Object: Tuberculosis(TB) infection after Transplantation, although not common, is regarded as one of the most serious clinical problems and threatening the safety of transplanted organs even for the recipients’ life.To simplify the culture procedures of Tuberculosis-specific T lymphocytes,  we  explored  a  novel  method  with a  rapid  expansion  protocol,  that  provided  a  new possibility for the treatment of life-threatening Tuberculosis infection after SOT.
Method: Peripheral blood mono-nuclear cells from T-spots positive 10 healthy donors were isolated and then stimulated and amplified with TB antigen pulsed dendritic cells（DCs） in the T cells conditioned medium. We used the same factors but different peptides of TB antigen,such as ESAT-6, CFP-10, 85Aga, 85Agb, to compare the impact of the specific T cell proliferation and function in vitro. We used the same concentrations of ESAT-6 peptides but different culture conditions to compare different cytokines and training environment on the influence of the specific T cells. In the optimization of culture conditions, we compare the immune response level of different subgroup of specific T cells caused by the ESAT-6 antigen stimulation.
Result: This study demonstrated that a mean 20.09±5.82 fold increase in cell numbers was obtained after two rounds of T cell amplification over 21 days of culture ( n =10). Resultant cultures were predominantly central memory T cells(CD45RO+/CD62L+/CCR7+/HLA-DR+), which produced T-helper (Th1) and Th17 cytokines when restimulated by TB-antigen. From the lymphocyte subgroup analysis ,we found that the vast majority of amplified specific T cells was CD3 + T cells (92.66±5.90%), including CD4 cells (34.32±5.21%) ,CD8 cells (53.43±7.79%), and a small amount of CD3 + CD56 + NKT cells (9.31±4.31) .Cultured cells exhibited a higher level of specific expansion and chemokine production pulsed with DC when restimulated by ESAT-6 antigen compared to Ag85a, Ag85b, CFP-10.It was superior to the other culture conditions with the cocktail of IL2 + IL12 + IL15 + IL7 from day7 to day21 on the cell amplification efficiency and function. From function test ,we found that CD4 cells group release higher cytokines(IFN-r/TNF/IL-2) rather than the CD8 and CD56 cells subgroup.
Conclusion: We select specific T cells effectively recognizing the peptide ESAT-6 to expand TB-specific T cells ex vivo in the optimization of culture conditions . 100ml of peripheral blood after three weeks culture,can meet therapeutic requirements for most recipients.Furthermore, the results demonstrate a reproducible and reliable procedure for adoptive therapy targeting TB infection after Transplantation. Key word：solid organ transplantation; tuberculosis; specific T cells;adoptive cell therapy