Human CD8+CD28-T Suppressor Cells Expanded By IL-15 In Vitro Suppress In An Allo-Specific And PD-1-dependent Manner

Background:CD8+CD28-T suppressor cells(Ts)have emerged as an important modulator of alloimmunity,which play a significant role in various diseases,such as cancer,chronic infections,autoimmune disease,and allogeneic transplantation.But the in vitro inducibility of Ts subsets is rather unclear.Here,we induced rapid expansion of human allospecific CD8+CD28" T cells in vitro by donor APCs plus IL-15,and investigated its immunosuppressive function both in vitro and in vivo circumstances.Methods:Purified CD8+T cells from human peripheral blood mononuclear cells(PBMCs)stimulated by APCs(CD2 depleted PBMCs)from HLA-A,B and DR mismatched volunteers were cultured for 9 days in the presence of IL-15.These cells were then analyzed for phenotypic characteristics and the CD28-population was isolated for functional experiment.A CFSE-based CD4+T cells proliferation assay in vitro was used to assess inhibition of proliferation by CD8+CD28-Ts.The expanded CD8+CD28-Ts cells were adoptive transfered into NOG mice via i.p.to assess allospecific immunosuppressive function of CD8+CD28-Ts in vivo.Suppression mechanisms of CD8+CD28’ Ts were also investigated by anti-PD-1 or anti-PD-L1.Results:In the presence of IL-15,the fraction and total number of CD8+CD28-T cells in CD8+T cells increased dramatically,the number increased by 23.42±5.43 times after 9 days culture.Moreover,these expanded CD8+CD28-T cells could vigorously inhibit the proliferation of CD4+T cells in contact dependent and donor-specific manner in vitro,and when transferred into NOG mice,their suppressive capacity kept steady in vivo.The expression of CD 132,CD25 and PD-1 for CD8+CD28-Ts cells were up-regulated,while CD 122,GZM-B and perforin expression were down-regulated.Interestingly,when anti-PD-1 or anti-PD-L1 was added into the culture system,suppress effect mediated by CD8+CD28’ Ts cells was partially abolished when compared with isotype control.Conclusions:Human allospecific CD8+CD28-Ts could be generated by IL-15 plus donor APCs in a relative short period of time in vitro,these cells could steadily exert their immunosuppressive function both in vitro and in vivo circumstances.Co-inhibitory signals PD-1:PD-L1 pathway play a role in the suppression mechanism.