Epicutaneous immunization with DNP‐BSA induces CD4⁺ CD25⁺ Treg cells that inhibit Tc1‐mediated CS

Abstract

As we have shown previously that protein antigen applied epicutaneously (EC) in mice inhibits TNP-specific Th1-mediated contact sensitivity (CS), we postulated that the maneuver of EC immunization might also suppress Tc1-dependent CS response. Here we showed that EC immunization of normal mice with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) applied on the skin in the form of a patch induces a state of subsequent unresponsiveness due to regulatory T cells (Treg) that inhibited sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by TCRab + CD4 + CD25 + lymphocytes harvested from lymph nodes (LNs) of skin-patched animals. Flow cytometry revealed that EC immunization with DNP-BSA increased TCRab + CD4 + CD25 + FoxP3 + lymphocytes in subcutaneous LNs, suggesting that observed suppression was mediated by Treg cells. Further, in vitro experiments showed that EC immunization with DNP-BSA prior to 1-fluoro-2,4-dinitrobenzen sensitization suppressed LN cell proliferation and inhibited production of TNF-a, IL-12 and IFN-c. Using a transwell system or anti-CTLA-4 mAb, we found that EC induced suppression required direct Treg-effector cell contact and is CTLA-4-dependent. Contact sensitivity (CS), also known as allergic contact, dermatitis is a serious health problem and one of the most common occupational diseases in developed countries. 1 CS responses to contact-sensitizing haptens, such as picryl chloride, oxazolone or dinitrofluorobenzene (DNFB), are classical manifestations of T-cell-mediated immunity in vivo. In previously sensitized hosts, CS and the related delayed-type hypersensitivity reaction are manifested as macroscopically measurable inflammation (skin swelling) that peaks at 24-48 h after topical cutaneous hapten challenge. 2 CS can be mediated by either CD4 + Th1, MHC class II-restricted cells locally releasing IFN-g to recruit a characteristic inflammatory infiltrate, 2 or CD8 + MHC class I-restricted Tc1 cells, which can similarly release IFN-g but predominately mediate cytotoxic damage to local skin cells, such as keratinocytes, 3,4 and by IL-17-producing Th17 cells. 5 Tc1-mediated CS to DNFB in BALB/c mice also requires participation of B-1, NKT and TCRgd lymphocytes that enable recruitment of Tc1 effector cells. 6 CS response to DNFB is mediated by CD8 + T cells that are regulated by CD4 + T lymphocytes. 7 It was already shown that Tc1-mediated CS to DNFB was negatively regulated by antigen (Ag)-specific CD4 + CD25 + FoxP3 + CTLA-4 T regulatory T cells (Treg), which are induced by hapten skin sensitization. 8 This population of regulatory cells expresses high level of inducible costimulator and are distinguishable from other Treg cells by the expression of IFN-g, IL-10 and IL-17. 8 On the other hand, suppression of Tc1-mediated CS can be induced via gavage with the hapten. 9 This type of hapten immunization induces deletion of a large fraction of Ag-specific CD8 T cells in the liver, and mesenteric lymph nodes (LNs) also trigger the suppressive fu...

Epicutaneous Immunization with Collagen Induces TCRαβ Suppressor T Cells That Inhibit Collagen-Induced Arthritis

“…These data suggest that EC-induced suppressor cells are very efficient regulators. This is consistent with our previous observation that small numbers of purified TCRαβ + suppressor cells can ameliorate the inflammatory response, since only 2.5 × 10 3 sorted cells efficiently inhibits CHS and EAE [15][16][17] .…”

“…In summary, induction of tolerance via EC immunization with protein antigen can inhibit cell-mediated immune responses in an animal model of RA, as we have previously demonstrated in CHS [14,15] , allogeneic skin graft rejection [19] and TNBS-induced colitis [18] , as well as in an animal model of multiple sclerosis [16,17,58] and in patients with multiple sclerosis [20,21] . This method may therefore be useful for the treatment of a variety of inflammatory diseases.…”

“…Our previous work both in contact hypersensitivity (CHS) [14,15] , EAE [16,17] and TNBS (trinitrobenzene sulfonic acid)-induced colitis [18] has demonstrated that EC immunization with protein antigen prior to the induction of T-mediated immune response causes strong suppression of the response. Our further work employing allogeneic skin grafts has shown that EC immunization with a protein antigen delays graft rejection [19] .…”

Epicutaneous immunization with protein antigen TNP-Ig alleviates TNBS-induced colitis in mice

“…Then, we found that EC immunization with myelin basic protein (MBP) prior to induction of experimental model of multiple sclerosis (EAE) resulted in protection from developing disease [9,24,25]. More recently, we showed that EC immunization with protein antigen suppresses Tc1-dependent and NK cell mediated CS to DNP [10,11] and an animal model of skin graft rejection [8]. Then, experiments in an animal model of arthritis [collagen induced arthritis (CIA)] showed that EC immunization with collagen prior to CIA induction alleviates disease [21].…”

Complementary methods for contact hypersensitivity (CHS) evaluation in mice

“…Ear punches were transferred to tubes containing 1 ml of formamide. After 18 h incubation at 37°C the samples were centrifuged and the optical density (OD) of the supernatant fluids was read at 565 nm against blank containing formamide alone (Majewska- Szczepanik et al, 2012).…”