Figure 6
Figure 6. Programming of human B cells to produce the broadly neutralizing anti-HIV antibody, b12-IgG1. (A) B12 levels during stage 2 were measured using b12-specific ELISA and are shown by weeks. Note that the culture media were changed biweekly; thus, the data represent the accumulation of the antibody in 3 to 4 days. The results are mean plus or minus SE from 3 independent cultures. * indicates significant difference from week 2 (P < .05). (B) A total of 50 000 cells of each treatment from stage 2 were transferred to stage 3 of 500 μL per well. Total IgM, IgG, and IgA levels were measured, and the results are mean plus SE from 3 independent cultures. “Vector or GFP” represents the averaged effect of empty vectors (FMHW and FEEKW) and GFP-containing vectors (MH-GFP and EEK-GFP). (C) The levels of b12-IgG1 at the end of stage 3, assayed using b12-specific ELISA, are shown (*P < .02; **P < .002). The bars are color-coded as in panel B. (D) MH-GFP- or MH-b12-transduced HSPCs were cultured through stage 2 and stage 3, and surface-stained with anti-CD19 antibody and intracellularly stained with anti-IgG antibody and monomeric gp120. Monomeric gp120MN was labeled using the Alexa-Fluor-647 Protein Labeling Kit from Invitrogen (Carlsbad, CA). Data were analyzed using flow cytometry and pregated on CD19+ cells. Intracellular proteins recognizing gp120 are identified as α-gp120. (E) Genomic DNA from progenitor cells (at week 2 of stage 2) derived from CD34+ cells uninfected or infected by MH-b12 or EEK-b12 virus was extracted and virus integration was determined as described in “Detection of virus integration.” Provirus copy numbers per cell are shown as mean plus SE from 3 independent cultures. (F) Dakiki plasmacytoma cells were transduced by U-GFP, MH-GFP, or EEK-GFP lentiviruses. Flow cytometry was performed after 72 hours of incubation and the fold of GFP intensity over the effect of U-GFP (defined as 1) is shown. The difference between MH-GFP and EEK-GFP was significant (P < .05). Student 2-tailed t tests were performed using Microsoft Excel statistical software module and the difference was considered statistically significant when the P value was less than .05.

Programming of human B cells to produce the broadly neutralizing anti-HIV antibody, b12-IgG1. (A) B12 levels during stage 2 were measured using b12-specific ELISA and are shown by weeks. Note that the culture media were changed biweekly; thus, the data represent the accumulation of the antibody in 3 to 4 days. The results are mean plus or minus SE from 3 independent cultures. * indicates significant difference from week 2 (P < .05). (B) A total of 50 000 cells of each treatment from stage 2 were transferred to stage 3 of 500 μL per well. Total IgM, IgG, and IgA levels were measured, and the results are mean plus SE from 3 independent cultures. “Vector or GFP” represents the averaged effect of empty vectors (FMHW and FEEKW) and GFP-containing vectors (MH-GFP and EEK-GFP). (C) The levels of b12-IgG1 at the end of stage 3, assayed using b12-specific ELISA, are shown (*P < .02; **P < .002). The bars are color-coded as in panel B. (D) MH-GFP- or MH-b12-transduced HSPCs were cultured through stage 2 and stage 3, and surface-stained with anti-CD19 antibody and intracellularly stained with anti-IgG antibody and monomeric gp120. Monomeric gp120MN was labeled using the Alexa-Fluor-647 Protein Labeling Kit from Invitrogen (Carlsbad, CA). Data were analyzed using flow cytometry and pregated on CD19+ cells. Intracellular proteins recognizing gp120 are identified as α-gp120. (E) Genomic DNA from progenitor cells (at week 2 of stage 2) derived from CD34+ cells uninfected or infected by MH-b12 or EEK-b12 virus was extracted and virus integration was determined as described in “Detection of virus integration.” Provirus copy numbers per cell are shown as mean plus SE from 3 independent cultures. (F) Dakiki plasmacytoma cells were transduced by U-GFP, MH-GFP, or EEK-GFP lentiviruses. Flow cytometry was performed after 72 hours of incubation and the fold of GFP intensity over the effect of U-GFP (defined as 1) is shown. The difference between MH-GFP and EEK-GFP was significant (P < .05). Student 2-tailed t tests were performed using Microsoft Excel statistical software module and the difference was considered statistically significant when the P value was less than .05.

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