HLA-restricted and Antigen-specific CD8+ T Cell Responses by K562 Cells Expressing HLA-A*0201

BACKGROUND: Identification of antigen-specific T cells has yielded valuable information on pathologic process and the disease state. Assays for quantification of inflammatory cytokines or lytic-granule molecules have been generally used to evaluate antigen specific T cell response, however their applicability have been hampered due to the limited source of autologous antigen-presenting target cells (APC). METHODS: K562, a leukemic cell line deficient of human leukocyte antigen (HLA), was transfected with a gene encoding HLA-A*02 (K562/A*02) and its function as stimulator cells in inducing activation of HLA-matched T cells was evaluated by IFN-gamma enzyme linked immunospot (ELISPOT) assay. RESULTS: The stable transfectant K562/A*02 pulsed with HLA- A*02 restricted peptide could specifically induce IFN-gamma secretion by CD8+ T cells compared to no detectable secretion by CD4+ T cells. However, CD56+ NK cells secreted IFN-gamma in both K562/A*02 with peptide and without peptide. The number of IFN-gamma secreted CD8+ T cells was increased according to the ratio of T cells to K562 and peptide concentration. Formalin-fixed K562/A*02 showed similar antigen presenting function to live K562/A*02. Moreover, K562/A*02 could present antigenic- peptide to not only A*0201 restricted CD8+ T cells but also CD8+ T cells from A*0206 donor. CONCLUSION: These results suggest that K562/A*02 could be generally used as target having specificity and negligible background for measuring CD8+ T cell responses and selective use of K562 with responsder matched HLA molecules on its surface as APC may circumvent the limitation of providing HLA-matched autologous target cells.

Induction of Aspergillus fumigatus Specific T Cells using Dendritic Cells Pulsed with Asp f16 Recombinant Protein in vitro

Invasive aspergillosis is the most common Aspergillus fumigatus infection in immunocompromised patients. Although the treatment of the invasive aspergillosis has been mostly relied on antifungal agents, there still exists the need for more effective therapy. To develop cellular immunotherapy specific for Aspergillus fumigatus, we generated specific T cells using dendritic cells (DCs) pulsed with an Aspergillus fumigatus derived recombinant protein in vitro and examined their functions. The f16. p2+3 region containing the conserved region of Asp f16 gene was cloned from Aspergillus fumigatus and the recombinant protein was produced in E. coli. IFN-gamma secretion from the T cells stimulated with recombinant f16. p2+3 (rf16. p2+3) was measured by enzyme linked immunospot (ELISPOT) assay and the cytolytic activity of the stimulated T cells by 51Cr release assay. The number of IFN-gamma secreting cells were significantly increased in the peripheral blood mononuclear cells (PBMCs) stimulated with the rf16. p2+3 pulsed DCs (31+/-12 spots/10(4) cells), compared to that of PBMCs directly stimulated with rf16. p2+3 (83+/-15 spots/10(6) cells). IFN-gamma ELISPOT assay using purified CD4+ or CD8+ as responder cells showed that CD4+ T cells (43 spots/10(4) cells) mainly produced IFN-gamma compared with CD8+ T cells (7 spots/10(4) cells). Furthermore, helper T cells specific for rf16. p2+3 could be efficiently generated by the stimulation with DCs for two weeks. However, cytotoxic T lymphocyte activity was not induced. Our results suggest that the rf16. p2+3 protein could be used for the generation of helper T cells in vitro.

The Study of MHC class I Restricted CD8+ T Cell Mediated Immune Responses against Mycobacterium tuberculosis Infection: Evidence of M. tuberculosis Specific CD8+ T Cells in TB Patients and PPD+ Healthy Individuals

BACKGROUND: The protective immunity against tuberculosis (TB) involves both CD4+ T cells and CD8+ T cells. In our previous study, we defined four Mycobacterium tuberculosis derived peptide epitopes specific for HLA-A*0201 restricted CD8+ T cells (ThyA30-38, RpoB127-135, 85B15-23, PstA175-83). In this study, we investigated the immune responses induced by these peptide specific CD8+ T cells in latently and chronically infected people with TB. METHODS: We characterized these peptide specific CD8+ T cell population present in PBMC of both TB patients and PPD healthy people using IFN-gammaelispot assay, intracellular staining and HLA-A2 dimer staining. RESULTS: The frequency of peptide specific CD8+ T cell was in the range of 1 to 25 in 1.7x10(5) PBMC based on ex vivo IFN-gamma elispot assay, demonstrating that these peptide specific CD8+ T cell responses are induced in both TB patients and PPD people. Short term cell lines (STCL) specific for these peptides proliferated in vitro and secreted IFN-gamma upon antigenic stimulation in PPD+ donors. Lastly, HLA-A*0201 dimer assays indicated that PstA175-83 specific CD8+ T cell population in PPD+ healthy donors is heterogeneous since approximately 25~33% of PstA175-83 specific CD8+ T cell population in PPD+ healthy donors produced IFN-gamma upon peptide stimulation. CONCLUSION: Our results suggest that MHC class I restricted CD8+ T cell mediated immune responses to M. tuberculosis infection are induced in both TB patients and PPD+ people; however, the CD8+ T cell population is functionally heterogeneous.

CTL Response to Pre-erythrocytic Stage Vaccine Candidate of Plasmodium falciparum in HLA-A*0201 Transgenic Mice Detected by ELISPOT Assay / 中国寄生虫学与寄生虫病杂志

The importance of cytotoxic T-lymphocyte(CTL)against malaria parasite in pre-erythrocytic stage has been presented in relevant researches. In order to investigate whether one CTL epitope(YLNKIQNSL)involved in a chimeric pre-erythrocytic stage vaccine candidate of Plasmodium falciparum which was expressed and purified in the laboratory can stimulate in vivo CTL response,HLA-A*0201 transgenic mice were immunized with this vaccine candidate. Enzyme-linked immunosorbent spot(ELISPOT)assay was performed on the splenocytes from the immunized transgenic mice. Positive result indicated that this CTL epitope can be in vivo processed and correctly presented.