Background: CD8⁺CD28^– T suppressor (Ts) cells were recently reported to play a critical role in transplant tolerance, harnessing the potential for clinical use in transplantation. Here, we report a novel strategy to generate plenty of human CD8⁺CD28^– Ts cells and debate their immune suppression capacity in vitro and in vivo, their origin and phenotypic characteristics.

Methods: Purified CD8⁺ T cells from PBMCs of individual A were stimulated with APCs (CD2^– cells from PBMCs) from HLA-A, -B, -DR mismatched individual B in the presence of IL-2, IL-7, IL-15. After 9 days co-culture, cells were further selected and defined as CD8⁺CD28^– Ts cells. Then, the CFSE-labeled CD4⁺ T cells from individual A were co-cultured with B-APCs or I-APCs (HLA-A, -B, -DR mismatched with individual A and B) in the presence of CD8⁺CD28^– Ts cells. After 7 and 11 days, CFSE dilution assay was used to assess the suppressive capacity of CD8⁺CD28^– Ts cells. Similarily, different cell populations were injected into NOG mice via i.p. to assess the in vivo suppressive capacity of CD8⁺CD28^– Ts cells by flow cytometry and immunohistochemistry staining methods. At last, the phenotypic characteristics of CD8⁺CD28^– T cells before and post-expanded were analyzed by flow cytometry.

Results: Following exposure to B-APCs, there was an evident decrease in the proliferating of CD4⁺ T cells on day 7 and day 11 in the presence of Ts cells, compared with in absence of CD8⁺CD28^– Ts cells (P<0.01). Additionally, CD8⁺CD28^– Ts cells did not obviously inhibit the proliferation of CD4⁺ T cells stimulated by I-APCs (P<0.01). Moreover, when CD8⁺CD28^– Ts cells were transferred into NOG mice, their allospecific suppressive capacity kept steady and they still kept alive on day 11. Besides, the CD8⁺CD28^– Ts cells up-regulated co-inhibitory molecule TIM3, whereas down-regulated cytotoxicity related molecules perforin, granzyme B, etc., implying possible suppressive mechanisms.

Conclusions: In vitro expanded human CD8⁺CD28^– Ts cells by common γc cytokines with donor APCs possessed remarkable donor specific suppressive capacity not only in vitro but also in vivo circumstances. These findings may facilitate CD8⁺CD28^– Ts cells based immune tolerance induction and monitor in clinical transplantation in future.

CITATION INFORMATION: Liu G., Feng F., Zhu P., Liu J., Liu Y., Yu Y. Human CD8⁺CD28^– Ts Cells Expanded by Common Gamma Chain (γc) Cytokines Keep Steady Allospecific Suppressive Capacity In Vivo Am J Transplant. 2017;17 (suppl 3).

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