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Method Article

Murine Model of Epicutaneously-Induced Immunomodulation

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DOI:

10.3791/67578

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June 24th, 2025

 ,  ,  ,  ,  , 
1Department of Medical Physiology, Chair of Biomedical Sciences, Institute of Physiotherapy, Faculty of Health Sciences, Jagiellonian University Medical College, 2Department of Clinical Immunology, Wroclaw Medical University, 3Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College

* These authors contributed equally

In This Article

Summary

The study demonstrates the role of the skin in modulating immune responses in mice through epicutaneous (EC) immunization. EC immunization with a protein antigen can suppress immune responses in contact hypersensitivity or reverse skin-induced suppression when combined with pathogen-associated molecular patterns. EC immunization shows promise as an effective, needle-free approach to immunotherapy.

Abstract

The skin, as the largest organ in the human body interfacing with the external environment, plays crucial physiological roles, including (1) protecting the organism from xenobiotics and microorganisms, (2) facilitating thermoregulation, (3) maintaining water and electrolyte balance, (4) synthesizing vitamin D, and (5) participating in immunity. This paper highlights the skin's pivotal involvement in immunological mechanisms, showcasing its capacity to immunomodulate by suppressing or potentiating the immune response. Previous findings reveal that, in mice, epicutaneous (EC) immunization with an antigen, either alone or in conjunction with pathogen-associated molecular patterns (PAMPs), can suppress or potentiate immune responses, respectively. Specifically, it was observed that EC immunization with an antigen can suppress immune responses in various murine models of autoimmune diseases or allergic contact dermatitis. The results indicate that EC immunization with an antigen combined with PAMPs could serve as an effective strategy to boost immunity against pathogens or alleviate T helper type 2 lymphocyte (Th2)-dependent allergies. This protocol outlines a method for inducing skin suppression via the epicutaneous application of an antigen in a murine model of human allergic contact dermatitis (ACD)-contact hypersensitivity (CHS) mediated by Th1 cells. However, it can also be applied to various murine disorder models. The article presents EC-induced immunomodulation as an attractive murine model for needle-free immunotherapy across various disorders.

Introduction

This article presents a method for inducing tolerance (referred to as 'suppression' throughout this text) in a mouse model of contact hypersensitivity (CHS) and for reversing this tolerance (referred to as 'contrasuppression' throughout this text) via epicutaneous (EC) application of an antigen alone or in combination with ligands for pathogen recognition receptors (PRRs), respectively1. In described protocol (a) haptenized mouse immunoglobulins (TNP-Ig) are used as antigen to suppress contact hypersensitivity or (b) antigen with pathogen-associated molecular patterns (PAMPs), ligands for Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-containing protein (NOD)-like receptors (NLRs), to reverse skin-induced suppression2,3,4. The findings indicate that EC-induced immunomodulation is mediated by antigen-non-specific T suppressor (Ts) cells and antigen-specific T contrasuppressor (Tcs) cells. These cells can be examined in a mouse model of adoptive cell transfer to determine the mechanism of the tested reactions. The current protocol describes two models of cell transfer, "adoptive transfer IN" and "adoptive transfer OUT". Adoptive transfer IN is useful for testing the immunomodulation in the afferent and efferent phases of CHS. On the other hand, "adoptive transfer OUT" reflects the effect of immunomodulation only in the efferent phase of CHS.

The article describes a method of skin-induced suppression via the epicutaneous application of an antigen in a murine model of human allergic contact dermatitis (ACD) - contact hypersensitivity (CHS) mediated by T helper type 1 lymphocytes (Th1)5. However, this approach was also tested for its efficacy in regulating (suppressing, tolerance) experimental models of T cytotoxic lymphocyte type 1 (Tc1)-mediated CHS6,7, autoimmune diseases such as collagen-induced arthritis (CIA)8,9 , experimental autoimmune encephalomyelitis (EAE)10,11, and ulcerative colitis12. Moreover, the protocol describes the method of skin-induced contrasuppression (reversed suppression) via EC application of an antigen together with PRR ligands in a murine model of CHS13,14,15,16. The presented results indicate that EC-induced antigen-nonspecific suppression can be reversed by purifying TLR2, TLR3, TLR4, TLR9, and NOD2 ligands. The method of skin-induced contrasuppression was also employed in an animal model of atopic dermatitis (AD), showing that EC treatment with an antigen in the presence of synthetic single-stranded DNA molecules containing unmethylated oligodeoxynucleotides (CpG) - ligand for TLR9 can suppress Th2-mediated responses in an antigen-specific manner (shifts the immune response toward a Th1/Th17 phenotype)17. The described method was also tested in pneumococcal pneumonia, demonstrating that EC immunization with pneumococcal polysaccharide conjugated to bovine serum albumin (PC-BSA), together with CpG, could potentially enhance immunity to Streptococcus pneumoniae18.

A pivotal aspect of EC-induced immunomodulation is the selection of the model and the nature of the antigen. The article includes a table detailing the antigens used, the antigen exposure duration, and the tested models' T-cell specificity (see Table 1). The described method for immunomodulation is targeted towards diseases involving antigen-specific T lymphocytes. The effectiveness of this method was assessed by utilizing a compatible EC-applied antigen in the process. The compatibility of the antigen is crucial to ensure that it effectively interacts with the T lymphocytes involved in the disease process. The application of a protein or peptide patch on intact skin facilitates antigen penetration through the epidermis19,20. This increased antigen penetration is attributed to heightened perspiration beneath the dressing. Alterations in epidermal permeability occur within 4-10 h of patch application, while EC-induced immunomodulation may remain effective for up to two weeks5,14.

Although EC treatment is widely used, the existence of many variations of this method could raise questions. Current understanding suggests that the extent of epidermal damage prior to antigen administration may influence the nature of the resulting immune response21. Superficial epidermal damage triggers the release of anti-inflammatory cytokines by Langerhans cells, favoring the initiation of the Th2 response22. In contrast, more extensive epidermal damage promotes the release of pro-inflammatory cytokines and facilitates antigen delivery to dermal dendritic cells23.The presented protocol highlights (a) the recognition of challenges when applying this method in small and active animals, particularly mice; (b) considering factors such as skin condition after razor shaving, proper contact of substances with the skin, exposure time, and hygienic conditions of the skin during EC treatment which can determine the treatment's effectiveness. It is crucial to consider the method of skin depilation, whether physical or chemical, before antigen application, as different skin preparations can affect the skin's condition in various ways. It is acknowledged that all these procedures can activate skin immune cells, potentially leading to skin infections or influencing disease progression. The potential side effects on skin conditions after depilation should be a primary consideration when selecting the depilation method. The researchers described a method of T-cell-mediated immune suppression induced via the EC application of an antigen on razor-shaved dorsal skin, demonstrating its effect on various mouse models of immune responses. The antigen's EC application, along with PAMPs, induces antigen-specific T contrasuppressor cells. Moreover, the presented method of EC application, particularly with repeated exposure to antigens or PAMPs, does not cause concerning side effects.

The described protocol underlines the importance of various factors in achieving reliable and repeatable results with this method, especially in small animals like mice. The non-invasive nature of this approach typically results in a lower risk of adverse reactions (such as pain, infection, or scarring) compared to intradermal or injectable immunization techniques. Epicutaneous immunization is user-friendly and easily administered in non-clinical settings, improving accessibility and compliance compared to more invasive methods.

Table 1: EC-induced modulation of immune response in different mouse disease models. Abbreviations: ACD = allergic contact dermatitis; AD = atopic dermatitis; Ag = antigen; CIA = collagen-induced arthritis; CHS = contact hypersensitivity; CFA = complete Freund's adjuvant; COLL II = type II collagen; DNFB = 2,4-dinitrofluorobenzene; DNP-BSA = dinitrophenyl conjugated to bovine serum albumin; EAE = experimental autoimmune encephalomyelitis; EC = epicutaneous; LPS = lipopolysaccharide; OVA = ovalbumin; PAMP = pathogen-associated molecular pattern; PC-BSA = pneumococcal polysaccharide conjugated to bovine serum albumin; Th1 = T helper type 1 lymphocyte; Tc1 = T cytotoxic type 1 lymphocyte; TNCB = 2,4,6-trinitrochlorobenzene; TNP-Ig = 2,4,6-trinitrophenyl-conjugated mouse immunoglobulins; Ts = T suppressor cells; Tcs = T contrasuppressor cells. Please click here to download this Table.

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Protocol

All experiments presented in this article were approved and conducted in accordance with the guidelines of the 1st and 2nd Local Ethical Committee on Animal Testing in Krakow, Poland. The procedures complied with local recommendations, particularly regarding the administration of ketamine/xylazine or isoflurane as anesthetics, the shaving of mouse skin using razor blades, the application of substances/haptens on the shaved skin and both ear sides, and the collection of peripheral immune organs. The study used CBA/J (H-2k) male and female mice, 6-12 weeks old, with body weights ranging from 20-25 g. Details on the reagents and equipment used are listed in the Table of Materials.

1. Animal preparation

  1. House mice together; it is important to prevent the negative effects of isolation on their behavior and well-being. For statistical significance, use 10-12 animals per group.

2. Preparing the operating area

  1. Disinfect the operating table with a 70% ethanol solution before and after all procedures. If using mice requires sterile conditions, conduct all operations within a biosafety cabinet.

3. Induction of suppressor cells (Ts) for Th1-mediated CHS reaction

NOTE: This procedure is depicted in Figure 1A.

  1. On the day "0" anesthetize the mice with 30% isoflurane in propylene glycol by an open drop method. Hold the mouse in the left hand, exposing the dorsal side of its body. With the right hand, lather the grey soap with copious water and apply it to the skin.
    1. Using a sterile razor blade, gently shave a 2 cm² skin area. Avoid injuring the skin. Any skin lesions will disqualify the mice from further procedures.
      NOTE: The grey soap is a natural and hypoallergenic product made from a gentle vegetable base. It effectively regenerates and moisturizes dry and sensitive skin. This soap does not contain any artificial colors, synthetic fragrances, or ingredients of animal origin. The ingredients include Sodium Palmate, Sodium Palm Kernelate, Aqua (Water), Glycerin, Taraxacum Officinale (Dandelion) Flower/Leaf/Stem Extract, Palm Acid, Palm Kernel Acid, Sodium Chloride, Propylene Glycol, Tetrasodium EDTA, Etidronic Acid. The gray soap is designed for sensitive and delicate skin that is prone to irritation and psoriasis. It effectively cleanses problematic skin while preventing excessive dryness and a tight feeling. This soap also soothes irritations, hydrates, and promotes faster skin regeneration. Importantly, it does not compromise the skin's protective barrier.
  2. Place the mice into the cage. After shaving, wait at least six hours before applying the antigen to allow the skin to dry and be cleaned by the animal.
  3. On day "+1", prepare a 1 mg/mL solution of antigen 2,4,6-trinitrophenyl-conjugated mouse immunoglobulin (TNP-Ig), in sterile Dulbecco′s phosphate-buffered saline (DPBS). Prepare the volume of an antigen 1200 µL for ten mice. Mix the solutions just before use in a 2 mL vial. Protect TNP-Ig from light by covering the vial with aluminum foil.
    NOTE: Include a CHS control group of mice treated only with the vehicle (DPBS) to assess non-specific inflammatory reactions caused by shaving and applying the solvent on the shaved spot. A different type of antigen should be used for Ts induction to test their antigen specificity. Mouse immunoglobulins (Ig) were prepared from mouse sera and conjugated with TNP hapten (Supplementary File 1). A single preparation with the substitution level of 40 TNP per Ig molecule (TNP40-Ig) was used throughout24,25.
  4. On the same day, anesthetize mice with 30% isoflurane in propylene glycol. Apply the antigen on the previously shaved spot by placing a 1 cm2 gauze patch soaked in 100 µL of the antigen (prepared in step 3.3). Cover this with a thin plastic patch (2 cm²) and secure it with fabric adhesive tape. In the CHS control group, apply only DPBS.
    NOTE: 30 µm transparent document sleeves can be used for the plastic patch. The fabric adhesive tape should be easily removable, flexible, conform to body shapes, and allow air and water vapor permeability.
  5. On the day "+5," anesthetize the mice with 30% isoflurane in propylene glycol. Carefully cut the patch with safety scissors featuring rounded corners, then gently remove the adhesive tape. Allow the mice to move freely and naturally clean their skin for at least 4 h. If necessary, shave any regrown hair at the site of antigen application, though this is usually not required.
    NOTE: Cut the adhesive tape only at the area of antigen administration (gauze and plastic area), where it adheres tightly only at the edges of the bandage. Wait at least 4 h before applying the antigen to allow the skin to dry and be cleaned by the animal.
  6. On the day "+5," repeat step 3.3 and step 3.4.
  7. On the day "+8," repeat step 3.5. This marks the completion of a week-long skin exposure to the antigen. Following this period, use the mice to test suppression of the CHS reaction -in vivo active models (step 6) or euthanize them to serve as donors of Ts cells in "adoptive transfer IN" (step 8) or in "adoptive transfer OUT" (step 9).

4. Induction of contrasuppressor T-cells (Tcs) for Th1-mediated CHS reaction

NOTE: This procedure is depicted in Figure 1B.

  1. On the day "0" execute the steps 3.1-3.2.
  2. On the day "+1," prepare a mixture of 100 µg of antigen TNP-Ig and 100 µg LPS (example of PAMP) in 100 µL of DPBS. Prepare the solution immediately before use in a 2 mL vial and shield it from light by wrapping the vial in aluminum foil if the antigen is photosensitive.
    NOTE: To evaluate contrasuppression (reversal of skin-induced suppression), a control for suppression (Ts control group) must be included, where mice are treated only with the antigen TNP-Ig. Furthermore, mice treated solely with DPBS (CHS control) and LPS alone (PAMP control) should be included to determine the extent of non-specific modulation of immune responses. LPS is an example of an immune-stimulatory particle26. The specific characteristics of any immune stimulator depend on the mouse model used, the types of immune cells involved in the immune response being studied, and the pharmacokinetics of the substances under investigation.
  3. On the same day, anesthetize mice with 30% isoflurane in propylene glycol. Place a 1 cm2 gauze patch on the previous shaved skin, saturated with a solution containing 100 µL of the mixture (prepared in step 4.2). Secure the gauze with a thin plastic patch (2 cm²) and affix it in place using fabric adhesive plaster. In the control groups, apply DPBS (CHS control) or LPS (PAMP control), or TNP-Ig alone (Ts control group).
    NOTE: 30 µm transparent document sleeves can be used for the plastic patch. The fabric adhesive tape should be easily removable, flexible, conform to body shapes, and allow air and water vapor permeability.
  4. On the day "+5," execute step 3.5.
  5. On the day "+5," repeat steps 4.2-4.3.
  6. On the day "+8," repeat step 3.5. A week of skin exposure to the antigen mixed with LPS is finished. Following this period, test mice in CHS reaction - in vivo active models (step 6), or euthanize to serve as donors of Tcs cells in "adoptive transfer OUT" (step 10).

5. Induction of T effector cells (Teff) for Th1-mediated CHS reaction (sensitization phase of CHS)

  1. On day "0," shave the chest and abdomen of the mice (2 × 2 cm area) using grey soap with water followed by a razor blade. Allow the skin to rest for 6 hours before applying the hapten to ensure the absence of irritation.
  2. On the same day, just before use, prepare the hapten solution (5%) in a glass vial and cover it with aluminum foil to protect it from light: 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture (ratio 1:3).
  3. On the same day, sensitize mice by applying 150 µL of hapten on the previously shaved spot. Apply the vehicle alone (acetone-ethanol mixture) in the negative control group to assess the non-specific inflammatory reaction. Before returning the animal to the cage, wait 30 s to let the skin dry.
    CAUTION: Use gloves; TNCB causes severe allergic reactions in most people.

6. Active model: Testing of Ts or Tcs cells in Th1-mediated CHS

NOTE: This procedure is depicted in Figure 2.

  1. Induce the Ts or Tcs cell by following step 3 and step 4. This procedure will last from day "0" up to day "+8".
  2. On the day "+8", start to induce Teff cells by following step 5.
  3. Four days later, evaluate the CHS response by performing the elicitation phase (challenge). In brief, apply TNCB to both sides of the ears. Then, quantify the thickness of the ears by measuring them with a micrometer. A detailed description of this test and other possible endpoints is reported elsewhere (Zemelka-Wiacek M. et al.27).

7. Isolation of Teff, Ts, or Tcs cells from donor mice

  1. Disinfect the operating table with a 70% ethanol solution both before and after every procedure. Conduct all procedures within a biosafety cabinet to maintain sterility and use sterilized instruments. To isolate Ts, Tcs and Teff cells on the day "+8", use EC-immunized mice described in steps 3, 4, and 5, respectively.
    NOTE: Adhere to a donor-to-recipient ratio of 1:1.
  2. Anesthetize donors with an intraperitoneal (i.p.) injection of a mixture of ketamine (90-120 mg/ kg) and xylazine (5-10 mg/ kg). Provide thermal support throughout the procedure. Ensure the mouse is fully anesthetized for at least 5 min.
  3. Disinfect the entire abdominal area of the anesthetized mouse with 70% ethanol, then incise the abdominal layer (3-4 cm) to expose the peritoneal cavity. Isolate the auxiliary and inguinal lymph nodes (ALNs) and the spleen (SPL) with the sterile forceps into tubes filled with sterile DPBS that is supplemented with 1% fetal bovine serum (FBS). Pool the ALNs from all donors in one vial and SPLs in another and keep the vials on ice. Each axilla contains two axillary lymph nodes, while a single inguinal lymph node is situated in the hip region adjacent to three blood vessels. SPLs are positioned on the left side of the body, behind the intestine and stomach.
    NOTE: Work with sterile tools under the biosafety cabinet to maintain sterile conditions. After this procedure, mice must be euthanized following institutionally approved protocols.
    1. Mash tissue between the frosted ends of two microscope slides. Strain the cell suspension through a cell strainer with a 70 µm pore size.
  4. Rinse the cells with 30 mL of DPBS supplemented with 1% FBS. Centrifuge at 300 x g for 10 min at 4 °C. Remove the supernatant and resuspend the cell pellet in 1-5 mL of DPBS.
  5. Count the cell's viability using a hemocytometer and Trypan Blue. Mix 10 µL of cell suspension with 90-990 µL of Trypan Blue, adjusting the volume based on the cell count. Incorporate this dilution factor into the viability calculation.
  6. Prepare a mixture of ALNs and SPLs (ratio 1:1). Adhere to the number of cells: use 1 up to 5 x 107 Ts or Tcs cells per recipient in 1 mL of DPBS supplemented with 1% FBS and 7 x 107 Teff cells/ recipient in 1 mL of DPBS. Keep the donor-recipient ratio for all cell populations 1:1 and also the donor-DPBS ratio 1:1. Keep the vials with cells on the ice.
    NOTE: The execution of further in vitro experiments necessitates a proportional increase in the number of cell donors.
    NOTE: Additionally, isolated Teff, Ts, and Tcs cells can undergo additional purification or analyses using flow cytometry, such as phenotyping or cytokine secretion assessment. It is also feasible to initiate cell cultures to explore cell proliferation capacity or quantify cytokine levels in culture supernatants. Conducting further experiments necessitates a proportional increase in the number of cell donors.

8. Adoptive transfer IN: Testing of Ts in Th1-mediated CHS

NOTE: This procedure is depicted in Figure 3. Work sterile and use 70% ethanol solution both before and after every procedure. Ensure all cell operations are conducted within a biosafety cabinet to maintain sterility and use sterilized instruments.

  1. Induce and isolate Ts cells (donors) by following step 3 and step 7. Rinse the cells with 30 mL of DPBS. Centrifuge at 300 x g for 10 min at 4 °C. Remove the supernatant and resuspend the cell pellet in 200 µL of DPBS per recipient (donor-recipient ratio 1:1).
  2. Recipients (naïve syngeneic mice): anesthetize recipients with an intraperitoneal (i.p.) injection of a mixture of ketamine (90-120 mg/ kg) and xylazine (5-10 mg/ kg). Ensure the mouse is fully anesthetized for at least 5 min.
  3. Administer an intravenous (i.v.) injection of a prepared mixture of 1-5 x 107 Ts cells (step 8.2) in 200 µL into the anesthetized recipient.
  4. On the same day, induce Th1-mediated CHS reaction. In brief, induce Teff cell (sensitization phase) described in steps 5.1-5.3). Four days later evaluate the CHS response by performing the elicitation phase (challenge) described in step 6.3.

9. Adoptive transfer OUT: Testing of Ts in Th1-mediated CHS

  1. Induce and isolate Ts and Teff cells (donors) by following steps 3, 5, and 7. Keep the isolated Ts and Teff in two separate vials. Divide the Teff cells into two vials: one for the "Ts test group" and one for the "CHS control".
    NOTE: Work sterile and use a 70% ethanol solution both before and after every procedure. Ensure all cell operations are conducted within a biosafety cabinet to maintain sterility and use sterilized instruments. Include a control group of recipients to whom only Teff cells will be administered ("CHS control"). For this group, extra donors should be used to adhere to the donor-recipient ratio of 1:1. To enhance the quality of results, recipients receiving control cells transferred from donors EC-treated with a solvent solution should be added. The decision to add any additional group of recipients and donors needs to be carefully planned, because this approach requires many mice, which does not comply with the 3Rs principle (Reduce, Refine, Replace).
  2. Add Ts cells to Teff cells for the "Ts test group". Add adequate DPBS to Teff cells in the "CHS control" vial.
    NOTE: Keep the number of 1-5 x 107 Ts cells and 7 x 107 Teff cells per recipient.
  3. Mix all vials gently. Keep the vials in the water bath (37 °C) for 30 min, mixing gently every 5 min.
  4. Rinse each vial of cells with 30 mL of DPBS supplemented with 1% FBS. Centrifuge at 300 x g for 10 min at 4 °C. Decant the supernatant and resuspend the pellet of cells in 200 µL of DPBS/ recipient.
  5. Pass each cell suspension through a 70 µm pore cell strainer. If necessary, adjust the volume of 200 µL of DPBS/ recipient.
  6. Recipients (naïve syngeneic mice): anesthetize recipients with an intraperitoneal (i.p.) injection of a mixture of ketamine (90-120 mg/ kg) and xylazine (5-10 mg/ kg). Ensure the mouse is fully anesthetized for at least 5 min.
  7. Administer an intravenous (i.v.) injection of prepared cells.
  8. On the same day, induce the elicitation phase of CHS (challenge) described in step 6.3. Include "negative control", where conduct only CHS challenge.

10. Adoptive transfer OUT: Testing of Tcs in Th1-mediated CHS

  1. Induce and isolate Ts, Tcs, and Teff cells (donors) by following steps 3-5, and step 7.
    NOTE: Work sterile and use a 70% ethanol solution both before and after every procedure. Ensure all cell operations are conducted within a biosafety cabinet to maintain sterility and use sterilized instruments. Include two control groups of recipients: a "CHS control" in which only Teff cells will be administered and a "Ts control" in which Teff cells will be incubated solely with Ts cells. For these groups, use extra donors to adhere to the donor-recipient ratio of 1:1.
  2. Keep the isolated Ts and Tcs cells in separate vials. Divide the Teff cells into three vials: one for the "Tcs test group", one for the "Ts control", and one for the "CHS control".
  3. First incubation: For the (a) "Tcs test group" add Tcs cells to Teff cells. For the (b) "Ts control" and (c) "CHS control", add an adequate amount of DPBS to Teff cells (see Table 2).
    NOTE: Keep the number of 1-5 x 107 Tcs cells and 7 x 107 Teff cells per recipient.
  4. Mix all vials gently. Keep in the water bath (37 °C) for 30 min, mixing gently every 5 min.
  5. Rinse each vial of cells with 30 mL of DPBS supplemented with 1% FBS. Centrifuge at 300 x g for 10 min at 4 °C. Decant the supernatant and resuspend the pellet of cells in 1 mL of DPBS supplemented with 1% FBS per recipient. Maintain a cell mixture-DPBS ratio 1:1.
  6. Second incubation: For the (a) "Tcs test group" add Ts cells to the mixture of preincubated Teff and Tcs cells. For the (b) "Ts control", add Ts cells to the mixture of preincubated Teff cells with DPBS. For the (c) "CHS control", add adequate DPBS to the preincubated Teff cells.
    NOTE: Keep the number of 1-5 x 107 Ts cells per recipient. Conduct incubations across all experimental groups (a, b, and c) to ensure uniformity in cell loss during the washing and centrifugation procedures.
  7. Mix all vials gently. Keep in the water bath (37 °C) for 30 min, mixing gently every 5 min.
  8. Rinse each vial of cells with 30 mL of DPBS. Centrifuge at 300 x g for 10 min at 4 °C. Decant the supernatant and resuspend the pellet of cells in 200 µL of DPBS/ recipient.
  9. Pass each cell suspension through a 70 µm pore cell strainer. If necessary, adjust the volume of 200 µL of DPBS/ recipient.
  10. Recipients (naïve syngeneic mice): anesthetize recipients with an intraperitoneal (i.p.) injection of a mixture of ketamine (90-120 mg/ kg) and xylazine (5-10 mg/ kg). Ensure the mouse is fully anesthetized for at least 5 min.
  11. Administer an intravenous (i.v.) injection of prepared cells.
  12. On the same day, induce the elicitation phase of CHS (challenge) described in step 6.3. Include "negative control", where conduct only CHS challenge.

Table 2: Schedule of cell incubations during Tcs "transfer OUT". Please click here to download this Table.

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Results

This study examines whether antigens applied via skin alone or combined with PAMP can induce Ts or Tcs cells, respectively, which modulate the CHS reaction. The immunization of CBA/J mice via EC with 100 µg of TNP-Ig or TNP-Ig plus 100 µg of LPS induces Ts or Tcs cells, respectively. The mice were then sensitized with 5% TNCB and challenged with the same hapten 4 days later. The reaction was evaluated by measuring ear swelling before and after the challenge. Each experiment has a negative control, which...

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Discussion

The investigations were conducted on many different animal models and using many mouse strains like BALB/c, C57BL/6, HLADR4-Tg, DBA 1, and B10.PL and SJL demonstrate that EC immunization with an assigned antigen can serve as a versatile and potentially limitless means of creating tolerance and controlling unwanted immune responses. The current protocol outlines two cell transfer models: (1) 'adoptive transfer IN,' which is useful for assessing immunomodulation during both the afferent and efferent phases of CHS, ...

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Disclosures

Authors have nothing to disclose.

Acknowledgements

This study was supported by grants from Polish Committee of Scientific Research Grants 3 PO5B 091 25, 2PO5A 204 29 and from National Science Center N N401 3554 33 to M.S. The graphs were drawn using GraphPad Prism software 10.2.3, and the figures 1-3 were created in BioRender. Zemelka-Wiacek, M. (2025): (1) https://BioRender.com/3ux2px ; (2)  https://BioRender.com/s0y8zs7 ; (3) https://BioRender.com/0qziglw. The authors thank Dorota Woźniak for taking care of the mouse colonies.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2,4,6-trinitrochlorobenzene (TNCB)Tokyo Chemical Industry CO., LTD, JapanC0307
2,4,6-trinitrophenyl-conjugated mouse immunoglobulin (TNP-Ig)Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College, Krakow, Poland
acetone (ACS reagent, ≥99.5%)Merck KGaA, Darmstadt, Germany179124
aluminum foilMerck KGaA, Darmstadt, GermanyZ185140  
analytical balance Sartorius Weighing Technology GmbH, Goettingen, GermanyPRACTUM224-1s, 29105177
bovine serum albumine (BSA)Merck KGaA, Darmstadt, GermanyA9418
CBA/J (H-2k) Breeding unit of the Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College, Krakow, Poland
cell strainer, pore size 70 μmBIOLOGIX, China15-1070
disposable pipettes capacity: 5 mL, 10 mL, 25 mLMerck KGaA, Darmstadt, GermanyZ740301, Z740302, Z740303
Dulbecco′s phosphate buffered saline (DPBS)ThermoFisher Scientific,  Waltham, Massachusetts, USA14190144
eppendorf Safe-Lock Tubes 1.5mlEppenforf, Germany30120086
eppendorf Safe-Lock Tubes, 2.0 mlEppenforf, Germany30120094
ethanol 100% (absolute alcohol)Merck KGaA, Darmstadt, Germany1.07017
ethanol 96%Merck KGaA, Darmstadt, Germany1.59010
fetal bovine serum (FBS)ThermoFisher Scientific,  Waltham, Massachusetts, USAA3160802
glass microscope slides (sandblasted single frosted)ThermoScientific, Portsmouth, NH, USA421-004T
Graph Pad PrismGraphPad Software Inc.v. 10.2.3
grey soap (Bialy jelen soap bar)Pollena Ostrzeszów, Producent Chemii Gospodarczej Sp. Z.o.o. , Sp. K., Poland163057
hemocytometerVWR, Avantor, U.S.A612-5719
incubator Heracell 150iThermo Electron  LED Gmbh, Germany41071068
isoflurane (Aerrane)Baxter, Deerfield, IL, USA8AGG9623
ketamine 100 mg/ mL, solution for injectionBiowet Pulawy Sp. z o.o., Pulawy, Polandcat.# not avaliable
laboratory Centrifuge (Heraeus Fresco 21)Thermo Scientific, Germany75002425
laboratory Centrifuge (Heraeus Megafuge 1.0R) Thermo Scientific, GermanyB00013899
lipopolysaccharide (LPS)Merck KGaA, Darmstadt, GermanyL-8274
mask (FFP2)VWR, Radnor, Pennsylvania, United States111-0917
micrometerMitutoyo, Tokyo, Japan193-111
microscope with objectivesLeica Microsystems CMS GmbH, GermanyDM1000, 294011-082007
propylene glycol Chempur, Piekary figure-materials-1 Poland114489005
razor bladeVWR, Radnor, Pennsylvania, United StatesPERS94-0462
transparent document sleeves 30 µmSCHEMAT5906961126542
trypan blueMerck KGaA, Darmstadt, GermanyT8154
tubes 15 mL sterileMerck KGaA, Darmstadt, GermanyCLS430055 (Corninig)
tubes 50 mL, sterileMerck KGaA, Darmstadt, GermanyCLS430290 (Corning)
vials, screw top, clear glass (vial only) 22 mLMerck KGaA, Darmstadt, Germany27173
Viscoplast Polovis, Silk adhesive, 5x0.0125m3M Poland, Nadarzyn, Poland7100231620
water bath AJL Electronic, PolandLW102
xylazine (xylapan 20 mg/ mL) solution for injectionVetoquinol Biowet Sp. z o.o., Gorzow Wielkopolski, Polandcat.# not avaliable

References

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Epicutaneous ImmunizationImmunomodulation ProtocolContact HypersensitivityT Suppressor CellsT Effector CellsAdoptive TransferSkin ImmunityAllergic Contact DermatitisPathogen Associated Molecular Patterns
Murine Model of Epicutaneously-Induced Immunomodulation
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