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