Team:TECHNION-ISRAEL/Results

Results

• Introduction
• “Tri-Display” Optimization
• Tet-Off
• “TRE-Tri-Display”
• Apoptosis test with soluble anti-IgM
• Testing the assay plasmid
• Proof of concept
• Discussion

Introduction

The goal of our project is to prevent allergies and autoimmune diseases by displaying their characteristic epitopes on the membrane of hematopoietic stem cells. This will, theoretically, lead to deletion of the harmful immune cells, thus precluding immune reaction towards harmless substances or “self.”

Experiments

Our experiments can be divided into three categories:

1. Tri-Display
2. Timing
3. Tolerance assay

Before reading the results, it is important to note that all our experiments were run in duplicate, and the results below are based on a weighted median fluorescence. This means we multiplied the median fluorescent intensity by the percentage of positive cells. This was done in order to obtain consistent and reliable results.

“Tri-Display” Optimization experiment

The full protocols for this experiment can be seen here.

One of our goals was to successfully express three different proteins on the cellular membrane using our modular plasmid. We tested expression in our model cell line HEK293. In order to quantify the expression level of each protein on the cellular membrane we used 3 different antibodies conjugated to fluorophores (APC, PE and FITC) and measured the fluorescence using a flow cytometer (Figure 3).

Figure 3: Antibodies conjugated to fluorophores bind to the tags on the membrane.

In this experiment we tested four potential “Tri-Display” plasmids we designed, in order to determine which worked best. For positive control we used the commercial pDisplay vector, which contains one epitope with all three tags (figure 4).

Figure 4: Median fluorescence of four different optimized “Tri-Display” constructs

We can see that the best "Tri-Display” vector, capable of sufficiently expressing all three proteins in almost perfectly equal ratios, is the p2a plasmid. Accordingly, we proceeded to insert the P2A based display plasmid into our final ToloGen construct, the “TRE-Tri-Display”.

Tet-Off experiment

The full protocols for this experiment can be seen here.
In order to test our delay mechanism, we co-transfected the TRE-GFP plasmid (BBa_K2520006) with the CMV-tTA plasmid (BBa_K2520009) into HSC (Hematopoietic stem cells) model. After induction, we expected to see that the florescent intensity is inversely proportional to the Doxycycline (analog to Tetracycline inducer) concentration. Meaning a higher concentration of Doxycycline, would correspond to a lower fluorescent intensity. For negative control we co-transfected TRE-GFP and Puc19 into HSC model. This allowed us to test the basal expression of our system.

Figure 1: co-transfection of TRE-GFP plasmid and CMV-tTA plasmid in HSC model.

After induction, we expected to see that the florescent intensity is inversely proportional to the Doxycycline concentration. Meaning a higher concentration of Doxycycline, would correspond to a lower fluorescent intensity. For negative control we co-transfected TRE-GFP and Puc19 into HSC model. This allowed us to test the basal expression of our system.

Figure 2: weighted median fluorescence of cells expressing GFP with different concentrations of Doxycycline.

The results shown in Figure 2 are in line with our expectations. A clear decrease in fluorescence intensity can be seen with increased concentrations of Doxycycline. At a concentration of 10ng/ml fluorescence intensity stabilizes. The negative control, TRE-GFP plasmid with Puc19, shows no fluorescent intensity for GFP, indicating that the leakiness of our Tet-Off system is very low.

“TRE-Tri-Display” experiment

The full protocols for this experiment can be seen here.
Our final experiment was to test the ToloGen construct we created (figure 5).

Figure 5: Modular ToloGen plasmid “TRE-Tri-Display”

We can see that the best "Tri-Display” vector, capable of sufficiently expressing all three proteins in almost perfectly equal ratios, is the p2a plasmid. Accordingly, we proceeded to insert the P2A based display plasmid into our final ToloGen construct, the “TRE-Tri-Display”.
This experiment combined both induction and immunofluorescent staining. We co-transfected the “TRE-Tri-Display” (BBa_K2520007)and CMV-tTA (BBa_K2520009) plasmids into HSC model. After 24 hours we made induced the cell with 6 different concentrations of Doxycycline. After 48 hours, we stained the cell with antibodies analyzed them using a flow cytometer (Figure 6). We expected to see a decrease in the fluorescent intensity of all three proteins (as we saw in the delay mechanism experiment).

Figure 6: weighted median fluorescence of the cells in three different channels (APC, FITC and PE), corresponding to the three different proteins, in different concentrations of Doxycycline

From the results shown in Figure 6 we can see a general decrease in the membrane expression of all three proteins as the concentration of doxycycline is increased.
Still, the results of this experiment are not fully satisfactory, as the expression is inconsistent with expectations at two specific concentrations, and the general fluorescence intensity is lower than we expected. We believe this may be due to the very long nature of the experiment, the dependence on co-transfection combined with the complexity of the construct, and certain complications that delayed our flow cytometry analysis. Unfortunately, there was no time to conduct this experiment again.
In the future, we plan on repeating this experiment, possibly while using a cell line that constitutively expresses the tTA protein. This would allow us to forgo co-transfection and achieve more consistent results.

Tolerance assay

Apoptosis test with soluble anti-IgM

In this experiment we confirmed that our B cell model- WEHI-231 cells can undergo apoptosis with the addition of soluble anti IgM. We checked the percentage of apoptotic cells using the commercial “Dead Cells Apoptosis Kit” (Invitrogen). Apoptosis was measured at 24 hours, compared to a control (B cell model without anti-IgM).
We conducted two important apoptosis assays:
A) Assay using soluble anti-IgM
B) Assay using membranal anti-IgM

1. Anti-IgM display test
2. Co-cultured tolerance assay

Figure 7: Detection of apoptosis using Annexin V FITC and PI assay

As can be seen from the above graphs, the percentage of B cell model that were double positive (upper right quadrant, signifying dead cells) was higher in the experiment plates (with anti-IgM) as compared to the control (without anti-IgM). In addition, there were no positive cells for Annexin V alone, meaning that there were no early apoptotic cells.

Testing the assay plasmid

After we proved that our B cell model undergoes apoptosis when exposed to soluble anti-IgM, we moved on to our final assay- proof that our B cell model undergo apoptosis after exposure to a Hematopoietic stem cell model (HSC model)- HEK-293, transfected with our assay plasmid.
First, we transfected HSC model with our assay plasmid in order to verify that the anti-IgM construct we created was functional. The anti-IgM was expressed under two promoters- a mutant EF1a ( BBa_K2520023) promoter and a CMV promoter. We tested the functionality of our anti-IgM scFv-FC-Fusion using soluble murine IgM conjugated to a fluorophore.

Figure 8: fold change of the weighted average after extracellular staining with IgM conjugated to FITC, compared with negative control (non-transfected cells).

As can be seen, our construct was able to bind IgM more specifically than the control, though further optimization is clearly necessary. It is important to mention that although the experiment plates bound IgM more specifically than the control, the percentage of cells expressing anti-IgM was very low (0.0175% in the control, 1.535% under the CMV promoter and 1.46% under the EF1a promoter). We believe this is due to the complex nature of the antibody being displayed, and the need for dimerization on the membrane. For more on how we designed this experiment please see our Proof of Concept page.

Tolerance Assay

Induce apoptosis in an immature B cell model with anti-IgM presenting Hematopoietic stem cell model

We transfected the HSC model with our assay plasmid. After 24 hours, we washed the cells, and cocultured them with our B cell model. After an additional 24 hours (48 hours after transfection) we quantified the apoptosis of the B cell model using a “ Dead Cells Apoptosis Kit” (Invitrogen) and compared the results to a control plate of our B cell model in monoculture.

Figure 9: Detection of apoptosis using Annexin V FITC and PI assay

As can be seen from the above graphs (Figure 11), the percentage of dead B cell model was nearly twice that of the control (cocultured with non-transfected HSC model). Therefore, we can conclude that B cell model underwent apoptosis as a result of our model HSCs presenting anti-IgM. This supports the claim that our display system is capable of presenting epitopes effectively and can induce tolerance.

Discussion

When we began working in the lab, we had two goals:

1. Create a functional and inducible Tri-Display plasmid that incorporates the elements of our design.
2. Demonstrate that our system induces immune tolerance

We believe that we have achieved both these goals. We successfully tested our display mechanism and inducible promoter separately, demonstrated that cells transfected with our modular display plasmid can induce immune tolerance, and lastly, we showed that the inducible Tri-Display plasmid showed promising results, but still requires further testing and optimization.
In the future, we would like to further optimize both the display and delay mechanisms in order to achieve more robust and consistent expression. Furthermore, because of an unfortunate shipping delay, we did not have time to test our “Kill Switch” mechanism. In the future we plan on combining all three systems, the inducible promoter, Tri-Display system, and “Kill Switch,” thus creating the final ToloGen plasmid.
Finally, a tremendous amount of work went in to working with our second model cell line, HPC-7 cells. Not every endeavor can be successful, and within the time frame of iGEM we were not able to conduct our project experiments on these cells. Towards the end of our project we managed to successfully transfect these cells, but time ran out before we could conduct further experiments. In an effort to aid future teams and scientists we documented our efforts in detail HERE.

1. Gottschalk, Alexander R., and José Quintáns. "Apoptosis in B lymphocytes: the WEHI-231 perspective." Immunology & Cell Biology 73.1 (1995).