Background: CD8⁺CD28^– T suppressor cells (Ts) have emerged as an important modulator of alloimmunity, 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.

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 23.4±5.4 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 CD132, CD25 and PD-1 for CD8⁺CD28^– Ts cells were up-regulated, while CD122, 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.

CITATION INFORMATION: Feng F., Liu G., Zhu P., Zhu M., Lu X., Liu J., Luo X., Liu Y., Yu Y. Human CD8⁺CD28^– T Cells Driven In Vitro by IL-15 Suppress in Allo-Specific Manner Partially through PD-1:PD-L1 Pathway Am J Transplant. 2017;17 (suppl 3).

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