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. Author manuscript; available in PMC: 2022 May 7.
Published in final edited form as: Am J Transplant. 2021 Sep 27;22(2):504–518. doi: 10.1111/ajt.16842

Figure 7. CSB-Treg from post-LTx and HC PBMC effectively suppress autologous CD8+ T cells.

Figure 7.

CSB-Treg from post-LTx patients (A) or HC samples (B) generated using the new conditions were evaluated for their capacity to suppress autologous CD8+ T cells using an in vitro MLR between the original (autologous) CTV-labeled responder PBMCs and the γ-irradiated stimulator pair. Different ratios of Treg:Responder were evaluated. At day 7, autologous responder PBMC cells were stained and analyzed using flow cytometry to assess CD8+ T cell proliferation (CTV dilution), as well as frequency of CD8+ T cells expressing activation (CD25+ and/or HLA-DR+) and cytotoxic markers (granzyme B+perforin+). Data is shown in representative flow cytometry plots from single experiments (independent triplicate experiments were performed for each condition, using n=3 post-LTx and n=3 HC samples). All samples were gated within CD3+CD8+ viable lymphocytes. Gates of interest are highlighted in red. Resting responder cells (R) cultured for the same period were used as a control.

(A) CD8+ T cells from post-LTx samples showed little proliferation whereas they demonstrated significant proliferation, and an increased proportion expressed activation markers and cytotoxic markers after MLR (RS). Addition of CSB-Tregs to the MLR (Treg + RS) was associated with robust suppression of autologous responder CD8+ T cell proliferation and a decrease CD8+ T cells expressing activation and cytotoxic molecules. (B) Similar results to (A) were obtained using HC PBMC responders, stimulators and HC CSB-Treg.

R: responder PBMC; S: Stimulator SPL or PBMCs; RS: responder PBMCs cocultured with stimulator cells (SPL cells for post-LTx and PBMC for HC); RS+Tregs: RS plus autologous Treg (isolated from a CSB-MLR using the same R and S) at either 1Treg:5R or 1Treg:10R ratios. HC: healthy controls; PBMC: peripheral blood mononuclear cells; CSB: costimulatory blockade; post-LTx: post liver transplant; SPL: splenocytes; Treg: regulatory T cell; CTV: CellTraceTM dye; MLR: mixed lymphocyte reaction