Differential graft-versus-leukaemia effect by CD28 and CD40 co-stimulatory blockade after graft-versus-host disease prophylaxis

Abstract

Co-stimulatory blockade may be a promising strategy for tolerance induction in transplantation. In allogeneic bone marrow transplantation (BMT) for leukaemia treatment, however, preservation of the graft-versus-leukaemia (GVL) effect is another critical requirement for clinical application. In this study, we have compared the effect on GVL of using CD28 and CD40 co-stimulatory blockades as graft-versus-host disease (GVHD) prophylaxis in a murine allogeneic BMT model with simultaneous transfer of BCL1 leukaemia. Despite the relative improvement of GVHD as assessed by survival and body weight in both treatment regimes, treatment with anti-CD154 moAb clearly diminished the GVL effect, whereas treatment with anti-CD80 and CD86 MoAbs maintained this effect. Although T cell-mediated effector function at 14 days post-BMT assessed by IFNgamma expression and cytotoxicity against host alloantigen was comparable between both co-stimulatory blockades, IL-12 mRNA expression was preferentially reduced by CD40 blockade. Our results suggest the differential involvement of the CD28 and CD40 co-stimulatory pathways in the development of GVHD and GVL effects. CD28 blockade may be a favourable strategy for tolerance induction in leukaemia patients undergoing BMT.

Fig. 1

Effects of treatment with either anti-CD154 or anti-CD80/86 MoAbs in a complete allogeneic GVHD model. Each group of sublethally (6·5Gy) irradiated BALB/c mice received 2·0 × 10⁶ splenocytes and 2·5 × 10⁷ TCD-BM cells from wild-type B6 mice, and treated with either control reagents (BMS, •) anti-CD80/86 (RM80/PO3) MoAbs (BMS CD80/86, ▪), or anti-CD154 (MR1) MoAb (BMS CD154, ▴). One group of mice received TCD-BM cells alone (BM, ○). Each group consists of 8–19 mice. Survival rates (a) and mean body weight (b) are plotted. Data shown are representative of three experiments. BM versus BMS, P = 0·013, BMS CD80/86 versus BMS, P = 0·043, BMS CD154 versus BMS, P = 0·043. (c) Peripheral blood cells and splenocytes were analysed at day 28 post-BMT. ALC; absolute lymphocyte count. Splenocytes were stained with FITC-anti-CD4, PE-anti-CD8 and PerCP-anti-CD3 MoAbs. CD3⁺, CD3⁺4⁺ and CD3⁺8⁺ cells were counted as donor T, CD4⁺ T, CD8⁺ T cells, respectively. More than 99% of splenocytes were H-2d⁻ donor-derived cells in all groups. Data represent the mean ± s.d. from five mice in each group. *Statistically different (P < 0·05).

Fig. 2

Treatment with anti-CD154 MoAb permits growth of BCL1. Sublethally irradiated BALB/c recipient mice received TCD-BM (2·5 × 10⁷) and either whole (a) or asialo GM1⁺ cell-depleted (b) splenocytes (2·0 × 10⁶) from B6 mice with 1 × 10⁵BCL1 cells. In (c), BALB/c nude mice were inoculated with 1 × 10⁵ BCL1 cells. In all experiments, MoAb treatment was performed as described in Fig. 1. Recipient mice were killed on day 28 and each spleen weight was measured. †One mouse treated with anti-CD154 MoAb died by leukaemia before day 28 in (b). The bars represent the mean ± s.d. *Statistically different (P < 0·05).

Fig. 3

IFNγ expression by CD4⁺T cells and T cell expansion are inhibited comparably by both treatments. (a) Expression of IFNγ in individual splenocytes from each group of mice at 14 days post-BMT was assessed by immunofluorescent staining of intracellular cytokines by flow cytometry. Cells were stained with either FITC-anti-CD4 or anti-CD8, PerCP-CD3 and PE-anti-INFγ or with appropriated fluorochrome-conjugated control Ig. Samples were analysed by flow cytometry. An electronic gate was set on either CD3⁺4⁺ or CD3⁺8⁺ lymphocytes, and the expression of IFNγ was analysed. (b) Splenocytes from each recipient at 14 days were used as effector cells. Cytotoxicity against BCL1, A20 and BALB/c PHA blasts was measured. Each bar represents the mean ± s.d. from five mice; •, cont. Ig; ▪, CD80/86; ▴, CD154. (c) Cells were stained with FITC-anti-CD4, PE-anti-CD8 and PerCP-anti-CD3 MoAb or with appropriated fluorochrome-conjugated control Ig. Samples were analysed by flow cytometry. CD3⁺, CD3⁺4⁺ or CD3⁺8⁺ lymphocytes were counted as T, CD4⁺ T and CD8⁺ T cells, respectively. Over 98% of splenocytes were H-2d⁻ donor origin in all groups (not shown). *Statistically different (P < 0·05). Data are representative of two experiments.

Fig. 4

Treatment with anti-CD154 MoAb significantly inhibits IL-12 mRNA. B6 mice received 2 × 10⁷ irradiated BALB/c splenocytes and MoAb treatment as described in the Methods. Total RNA from splenocytes at the indicated time after transfer was isolated and expression of IL-12 p40 mRNA was assessed by RT-PCR as described in the Methods. (a) Kinetic change in IL-12 p40 mRNA expression after transfer. The predicted product sizes for IL-12 p40 and β2-microglublin were 384 and 222 bp, respectively. (b) Comparison of IL-12 p40 mRNA at 6 h after transfer. The amount of mRNA for IL-12 p40 and β2-microglobulin in individual samples at 6 h after transfer was measured by densitograph and the unit values were normalized to the amount of mRNA for β2-microglobulin. The bars represent the mean ± s.d. from each group of seven to eight mice. *Statistically different (P < 0·05).