Peptide/MHC-I Specific T-cell Induced By Artificial Antigen-presenting Cell And Its Application In The Study On T-cell Allorecognition And Tumor Immunotherapy

Activation of antigen-specific T cell plays a central role in initiating adaptive immune response. Antigen-presenting cell (APC) provides two signals for T-cell activation. The signal one is through T cell receptor (TCR) engaging the peptide/MHC complex on the surface of APC, which provides the basis for antigen specificity. The signal two through the other receptor on T cell (such as CD28, LFA-1 and CD2) provides a costimulatory signal following engagement of the ligand on the surface of APC (such as B7, ICAM-1 and LFA-3). TCR and other receptor on the surface of T cell upon ligand binding can initiate T cell activation by mediating signal transduction pathways and improving the total affinity of T cell-APC interaction. CD28 expressed on resting and activated T cells, the most potent costimulatory receptor, can provide costimulatory signal by binding B7 or anti-CD28 antibody. According to these, the artificial antigen-presenting cells (aAPCs) can be prepared by coating peptide/MHC complex and anti-CD28 antibody to the cell-size beads, which can provide signal one and signal two for T cell activation. Use of the aAPCs enabled stringent control of the antigens presented to T cells. So the aAPCs can be used for the study of alloreactive T cell recognition. Our research results showed alloreactive T cell is pMHC specific. Based on this, tumor specific alloreactive T cells induced by H-2Kb-Ig/TRP2180-188-aAPCs can be prepared which delay the growth of melanoma specifically. The following is a brief introduction to this research:1. The establishment of techniques for preparation recombinant peptide/MHC class I complexesBACKGROUND: Recombinant peptide/MHC class I complexes is a useful tool for the study of T cell recognition. AIM: To build the techniques for preparation of recombinant all sorts of peptide/MHC class I complexes and construct an HLA-A*2402/BRLF1198-206 tetramer. Since HLA-A*2402 is one of the most common human MHC class I allele in East Asia, we choose HLA-A*2402 and its binding peptide (EBV antigen BRLF1 198-206), to form the HLA-A*2402/BRLF1198-206 complexes and construct HLA-A*2402/BRLF1198-206 tetramers.METHODS: the HLA-A*2402-BSP-pET21d recombinant plasmid was cloned, and the HLA-A*2402-BSP fusion protein was induced by IPTG to expressed highly as insoluble aggregates in E.coli. After initial purification, the aggregates were dissolved in 8 M urea. Then the two subunits refolded to form an HLA-A*2402/BRLF1198-206 complex by dilution method in the presence of an antigenic peptide [NH2-TYPVLEEMF-COOH of EB virus BRLF1 198-206]. After concentrate and buffer exchange of refolding products, the BirA enzyme was used to biotinylate the refolded complex. The refolded and biotinylated products were detected by ELISA and Werstern blot. HLA-A*2402/BRLF1198-206 tetramers was prepared by mixing the biotinylated peptide-HLA complex with phycoerythrin (PE) labeled avidin at a molar ratio of 4:1.To the end, the HLA-A*2402/BRLF1198-206 tetramer is applied to detect antigen-specific cytotoxic T lymphocytes (CTLs) induced by aAPCs in vitro.RESULTS: The HLA-A*2402-BSP-pET21d recombinant plasmid was cloned successfully. ELISA and Werstern blot results demenstrate the refolded complex has been refolded and biotinylated successfully. FACS results show HLA-A*2402/BRLF1198-206 tetramers we prepared can bind its targets specifically and efficiently(10.7% vs 0.11%). CONCLUSION: HLA-A*2402/BRLF1198-206 tetramers can be prepared successfully.The techniques and methods can be used for preparation of recombinant complexes of other types of MHC class I molecules with the corresponding peptides, and lay the foundation for the construction of artificial antigen-presenting cells and the following study of alloreactive T cell recognition.2. The establishment of techniques for preparation aAPCsBACKGROUND: According to the principle of T cell activation, the artificial antigen-presenting cells (aAPCs) are designed to stimulate antigen-specific T cells. Use of the aAPCs enabled stringent control of the antigens presented to T cells. So the aAPCs can be used for the study of alloreactive T cell recognition.AIM: To construct HLA-A2/LMP2A426~434-aAPCs and establish the technology for preparation of aAPCs.METHODS: HLA-A2/LMP2A426~434-aAPCs were generated by coating HLA-A2/LMP2A426~434 complexes and anti-human CD28-specific antibodies to cell-sized latex beads. The immunophenotype of HLA-A2/LMP2A426~434-aAPCs was analyzed by FACS. Then by co-culture of the HLA-A2/LMP2A426~434-aAPCs and peripheral blood lymphocytes from HLA-A2 positive healthy donors, LMP2A426~434 antigen-specific CTLs were induced and expanded in vitro. The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2/LMP2A426~434 tetramer staining, cytotoxicity against HLA-A2/LMP2A426~434 bearing T2 cell and the cytotoxicity inhibited by the anti-HLA class I antibody (W6/32).RESULTS: The analysis of FACS shows that HLA-A2/LMP2A426~434 complexes (85.48% vs 12.02%) and anti-CD28 antibody (72.38% vs 10.54%) are immobilized on microbeads. Tetramer staining and cytotoxicity assay results showed that LMP2A426~434 antigen-specific CTLs could be induced and expanded in vitro by the HLA-A2/LMP2A426~434-aAPCs.CONCLUSION: HLA-A2/LMP2A426~434-aAPCs can be constructed successfully. The techniques and methods can be used for construction of other types of aAPCs and lay the foundation for the study of antigen specific T cell.3. Study of Peptide Involvement in T Cell Allorecognition using recombinant peptide/MHC class I and aAPCsBACKGROUND: Allogeneic transplantation causes acute rejection when MHC molecules of donor and receptor were mismatched (allo-reaction). The major mechanism is that alloreactive T cells recognize the graft cell-expressed allogeneic tissue antigens (alloantigens), and respond to them. The phenomenon of T-cell alloreactivity is a paradox. Alloreactive T cells respond to MHC molecules with exceptional vigor. Typically, 1–10% of T cells respond to allogeneic MHC molecules. This is orders of magnitude above the frequency of T cells specific to antigen presented by self-MHC. The molecular basis for exceptionally high frequency and diversity of T cells responding to allogeneic antigens remains elusive.AIM: To investigate CD8+ T cell allorecognition using recombinant peptide/MHC class I complexes and artificial Ag-presenting cells (aAPCs).METHODS: The frequency of CD8+ alloreactive T cell primed with skin graft was explored using tetramer staining tests by a panel of H-2Kd tetramers representing five self-peptides derived from ubiquitously expressed proteins known to be bound endogenously by this allotype. In addition, in the ELISPOT assay and CFSE proliferation assay, CD8+ alloreactive T cell with pMHC specific was explored using a panel of aAPCs that represent functional densities of H-2Kd molecules displaying antigen peptides.RESULTS: The analysis of FACS showed that peptide/H-2Kd complexes are immobilized on these different H-2Kd/peptide-aAPCs. Tetramer staining results showed the alloreactive populations generated using skin graft contain peptide specific subsets of T cells that respectively bind the H-2Kd/KYI, H-2Kd/SYF, H-2Kd/SFV, H-2Kd/YYV and H-2/Kd/NYA tetramers. They do not overlap. ELISPOT assay and CFSE proliferation assay results demonstrated skin grafting enriches for peptide-specific alloreactive CTL CONCLUSION: Peptide-specific recognition by alloreactive T cells predominates, supporting the proposal that the strength of the alloreactive response is primarily due to the large amount of T cells stimulated by the diverse array of peptides on the nonself MHC molecules.4. Exploitation of tumor-specific alloreactive T cells for Tumor Immunotherapy using aAPCsBACKGROUND: Autologous T cell responses to tumor Ags presented by self-MHC are usually weak and ineffective. The existence of the allorestricted repertoire raises the possibility of generating T cells reactive against synthetic self-peptides bound to nonself-MHC molecules, because tolerance to self-antigens is self-MHC restricted. Therefore, allorestricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy.AIM: To induce melanoma-specific H-2Kb-restricted alloreactive T cells from BALB/c mice (H-2Kd) using H-2Kb-Ig/TRP2180-188-aAPC in vivo for melanoma immunotherapy.METHODS: H-2Kb-Ig/TRP2180-188-aAPCs were constructed by coating H-2Kb-Ig/TRP2180-188 complexes and anti-mouse CD28-specific antibodies to cell-sized latex beads. CD8+ alloreactive T cells specific for a melanoma-specific, H-2Kb-restricted CTL epitope (TRP2180-188) derived from mouse tyrosinase-related protein 2 (TRP2) from BALB/c mice (H-2Kd) were prepared using H-2Kb-Ig/TRP2180-188-aAPC in vivo. The specificity of the aAPC-induced alloreactive T cells was demonstrated by H-2Kb-Ig/TRP2180-188 dimer staining, ELISPOT assay and cytotoxicity assay. Specific antimelanoma effects of the alloreactive T cells induced by H-2Kb-Ig/TRP2180-188-aAPCs was identified by intratumoral injection with alloreactive T cells.RESULTS: Dimer staining result showed the alloreactive T cells induced by H-2Kb-Ig/TRP2180-188-aAPCs can specifically bind H-2Kb-Ig/pTRP2 dimer instead of control dimer. ELISPOT assay result showed the induced-T cells can generate higher IFN-γspots when they were stimulated by H-2Kb-Ig/TRP2180-188-aAPCs. Cytotoxicity assay results determined the induced-T cells can specifically kill its target. The induced-T cells can delay the growth of melanoma specifically.CONCLUSION: Melanoma-specific H-2Kb-restricted alloreactive CTLs can be obtained from allo-restricted T cells repertoire of BALB/c mice by H-2Kb-Ig/TRP2180-188-aAPCs and can delay the growth of melanoma specifically. In this study, according to the principle of T cell activation, the artificial antigen-presenting cells HLA-A2/LMP2A426~434-aAPCs were constructed and T cells specific for HLA-A2/LMP2A426~434 were successfully prepared using HLA-A2/LMP2A426~434-aAPCs. The techniques and methods can be used for construction of other types of aAPCs. Use of the aAPCs enabled stringent control of the antigens presented to T cells. So the aAPCs was a very important tool for the study of antigen-specific T cell. Based on these, the study of alloreactive T cell recognition using a panel of H-2Kd /peptide-aAPCs determined the alloreactive T cell is pMHC specific. Because the alloreactive T cell is pMHC specific, Melanoma-specific H-2Kb-restricted alloreactive CTLs can be obtained from allo-restricted T cells repertoire of BALB/c mice by H-2Kb-Ig/TRP2180-188-aAPCs in vivo and can delay the growth of melanoma specifically. Therefore, our research lays the foundation for further study of antigen-specific T cell and offers experimental basis for the mechanism of allorecognition and tumor immunotherapy using tumor-specific allreactive T cells.