Difference between revisions of "Team:TECHNION-ISRAEL/Tcell assay"

The T cell central tolerance system is amazingly complex. One of the main mechanisms of T-cell tolerance is “clonal deletion". Through this process, bone-marrow-derived antigen-presenting cells (APCs) in the medulla of the thymus display self-antigens on their membrane as part of the major histocompatibility complex (MHC) class 2. MHC is a complex on the membrane of all antigen presenting immune cells. Immature (double positive) T cells with a T cell receptor (TCR) that can recognize the peptide-MHC complex, undergo apoptosis ^[1] , (see figure 1).

We aimed to show that our system can cause APCs (that differentiate from HSCs) to present our target epitopes on their MHC class II complexes, thus inducing tolerance in T cells.

Our assay was meant to demonstrate the following:

1. Cells engineered with our Display construct present epitopes on their membrane MHC complexes.
2. the peptide-MHC complex interacts with the T cell receptors and activates them.
3. The TCR – MHC complex interaction leads to apoptosis in immature T cells.

Accomplishing these tasks is extremely challenging for several reasons:

1. There is an extreme amount of variability between TCRs due to genetic recombination, and it is very challenging to isolate immature T cells with TCRs that can recognize our specific epitopes strongly.
2. There is great diversity in the MHC type between different strains of mice. Since the TCR binds the MHC molecule too, the desired TCR has to be matched to the specific strain of murine cells as well.
3. MHC complexes are presented on the membrane in low levels when the cells are not exposed to interferon gamma, making it more difficult to assess if TCR-MHC interaction is taking place.

We came up with two possible ways to adequately test our construct:

After much research and seeking out specific materials, we deemed these assays feasible. Unfortunately, due to time constrains, we were not able to conduct the experiments.
Even so, we think the assays we designed may be valuable to others, and so we have documented our plans and considerations below.

The TCR assay:

Several studies have shown that immature T cells undergo apoptosis in vitro, after exposure to soluble super antigens ^[2] , to soluble antigens presented on MHC class 1 complex ^[3] , and to soluble antigens presented on MHC class 2 complex ^[4] .

We propose the following options for presenting our epitopes on the MHC:

1. Differentiating our model cells into dendritic cells/macrophages, then transfecting the cells with our Display construct. The epitope will be displayed on the MHC class 2 complexes as a result of auto-phagocytosis (figure 3).
   
   

   Figure 3: Option 1

   
   
2. Transfecting HSC cells with the desired epitope. The epitope will be displayed on the MHC class 1 complexes as a result of the natural intercellular protein display mechanism (figure 4).
   
   

   Figure 4: Option 2

Although our HSC model cells,the HPC-7 cells , are multipotent/CMP (cell lines), and as such, are theoretically able to differentiate into dendritic cells or macrophages. We discovered that this is very difficult to do. For this reason, we chose to focus on the second option.
Our plan is to show that the model cells transfected with our display mechanism induce apoptosis in immature T cells derived from a TCR transgenic mouse (this means that all the mouse’s T cells have the exact same TCR). We will display the epitope that is specific to the TCR of the transgenic mouse, and thus determine if binding occurs between the MHC complex and the transgenic TCR by quantifying apoptosis.

Materials and methods:

Immature T cells:

A fetal thymus organ culture from a TCR transgenic C57BL/6 mouse, which was not scarified especially for our sake, was kindly offered to us by Prof. Steffen Jung's lab from Weizmann Institute. The TCR bears specificity for ovalbumin and MHC class 1. This strain of C57BL/6 was chosen in correspondence with the HPC-7 cells origin mouse strain, in order to address the second issue, mentioned above. Prof. Jakub Abramson's lab kindly offered to help us with the preparation and processing of the single cell thymocyte suspension.

APC cells:

We meant to use our model cell line, HPC-7, as APC cells.

Experiment:

Immature T cells are cocultured in Iscove’s modified Dulbecco’s medium (IMDM) with APC cells, transfected with our Display plasmid containing the ovalbumin epitope, for 24h and 48h. Apoptosis of immature T cells can be quantified by flow cytometry measurement using Annexin V and PI from an Invitrogen Dead Cell Apoptosis Kit. Interferons can be added in order to address issue 3 ^[5] .
Other acceptable systems for co-culturing thymocytes with the APC cells are RTOC or Matrigel.

The CD3 assay:

This assay demonstrates tolerance induction by indirect binding.
The CD3 complex is the T cell co-receptor, known for its role in transmitting intracellular signals when the TCR binds to a peptide-MHC complex during the T cell activation and signaling process ^[6] .
In a study from 1989, John Owen et al. showed that engaging the CD3/TCR complex of immature T cells with anti-CD3 antibodies leads to apoptosis ^[7] . This process is considered analogues to the process of clonal deletion ^{[7] [8]} .

Materials and methods:

Immature T cells:

Single cell suspension of thymocytes was kindly offered to be given by Prof. Jakub Abramson's lab.

Assay plasmid:

This plasmid contains the display system with the sequence of the anti-cd3 antibody gene, instead of an epitope. This plasmid requires special design, since displaying a functioning antibody on our model cells membrane is not an easy task (for further information, see B Cell Assay). Several studies ^{[9] [10]} have demonstrated that the scFv-FC fusion construct of anti- cd3, expressed on the cell membrane, can activate the T cell receptor efficiently. The scFv sequence was found in a paper from 1996 ^[11] (for a more detailed description of the construct's design, see B Cell Assay).

Experiment:

Thymocytes are cocultured with our model cells (HEK293/HPC-7), transfected with the assay plasmid, for 24h and 48h. Apoptosis of immature T cells can be quantified by flow cytometry using Annexin V and PI from an Invitrogen Dead Cell Apoptosis Kit.
Both the advantages and disadvantages of this assay result from the fact that, unlike the TCR assay, it does not involve the MHC complex. Therefore, this assay may be more feasible since any strain of mouse can be used for the preparation of the single cell thymocyte suspension, and it doesn't require a TCR transgenic mouse. The downside of this method is that due to the indirect binding, it does not achieve some of our assay goals. Nevertheless, as mentioned above, this assay shows the engagement of our model cells with the TCR complex analogous to the mechanism of central tolerance. Therefore, it can provide valuable information which may be helpful for additional studies.