Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Well, what's really unique about the Teer assay is that it allows you to directly visualize antigen specific T cells. The te, the teer is the main unique aspect of our assay. That's coupled with an amplification step that we do.

You know, we isolate the T cells and then we grow them in an environment that enriches the sample for the T cells that we're interested in. Then it's, it's, we're able to to specifically label just those cells and visualize them on a flow cytometer. And so with previous assays, things like CSFE or proliferation assays that have been around for decades, what you're getting is just kind of an overall bulk measurements of what's going on with the whole T cell population.

But you've got millions of T cells in the body and they see a whole diverse set of patterns. If you're lucky, maybe one in two to 300, 000 of cells of the cells in the sample will be the one that you're looking at. And that's why this assay, it does that enrichment steps.

And then with a test number, and because you're directly staining, you can actually visualize away from background a population that's less than 1%of the total number of cells. And so you can see very, very small differences and you can get a fairly precise count of the, the number of T cells that are in that blood sample, you know, that are in that individual that recognize your pattern of interest. But it doesn't stop there because the teters also allow you to actually not only label those T cells, but isolate them for further analysis.

And the teters that you use are actually made here at BRI, is that correct? Yes.We're one of a very small number of labs in the, in the country that have successfully made class two teters. Our intent is to distribute these reagents at costs to any laboratories that are interested in importing this technology.

What would you say are the most difficult or challenging aspects of this assay when it comes to replicating it in other labs? Well, as I think about it, there's really two, the growth of the T cells. It's something that really is a bit of an art.

You, you have almost daily decisions as far as whether the cells need additional nutrients or additional space to grow. And then the second part that I think is probably the most difficult for an inexperienced to operator is actually the data analysis. Kind of figuring out which part of the signal really is a positive response versus which part is just inherent to, you know, physics and attraction.

And the fact that everything has some degree of stickiness to it is something that again, takes a lot of experience, especially in context where the overall response is gonna be a little bit weak. How does the information you'll generate with this assay have potential to benefit patients At this point? One of the first hallmarks that comes up in any autoimmune patient is kind of a, a constellation of antibodies that are specific for that disease.

We know from the fundamental fundamentals of immunology that wherever there's an antibody response, there's always a T-cell response first. And so if we could look in at-risk individuals and see that jump in T-cell activity that would precede antibody formation, that would then help us kind of define really a new level of risk or individuals are headed towards having autoantibodies, which then hopefully would open up a window for interventive therapies. This procedure will purify and expand antigen specific CD four positive T cells and visualize them using teters reagents and equipment class two laminar flow biosafety cabinets.

This will be the primary workspace for the procedure. 50 milliliter conical tubes primary tube for PBMC isolation one XPBS. This should be calcium and magnesium free fol pack plus foil wrapped to protect from light drummed pipette aid CoStar 10 mil pipette aerosol canisters with 50 mil inserts used to prevent blood contamination of centrifuge.

GS six R centrifuge transfer pipettes, Pasteur pipettes hemolytic buffer containing 8.3 grams per liter. Ammonium chloride 1.0 grams per liter. Sodium bicarbonate 0.04 grams per liter DI sodium EDTA trian blue 0.2%solution in PBS hemo CYTOMETER 15 milliliter conical tube running buffer containing PBS supplemented with two millimolar EDTA plus five grams per liter.

BSA max CD four isolation kit two purchased from Milin biotech EP magnet block purchased from stem cell technologies. Other magnetic cell enrichment platforms could be used with equal efficacy. Five milliliter polypropylene tube RPMI 1640 supplemented with 25 millimolar hippies used to make T-cell medium pooled human serum used to supplement T-cell medium pen strip used to supplement T-cell medium 500 milliliters 0.22 micron bottle top filter sterile 48 well plate used for in vitro expansion culture, step 37 degree CO2 incubator, 20 milligram per milliliter solution of synthetic peptide containing the epitope of interest.

Five milliliter polystyrene fax tube peptide loaded class two teer used to visualize antigen specific T cells, anti CD three, anti CD four and anti CD 25 antibodies used for coating antigen-specific T cells. Fax caliber flow cytometer or equivalent before starting the procedure. Assemble all materials and equipment in your workspace.

The following demonstration will cover the following steps. PBMC isolation, CD four positive T-cell separation in vitro expansion culture visualizing T cells by teem or staining. Step one PBMC isolation.

Obtain a blood sample. Blood should be collected in syringes or blood tubes and anticoagulated with heparin at a one to 50 ratio to prevent clotting, expect a yield of about 1 million PBMC per milliliter of blood. About 40%of which will be CD four positive T cells.

Aliquot the blood into 50 milliliter conical tubes. 25 milliliters of blood per tube. Gently mix the blood before ALI clotting to distribute the plasma.

Add PBS bringing the total volume to 40 milliliters and underlay by drying up 11 milliliters of fial inserting into blood tube and carefully removing the pipette aid from the pipette. The fial will slowly drain into the bottom of the tube. Once the level has equalized, slowly remove the pipette from the blood tube and discard cap the blood tubes and move into the aerosol of canisters.

Firmly close the canisters and centrifuge at 900 times G for 20 minutes with no break. It is critical that no break is applied at the end of this spin as the breaking force will disturb the layer of cells after the spin. The pbmc should form a distinct white layer between the yellow plasma above and the transparent fial below.

Gently skim off white blood cell layer using a transfer pipette and transfer to new tube. Some plasma and fial may be drawn up with the pbmc, but avoid drawing up any of the red cell layer. Typically two blood tubes can be combined into one cell tube at this step.

To increase the pellet size, add PBS bringing the total volume to 15 milliliters and centrifuge at 500 times G for 15 minutes on low break and aerosol of canisters aspirate liquid using a paste pipette, taking care not to disturb the pellet. Treat with hemolytic buffer by adding five to six milliliters to each tube and gently mixing to remove clumps. Incubate for no more than five minutes.

Add PBS bringing the total volume to 50 milliliters and centrifuge at 230 times G for 10 minutes. On low break aerosol canisters are no longer needed for these slower spins. Wash two times aspirate liquid using a pasture pipette filled tube with PBS and centrifuge at 230 times G for 10 minutes prior to the final wash step, remove an aliquot of resuspended cells, dilute with trian blue and count using a hemo cytometer.

This cell count will dictate the reagent volumes used in the T-cell separation. Step step two, CD four positive T-cell separation, aspirate liquid and add running buffer to bring the total volume up to 40 microliters per 10 million cells. Usually this requires 30 microliters of buffer plus the residual pellet volume.

Transfer this volume to a 15 milliliter conical tube. Add antibody cocktail from the CD four isolation kits, 10 microliters per 10 million cells cap tube and place on ice for 10 minutes. Remove tube from ice, remove cap and add 30 microliters of running buffer per 10 million cells.

Add magnetic beads from the CD four isolation kit. 20 microliters per 10 million cells cap tube and place on ice for 15 minutes. Add 10 volumes of running buffer to wash and centrifuge at 230 times G for 10 minutes.

During the spin, set up the magnet and five milliliter polypropylene tube in your workspace. Aspirate liquid from cell pellet. Add two milliliters running buffer and remove clumps using a transfer pipette.

Using the same transfer pipette transfer cells into the five milliliter tube and incubate in the magnet for 15 minutes. Carefully decant the CD four positive T-cell fraction into a marked 15 mil tube by inverting the magnet and emptying all but the last drop. Add another two milliliters of running buffer to the five milliliter tube and remove from the magnet.

Gently rinse the CD four negative cells off the sides of the tube and transfer to a marked 15 mil tube. Add two milliliters T-cell medium to each tube. Remove aliquots for cell counting and centrifuge at 230 times G for 10 minutes.

Dilute cell aliquots with trian blue and count using a hemo cytometer. These cell counts will dictate the number of wells used for the expansion culture. Step step three.

In vitro expansion culture aspirate liquid from the CD four positive and CD four negative cell pellets based on the cell counts. Add media to the CD four positive cells to adjust the volume to 3 million cells per mil. Add media to the CD four negative cells to adjust the volume to 10 million cells per mil.

The expansion culture requires 3 million CD four negative cells per well and two to 3 million CD four positive cells per well. Typically, CD four positive cells are the limiting population. Aliquot CD four negative cells into the appropriate number of wells on a 48 well plate.

By adding 300 microliters to each place the plate in a 37 degree incubator for one hour. Remove the plate, the incubator, wash the wells by adding 500 microliters of fresh media and gently pipetting up and down 12 to 20 times using a transfer pipette and removing all of the liquid. This wash will leave behind a layer of adherent antigen presenting cells.

When the washing is complete, add just enough media to wet the bottom of each, well roughly 100 microliters. Otherwise the A adherent cells will dry out. Add two to 3 million CD four positive cells to each well if necessary.

Add additional media to bring the total volume in each well up to one milliliter. Add the desired peptide to each.Well. The typical concentration for stimulation is 10 micrograms per milliliter.

Final place plate in a 37 degree incubator for seven days. After seven days. Add 50 microliters of hemo IL two to each well on each subsequent day.

Monitor cell growth using a microscope feed wells that are not yet confluent by removing half of the media, adding fresh media and 50 microliters of IL two split confluent wells by resus suspending the cells moving half of the cells to an empty well. Adding fresh media and 50 microliters of IL two return cells to incubator. The expanding cells will be ready for teer staining after 13 to 15 days of culture.

Step four, visualizing T-cells by teer staining, purchase or assemble teters to match the peptide. MHC combinations used to stimulate the CD four positive T cells. Remove plate from incubator.

Carefully draw off half of the media from each. Well transfer an aliquot of cells from each well typically 75 microliters or about 50, 000 cells into a five milliliter polystyrene Fax tube add teer typically one microliter per 50 microliters of total volume and place in 37 degree incubator for one hour. It is also advisable to stain a second aliquot of each well with a mismatched teer as a negative control label cells with anti CD three, anti CD four, and anti CD 25 antibodies.

Additional or alternative antibody markers can be used. However, do not use PE labeled antibodies since this channel must be reserved for the teems at this time. Also, label any single color or isotype controls using extra cells.

Incubate antibody labeled cells for 15 to 30 minutes at four degrees. Wash each tube with 0.5 to one milliliter of running buffer and centrifuge at 230 times G for 10 minutes. Carefully decant liquid from the fax tubes, calibrate flow cytometer using reference beads, single color control tubes, isotype controls, and et cetera.

Analyze each tube using the flow cytometer. The tetra positive cells should be a distinct population among the expanded CD four positive T cells. Tetra positive cells are usually CD 25 positive and often CD four high.I.