一种研究人体 T 细胞召回反应的吸水泡协议

The overall goal of this video is to demonstrate the suction blister cutaneous recall method which allows us to harvest antigen-specific T-cells and cytokines representative of the adaptive immune response in vivo. This method can be used to study cellular responses and adaptive immunity in humans. The main advantage of this technology is that it allows us to harvest antigen-specific T-cells directly from the site of the immune reaction using a relatively non-invasive and safe method.

The basic principle of cutaneous recall is to deposit antigen into the dermal layer of the skin. In individuals with existing immunological memory of the antigen, T-cells will migrate to the site of reaction and induce a local immune response where specific T-cells are clonally expanded and differentiated in situ. Induction of suction blisters over this reaction allows us to harvest these specific T-cells as well as the cytokines produced in vivo.

In this video demonstration, we use PPD as our recall antigen. PPD is commonly used for the diagnosis of mycobacterium tuberculosis infection. But, it also induces skin reactions in people previously vaccinated with the BCG vaccine, as you will see in this video.

Materials for the tuberculin skin test include alcohol swabs, a one milliliter syringe with a 27 to 30 gauge short needle, and a solution of PPD approved for human use. Use a PPD solution of 20 tuberculin units per milliliter. Disinfect the vial using an alcohol swab.

And please note the composition may vary between manufacturers. Then locate the injection site on the ventral side of the volunteer's forearm and disinfect the area using an alcohol swab. Use a one milliliter sterile syringe with a 29 gauge, 12 millimeter fixed needle.

Gently mix and aspire 0.1 milliliters of the PPD solution. Make sure that there as no visible bubbles. Stress the skin and point the needle at a five to fifteen degree angle with the bevel facing upwards.

Insert the tip of the needle into the dermal layer of the skin and make sure the tip is almost visible through the epidermis. Slowly inject 0.1 milliliter PPD solution. If administered correctly, a papule of six to ten millimeters will appear immediately.

The papule disappears after approximately ten minutes. Multiple depositions of PPD is possible, but could require additional ethical approval. If two injections are administered, aim for maximal separation while keeping in a good distance from the elbow and wrist area.

The clinical skin reaction appears within the first 24 hours and is accompanied by an itching sensation. Measure the size of the skin reaction 48 to 72 hours post-injection. When evaluating the response, it's important to measure the induration and not the erythema.

Palpate the hat swelling using your finger. Then mark the edges of the induration using a ball point pen. Finally, use a soft ruler to measure the induration diameter and note the results in millimeters.

After seven days, it's time to induce the suction blister. For induction of the skin suction blister, use a vacuum pump with an attached suction chamber. Use either a commercially available or a customized device.

Importantly, the pump needs to be adjustable within the range of minus 20 to minus 40 kilopascal and provide a steady and reliable negative pressure. The bottom part of the suction chamber consist of a plate with a small hole that is in contact with the skin. Plates may have single or multiple holes and different sizes can be applied in order to fit the skin reaction.

The top of the chamber should allow visual monitoring of the blister. Prior to induction, disinfect the skin and the chamber plate. Make sure the arm is resting comfortably.

Attach and secure the chamber, here by using adjustable straps. The center of the skin reaction should be visible through the bottom plate hole. Turn on the suction device and adjust the negative pressure to minus 20 kilopascal.

After thirty minutes, increase the pressure to minus 25 kilopascal. After 60 minutes, further increase the pressure to minus 30 kilopascals. And keep the pressure here until a single blister is fully formed.

The blister induction phase normally takes around one to three hours. With the actual blister formation occurring gradually within the last 30 minutes, as demonstrated here in high speed. If there are signs of blister rupture or hemorrhage, it's advised to slowly release the pressure and stop the induction early.

Blisters may appear as single or multiple merging blisters. The process is often associated with an itchy sensation. After the blister is fully formed, release the pressure and carefully remove the suction chamber.

In correctly induced blisters, the epidermal layer is fully separated from the dermal layer of the skin, leaving a cavity filled with fluid. Protect the blister by applying a soft dressing on the surrounding skin area and placing a hard cap over the blister, here from a a 15 milliliter plastic tube. Secure the cap by a non-allergenic tape followed by a soft, stretchy bandage.

Instruct the volunteer to keep the protective dressing overnight. On the next day, carefully remove the dressing and disinfect the blister area using spray. Use a 2 milliliter sterile syringe with a 23 gauge needle to harvest the blister content.

Insert the needle into the top lateral side of the blister roof and slowly aspire the fluid. Avoid touching the floor of the cavity, but make sure to harvest all the fluid as blister volume may be low, as in this example. The collapsed blister will heal without scarring.

Add a protective dressing over the blister. Then transfer the blister fluid to a sterile tube. Spin down for four minutes at 600 G using a tabletop centrifuge.

After, harvest the supernatant and re-suspend the cell pellet in 500 microliters of cell medium. The fresh cells are now ready for counting and further analysis. Suction blister cells can be used for in vitro analysis, such as flow cytometry.

In this experiment with BCG-vaccinated volunteers, cell yield ranged from 15, 000 to over 200, 000 cells per blister. Here, we isolated cells from a single suction blister and stimulated the cells with PPD in vitro followed by a staining and flow cytometry. For comparison, we did a parallel analysis on PBMCs from the same PCG-vaccinated volunteer.

In the panels, you see populations of CD4 positive T-cells stained for intercellular production of TNF-alpha, interferon-gamma, and IL2. In this volunteer, over 30%of the CD4 positive T-cells isolated from the skin were antigen-specific. In PBMCs, this fraction was less than 1%Furthermore, the individual cytokine profiles of blister cells were representative of both central memory T-cells and effector T-cells, illustrating how this method allows us to study long-lived memory responses generated in vivo.

Here, figure A illustrate that fluid obtained from suction blisters can be analyzed for cytokines produced in vivo. In figure B, we cultured cells obtained from suction blisters for four days in the presence of mycobacterium tuberculosis, illustrating how suction blister cells are capable of producing interferon-gamma and TNF alpha ex vivo. After watching this video, you should have a good understanding on how to induce and harvest cells from suction blisters following cutaneous antigen deposition.

In this demonstration, we used an antigen of special relevance within the field of tuberculosis research. But, the basic principles of this methodology can be of use within the fields of adaptive immunity, or in trials investigating T-cell targeting agents. Also, please remember that ethics and safety is a first priority when working with human volunteers.

Also remember that working with human samples can be hazardous and that safety precautions should always be followed in the lab.