Donor Toll-Like Receptor 9 Genetic Factors in Susceptibility to Acute Graft-Versus-Host Disease and Cytomegalovirus Infection After Related and Unrelated Hematopoietic Stem Cell Transplantation

Toll-like receptors (TLRs) are transmembrane proteins on the surface of immune cells that detect conserved molecular motifs known as “microbe-associated molecular patterns” from a variety of organisms. They interact with several adapter proteins to activate transcription factors, leading to the production of inflammatory cytokines and the activation of adaptive immunity. Current evidence suggests that common polymorphisms in TLR genes have been associated with the susceptibility to autoimmune disease and several infections, their association with outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has yet to be explored.

We unselectively included all patients undergoing allo-HSCT in our Bone Marrow Transplantation Center between January 2001 and March 2009 if donor and patient DNA were available. All the patients and their donors were from a homogeneous genetic background. We analyzed 10 single nucleotide polymorphisms (SNPs) in the TLR1gene (rs4833095 C/T, rs5743565 A/G), TLR2gene (rs11938228 A/C, rs3804099 C/T), TLR3 gene (rs3775291 A/G, rs3775296 G/T), TLR8gene (rs3764880 A/G, rs2159377 C/T) and TLR9 gene (rs352139 G/A, rs352140 C/T) in 2 independent cohorts. The initial cohort consisted of 138 pairs of patients and their unrelated donors (URDs). The second cohort consisted of 102 pairs of patients and their HLA-identical sibling donors.

(1) We found that two SNPs in donor side, TLR9 +1174 G/A (rs352139) and TLR9 +1635 C/T (rs352140), influenced the risk of aGVHD. The association was particularly strong in the URD transplantation cohort. Multivariate analysis confirmed that an unrelated donor with the TLR9 +1174 variant allele (A allele) was an independent risk factor for aGVHD (P= 0.009, RR= 3.123). In contrast, an unrelated donor with the TLR9 +1635 variant genotype (TT) was protective (P= 0.067, RR= 3.457). The same effect was observed in the sibling transplantation cohort, although the incidence of clinically significant aGVHD in this cohort was low overall and the association was not statistically significant. (2) Since cytomegalovirus (CMV) reactivation and disease continued to be important complications post-HSCT, and CMV infection is of special concern in the Chinese population. The incidence of asymptomatic infection is high. The episodes of major infection were focused on early CMV infection post-HSCT. Early CMV infection refers to antigenemia or disease with onset by day 100 after transplantation. In the present study cohorts, all patients and almost all donors were CMV seropositive (CMV-specific immune globulin G positive, CMV-IgG(+)) before HSCT and only one donor from the Taiwan Tzu Chi Stem Cells Center was CMV seronegative. All patients received CMV prophylaxis treatment and those experiencing pre-transplantation CMV recurrent infection received preemptive treatment. Of the total of 240 patients, 134 (55.8%) had experienced early CMV infection with a median onset of 27 days (range 2–64) post-HSCT. Patients who received stem cells from donors with the TLR9 +1635 variant genotype (TT) had a reduced incidence of grades II–IV aGVHD, however, they experienced early CMV infection more frequently than those with the wild-type genotype (CT or TT) in both the URD transplantation cohort (TT: 80% vs. CT or CC: 59.3%, Gray's test p =0.02) and the sibling transplantation cohort (TT: 66.7% vs. CT or CC: 41.7%, Gray's test p=0.03), although a higher incidence of early CMV infection was observed in the URD transplantation cohort (63% vs. 46%, Gray's test p = 0.025). Multivariate analysis confirmed unrelated donors (p =0.046, RR = 1.438, 95%CI,1.007–2.053), patients experiencing recurrent CMV infection pre-transplantation (p = 0.004, RR = 0.597, 95%CI, 0.419–0.849) and donors with the TLR9 +1635 TT genotype (p=0.005, RR=0.561, 95%CI, 0.376–0.836) all contributed to the development of early CMV infection.

There is increasing evidence for a role for TLR9 in the pathogenesis of aGVHD and viral infection. These result is the first report of donor TLR9 gene polymorphic features with the risk of aGVHD and early CMV infection, which are located within the promoter region and coding polymorphisms and may influence transcriptional regulation and the amino acid exchange of the TLR9 gene.

No relevant conflicts of interest to declare.