Polymorphisms in CXCR3 ligands predict early CXCL9 recovery and severe chronic GVHD

Abstract

Chronic graft-versus-host disease (cGVHD) is a major cause of mortality and morbidity after allogeneic stem cell transplantation (alloSCT). The individual risk of severe cGVHD remains difficult to predict and may involve CXCR3 ligands. This study investigated the role of single-nucleotide polymorphisms (SNPs) of CXCL4, CXCL9, CXCL10, and CXCL11, and their day +28 serum levels, in cGVHD pathogenesis. Eighteen CXCR3 and CXCL4, CXCL9-11 SNPs as well as peri-transplant CXCL9-11 serum levels were analyzed in 688 patients without (training cohort; n = 287) or with statin-based endothelial protection cohort (n = 401). Clinical outcomes were correlated to serum levels and SNP status. Significant polymorphisms were further analyzed by luciferase reporter assays. Findings were validated in an independent cohort (n = 202). A combined genetic risk comprising four CXCR3 ligand SNPs was significantly associated with increased risk of severe cGVHD in both training cohort (hazard ratio (HR) 2.48, 95% confidence interval (CI) 1.33-4.64, P = 0.004) and validation cohort (HR 2.95, 95% CI 1.56-5.58, P = 0.001). In reporter assays, significantly reduced suppressive effects of calcineurin inhibitors in constructs with variant alleles of rs884304 (P < 0.001) and rs884004 (P < 0.001) were observed. CXCL9 serum levels at day +28 after alloSCT correlated with both genetic risk and risk of severe cGVHD (HR 1.38, 95% CI 1.10-1.73, P = 0.006). This study identifies patients with high genetic risk to develop severe cGVHD.

Fig. 1. Association of single-nucleotide polymorphisms (SNPs) in CXCR3 ligand genes and serum chemokines with the incidence of severe chronic graft-versus-host disease (GVHD).

A Carriers of the variant allele for CXCL9 polymorphism rs884304 (AA + AG) were associated with a significantly higher incidence of severe chronic GVHD in the training cohort. B Effects of the three candidate SNPs for genetic risk group B on the incidence of severe chronic GVHD after alloSCT. Genetic risk group B was based on rs3733236, rs4282209, and rs655328. C Association of severe chronic GVHD incidence with genetic risk groups A and B. D Association of serum CXCL9 levels with a cumulative incidence of severe chronic GVHD after alloSCT in the training cohort. E Association of day +28 CXCL9 serum levels with genetic risk groups A and B. n.s. not significant; *P ≤ 0.05; **P < 0.01; ***P < 0.001.

Fig. 2. The effect of genetic risk (CXCR3 ligands) on severe chronic graft-versus-host disease (GVHD).

A Association of the combined genetic risk group with the incidence of severe chronic GVHD in the training cohort and validation cohort. B No difference could be observed between the genetic risk groups in patients with SEP in two consecutive cohorts (n = 144 and 159, respectively). C Association of serum CXCL9 levels at day +28 with the combined genetic risk group in patients without and with SEP. n.s. not significant; *P ≤ 0.05.

Fig. 3. The association of rs884304 with IFN-γ induced promoter activity and CXCL9 recovery.

A In patients, high-risk genotypes (AA + AG) of genetic risk group A (rs884304) were associated with significantly higher day +28 serum CXCL9 levels, but not with pre-transplant CXCL9. B In luciferase reporter assays, IFN-γ activation revealed that the constructs carrying variant allele of rs884304 (P < 0.001), rs884004 (P < 0.001), and the one carrying variant alleles of all the three SNPs (P = 0.001), but not rs2869462, were associated with significantly reduced suppressive effect of CsA compared to the wild-type construct. The results were collected from three independent experiments. Luciferase activity was normalized to 1 relative to the WT and all data were plotted as the mean ± SEM. C Similar to (B), with FK506 (tacrolimus) as calcineurin inhibitor. Results were collected from three independent experiments. Luciferase activity was normalized to 1 relative to the WT and all data were plotted as the mean ± SEM. D Recovery rates of CXCL9 serum levels in patients from the lowest value post alloSCT until day 28+ in the presence of calcineurin inhibitors (slope CXCL9) was higher in the genetic high-risk group. n.s. not significant; *P ≤ 0.05; ***P < 0.001.