Figure 1: Absence of memory of transplant rejection following acute rejection of tolerated allografts.

(a,b) BALB/c (B/c) hearts were transplanted into C57BL/6 (B6) recipients untreated (acute rejection (AR) group) or treated with anti-CD154+DST at the time of transplantation (tolerant (Tol) group). On day 60 (D60) post heart transplantation (HTx), a subset of tolerant mice were infected with Lm i.p. (Tol+Lm group). Untransplanted mice (naïve group) were used as controls. Splenocytes (a) and distal peripheral lymph nodes (distal LN) including cervical, brachial and axillary populations (b) from naïve, AR (D7 post HTx), Tol (D60 post HTx) and Tol+Lm (D7 post Lm) mice were stimulated with T-depleted B6 (syngeneic) or B6xB/c F1 (allogeneic) splenocytes. IFNγ-producing cells were analysed by intracellular flow cytometry on CD44⁺CD90⁺-gated events (naïve, Tol, Tol+Lm n=8 per group, AR n=4, experiment repeated twice, results pooled). The percentage of T cells specific for alloantigen (%allo–%syn) was multiplied by the number of live cells counted by the Trypan blue exclusion method to obtain the total number of IFNγ⁺ alloreactive T cells. Statistical significance at *P<0.05 by Kruskal–Wallis test with Dunn’s. (c) Experimental design for the transplantation of second allografts. (d) B6 mice treated with anti-CD154+DST at the time of B/c abdominal heart transplantation (on day minus 60), and that had rejected their allografts following Lm infection (3–5 × 10⁶ CFU i.p. on day 0 (D0); Tol+Lm) were re-transplanted in the neck on day 14 (D14) with B/c (n=6) or C3H (n=4) allografts, or on days >14 (21–42) with B/c allografts (n=4). As controls, animals transplanted with B/c hearts±Lm infection at the time of transplantation (non-Tol ±Lm+second B/c) received a second B/c heart 2 weeks later (n=4 each) in the neck. (e) Histology of 1st Tol B/c graft 60 days post transplantation (left), 1st Tol+Lm B/c graft at the time of rejection 8 days post infection (middle), and second B/c cervical graft D14 post second transplantation into mice that had rejected their first tolerated B/c heart following Lm infection (Tol+Lm, right), and composite histological scores. Scale bars, 100 μm. Data are presented as mean±s.e.m. of 3–6 hearts per group, repeated at least twice. Statistical significance by Student’s t-test at ***P≤0.001 or not significant (NS).