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Blood, Vol. 113, Issue 7, 1422-1431, February 12, 2009
Engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes.
Blood Luo et al.
113: 1422
Supplemental materials for: Luo et al
Files in this Data Supplement:
- Figure S1. Lentiviral transduction of HSPCs and testing of b12-encoding constructs in cell lines (JPG, 98.3 KB)
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(A) HSPCs were incubated for 24 h in the presence of IL-3 (10 ng/mL), Flt3 ligand (10 ng/mL), thrombopoietin (10 ng/mL), SCF (5 ng/mL) and G-CSF (5 ng/mL). Two sequential infections with mock or FUGW (i.e. U-GFP) virus at a multiplicity of infection of 1,000 were then given to the HSPCs. The same cytokines were added every other day during the infection. The infected cells were collected 3 days post-infection (a total of 5 days of incubation) and analyzed by flow cytometry. (B) All cloned lentiviral constructs were tested in two B-cell lines (Nalm-6 and Ramos) and one T-cell line (Jurkat) before being used to transduce HSPCs. The effect of the ubiquitin promoter (U) was compared to that of either MH (shown) or EEK (similar to the effect of MH; not shown) B-cell–specific promoters. None of the three cell lines express endogenous IgG; thus, the staining of intracellular IgG represents the transgenic b12-IgG1. (C) 293T cells were transfected with the lentiviral vector FUW-b12. Cellular proteins were extracted and analyzed under reducing (−β-mercaptoethanol; or β-ME) and non-reducing conditions (−β-ME) using SDS-PAGE, followed by Western blotting of IgG-Fc (γ heavy chain/HC) and the κ light chain/LC. The result showed that more than 90% of heavy (H) and light chains (L) were expressed and cleaved at the F2A site (uncleaved: H-2A-L), and that most heavy chains formed homodimers (H2; ~100 kD) through disulfide bonds and they picked up the light chains (H2L2) before secretion to the culture medium. (D) 293T cells were transfected with the lentiviral vector FUW-b12. The culture supernatant was collected and measured by Biacore assay, which gave the concentration of approximately 6 µg/mL of binding to gp120. The concentration of the purified b12 was 125 µg/mL. The culture supernatant and the purified b12 were diluted 5-fold to 1.2 µg/mL and 25 µg/mL of starting concentrations, respectively, when subjected to pseudovirus neutralization assay. The result showed that the b12-containing culture medium could neutralize the SF162 strain of pseudovirus as potently as the purified b12-IgG1. The neutralizing 50% inhibitory concentrations (IC50’s) were 0.040 µg/mL and 0.026 µg/mL for the culture medium and purified b12, respectively.
- Figure S2. Differentiation of HSPCs and generation of myeloid cells early in Stage-2 (JPG, 54.9 KB)
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HSPCs transduced with either U-GFP (upper panels) or MH-GFP (lower panels) lentiviral constructs were cultured on MS5 stromal cells for the indicated periods of time. (A) Upon exposure to MS5, the HSPCs lost the expression of CD34 and c-kit progenitor cell markers. Note that the percentages of double-negative cells increased over time. (B) After only 1 week of co-culture between transduced HSPCs and MS5, CD13− myeloid cells started to appear.
- Figure S3. Effect of the transgene on B-cell development during Stage-2 (JPG, 47.7 KB)
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(A) HSPCs uninfected (No virus) or transduced with different lentiviral constructs were cultured on MS5 stromal cells for 3–4 weeks (representing the stage of pro-B cells) or 6 weeks (representing the time when there is a mixture of pre-B and immature B cells). The percentages of B-cell populations among all live cells are shown as mean − standard error from at least three independent cultures. “Vector or GFP” represents the averaged effect of empty vectors (FMHW and FEEKW) and GFP-containing vectors (MH-GFP and EEK-GFP). (B) At the end of Stage-2 (6 weeks), both CD19−CD10− pre-B and CD19−CD10− immature B cells were generated and their proportions were similar regardless of the lentiviral constructs introduced (MH-b12 or EEK-b12). In these particular determinations, the percentage of total CD19− cells in the case of MH-b12 (left panel) is higher than that of EEK-b12 (right panel); however, the averaged effects of lentiviral constructs were the same statistically, as shown in (A).
- Figure S4. Effect of GFP on B-cell activation and differentiation into plasma cells during Stage-3 (JPG, 80.9 KB)
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(A) Cells carrying the MH-b12 or EEK-b12 transgene from Stage-2 were transferred onto the MS40L-low monolayer and incubated in the presence of IL-2 (10 ng/mL), IL-10 (100 ng/mL), and CpG (2 µM) for 2–3 weeks. GFP− and GFP− cell populations were gated in FlowJo for further analysis. (B) Regardless of the transgene (GFP), the percentage of CD19− cells increased but CD19 intensity decreased from before (lighter lines) to after (darker lines) Stage-3. Expression of CD86 did not differ between GFP− and GFP− populations. (C) Regardless of the transgene (GFP), both plasmablasts (CD20−CD38−∕−) and plasma cells (CD20−CD38−CD138−) were generated. The percentages shown are those of total live cells. (D) Class-switched B cells (surface IgG−) and memory B cells (CD19−CD27−) were also generated. The percentages shown are those of CD19− cells.
- Figure S5. Morphology of normal plasmablasts and plasma cells (JPG, 83.7 KB)
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(A) CD138− cells were enriched from normal human bone marrow mononuclear cells (BMMC) using magnetic isolation and analyzed by flow cytometry. Plasmablasts and plasma cells are shown in green and red gates, respectively. (B) Wright stains were performed on CD138-enriched populations of cells.
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