CD14 and CD19 positive cells, for the identification of monocytes and B cells, highlight a homogenous parent DC population. Middle and right panels: Plasmacytoid DC (PDC), myeloid DC type 1 (MDC1), and MDC2 subsets are identified via CD303, CD1c, and CD141, respectively. The numbers correspond to the percentage of cells within each respective gate. Data is representative of 2 buffy-coat donors. (C) Heat map of human HLA-DR-associated adalimumab peptides presented by moDCs and naturally existing blood DCs isolated from the same donor using 19 healthy buffy-coats. Eluted peptides were identified by LC-MS/MS using the Bruker TIMS ToF mass spectrometer. Identified peptide clusters are indicated below the horizontal axis of the relative adalimumab amino acid sequence position, and numbered relative to the clusters identified in Figures 1 and . The number of moDCs and DCs were adjusted to the same cell number: D21, 6.83x10 5 cells; D22, 5.46x10 5 cells; D23, 8.93x10 5 cells; D24, 3.60x10 5 cells; D25, 6.00x10 5 cells; D26, 6.83x10 5 cells; D27, 8.57x10 5 cells; D28, 9.83x10 5 cells; D29, 8.79x10 5 cells; D30, 4.00x10 5 cells; D31, 6.00x10 5 cells; D32, 10.00x10 5 cells; D33, 5.50x10 5 cells; D34, 6.03x10 5 cells; D35, 5.82x10 5 cells; D36, 7.00x10 5 cells; D37, 14.00x10 5 cells; D38, 10.00x10 5 cells; D39, 27.00x10 5 cells. The data was generated using a TIMS ToF Pro2 mass spectrometer.

Article Snippet: Lysates were incubated either with 10 μg (unless otherwise specified) anti-human HLA-DR biotin (L243 clone, RayBiotech; Cat: #150-10306) or with a mixed preparation of 10 μg anti-human HLA-DR biotin (L243 clone, RayBiotech), 15 μg anti-human HLA-DQ biotin (clone SPV-L3, Biotium; Cat: #BNCB0200-500) and 15 μg anti-human MHC-II pan biotin (clone WR18; Bio-Techne, Cat: #NB100-64358B).

Techniques: Flow Cytometry, Marker, Expressing, Isolation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Generated

Journal: Frontiers in Immunology

Article Title: Clinical MAPPs: a personalized healthcare-driven assay for the direct identification of potential T cell epitopes in patients

doi: 10.3389/fimmu.2026.1879253

Figure Lengend Snippet: Dendritic cell versus monocyte-derived dendritic cell (moDC) peptide presentation and activation time-course. (A) Heat map of HLA-DR-associated keyhole limpet hemocyanin (KLH) peptides in the MAPPs assay. HLA-DR-associated peptides were immunoprecipitated and eluted peptides were identified byLC-MS/MS using the TIMS ToF Pro2 mass spectrometer. Identified clusters are signified by the colored regions spanning from dark red to yellow, which represent the abundance of KLH-derived peptides from low to high, respectively. Donor number, cell type (moDC or DC), and stimulation time-point is denoted on the vertical axis. The number of moDCs and DCs were adjusted to the same cell number: donor 40, 2.17x10 5 cells; donor 41, 3.54 x10 5 cells; donor 42, 2.22 x10 5 cells (B) 95% bootstrapped confidence intervals of the percent of parent for the surface expression of each DC activation marker (CD40, CD80, CD83, CD86, and HLA-DR). Individual donors are denoted as thin lines, mean values as bold lines, and confidence intervals as transparent colored areas. moDCs or DCs were isolated from the same donor using 3 healthy buffy-coats, and both cell types were treated and stimulated with KLH and lipopolysaccharide (LPS) for either 12, 18, and 24 hours.

Article Snippet: Lysates were incubated either with 10 μg (unless otherwise specified) anti-human HLA-DR biotin (L243 clone, RayBiotech; Cat: #150-10306) or with a mixed preparation of 10 μg anti-human HLA-DR biotin (L243 clone, RayBiotech), 15 μg anti-human HLA-DQ biotin (clone SPV-L3, Biotium; Cat: #BNCB0200-500) and 15 μg anti-human MHC-II pan biotin (clone WR18; Bio-Techne, Cat: #NB100-64358B).

Techniques: Derivative Assay, Activation Assay, Immunoprecipitation, Tandem Mass Spectroscopy, Mass Spectrometry, Expressing, Marker, Isolation

Journal: Frontiers in Immunology

Article Title: Clinical MAPPs: a personalized healthcare-driven assay for the direct identification of potential T cell epitopes in patients

doi: 10.3389/fimmu.2026.1879253

Figure Lengend Snippet: Applying the clinical MAPPs assay protocol using cryopreserved PBMCs either from 43 patients enrolled in a clinical trial (upper panel) or from 30 healthy control donors (lower panel). The heavy chain of the biotherapeutic’s amino acid sequences is represented with the positions of the three hypervariable regions (CDR1, 2 and 3) indicated by the colored vertical lines. The PBMCs obtained from patients enrolled in a clinical trials were isolated from 10 mL whole blood prior to immediate cryopreservation. After thawing, CD14 + monocytes were isolated and differentiated to immature moDCs, which were challenged with the biotherapeutic and matured with LPS prior to the MAPPs assay. The same protocol was used to analyze cryopreserved PBMCs obtained from a healthy control donor cohort. Heat map of HLA-DR-associated keyhole limpet hemocyanin (KLH) peptides in the MAPPs assay. HLA-DR, DP and DQ-associated peptides were immunoprecipitated and eluted peptides were identified by LC-MS/MS using the TIMS ToF mass spectrometer For analysis, 1.35x10 6 monocytes were used for healthy controls while there was no cell number adjustment for patient’s samples (i.e., 100% of the obtained monocytes were used for the assay – see Supplementary Table 4 for detail). The stars denote samples for which there were no compound-related results obtained in the MAPPs assay. All data were collected using a TIMS ToF Pro2 mass spectrometer.

Article Snippet: Lysates were incubated either with 10 μg (unless otherwise specified) anti-human HLA-DR biotin (L243 clone, RayBiotech; Cat: #150-10306) or with a mixed preparation of 10 μg anti-human HLA-DR biotin (L243 clone, RayBiotech), 15 μg anti-human HLA-DQ biotin (clone SPV-L3, Biotium; Cat: #BNCB0200-500) and 15 μg anti-human MHC-II pan biotin (clone WR18; Bio-Techne, Cat: #NB100-64358B).

Techniques: Control, Clinical Proteomics, Isolation, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry