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: Cell Reports Medicine

Article Title: Debio 1562M CD37-targeting ADC is highly active and well tolerated in preclinical models of AML and MDS

doi: 10.1016/j.xcrm.2026.102749

Figure Lengend Snippet: Primary AML and MDS blasts show high sensitivity to Debio 1562M in vitro , as well as AML LSCs (A–E) Unsorted blood cells from patients with AML ( n = 31) were assessed for (A) levels of CD37 expression and (B) levels of internalization of labeled Debio 1562M (Debio 1562M-pHRodo MFI). AML blasts were identified using CD45 and CD34 markers, B cells using CD19, and T cells using CD3. (C) The same AML samples were cultured in the presence of increasing doses of Debio 1562M and the viability of the malignant blasts assessed. (D) AML patient samples classified by mutational or pre-treatment status versus sensitivity to Debio 1562M (IC50). (E) Viability of AML blasts and normal B and T cells at 100 nM dose of Debio 1562M are shown. (F) Relative colony-forming units of primary AML samples ( n = 13) following incubation with vehicle, an isotype antibody ADC, or Debio 1562M (1 or 10 nM) for 48 h. (G–J) Unsorted blood cells from patients with MDS ( n = 15) were assessed for (G) levels of CD37 expression and (H) levels of internalization of labeled Debio 1562M (Debio 1562M-pHRodo MFI). MDS blasts were identified using CD34, CD33, and HLA-DR markers; B cells using CD19; and T cells using CD3. (I) The MDS samples analyzed above were cultured in the presence of increasing doses of Debio 1562M and the viability of the malignant blasts assessed. (J) Viability of MDS blasts and normal B and T cells at 100 nM dose of Debio 1562M are shown. Shown are mean ± SEM. One-way ANOVA multiple comparison was used for statistical analysis ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also and and and .

Article Snippet: Combinations of the following antibodies were used for flow cytometry: naratuximab [20nM; Debiopharm], CD19-PE-Cy7 [0.5μg/ml; Beckman Coulter], CD33-APC [0.9 μg/mL Biolegend], CD3-APC-H7 [1.5 μg/mL; BD Biosciences], CD19-APC-Cy7 [1.255 μg/mL; Biolegend], CD33-BV450 [1 μg/mL; BD Biosciences], HLA-DR-PE [25 μg/mL Immunostep], CD45-BV510 [2.5 μg/mL; Fisher Scientific], CD64-BV605 [3.5 μg/mL; Biolegend], CD34-PE-Cy7 [1.5 μg/mL; Biolegend], CD123-APC [1.5 μg/mL; Biolegend], CD3-AF700 [3.5 μg/mL; Biolegend].

Techniques: In Vitro, Expressing, Labeling, Cell Culture, Incubation, Comparison

Journal: iScience

Article Title: Hypoxic-immune axis orchestrates metastatic dissemination via HIF isoform imbalance in pancreatic neuroendocrine tumors

doi: 10.1016/j.isci.2025.114340

Figure Lengend Snippet: Immune cell infiltration in KRAS G12C mutant PNETs (A) Quantitative analysis using Image Plus 6.0 software reveals a significant increase in Tregs (CD4 + , CD25 + , and FoxP3 + ) within KRAS- G12C mutant PNETs compared to wild-type KRAS PNET tissues, suggesting KRAS G12C -driven immunosuppressive cell recruitment. (B) Fluorescence intensity analysis demonstrates reduced infiltration of CD8 + cytotoxic T cells and HLA-DR + activated cells in KRAS G12C mutant tumors, indicative of impaired antitumor immunity. (C) Elevated MDSCs counts in KRAS G12C mutant PNETs correlate with enhanced immune evasion.

Article Snippet: PE Anti-Human HLA-DR Antibody [L243] , Elabscience , E-AB-F1111D.

Techniques: Mutagenesis, Software, Fluorescence

Journal: iScience

Article Title: Hypoxic-immune axis orchestrates metastatic dissemination via HIF isoform imbalance in pancreatic neuroendocrine tumors

doi: 10.1016/j.isci.2025.114340

Figure Lengend Snippet: Tregs, CD8 + T cells, and HLA-DR + cells in KRAS G12C -mutated PNETs (A) Flow cytometry plots and fluorescence intensity histograms demonstrate elevated CD4 + T cell proportions in KRAS G12C patient blood samples compared to wild-type KRAS tumors and healthy controls. (B) Quantification shows a significant enrichment of CD25 + T cells in KRAS G12C patients, surpassing both wild-type KRAS tumors and normal controls. (C) Quantitative data and histogram overlays confirm a substantial increase in FoxP3 + T cells frequency in KRAS G12C patients, with levels moderately elevated compared to wild-type KRAS and significantly higher than healthy individuals. (D) A slight decrease in CD8 + T cell frequency in KRAS G12C samples relative to wild-type KRAS, with levels significantly lower than those in healthy individuals (E) A moderate reduction in HLA-DR + cell frequency in KRAS G12C patients compared to wild-type KRAS, alongside a marked suppression relative to healthy controls.

Article Snippet: PE Anti-Human HLA-DR Antibody [L243] , Elabscience , E-AB-F1111D.

Techniques: Flow Cytometry, Fluorescence