Fig 2: DPP8/9 inhibitors activate the CARD8 inflammasome in T cells.a–c Human primary CD3+ T cells were pre-treated with the indicated compounds for 30 min prior to the addition of VbP (2 µM, 6 h). Cell viability was assessed using Cell-TiterGlo. Data are means ± s.e.m. of four or five biological replicates. ***p < 0.001 by two-sided Student’s t test. NS not significant. d Immunoblots of CARD8 KO CD3+ T-cell lysates treated with VbP (20 µM, 24 h). e, f CD3+ T cells (Donor 008) were primed with LPS (10 µg/mL, 4 h) before treatment with the indicated concentrations of VbP (6 h), LT (6 h), Lfn-flagellin (3 h), or nigericin (1 h). Lysates were then evaluated by immunoblotting (e) and supernatants were analyzed for LDH release (f) and IL-1ß secretion (g).

Fig 3: Local adaptive and innate immune response after 1 week from the implantation. CD68+ and CD163+ macrophages were particularly abundant in the xenogeneic group, especially in the spheroids infiltration. No differences were evident in CD206+ macrophages distribution whereas more iNOS + cells were infiltrating the cartilaginous spheroids. CD3+ lymphocytes were predominant in the osteotomy gap of the xenogeneic samples compared to the other groups. Brown color indicates the positive staining. i, tissue infiltration; c, cartilaginous spheroids; g, collagen gel.

Fig 4: VbP induces pyroptosis in resting but not activated T cells.CD3+ T cells were activated with CD3/CD28 costimulation for 48 h. Resting (rest) or activated (active) T cells were then treated with the indicated concentration of VbP for the indicated time before cell viability was assessed by Cell-TiterGlo (a, c) and lysates were evaluated by immunoblotting (b). Data are means ± s.e.m. of four biological replicates. ***p < 0.001 by two-sided Student’s t test. NS not significant.

Fig 5: T lymphocyte reaction to the non-autologous implants after 12 weeks. (A) Proliferation of the T cells isolated from the draining lymph nodes was assessed in a co-culture model with the donor cells. Specific proliferation induced by the donor cells was observed only in the xenogeneic group. Nil: lymphocytes that were not exposed to any other cell type; Aspecific: lymphocytes co-cultured with third-party Sprague Dawley MSCs; *p < 0.05; **p < 0.01. (B) CD3+ T lymphocytes (indicated by the arrows) were still prominent in the local immune response against the xenogeneic implants. Fewer CD3+ cells were spotted in the collagen and allogeneic samples. Brown color indicates the positive staining; i, tissue infiltration; b, bone; c, cartilaginous construct; *EBR.