Antigen Presentation Of Keratinocytes

Keratinocytes(KCs), the major component of the epidermis, is in direct contact with the external environment and also the first contact with the foreign antigen. It is reported that KCs not only act as the physical barrier of our body, but also has an important immune function. In the state of disease or directly be stimulated by cytokines such as IFN-γ, KCs can express the antigen presentation related molecules such as MHC-II, ICAM-1, and the expression of MHC-I also increase. The changes above make KCs have the essential condition to play the function of antigen presentation.However, previous studies just proved that KCs hold the condition to play the function of antigen presentation, for example, KCs can express some molecules that professional antigen-presenting cell(professional APC)have, or indirectly proved the function of antigen presentation of KCs, all with a regret of direct proof. This study proved KCs hold the function of antigen presentation by the assay of Flow Cytometry, Co-Immunoprecipitation, Mass spectrometric analysis and Elispot.Objective: To prove that KCs can play the function of antigen presentation in some certain condition, and to identify the sequences of the peptides MHC presented and the MHC restriction.Methods: 1. Stimulate the Ha Ca T cells to express the antigen presentation related molecules: To activate the human immortal keratinocytes(Ha Ca T cells) by IFN-γ. 2.Identifing the peptides by Mass spectrometric analysis: First,use the monoclonal antibody of MHC-I and MHC-II to get the MHC-peptide complexes in the cytoplasm or on the membrane of activated Ha Ca T cells by Co-Immunoprecipitation(Co-IP). Then, dissociate and separate the MHC-peptide complexes by the acidic liquid(PH3.0) and ultrafilter respectively. Use Silver staining and Western blot to confirm the specificity of the Co-IP. Third, identify the peptides presented by MHC and its protein sources by Mass spectrometric analysis. 3. Co-incubation assay: First, detect the types of the loca of MHC-I and MHC-II of the Ha Ca T cells. Then, predict the type of allele of the targeted MHC on the basis of the results of Mass spectrometric analysis, and sort the CD4+T cells and CD8+T cells of 7 psoriasis patients by flow cytometry. Co-incubate the Ha Ca T cells stimulated by IFN-γ or not with the CD4+T cells and CD8+T cells seperated from the PBMC of 7 psoriasis patients, and observe the activation of T cells by Elispot. 4. To verify the MHC-restriction: Compare the types of MHC of the positive patients in Co-incubation assay with the outcomes of the peptides identified by Mass spectrometric analysis, to verify the MHC-restriction of the antigen presentation of KCs.Results: After stimulated by IFN-γ, the Ha Ca T cellscan express MHC-II, ICAM-1 and higher MHC-I on the cell membrane. With the concentration of IFN-γ and the stimulated time increase, the Ha Ca T cells express more MHC-I and MHC-II on the membrane in a dose and time dependent manner. So we choose an appropriate concentration and stimulated time of IFN-γ(400U/ml, 48h) which can assure both a high expression of antigen presentation related moleculesand a good state of Ha Ca T cells. The Silver staining and Western blot results showed that the antibody of MHC could specifically catch MHC protein. The Mass spectrometric analysis successfully identified the sequences and protein sources of the peptides presented by MHC, including peptides from keratin, ribosomal protein, histone, et al. Co-incubation assay indicated that Ha Ca T cells stimulated by IFN-γ can specifically activate the CD4+T cells of one psoriasis patient, while the counterpart without the IFN-γ stimulation can not activate neither CD4+T cells nor CD8+T cells.Conclusion: After stimulated by IFN-γ, the KCs could express molecules, such as MHC-II and ICAM-1, which were exlusively expressed by the professional APC including Dendritic cells(DCs), Macrophages(MΦ) and B lymphocytes(B cells). At the same time, the expression of MHC-I was much higher than before. All the results above indicated that the activated KCs possess the essential condition to play the function of antigen presentation. The results of Mass spectrometric analysis indicated that there are some peptides presented in the antigen binding groove of MHC-I/II after KCs were stimulated by IFN-γ. The results of Co-incubation assay indicated that the activated KCs, which express the MHC-II loaded peptides, can activate antigen-specific T cells, while the unactivated KCs can not.