Exosomes Derived From Keratinocytes Support Superantigens-driven Proliferation Of Resting T Cells

Objectives:Staphylococcus aureus(S aureus)is one kind of common bacteria colonized on skin.Staphylococcus aureus not only correlates with infectious disease,but also is closely related to many inflammatory skin diseases(eczema,psoriasis,atopic dermatitis,etc.).The immune response induced by S aureus enterotoxin B(SEB),one kind of staphylococcal superantigen,is a vital pathogenic mechanism of S aureus.Keratinocytes,which account for 90% of epidermis,are also the main cells that SEB directly contact with.It has been proved that human keratinocytes were able to act as antigen presenting cells to process superantigen and induce the proliferation of T cells.However,all of the previous researches investigated the ability of presenting antigens of keratinocytes under the mixed culture condition.It is still unknown whether keratinocytes could directly present superantigens to T cells via a non-contact way.More recently,exosomes,one kind of nano-size membrane vesicles,have become the research focus of intercellular communication.Exosomes are nano-size membrane vesicles(diameter,30–100 nm)which are released into the extracellular medium by cells.It has been proved that exosomes could carry antigens or MHC–peptide complexes to T cells causing immune reactions.Therefore,we assumed that keratinocytes-derived exosomes,which could act as a “truck” between keratinocytes and T cells,may make it possible for keratinocyte to present superantigens to T cells via a non-contact way.In our study,we made use of the keratinocyte line(HaCaT cells)to detect whether keratinocytes could support superantigens-driven proliferation of resting T cells via an indirect contact way.Moreover,we further investigated the ability of HaCaT-exosomes in inducing the proliferation of T cells.Methods:Our study could be divided into two parts and the main method and conclusion were listed as follows:The first part: Transwell chamber was used to establish a co-culture system between HaCaT cells and CD3+ T cells under non-contact condition.The proliferation of CD3+ T cells induced by SEB-loaded HaCa T cells were detected by CCK8 assay and CFSE labeling assay,respectively.The second part:1.HaCaT cells-derived exosomes were isolated by differential centrifugation.The HaCaT cells-derived exosomes were observed for morphology by transmission electron microscope.Besides,the maker protein(CD63,Tsg101)and negative protein in exosomes were detected by Western blot.2.Interaction between HaCaT-derived exosomes and T cells was observed by confocal fluorescence microscope.3.The expression of MHC I?MHC II?ICAM molecules in HaCaT cells-derived exosomes were detected by Western blot for whether it had the molecular of presenting SEB.4.The proliferations of T cells induced by exosomes from SEB-loaded-HaCaT cells were detected by CFSE labeling assay.Results:The first part:Both of CCK8 assay and CFSE labeling assay indicated that the SEB-loaded HaCaT cells could induce T cells proliferation via an indirect contact way.The second part:1.Typical exosomes from HaCaT cells could be isolated with high purity by differential centrifugation.2.HaCaT-Exosomes could interact with T cells.3.HaCaT-Exosomes expressed low levels of MHC I but no MHC II molecules.When HaCaT cells were pretreated by IFN-?,HaCaT-Exosomes expressed low level of MHC II molecules and the MHC I molecules was further up-regulated.4.After IFN-? stimulation,Staphylococcal aureus enterotoxin B-loaded HaCaT cells secreted exosomes to induce the proliferation of both CD4+ and CD8+ T cells in vitro.Conclusion:Colonization of S aureus triggers the onset and aggravates the course of various inflammatory diseases.The immune response induced by staphylococcal superantigens(e.g.the S aureus enterotoxin B)is a vital pathogenic mechanism of S aureus.In our study,we have verified a new non-contact mechanism between keratinocyte and targeted T cells in the SEB related cutaneous immunity.Moreover,our study unveils that exosomes is another way of keratinocyte participating in superantigens related skin diseases.