Team:TECHNION-ISRAEL/Bcell assay

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Bcell

B cell Assay




The assay x we developed is meant to serve as proof of concept for our system and to demonstrate the following:

  1. Our system displays the chosen proteins correctly on the cellular membrane.
  2. Displayed proteins can interact with B cell receptors and activate them.
  3. Immature B cells (such as the ones we are targeting within the bone marrow) undergo apoptosis as a result of exposure to our antigen/epitope presenting cells.

Materials and Methods

WEHI-231

WEHI-231 is an immature murine B cell lymphoma cell line. This cell line was chosen specifically for our assay as it can provide valuable information about our system. Like all immature B cells, WEHI cells constitutively display immature antibodies of the IgM isotype on their membrane.



Multiple studies have shown [1] that when WEHI cells are exposed to anti-IgM antibodies, their B cell receptors (The membrane bound antibodies, also known as BCRs) are activated, and the cells undergo apoptosis. This process has been accepted as analogous to the process of Central Tolerance [1] [2] within the bone marrow, wherein immature B cells that are exposed to antigen capable of activating their BCRs undergo apoptosis (figure 1). While the antibodies displayed on the membrane of each WEHI cell have different affinities, they all possess the same constant IgM isotype which can be targeted with anti-IgM antibodies. These antibodies can activate and cross-link all the BCRs on the WEHI cells in a nonspecific manner, but the resulting intracellular cascade is identical to natural BCR activation.



Figure 1: central tolerance



HEK293

Throughout our project we used HEK-293 as a HSC model.


Assay plasmid

This plasmid contains our display system, but instead of an epitope we are displaying an anti-IgM antibody. This protein is designed to bind the IgM portion of the antibodies that are on the WEHI cells. We believe that this serves as a perfect model for our system wherein our epitopes are meant to bind the BCR’s of immature B cells within the bone marrow.



Figure 2: modified gBlock





Antibody display

For our assay to work, we need to display a functioning antibody on the membrane of our model cells. This is a herculean task for many reasons:

  1. Antibodies are complex protein heterodimers consisting of four separate proteins (two light chains and two heavy chains) from two completely different loci in the genome. Our platform is plasmid based and therefore less compatible with polycistronic genes.
  2. Antibodies undergo many post-translational processes that are unique to B cells in order to become functional. Our model cells are not B cells and therefore lack this ability.
  3. Since our display system uses a different transmembrane domain than naturally occurring membrane antibodies, correct folding of the antibody is even more unlikely.

As well as many other issues. After laborious research and multiple consultations with antibody display experts (Prof. Yoram Reiter) we set out to test two systems for membrane antibody display, namely, scFv display and scFv-FC fusion display.


scFv

Single Chain Variable Fragment (scFv) is a fusion protein consisting of both the heavy and light variable chains of an antibody connected by a short linker peptide (figure 3). After exhausting nearly all research options and crafting plans to create an anti-IgM scFv ourselves, we discovered a paper [3] in which an anti-IgM scFv sequence had already been created and tested. We then ordered a Gblock (figure 2) from IDT with the scFv sequence modified to fit our existing construct.


Figure 3: antibody



scFv-FC fusion

Singe Chain Variable Fragment – FC fusion is a protein that consists of a scFv attached to the FC region of an IgG antibody (Hinge-CH2-CH3) (figure 4). This amalgam leads to dimerization of the antibody fragments on the membrane which increases the stability and half-life of the synthetic antibody while also creating more distance from the membrane allowing for less steric hindrance (figure 5). Additionally the dimeric form of the antibody is more likely to allow for BCR cross linking of the WEHI cells which is necessary for apoptosis. We made two variants of this gene, one using the Hinge-CH2-CH3 region from rattus norvegicus, the source of our scFv, and another using the same region from human origin as has been described in publications [4] .


Figure 4: Singe Chain Variable




Figure 5: dimerization of the antibody



Experiment

HEK293 cells, transfected with our assay-plasmid, were cocultured with WEHI-231 cells (figure 6). Apoptosis of WEHI cells was quantified by flow cytometry measurement using Annexin V and PI from an Invitrogen Dead Cell Apoptosis Kit [5] according to the manufacturer’s instructions. Measurements are taken at 0 hours, 24 hours and 48 hours and compared with a control of WEHI cells cocultured with non-transfected HEK293 cells. In previous experiments [1] , 30% of the WEHI cells underwent apoptosis as a result of exposure to anti-IgM. If similar results are achieved in the cocultured WEHI cells, it can be concluded that our system presents antigen correctly and allows for BCR binding, cross-linking and activation. This will serve as proof of concept for our system and allow us to pursue animal experimentation licensure in the future.




Figure 6: A model HSC transfected with our assay plasmid inducing apoptosis in an immature B cell model by cross-linking the BCRs




  1. Gottschalk, Alexander R., and José Quintáns. "Apoptosis in B lymphocytes: the WEHI-231 perspective." Immunology & Cell Biology 73.1 (1995).
  2. Page, DAWNE M., and ANTHONY L. DeFranco. "Antigen receptor-induced cell cycle arrest in WEHI-231 B lymphoma cells depends on the duration of signaling before the G1 phase restriction point." Molecular and cellular biology 10.6 (1990): 3003-3012.
  3. Lee, Timothy K., et al. "Production of engineered IgM-binding single-chain antibodies in Escherichia coli." Journal of Industrial Microbiology & Biotechnology 14.5 (1995): 371-376.
  4. Jäger, Volker, et al. "High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells." BMC biotechnology 13.1 (2013): 52.
  5. Thermo Fisher Scientific, www.thermofisher.com/order/catalog/product/V13242.

My First Website

Department of Biotechnology & Food Engineering
Technion – Israel Institute of Technology
Haifa 32000, Israel

    • igem.technion.2017@gmail.com