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(A). (i) Expression of Pf GBP-130 ECD fused with Transferrin membrane domain on the membrane of CHO K1 cells by infecting the lentiviral vector; pMSCV Puro and its immunofluorescence analysis using anti-rabbit Pf GBP antibody. (ii) CHO K1 cells expressing Pf GBP-130 ECD bind LFA-1 αI-Fc. Binding of purified LFA-1 αI-Fc to Pf GBP-130 ECD expressing CHO cells was assessed by FACS using an PE-Texas red anti-human IgG antibody. (B) NK cells activation in the presence of CHO K1 cells expressing Pf GBP ECD. Human NK cells were purified (>95%) from fresh PBMC and co-cultured with CHO K1 cells expressing Pf GBP ECD in 2: 1 ratio (20,000 CHO-K1 cells:10,000 NK cells) and these cells were stimulated with (Poly I:C/lipofectamine 2000) for 24 h. NK cells were separated from adherent CHO K1 cells and NK cells activation was assessed by assaying the expression of activation markers <t>(CD69,</t> CD25) and a degranulation marker; CD107a. NK cells co-cultured with CHO K1 cells expressing Pf GBP-ECD protein showed significant increase in the expression of CD25 and CD69, as well as CD107a in comparison to the NK cells co-cultured with mock CHO cells. Addition of anti-CD11a (HI111 clone) antibodies reduced the expression of both activation and degranulation markers. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.
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(A). (i) Expression of Pf GBP-130 ECD fused with Transferrin membrane domain on the membrane of CHO K1 cells by infecting the lentiviral vector; pMSCV Puro and its immunofluorescence analysis using anti-rabbit Pf GBP antibody. (ii) CHO K1 cells expressing Pf GBP-130 ECD bind LFA-1 αI-Fc. Binding of purified LFA-1 αI-Fc to Pf GBP-130 ECD expressing CHO cells was assessed by FACS using an PE-Texas red anti-human IgG antibody. (B) NK cells activation in the presence of CHO K1 cells expressing Pf GBP ECD. Human NK cells were purified (>95%) from fresh PBMC and co-cultured with CHO K1 cells expressing Pf GBP ECD in 2: 1 ratio (20,000 CHO-K1 cells:10,000 NK cells) and these cells were stimulated with (Poly I:C/lipofectamine 2000) for 24 h. NK cells were separated from adherent CHO K1 cells and NK cells activation was assessed by assaying the expression of activation markers <t>(CD69,</t> CD25) and a degranulation marker; CD107a. NK cells co-cultured with CHO K1 cells expressing Pf GBP-ECD protein showed significant increase in the expression of CD25 and CD69, as well as CD107a in comparison to the NK cells co-cultured with mock CHO cells. Addition of anti-CD11a (HI111 clone) antibodies reduced the expression of both activation and degranulation markers. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.
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(A). (i) Expression of Pf GBP-130 ECD fused with Transferrin membrane domain on the membrane of CHO K1 cells by infecting the lentiviral vector; pMSCV Puro and its immunofluorescence analysis using anti-rabbit Pf GBP antibody. (ii) CHO K1 cells expressing Pf GBP-130 ECD bind LFA-1 αI-Fc. Binding of purified LFA-1 αI-Fc to Pf GBP-130 ECD expressing CHO cells was assessed by FACS using an PE-Texas red anti-human IgG antibody. (B) NK cells activation in the presence of CHO K1 cells expressing Pf GBP ECD. Human NK cells were purified (>95%) from fresh PBMC and co-cultured with CHO K1 cells expressing Pf GBP ECD in 2: 1 ratio (20,000 CHO-K1 cells:10,000 NK cells) and these cells were stimulated with (Poly I:C/lipofectamine 2000) for 24 h. NK cells were separated from adherent CHO K1 cells and NK cells activation was assessed by assaying the expression of activation markers (CD69, CD25) and a degranulation marker; CD107a. NK cells co-cultured with CHO K1 cells expressing Pf GBP-ECD protein showed significant increase in the expression of CD25 and CD69, as well as CD107a in comparison to the NK cells co-cultured with mock CHO cells. Addition of anti-CD11a (HI111 clone) antibodies reduced the expression of both activation and degranulation markers. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

Journal: bioRxiv

Article Title: LFA-1 Interaction with GBP-130 on Plasmodium falciparum -infected Red Blood Cells mediates NK Cell Activation and Parasite Control

doi: 10.1101/2025.08.20.671208

Figure Lengend Snippet: (A). (i) Expression of Pf GBP-130 ECD fused with Transferrin membrane domain on the membrane of CHO K1 cells by infecting the lentiviral vector; pMSCV Puro and its immunofluorescence analysis using anti-rabbit Pf GBP antibody. (ii) CHO K1 cells expressing Pf GBP-130 ECD bind LFA-1 αI-Fc. Binding of purified LFA-1 αI-Fc to Pf GBP-130 ECD expressing CHO cells was assessed by FACS using an PE-Texas red anti-human IgG antibody. (B) NK cells activation in the presence of CHO K1 cells expressing Pf GBP ECD. Human NK cells were purified (>95%) from fresh PBMC and co-cultured with CHO K1 cells expressing Pf GBP ECD in 2: 1 ratio (20,000 CHO-K1 cells:10,000 NK cells) and these cells were stimulated with (Poly I:C/lipofectamine 2000) for 24 h. NK cells were separated from adherent CHO K1 cells and NK cells activation was assessed by assaying the expression of activation markers (CD69, CD25) and a degranulation marker; CD107a. NK cells co-cultured with CHO K1 cells expressing Pf GBP-ECD protein showed significant increase in the expression of CD25 and CD69, as well as CD107a in comparison to the NK cells co-cultured with mock CHO cells. Addition of anti-CD11a (HI111 clone) antibodies reduced the expression of both activation and degranulation markers. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

Article Snippet: The following antibodies were used for staining: anti-human CD3 (biotin, OKT3; Elabsciences), anti CD16 (3G8; Biolegend), anti CD11a (HI111; BioLegend), CD56-PE (5.1H11; Elabsciences), Ultra-LEAFTM Purified Human IgG1 Isotype (Biolegend), Goat anti-Human IgG Alexa FluorTM 488 (Invitrogen), Goat anti-Human IgG Secondary Antibody, Alexa FluorTM 594 (Invitrogen), (CD107a (H4A3; Elabsciences), CD25-Alexa Fluor® 488 (BioLegend), and CD69 (FN50; Elabsciences).

Techniques: Expressing, Membrane, Plasmid Preparation, Immunofluorescence, Binding Assay, Purification, Activation Assay, Cell Culture, Marker, Comparison

(A) Activated human NK cells eliminate iRBCs in vitro. Human NK cells when co-cultured with iRBCs reduce parasitemia significantly after 96h and in the presence of anti- Pf GBP-130 antibodies this reduction in parasitemia was blocked. Presence of anti-GBP-130 abs resulted in parasitemia simialr to the control when NK cells were incubated with iRBCs alone. (B) NK cells activation in the presence of iRBCs. Human NK cells were were purified (>95%) from fresh PBMC and co-cultured with synchronized schizont stage iRBCs at a parasitemia of 0.5% in 10:1 ratio (NK: iRBC) for 48h. Quantification of activation and degranulation markers was performed after 48 hours. NK cells co-cultured with iRBCs showed significant increase in the expression of CD25 and CD69, the two activation markers as well as for the expression of CD107a, a degranulation marker in comparison to the NK cells co-cultured with RBCs alone. Addition of anti-rabbit PfGBP-130 antibodies reduced the expression of both activation and degranulation markers in these NK cells in comparison to rabbit IgG isotype control. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

Journal: bioRxiv

Article Title: LFA-1 Interaction with GBP-130 on Plasmodium falciparum -infected Red Blood Cells mediates NK Cell Activation and Parasite Control

doi: 10.1101/2025.08.20.671208

Figure Lengend Snippet: (A) Activated human NK cells eliminate iRBCs in vitro. Human NK cells when co-cultured with iRBCs reduce parasitemia significantly after 96h and in the presence of anti- Pf GBP-130 antibodies this reduction in parasitemia was blocked. Presence of anti-GBP-130 abs resulted in parasitemia simialr to the control when NK cells were incubated with iRBCs alone. (B) NK cells activation in the presence of iRBCs. Human NK cells were were purified (>95%) from fresh PBMC and co-cultured with synchronized schizont stage iRBCs at a parasitemia of 0.5% in 10:1 ratio (NK: iRBC) for 48h. Quantification of activation and degranulation markers was performed after 48 hours. NK cells co-cultured with iRBCs showed significant increase in the expression of CD25 and CD69, the two activation markers as well as for the expression of CD107a, a degranulation marker in comparison to the NK cells co-cultured with RBCs alone. Addition of anti-rabbit PfGBP-130 antibodies reduced the expression of both activation and degranulation markers in these NK cells in comparison to rabbit IgG isotype control. * Denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

Article Snippet: The following antibodies were used for staining: anti-human CD3 (biotin, OKT3; Elabsciences), anti CD16 (3G8; Biolegend), anti CD11a (HI111; BioLegend), CD56-PE (5.1H11; Elabsciences), Ultra-LEAFTM Purified Human IgG1 Isotype (Biolegend), Goat anti-Human IgG Alexa FluorTM 488 (Invitrogen), Goat anti-Human IgG Secondary Antibody, Alexa FluorTM 594 (Invitrogen), (CD107a (H4A3; Elabsciences), CD25-Alexa Fluor® 488 (BioLegend), and CD69 (FN50; Elabsciences).

Techniques: In Vitro, Cell Culture, Control, Incubation, Activation Assay, Purification, Expressing, Marker, Comparison