The overall goal of this experiment is to study the consequences on endothelial cell integrity and permeability upon interaction with activated T cells. This method helps to answer key questions in the neuroimmunology field, such as how does the direct interaction between T cells and endothelial cells affect blood-brain barrier integrity and permeability. The main advantage of this technique is that it enables automated, real-time assessment of barrier function upon targeted modulation of T cell and endothelial cell interaction.

The implications of this technique extend toward therapy of autoimmune CNS disorders, because it provides insight into T cell mediated blood-brain barrier breakdown, a crucial step into pathogenesis of such diseases. This method focuses on marine blood-brain barrier properties during EAE. But it can also be applied to red or human blood-brain barrier models, as well as studies of other autoimmune CNS disorders.

Generally, individuals new to this method may struggle because of possible difficulties with optimal timing of combined primary endothelial cell culture and T cell activation. We first developed this method when we set off to investigate effects of different T cell activation levels on the blood-brain barrier properties during EAE, an animal model of multiple sclerosis. Visual demonstration of this method is critical, as setting up T cell and endothelial cell co-cultures is difficult to learn, because initial variations in T between wells may need to subsequent misrepresentations of the results.

To begin this procedure, on day five after MBMEC isolation, precoat the permeable inserts with 80 microliters of MBMEC coating solution per insert. Incubate them at 37 degrees Celsius, and 5%carbon dioxide, for three hours. After removing the MBMEC coating solution from inserts, wash MBMECs by adding two milliliters of room temperature PBS to each well, and aspirating it after one minute, and then repeat this procedure once more.

Next, add 0.05%Trypsin EDTA at 300 microliters per well and incubate it for five to ten minutes. Subsequently, add two milliliters of MBMEC medium to each well to stop trypsinization. Then, transfer the collected cells to a 15 milliliter centrifugation tube and centrifuge the sample at 700 times g for eight minutes at four degrees Celsius.

After eight minutes, discard the supernatant and resuspend the cells in one milliliter of MBMEC medium without puromycin. To count the cells, mix 90 microliters of 0.04%trypan blue. With 10 microliters of cell suspension, pipette 10 microliters of the mixture between the glass cover and the hemocytometer.

Next, cede two times 10 to the fourth cells per insert. Add 810 microliters of MBMEC medium to the lower compartment of each well and place the plate with the inserts in the incubator until ready to proceed with TEER measurement. To stimulate CD4 positive T cells, precoat a round-bottomed 96 well plate with purified anti-mouse CD3 antibody at a desired final concentration for three hours at 37 degree Celsius.

After isolating the T cells, wash the precoated plate twice with PBS. Next, add purified anti-CD28 antibody to the isolated T cells at a desired final concentration and mix well. Then, cede the T cells and leave them at 37 degrees Celsius for two to three days.

To set up TEER measurement, place the 24 well module of the TEER instrument in the laminar flow hood. Remove the lids and place the inserts with MBMECs in the instrument, using forceps. Next, pipette 810 microliters of the fresh medium to the lower compartment of the module wells, added carefully between the insert and the wall of the module well.

After that, close the lids and place the instrument in the incubator. Connect the instrument to the computer. Then, turn on the instrument controller and open the software.

In the pop-up window, select New Measurement"Check show TER"and show Ccl"boxes. Then, press start. After completing the first measurement, select check all wells"in the Results tab to see all TEER and Ccl values.

Then, save the file. Next, choose the time point to co-culture MBMECs with T cells when Ccl is stable and lower than one microfarad per square centimeter and TEER has reached its maximum level. Carefully inspect the absolute TEER and Ccl values and exclude the wells in which MBMECs have not developed confluent enough monolayers by unchecking such wells.

Group the rest of the wells by right-clicking on the wells and selecting add well to new average well"Name it in the pop-up window and do the same for all individual wells to be grouped. Then check all average wells to confirm that all of them have the same initial conditions before the co-culture. In the experiment tab, press pause.

Disconnect the instrument and take it out of the incubator. Then, remove the lids in the laminar flow hood. Subsequently, remove some of the medium from the insert at the upper well compartment.

Add T cells to MBMECs by carefully pipetting 150 microliters of medium. Then, reconnect the instrument and press resume measurement. After 24 hours, press stop in the experiment tab and save the file.

Shown here are the spindle-shaped morphology in the immunofluorescent staining of PECAM-1 and Cldn-5 five days after MBMEC isolation. Here are the TEER and Ccl values before and after grouping of individual wells prior to the addition of T cells. The blue curve is not used for the experiment since MBMECs in this well have not developed TEER as high as in other wells.

In this experiment, mark 35 to 55 specific CD4 positive T cells were stimulated in an antigen-specific fashion for five days. Naive T cells and proinflammatory cytokines, interferon gamma and TNF-alpha served as a negative and positive control, respectively. Here, T cells were stimulated by the same amount of anti-CD3 and anti-CD28 antibodies and the ones cultured for three days exhibited a greater propensity for barrier disruption compared to the T cells cultured for only two days.

The results here show that stimulated T cells but not their supernatants caused a substantial damage to the MBMECs, indicating the direct contact between T cells and MBMECs is critical for barrier disruption. On the other hand, adding granzyme b inhibitor two to T cell culture restored MBMEC integrity to a greater extent, pointing to the cytolytic molecule as an important player in causing MBMEC damage and subsequent decrease in TEER Once mastered, this experiment can be done in 12 days if it's performed properly. While attempting this procedure, it's important to remember to choose the correct time point for setting up the co-culture of T cells and endothelial cells.

Following this procedure other essays can be employed, such as immunofluorescence microscopy, flow cytometry, and probability essays. In order to address additional questions, such as How does this interaction affect tight junction molecule expression permeability and the extent of T cell transmigration? After its development, this technique paved a way for researchers in the field of neuroimmunology to explore autoimmune disorders of the CNS in mice.

After watching this video, you should have a good idea of how to assess the direct effects between activated T cells and endothelial cells with regard to blood-brain barrier integrity and permeability.