Murine Model of Epicutaneously-Induced Immunomodulation

This protocol outlines a method for skin-induced immunomodulation in a murine model of human allergic contact dermatitis mediated by T helper one lymphocytes. Recent studies showed that epicutaneous immunization with protein antigen alone suppresses T cell-mediated immune responses while coadministration with pumps reverses skin-induced suppression. This highlights the potential of skin-based, needle-free strategies for generating regulatory cells to modulate immune responses.

It is well known that the degree of epidermal damage prior to antigen administration may determine the type of the induced immune response. Our study has established that epicutaneously induced immunomodulation is mediated by antigen-nonspecific T suppressor cells and antigen-specific T contrasuppressor cells. Despite widespread use of the epicutaneous immunization, the lack of standardized protocols and variations in application methods create inconsistencies in outcomes.

Our protocol addresses the challenges by offering a reproducible and controlled murine model. To begin, shave the back skin of the anesthetized mouse with a razor on day zero. On day one, prepare a one-milligram-per-milliliter solution of antigen 2, 4, 6-trinitrophenyl conjugated mouse immunoglobulin in sterile DPBS.

Protect the antigen from light by covering the vial with aluminum foil. Mix the solutions just before use in a two-milliliter vial. Apply the antigen solution to the shaved skin by placing a one-square-centimeter gauze patch soaked in 100 microliters of antigen.

Cover this gauze with a two-square-centimeter thin plastic patch and secure it with fabric adhesive tape. Once the mouse recovers from anesthesia, return it to its cage. On day five, after anesthetizing the mouse, use safety scissors with rounded corners to carefully cut the patch.

Gently remove the adhesive tape. After the mouse regains consciousness and is returned to its cage, allow it to move freely and clean its skin naturally for at least four hours. On day eight, to test EC-induced T suppressor cells in Th1-mediated contact hypersensitivity reaction, apply 150 microliters of the freshly prepared TNCB hapten solution to the shaved abdominal skin of the anesthetized mouse to induce T effector cells.

Four days after inducing T effector cells, evaluate the contact hypersensitivity response by measuring the ear baseline and applying TNCB hapten to both sides of the ears. Measure the ear thickness by using a micrometer 24 hours later. For the isolation of seven-day T suppressor or four-day T effector cells from the donor mice, disinfect the entire abdominal area of the anesthetized mouse with 70%ethanol.

Make a one-centimeter incision to expose the peritoneal cavity. Using sterile forceps, isolate the auxiliary and inguinal lymph nodes and spleen into tubes filled with sterile DPBS supplemented with 1%fetal bovine serum, or FBS. Pull the lymph nodes from all donors in one vial and spleens in another and keep the vials on ice.

Mash lymph nodes and spleens between the frosted ends of two microscope slides to create a cell suspension. Strain the cell suspension through a cell strainer with a 70-micrometer pore size. Rinse the cells with 30 milliliters of DPBS supplemented with 1%FBS and centrifuge at 300g for 10 minutes at four degrees Celsius.

Discard the supernatant and resuspend the pellet in one to five milliliters of DPBS. To count the cell viability, mix 10 microliters of cell suspension with 90 to 990 microliters of trypan blue, depending on the cell count, and count the cells using a hemocytometer. Next, prepare a 1:1 mixture of auxiliary lymph node and spleen cells to obtain 1 to 5 times 10 to the power of 7 T suppressor cells per recipient in one milliliter of DPBS with 1%FBS.

For T effector cells, prepare 7 times 10 to the power of 7 cells per recipient in one milliliter of the same buffer. Rinse the isolated cells with 30 milliliters of DPBS, then centrifuge them at 300g for 10 minutes at four degrees Celsius and resuspend the pellet in 200 microliters of DPBS per recipient. For the adoptive transfer in, administer an intravenous injection of the prepared mixture containing 1 to 5 times 10 to the power of 7 T suppressor cells in 200 microliters to the anesthetized recipient before inducing Th1-mediated contact hypersensitivity reaction.

On the same day, sensitize anesthetized mice by applying 150 microliters of HAp10 on the previously shaved spot. Four days later, evaluate the contact hypersensitivity response by measuring the ear baseline and applying HAp10 to both sides of the ears. Measure the ear thickness 24 hours later.

For the adoptive transfer out, after inducing and isolating T suppressor and T effector cells from donors, divide the T effector cells into two separate vials, labeled as TS test group"and CHS control. Add T suppressor cells to T effector cells for the TS test group and mix gently. Then add DPBS supplemented with 1%FBS only to the T effector cells in the CHS control vial and mix it.

Place the vials in a 37-degree Celsius water bath for 30 minutes, mixing the contents gently every five minutes. Rinse each vial with 30 milliliters of DPBS, then centrifuge at 300g for 10 minutes at four degrees Celsius. Decant the supernatant and resuspend the pellet in 200 microliters of DPBS per recipient.

Now, pass each cell suspension through a 70-micrometer pore cell strainer. Administer an intravenous injection of the prepared cells into recipient mice. Epicutaneous exposure to antigen with or without pathogen-associated molecular pattern influences the induction and modulation of contact hypersensitivity reaction.

Epicutaneous immunization with TNP-Ig antigen significantly suppressed contact hypersensitivity as shown by reduced ear swelling compared to the contact hypersensitivity control group. Coapplication with lipopolysaccharide reversed the suppression, restoring swelling levels similar to the control. Intravenous transfer of lymph node and spleen cells from mice tolerized with TNP-Ig significantly reduced TNCB-induced ear swelling in recipient mice compared to the contact hypersensitivity control, confirming the suppressive role of T suppressor cells.

Transfer of T effector cells incubated with T suppressor cells significantly suppressed the contact hypersensitivity response in recipients, while transfer of T effector cells alone triggered the contact hypersensitivity response.