Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming

Abstract

The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced by follicular helper T cells, CD40 ligand (CD40L) and interleukin-21 (IL-21), we convert them to highly proliferating, cell surface B cell receptor (BCR)-positive, immunoglobulin-secreting B cells with features of germinal center B cells, including expression of activation-induced cytidine deaminase (AID). We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study B cell biology and signal transduction through antigen-specific B cell receptors and for the rapid generation of high-affinity human monoclonal antibodies.

Figure 1

Overexpression of BCL6 and Bcl-xL confer a high proliferative capacity and fixed differentiation phenotype to peripheral blood CD27⁺IgG⁺ memory B cells (a) Flow cytometry identification of BCL6-transduced (ΔNGFR⁺), Bcl-xL-transduced (GFP⁺) and BCL6+Bcl-xL transduced (GFP⁺ΔNGFR⁺) CD19⁺ B cells in culture with CD40L-L cells and IL-21 four days post transduction. (b) Percentages of the individually transduced populations over time within a single non-sorted culture maintained with CD40L-L cells and IL-21. BCL6+Bcl-xL ○, Bcl-xL-only ■, BCL6-only □ transduced cells or non-transduced cells (●). (c) Cultured double-transduced cells were left unsorted (left panel) or the individual populations were sorted (right panel) and cultured separately. The cumulative cell number was calculated based on the original number of transduced B cells at input and illustrates the theoretical absolute cell numbers produced in culture. (d) CD19⁺ cells from non-sorted double transduced cultures were analyzed for CD38, HLA-DR, and surface Ig kappa (IgL-κ) or lambda (IgL-λ) light chain expression. The gates in the upper panel show the percentage terminally differentiated cells. Phenotype shown of cells 7 days post transduction. Data are representative of four separate experiments.

Figure 2

CD27⁺ memory peripheral blood cells acquire a stable GC-like phenotype following transduction with BCL6 and Bcl-xL and subsequent culture. (a) Phenotype of BCL6+Bcl-xL transduced CD27⁺ memory B cells (6XL, black histogram line) compared to tonsil GC cells (GC, CD38⁺CD20⁺, shaded grey), tonsil naïve and memory cells (N/M, CD38⁻CD20^low, red histogram), and tonsil plasma cells (PC, CD38⁺⁺CD20^low, blue histogram). BCL6+Bcl-xL transduced monoclonal cell lines show an identical phenotype (not shown). (b) Relative mRNA levels of AICDA (encoding AID) in CD19⁺IgG⁺CD27⁺ PB memory B cells and CD19⁺CD38⁺CD20⁺IgD⁻ tonsillar GC B cells (value set as 1) compared to BCL6+Bcl-xL transduced bulk CD27⁺ memory cells using quantitative 5 RT-PCR.

Figure 3

Generation and characterization of Tetanus toxoid (TT)-specific monoclonal human B cell lines. (a) Cell sorting of TT-specific B cells from bulk CD19⁺ BCL6+Bcl-xL transduced memory B cell culture using TT-PE staining. (b) Monoclonal cell lines express surface IgG and are either IgL-λ or IgL-κ light chain positive. Shaded histograms are CD19⁺ tonsil B cells. (c) The cumulative cell number (as in Fig. 1c) of two representative B cell clones in time. (d) TT-specific and control TT negative cell lines were incubated with TT or F(ab)₂ -human IgG antibody stimulation and phospho-ERK1/2 was determined by immunoblot analysis. STAT3 blotting was used to verify equal loading. Results are representative of two experiments (e) Calcium flux in TT specific IgG positive B cell lines. Cells were loaded with Indo-1 AM and were subsequently stimulated with TT (dashed black trace), IgG F(ab)₂ antibody (solid black trace), or control IgG (solid grey trace). The Indo-1 response of a TT-negative clone stimulated with TT antigen is shown as an antigen specificity control (dashed grey trace). Shown is the ratio bound versus unbound Indo-1. Results are representative of three experiments.

Figure 4

Isolation of high affinity, broadly RSV neutralizing antibodies from RSV-specific B cell clones. (a) IgM or IgG production of individually cloned BCL6+Bcl-xL-immortalized cell lines, from the CD19⁺CD27⁺IgA⁻IgM⁻ and CD19⁺CD27⁺IgA⁻IgG⁻ memory B cell pool respectively, maintained on CD40L-L cells and IL-21 in a 3 day culture. (b) Generation of RSV neutralizing antibodies. Neutralization dose response curve against the RSV A2 virus for the newly generated RSV antibodies D25 (●), AM14 (), AM16 (○), and AM23 () compared to palivizumab (Δ). (c) Neutralization dose response curve of the RSV antibodies D25, AM14, AM16, AM23 and palivizumab against the primary RSV isolate X. Symbols as in 4b (d) Effects of rD25 (●), palivizumab (Δ) and a control IgG1 (×) on RSV replication in cotton rat lungs. Antibodies were administered i.m. one day before intranasal challenge with RSV X (10⁶ TCID₅₀/animal). Lung virus titers were calculated per gram of lung tissue; control prophylaxis reached a titer of 4.7 log TCID₅₀ g⁻¹, while the lower limit of detection was 2.1 log₁₀TCID₅₀ g⁻¹.

Figure 5

Expression and activity of AID in BCL6+Bcl-xL transduced cells. (a) mRNA levels of AICDA (encoding AID) in CD19⁺CD38⁺CD20⁺IgD⁻ tonsillar GC B cells and CD19⁺IgG⁺CD27⁺ PB memory B cells compared to 23 BCL6+Bcl-xL transduced monoclonal cell lines and monoclonal cell line D25 using quantitative RT-PCR. (b) Percentage of subclones with indicated number of VH mutations, as percentage of total number of subclones sequenced. (c) Location of VH mutations, percentage of mutations per VH region per basepair. (d) Binding of D25-subclone Ig to RSV infected HEp2 cells. Red squares are individual D25 subclones, black circles rD25. Blue circles indicate clones with deviating binding activity. (e) mRNA levels of AICDA in the monoclonal cell line D25 transduced with Control-YFP or Id3-YFP using quantitative RT-PCR.