Expansion Of CMV-Specific-CTL With G-CSF Mobilized Peripheral Blood Stem Cells In The Presence Of IL-2,IL-15 And IL-21

Objective To establish a method for in vitro expansion of cytomegalovirus specific cytotoxic T lymphocytes（CMV CTL）and prove the anti-cytomegalovirus effect ofCMV CTL,which will provide theoretical basis for clinical extensive adoptive immunotherapy and treatment of cytomegalovirus infection.Methods Full-length cytomegalovirus pp65（CMV pp65）polypeptide1μg/mL was used to repeat rounds of in vitro stimulation of peripheral blood mononuclear cells（PBMC）isolated from peripheral blood stem cell collections mobilized by granulocyte-colony stimulating factor（G-CSF）,and interleukin 2（IL-2）,IL-15 and IL-21 were also added for amplification for 20 days.On the 7th day of culture,autologous PBMCs loaded with CMV pp65 antigen peptide were added after being processed by mitomycin.On day 10,γray-radiated PBMCs loaded with CMV pp65 antigen peptide and CD3（OKT3）were supplemented.For the control group,no antigen was added into PBMCs for amplification.The phenotypes of T lymphocytes and the secretion levels of tumor necrosis factor alpha（TNF-α）and interferon gamma（IFN-γ）before and after amplification were analyzed by polychromatic flow cytometry.Meanwhile,the degranulation and killing function of CD107a in each group were detected as well.The titer of serum CMV IgM/IgG antibody of the donors was detected by ELISA.Results（165.26±6.14）×10⁶ cells were harvested after culture,including CD3⁺T cells 89.21%,CD8⁺T cells accounted for（43.54±28.03）%of CD3⁺T cells,and CD4⁺T cells account for（34.23±26.18）%.The CD3⁺T cells obtained were predominated by effector memory T cells(T_EM),and the proportions of T_EMM in CD8⁺T cells and CD4⁺T cells after amplification culture were significantly higher than those before culture.Additionally,the proportions of stem cell-like memory T cells(T_SCM)and tissue resident memory T cells(T_RM)after culture were obviously raised than those before culture.In the functional experiment,the proportion of IFN-γ⁺secreting CMV-specific CD8⁺T cells after amplification was much higher than that before amplification,and the proportion of TNF-α⁺CMV-specific CD8⁺T cells was on the rise;the proportions of CMV specific CD4⁺T cells that could secrete IFN-γand TNF-αhad no obvious changes.The CD107a expression level of CMV-specific CD8⁺T cells was（3.28±1.97）%,which was not significantly different from that before culture.Furthermore,the level of CMV IgG was positively correlated with the donor’s ages,and in such a culture system,the proportions of amplified IFN-γ⁺and TNF-α⁺secreting CMV-specific T cells were negatively correlated with the donor’s ages.Conclusion In the present research,we have successfully established an effective method to amplify CMV CTL in vitro,and proved that CMV CTL amplified by G-CSF mobilized peripheral blood stem cells has anti-cytomegalovirus effect.