Fig 1: Wnt activation increases T cell survival and CMV-specific polyfunctionality in CMV D+/R– LT patients.(A) Human memory CD8+ T cells were stimulated with CD3/CD28 and treated with DMSO, TWS119, or SKL2001. On day 7, cells were replated at the same concentration in regular culture medium in the absence of further TCR stimulation and Wnt agonists for 7 days. Viability was analyzed on day 14 (n = 8). Dunn’s test for multiple comparisons. (B) Memory cells in different treatment conditions for 7 days were treated with 0 µM or 50 µM of cisplatin for 6 hours to induce apoptosis. Annexin V and 7-AAD were stained to analyze apoptotic cells. Percentages of Annexin V+/7-AAD+ in each condition were plotted (n = 4). (C) Antiapoptotic proteins Bcl-xL and Bcl-2 were analyzed by flow cytometry in memory CD8+ T cells with or without Wnt agonists for 7 days (n = 5). Dunn’s test for multiple comparisons. (D) Schematic showing the design of adoptive transfer experiment. Healthy donor memory CD8+ cells were sorted from PBMCs and treated with or without SKL2001 for 7 days; 5 × 106 autologous CD4+ T cells and 2 × 106 cultured CD8+ T cells were cotransferred into SCID mice (n = 5). CD8+ cells were monitored periodically. Thirty days after injection, T cells were harvested and a polyfunctionality assay was performed. (E) CD8+ T cell persistence was assessed over 1 month after adoptive transfer. (F) Polyfunctionality assay of transferred T cells on day 30. (G) PBMCs from CMV-infected (D+R–) LT recipients were isolated and stimulated with CMV pp65 peptide pool for 7 days. On day 7, PBMCs were restimulated with pp65 peptide pool for 8 hours. CD107a, TNF-a, IFN-?, and IL-2 were analyzed for the polyfunctionality assay. (H) CMV-specific polyfunctionality was improved in the cells of 15 CMV D+/R– LT patients following DMSO or SKL2001 treatment (n = 15). Mann-Whitney U test. *P < 0.05; ***P < 0.001. #P < 0.05 by Dunn’s test.