Published online Dec 7, 2017. doi: 10.3748/wjg.v23.i45.8017
Peer-review started: July 20, 2017
First decision: September 6, 2017
Revised: September 22, 2017
Accepted: October 26, 2017
Article in press: October 26, 2017
Published online: December 7, 2017
A critical consideration in pediatric liver transplantation (LT) is the management of risks associated with life-long immunosuppression. Tolerance induction is utilized in order to enable discontinuation of immunosuppressant use in pediatric LT recipients. Donor-specific microchimerism (MC) is considered to underlie tolerance induction and graft acceptance. During pregnancy, exposure of the fetus to maternal cells results in natural MC to non-inherited maternal antigens (NIMAs) (NIMA-MC). Recent clinical studies have suggested the association of NIMA-MC with acquired immunologic hyporesponsiveness to NIMAs; this has been termed the “NIMA effect”. However, reports of this effect in LT are scarce.
To investigate the pretransplantation status of NIMA-MC and its effect on cellular rejection in children receiving maternal liver grafts, HLA-DRB1 MC between the child and mother was analyzed. This blood DNA test was already reported by this research team, and we found that the presence of pretransplantation MC was associated with beneficial effects on rejection-free survival in family donor renal transplantation.
In the present study, we aimed to investigate the rate of NIMA-MC in pretransplantation blood samples from pediatric recipients of LT, and examine the effect of NIMA-MC on rejection-free graft survival in patients with a maternal donor.
The presence of pretransplantation NIMA-MC in the peripheral blood was tested using nested PCR-single-strand conformation polymorphism (SSCP) analysis for the HLA-DRB1 alleles. At first, low-to-intermediate resolution HLA-DRB1 typing was performed via the PCR-sequence specific oligonucleotide (SSO) typing method, using either the Dynal RELITM SSO HLA typing kit (Dynal Biotech Ltd., Wirral, UK) or the Luminex-based, WAKFlow HLA typing kit (Wakunaga, Hiroshima, Japan). For HLA-DRB1 MC analysis, high-resolution HLA-DRB1 allelic data are required for mismatched HLA-DRB1 alleles between the mother and child. Allele-level HLA-DRB1 typing was performed via the PCR- SSCP method. For nested PCR amplification of the HLA-DRB1 gene, we used primers to amplify exon 2 of the HLA-DRB1/B3/B4/B5 genes for the first-round PCR, and HLA-DR group-specific primers (for eight different groups) to amplify exon 2 of HLA-DRB1 for the second-round PCR. The first-round PCR products were diluted to 1:200 in water and used as templates for the second-round PCR. The nested PCR product was analyzed via SSCP analysis, and single-strand DNA fragments separated in the gel were visualized with silver staining. For the determination of the presence or absence of NIMA-MC, the band pattern of DNA from pediatric LT recipients was compared with those of maternal DNA and reference DNA with known DRB1 allele types; these were simultaneously analyzed by gel electrophoresis. The rate of pretransplantation NIMA-MC in the pediatric recipients was calculated. To evaluate the effect of NIMA-MC on transplantation outcome, the rate of biopsy-proven cellular rejection (BPCR) among different groups of NIMA-MC and/or HLA-DR match status was compared in patients with a maternal donor using the Fisher’s exact test.
In this small cohort study, pretransplantation NIMA-MC tended to be associated with a lower risk of cellular rejection in pediatric LT recipients with maternal donors. BPCR rate in the patients with pretransplantation NIMA-MC or HLA-DR identity with the mother was significantly lower than those with NIMA-MC negativity (0% vs 50%). This is a novel finding, and further study for large cohort validation is required.
The presence of pretransplantation NIMA-MC or HLA-DR identity with the mother could be associated with BPCR-free survival in pediatric recipients of LT from maternal donors. NIMA effect has been validated in HLA-haploidentical hematopoietic stem cell transplantation. Pretransplantation NIMA-MC could also exert a beneficial effect on rejection-free graft survival in pediatric LT with a maternal graft. Most studies on the NIMA effect in organ transplant recipients have focused on the donor-recipient relationship in the family or the presence or absence of NIMAs in the donor. By actual testing of the presence or absence of NIMA-MC in peripheral blood of the patients, the NIMA effect on outcomes of solid organ transplantations, including LT can be better investigated. Investigation of donor-specific MC in pretransplantation blood samples has rarely been performed. Presence of pretransplantation NIMA-MC could have a beneficial effect on the rejection-free survival in pediatric LT recipients with maternal grafts. Laboratory testing of pretransplantation NIMA-MC in the peripheral blood using nested PCR-single-strand conformation polymorphism analysis for the HLA-DRB1 alleles was previously reported by this research team in renal transplantation (Transplantation 2013; 95(11): 1375-1382). Pretransplantation NIMA-MC for HLA-DRB1 alleles was detected in 23.1% of the pediatric LT recipients; the presence of pretransplantation NIMA-MC or HLA-DR identity with the mother could be associated with BPCR-free survival in pediatric recipients of LT from maternal donors. The presence of pretransplantation NIMA-MC could be associated with BPCR-free survival in pediatric recipients of LT from maternal donors. If the beneficial effect of pretransplantation NIMA-MC on allograft outcomes can be confirmed via larger scale multi-center studies, it would have implications for donor selection when both parental donors are available and the development of protocols, including reduced immunosuppression, for managing pediatric LT recipients.
Presence of microchimerism (MC) to non-inherited maternal antigen (NIMA) is known to be associated with acquired immunologic hyporesponsiveness to NIMA, which is called “NIMA effect”. NIMA effect has been suggested in pediatric liver transplant patients with biliary atresia receiving maternal grafts. However, actual presence or absence of NIMA-MC has never been verified. This is the first study showing possible beneficial effect of NIMA-MC on rejection-free survival in pediatric liver transplant patients receiving maternal grafts with actual laboratory determination of the presence or absence of NIMA-MC. This study is based on a small number of patients and could not get a strong evidence of beneficial effect of NIMA-MC in pediatric liver transplant patients receiving maternal grafts. Further studies using a larger number of patients, and preferentially multicenter studies are warranted. If beneficial effects of NIMA-MC in pediatric liver transplant patients is verified, it will have useful clinical implications of selecting family donors and modifying life-long immunosuppression. MC detection via the amplification of HLA-DRB1 gene was used in this study, which is not a robust method applicable to all transplant cases, especially between one haplotype-matched mother and child pairs. MC detection methods using robust genetic markers other than HLA-DRB1 gene with good enough sensitivities had better be developed and used in the future research. In the present study, only the presence or absence of pretransplant NIMA-MC has been investigated and future studies are required to investigate the effect of posttransplant persistence of NIMA-MC on clinical outcome.