Title: Mechanisms of skin-induced suppression of Tc1 CD8+ mediated contact sensitivity in mice.


It is well known that immunization via the digestive tract or nasal mucosa may induce a strong local response and simultaneously leads to a state of profound immunosuppression in the periphery. For many years, the skin was considered to be an organ where immune responses such as contact hypersensitivity (CHS) were easily induced. However, skin as a site for the induction of tolerance has received very little attention. Experiments performed at the Department of Medical Biology at Jagiellonian University School of Medicine showed that, indeed, epicutaneous (EC) application of protein antigens resulted in the induction of T regulatory cells (Treg) which were able to inhibit murine Th1 CD4+ -mediated contact sensitivity to a TNP-coupled protein. EC immunization with a protein antigen prior to active contact sensitization was shown to be mediated by TCRαβ+ CD4+ CD8+, in an antigen non-specific manner. The mechanism of skin-induced suppression relies on the action of the anti-inflammatory cytokine TGF-β. Recently it was shown that EC application of a protein antigen reduced inflammatory responses and decreased disease incidence in two different experimental models: experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA). Further work employing allogeneic skin grafts showed that EC immunization with a protein antigen delayed graft rejection in mice. The effector and regulating mechanisms of CHS mediated by Th1 CD4+ lymphocytes are well known, which may contribute to the development of new methods of contact skin inflammation treatment.In this dissertation, it was investigated whether the EC immunization with a DNP-coupled protein antigen can suppress Tc1 CD8+-mediated contact hypersensitivity in BALB/c mice. We have found that EC immunization with DNP-conjugated bovine serum albumin (DNP-BSA) prior to sensitization with 0.5% dinitrofluorobenzene (DNFB) did indeed suppress Tc1 CD8+-mediated CHS in vivo when compared with positive control. The observed reduction of ear swelling was correlating with the depression of vascular permeability, ear weight, IFN-γ concentration and MPO activity in ear homogenates. These results were confirmed by an in vitro model, where EC deposition of a protein antigen suppressed proliferation of Tef cells. It was also shown that DNP-BSA application prior to sensitization with DNFB significantly inhibited production of pro-inflammatory IFN-γ, IL-12 and TNF-α.In a further experiment it was determined that the optimal dose of EC-applied DNP-BSA which could suppress CHS ranges between 100 and 3 µg of antigen / mouse and that the state of unresponsiveness declined with time and lasted for around three weeks. ; Experiments in a model of an active immunization, “transfer in” and adoptive cell transfer of CHS employing four non-cross-reacting antigens - DNP-BSA, MBP, OVA or OX-Ig - showed that suppression induced via EC immunization with an antigen is antigen non-specific.Further experiments, in a model of “transfer in” and adoptive cell transfer of CHS, demonstrated that suppressive activity could be transferred by cells isolated from the axillary and inguinal lymph node cells, spleen and thymus of EC-immunized mice. The lowest number of EC-induced Treg cells which could effectively inhibit Tc1 CD8+-dependent CHS was 1.35x107 unseparated LNC and only 2x106 of purified Treg cells. Finally we showed that transfer of EC-induced Treg cells either at the time of immunization or challenge significantly suppressed CHS response.The phenotype of the Treg lymphocyte was found using the negative selection experiment with monoclonal antibodies and rabbit complement, FACS sorting and TCRδ-/- (mice lacking T lymphocytes with the TCRγδ receptor), CD1d-/- (mice lacking NKT lymphocytes DC1d dependent) and β2m-/- (mice lacking CD8 lymphocytes) mice. These data suggested that EC immunization with DNP-BSA induced a population of Treg cells with a TCRαβ+CD4+CD25+Foxp3+ phenotype.In the final section of this dissertation, the mechanism of suppression induced via EC application of DNP-BSA was discussed. Four different approaches were used: the expression of cytokine production by lymph nodes, the influence of DNP-BSA-induced Treg cells on the in vitro proliferation of Tef CHS cells, adoptive transfer of CHS after the incubation of Treg and Tef cells with application of the trans-well chambers, and inhibition of interaction between Treg and Tef cells through blockage of the CTLA-4 molecule. These experiments showed that the mechanism of suppression requires direct contact between Treg and Tef cells and that the participation of the CTLA-4 molecule is necessary. Moreover, immunization with DNP-BSA does not affect production of anti-inflammatory cytokines IL-4, IL-10, IL-17E or TGF-β.In summary, this dissertation describes a novel method of evoking immune tolerance via EC immunization of DNP-BSA. Because of its effectiveness, ease of induction and non-invasive protein antigen application, this immunization route may be effective for a new method of Tc1 CD8+-dependent contact hypersensitivity reaction therapy.

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2 - studia doktoranckie

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Uniwersytet Jagielloński. Collegium Medicum. Wydział Lekarski.


Marian Szczepanik

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Biblioteka Medyczna Uniwersytetu Jagiellońskiego- Collegium Medicum

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Oct 30, 2019

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Jul 19, 2013

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ZB-118304 Oct 30, 2019


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