Part:BBa_K1391100:Experience
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B-cell receptors (BCRs) are multiprotein immune receptors found exclusively on the surface of B cells. The BCR multiprotein complex is centered around a membrane-bound IgM antibody formed from two light chains and two heavy chains. This antibody is bound to two accessory proteins CD79A and CD79B. When the antibody binds to an extracellular antigen, receptors dimerize resulting in the phosphorylation of the intracellular tails of CD79A and CD79B by the tyrosine-protein kinase Lyn. In response, another cofactor, spleen tyrosine kinase (Syk), is recruited to the receptor and phosphorylated, initiating a signalling cascade that results in the proliferation of the activated B cells. This receptor is important in clonal selection of B cells during human immune response.
This particular sequence is the heavy chain for an antibody against beta amyloid meant to be used in a B-Cell Receptor. It is meant to be used in conjunction with an associated light chain as well as CD79A, CD79B, Lyn, and Syk.
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In our first experiment, we aimed to determine if the engineered B-cell receptor components (CD79A, CD79B, IgM Heavy Chain, and Kappa Light Chain) were able to assemble to form a receptor complex and then localize to the cell membrane. Since the beta-amyloid oligomers characteristic of Alzheimer's disease accumulate in the extracellular matrix of the brain, it is important that the receptor membrane localize so that it can detect these plaques outside the cell.
To determine the localization of the receptors, we immunostained using IgM specific antibodies. We analyzed the immunostained samples in two ways. The first was through flow cytometry analysis. This method enabled us to determine whether the antibodies bound to the outside of our cells, which would indicate that the B-cell receptor's IgM component had reached the membrane. We also used confocal microscopy to visualize the localization of our receptor inside our cells by permeabilizing the cells and incubating them with anti-IgM antibodies.
For samples that were analyzed using flow cytometry, we transiently transfected HEK293 cells with plasmids encoding constitutive expression (hEF1a promoter) of the engineered B-cell receptor components along with hEF1a:mKate2 (constitutive red fluorescent protein) as a transfection marker. The transfection marker provides an indication of approximately how many plasmids are uptaken by a particular cell, which helps to connect plasmid number to observed output levels. We then treated cells with anti-IgM antibodies conjugated to Alexa Fluor 488 (yellow fluorescent dye). By measuring yellow output relative to red output using the flow cytometer, we hoped to be able to compare plasmid number to anti-IgM antibody binding, where high levels of red fluorescence (many plasmids) would correspond to high levels of yellow fluorescence (high levels of antibody binding, meaning a high level of BCR surface expression).
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Determining membrane localization was complicated by bleedthrough from mKate into the yellow fluorescence channel. Samples were stained with Alexa fluor 488 (yellow) conjugated anti-IgM antibodies and evaluated using flow cytometry. Red fluorescence refers to the amount of mKate (transfection marker) in the cells whereas yellow fluorescence measures the amount of anti-IgM binding (indicating the presence of B-cell receptors on the cell surface). (A) Untransfected, unstained HEK293; (B) HEK293 transfected with dummy DNA, stained; (C) HEK293 transfected with mKate, stained; (D) HEK293 transfected with mKate and BCR components, stained. |
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In our first trial of this experiment, we saw a significant increase in yellow fluorescence between untransfected cell populations (A,B) and transfected ones (C,D). However, we saw similar amounts of yellow fluorescence between cells that were transfected with just hEF1a:mKate2 and those transfected with both hEF1a:mKAte2 and the B-cell receptor DNA. Additionally, there was a very strong one-to-one correlation between yellow and red fluorescence - a tighter distribution than would generally be expected for this kind of experiment. This led us to believe that our results were actually stemming from bleedthrough of the mKate2 fluorescent protein into the FITC channel that we used to detect yellow fluorescence. If both the red and yellow channels registered signal from the same protein, this would explain the tight, one-to-one correlation.
To address this problem, we decided to repeat the transfection without using a transfection marker since we determined that all of the fluorescent proteins that we had available to us would produce the same, if not a greater, bleedthrough effect.
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Flow cytometry demonstrates synthetic B-cell receptor membrane localization in HEK293 cells. Cells were stained with Alexa Fluor 488 conjugated anti-IgM antibodies. Typical HEK293 cells do not express B-cell receptors, whereas Ramos cells (a positive control) are derived from B cells and do express B-cell receptors. (A) HEK293 transfected with dummy DNA, stained; (B) HEK293 transfected with synthetic B-cell receptor, stained; (C) Ramos, unstained; (D) Ramos, stained |
By transfecting without a transfection marker, we were able to identify a population of HEK293 cells that were expressing our B-cell receptor on the cell membrane. However, less than 2% of transfected cells showed this kind of expression, suggesting that either our transfection efficiency was low or that expressing our receptor resulted in harmful effects to the cells.
Our second method of determining membrane localization was using confocal microscopy. To do this we once again transfected HEK293 cells with plasmids encoding constitutive expression of the receptor and a constitutive color transfection marker (hEF1a:eYFP). Our choice of transfection marker here was not important since any fluorescence would be quenched when the cells were fixed. Instead, we used the transfection marker to determine if we had a high enough transfection efficiency to proceed with immunostaining. After transfecting, we then fixed the samples and stained them with the same antibodies that we used for the flow cytometry analysis and added DAPI to stain the nucleus for better visualization of the cells.
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Fluorescent microscopy suggests membrane localization of synthetic B-cell receptor in HEK293 cells. Cells were stained with Alexa Fluor 488 conjugated anti-IgM antibodies and DAPI was used as a nuclear stain. (Left) Untransfected HEK293 control, stained; (Right) HEK293 transfected with synthetic B-cell receptor, stained |
In the resulting microscopy images, we saw a clear increase in yellow fluorescence between cells that were transfected with the receptors and those that were not. We also saw halos of yellow around the blue nuclei in the transfected cells, suggesting that the receptor may have been localizing to the cell membrane. However, we also observed some cytosolic expression, potentially from receptor being held in endoplasmic reticulum during processing. Results of further experiments suggested that the receptors might have been getting overexpressed, given the large mass of receptor DNA that we were transfecting and the fact that we were using a strong constitutive promoter to express the receptors.
Based on the results from flow cytometry and microscopy, we concluded that the B-cell receptor was reaching the surface of the transfected HEK293 cells.
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Experiment 2:
Beta-amyloid binding to the receptor
In this experiment we aimed to determine whether or not the B-cell derived receptor in our system was in fact binding to beta-amyloid oligomers. To do this, we transfected HEK293 cells with plasmids encoding our receptor components and hEF1a:eBFP2 (a transfection marker). We then treated the cells with biotinylated beta-amyloid oligomers and red Alexa Fluor 594-conjugated streptavidin. If the receptor bound to the beta-amyloid, the streptavidin would, in turn, bind to the biotin on the beta-amyloid oligomers leading to a higher level of red fluorescence. Similar to the first experiment, we analyzed the cells using both flow cytometry and confocal microscopy, looking for increased red fluorescence in cell populations that were transfected with the receptors.
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Flow cytometry was inconclusive about beta-amyloid binding. Cells were incubated with biotinylated beta-amyloid and Alexa fluor (red) conjugated streptavidin. Red fluorescence indicates beta-amyloid binding and blue fluorescence is the transfection marker (eBFP). (A) HEK293 transfected with eBFP; (B) HEK293 transfected with eBFP, stained with beta-amyloid and streptavidin; (C) HEK293 transfected with eBFP and synthetic B-cell receptor, stained with streptavidin; (D) HEK293 transfected with eBFP and synthetic B-cell receptor, stained with beta-amyloid and streptavidin |
The flow cytometry results that we obtained through this experiment did not lead us to conclusive results as to whether our receptor was binding beta-amyloid oligomers. Though there were an increased amount of cells showing red fluorescence in after beta-amyloid/streptaviding staining, this increase did not correlate with an increase in plasmid count (as measured by the blue transfection marker). Additionally, the truncation in blue fluorescence observed in cell populations transfected with the B-cell receptor (C, D) suggested that at a certain level of expression our receptor was becoming toxic to the cells or inducing too high of a metabolic load.
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Fluorescent microscopy to determine beta-amyloid binding to synthetic B-cell receptor was inconclusive. Cells were incubated with oligomerized biotinylated beta-amyloid and red Alexa fluor conjugated streptavidin. Blue indicates DAPI nuclear staining and red indicates Alexa fluor. (Left) Untransfected HEK293 control, stained with streptavidin and beta-amyloid; (Right) HEK293 transfected with synthetic B-cell receptor, stained with streptavidin and beta-amyloid |
Like cytometry, microscopy results for beta-amyloid binding were also inconclusive. Both samples of cells expressing the receptor and untransfected cell samples showed some degree of red fluorescence (indicating the presence of streptavidin), but there was no clear difference between the two and no particular localization was observed.
Given the cytometry and microscopy results from our beta-amyloid binding experiment, it is unclear whether beta-amyloid does in fact bind our synthetic B-cell receptor. However, this inconclusive result does not necessarily keep us from testing activation of the receptor, since anti-IgM antibodies have also been shown to cause receptor dimerization, activating the BCR.
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Experiment 3:
Evaluating relative levels of Syk-TEVp and endogenous Syk
In this experiment, we wanted to compare the levels of endogenous cofilin and Syk-TEV protease (TEVp) expressed under an inducible promoter with different levels of induction. Different levels of Syk-TEVp expression were achieved using an rtTA/TRE system, where rtTA is a transcription factor activated by doxycycline (a small molecule) that activates genes under the regulation of a TRE promoter. High concentrations of doxycycline correspond to high levels of gene expression in this system. By examining differences in expression levels between endogenous and exogenous Syk, we hoped to gain insight into what level of Syk-TEVp expression would lead to the best signal:noise ratio of exogenous to endogenous Syk. We transfected HEK293 cells with DNA encoding inducible expression of our Syk-TEVp fusion construct and hEF1a:eYFP (a transfection marker) as well as hEF1a:rtTA, which is required for the doxycycline-inducible activation of our Syk-TEVp construct. We added different concentrations of doxycycline to various cell populations, and subsequently analyzed the cell lysates by probing for Syk in a Western blot analysis.
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Exogenous Syk-TEVp under high levels of doxycycline induction is expressed at comparable levels to endogenous Syk in HEK293 cells. Cell lysates were examined from various cell populations: untransfected HEK293 cells, Ramos cells, HEK293 cells transfected with dummy DNA, HEK293 cells transfected with hEF1a:eYFP only, and HEK293 cells transfected with hEF1a:eYFP and TRE:Syk-TEVp under varying levels of doxycycline (dox) induction. Primary antibody probes against Syk, GAPDH (a loading control), and eYFP (a transfection efficiency control) were used along with IR dye conjugated secondary antibodies, and the blots were imaged using an IR scanner. The two copies represent different blocking conditions: (A) blocked in 5% BSA and (B) blocked using Odyssey Blocking Buffer. </td> </table>
Experiment 4:Quantifying cleavage levels with non-activated receptor
<a name="ex5"></a> Experiment 5:Cleavage levels in active versus non-activated receptor
Citations [1] Gilad Barnea, Walter Strapps, Gilles Herrada, Yemiliya Berman, Jane Ong, Brian Kloss, Richard Axel, Kevin J. Lee.The genetic design of signaling cascades to record receptor activation. PNAS (2007) Print
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