Fig 2: 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.

Fig 3: Cis versus trans CD137-costimulation by CD137L-Fc coated beads on human CD8+ T lymphocytes.a, schematic representation of microbeads coated with CD137L-Fc used to stimulate CD8+ T cells. Human primary CD8 T cells from healthy donors were activated with microbeads for 96 h. b, representative dot plots of cell-surface CD25 (n = 11) and c, representative histograms (n = 10) of geometric mean fluorescence intensity (gMFI) for CD25 surface expression on CD8+ T cells. d, intracellular Bcl-xL (n = 8) expression analysed by flow cytometry in the indicated conditions. Summary data are given as delta (?), the difference of the value between cis and trans conditions to which the anti-CD3 background has been subtracted in each case. e, concentrations of human IFN? (n = 5) in the culture supernatants of the indicated conditions. Data are given as mean ± s.e.m. Statistical significance was determined with the Wilcoxon test (two-sided). Source data are provided as Source Data file.