MICB Genetic Variants and Its Protein Soluble Level Are Associated with the Risk of Chronic GvHD and CMV Infection after Allogeneic HSCT

According to the European Society for Blood and Marrow Transplantation registry, a total of 47,412 hematopoietic stem cell transplantation (HSCT) procedures were performed in 2021. Allogeneic HSCT constituted 42% of them (19,806) (Passweg et al. 2023). Although HSCT has a high success rate, it may still lead to some post-transplant complications, including graft-versus-host-disease (GvHD) and cytomegalovirus (CMV) infection (Eberhardt et al. 2023; González-Cruz et al. 2023; Holtick et al. 2024; Sulaiman et al. 2024).

Natural killer (NK) cells are the first cell subset to reconstitute after HSCT. They belong to the innate lymphoid cells family and are one of the most important parts of the human innate immunity (Peterson and Barry 2021; Blunt and Khakoo 2023; Prokopeva et al. 2023). The NK cells are regulated by their activating and inhibitory receptors, i.e., killer Ig-like receptors, natural cytotoxicity receptors, or C-type lectin-like proteins, with the activating NKG2D being one of the most studied receptors (Patil and Schwarer 2009; Bogunia-Kubik and Łacina 2021; Siemaszko et al. 2023).

In humans, ligands for the NKG2D receptor are the MHC class I chain-related A and B molecules (MICA and MICB) and the UL-16 binding proteins (Siemaszko et al. 2021). These molecules serve as natural biomarkers, as they are typically not expressed on normal cells but are often overexpressed on stress-induced or transformed cells (Goulding et al. 2023; Sánchez-Cerrillo et al. 2023). Both MICA and MICB can be expressed in serum in their soluble forms (Nagai et al. 2022). It was reported that the soluble MICA (sMICA) levels were decreased in healthy individuals compared with its elevated levels in patients suffering from various diseases and malignancies, e.g., ankylosing spondylitis, non-small cell carcinoma, pancreatic cancer, breast cancer as well as SARS-CoV-2 infection (Wang et al. 2015; Onyeaghala et al. 2017; Wang et al. 2021; Farzad et al. 2022; Kshersagar et al. 2022). The impact of the serum soluble MICB (sMICB) molecule has been, however, less studied. A study conducted on cancer patients (individuals diagnosed with various malignancies, including prostate cancer, gastrointestinal cancers, breast cancer, or lung cancer) revealed that they had increased sMICB levels in the serum when compared with healthy individuals. A significant association was also observed between the sMICB level and metastasis (Holdenrieder et al. 2006b). As MICA is the most polymorphic of all nonclassical MHC and MHC-like molecules, another frequently studied factor is its genetic variability. One of the most-frequently studied MICA single nucleotide polymorphisms (SNPs) is the methionine (Met)-to-valine (Val) amino acid exchange at position 129. Contrary to the Val isoform, the Met variant is associated with higher NKG2D receptor signaling, resulting in not only more efficient NK cell cytotoxicity but also with faster NKG2D downregulation on NK and T cells (Isernhagen et al. 2015). The impact of MICA Met129Val polymorphism was described in many studies, indicating the protective effects of the Met allele and describing the Val variant (and especially the Val/Val genotype) as a risk factor (Ouni et al. 2017; Zingoni et al. 2018; Ouni et al. 2020). The other significant factor is MICA Met129Val compatibility between the recipient and the donor. It was reported that in unrelated transplantations, a MICA Met129Val mismatch is a risk factor for acute graft-versus-host disease (aGvHD) (Parmar et al. 2009; Fuerst et al. 2016). MICB polymorphisms are less studied. It was reported that in acute myeloid leukemia patients receiving HSCT, the MICB-58 (Lys58Glu) polymorphism had a negative effect on relapse-free survival (Machuldova et al. 2021). The MICB rs1051788 polymorphism was associated with reduced risk of primary graft dysfunction after lung transplantation (Aguilar et al. 2024). The impact of MICB polymorphisms was also investigated in different diseases, e.g., leukemia, dengue fever, rheumatoid arthritis, or systemic lupus erythematosus (Yu et al. 2017; Baek et al. 2018; Wang et al. 2019; Faridah et al. 2023).

In our present study, HSCT recipients and donors were genotyped for two MICB SNPs (rs1065075; G to A substitution resulting in Lys48Glu amino acid exchange and rs3828903; G to A nucleotide substitution within intronic sequence). In addition, serum sMICB levels were determined in patients' sera 30 days after transplantation. Our findings showed significant associations between the MICB polymorphisms and sMICB serum levels and risk for development of CMV infection or cGvHD.

The MICA molecule is the most polymorphic of all non-classical HLA and HLA-like molecules, whereas MICB is characterized by more limited genetic variability. Its polymorphic variants were reported to have an impact on relapse-free survival or mortality in HSCT recipients, dengue severity, and immunosurveillance in oral squamous cell carcinoma (Ivanova et al. 2021; Machuldova et al. 2021; Faridah et al. 2023; Petersdorf et al. 2023). An interesting MICB polymorphism is the Ile/Met amino acid exchange at position 98. Carapito et al. (2020) reported that the donor/recipient mismatch for Ile98Met was associated with an increased risk of developing severe aGvHD grades II–IV or cGvHD. Here we focused on two other SNPs, not previously investigated in the context of HSCT, to study their potential associations with the risk of post-transplant complications. Both SNPs chosen for this study might be related to the ligand–receptor interactions and are located in a similar region of the MICB gene. The rs1065057 polymorphism (exon 2) is localized within the region responsible for encoding of the α1 domain of MICB protein, which is exposed to the corresponding NKG2D receptor. The other polymorphism (rs3828903) is localized in intron 1, which is placed between the leader sequence and exon 1.

Our results on the genetic distribution of two MICB polymorphisms revealed that in both rs1065057 (Lys48Glu) and rs3828903 (intronic substitution) SNPs, the G allele plays a favorable role as it was associated with a decreased incidence of cGvHD development after HSCT. Additionally, we showed that the presence of the MICB rs1065075 G allele in recipients and donors may play a protective role against CMV infection after HSCT. The effect of recipient genotype was confirmed in a multivariate analyses together with HLA compatibility and lack of pre-transplant anti-CMV IgG anti-bodies as protective factors.

Both MICA and MICB molecules can be shed in their soluble forms from the cell surface, indicating immune evasion and escape from detection by NK cells (Chitadze et al. 2013; Suresh 2016). Being one of the most characteristic tumor immune escape mechanisms, shedding of these two molecules is possible due to metalloproteinases (ADAMs and MMPs families) and disintegrins (Zocchi et al. 2015). When expressed on the surface of target cells, MICA and MICB serve as ligands for NKG2D activating receptor, allowing their recognition by NK cells. Blocking this ligand–receptor interaction compromises cytotoxic properties of the NK cells. Increased levels of soluble NK cell ligands (sMICA, sMICB, and sULBPs) had been associated with poor prognosis in cancer patients (Groh et al. 2002; Doubrovina et al. 2003; Wu et al. 2004; Holdenrieder et al. 2006a; Nuckel et al. 2010; Vela-Ojeda et al. 2021). Interestingly, the MICB molecule can be found in its soluble form in the tumor microenvironment but is not expressed directly on the surface of tumor cells (Raffaghello et al. 2004; Holdenrieder et al. 2006b; Boutet et al. 2009; Kaidun et al. 2023). NKG2D ligands are overexpressed during CMV infection, which helps with the recognition and clearance of the infected cells. It was reported that the human CMV-encoded UL16 protein binds specifically to MICB, competing with NKG2D. This leads to decreased binding to the activating receptor and, as a result, decreased NK cell activity (Spreu et al. 2006).

In accordance with these observations, we detected higher sMICB serum concentrations in patients who developed CMV after HSCT. Moreover, increased sMICB serum levels were found in patients who suffered from cGvHD. This is a novel observation that has not been previously described. Furthermore, our study has also revealed associations between the MICB polymorphism and sMICB concentrations in patients' sera. Patients homozygous for rs1065057 AA and rs3828903 AA were characterized with higher sMICB levels compared to recipients carrying at least one G allele, further confirming the protective role of those genetic variants. Since soluble forms of non-classical HLA and HLA-like molecules may serve as decoy ligands for NK cell receptors, blocking cytotoxic properties, their increased levels are considered as a negative factor for disease development and worse outcome (Salih et al. 2006; Ribeiro et al. 2016; Siemaszko et al. 2021). Results obtained in this study support these findings. Nevertheless, it should be noted that the number of tested samples is relatively small and a study on a larger cohort could be needed to confirm our findings. Furthermore, the biological material was not available for some of the donors, limiting our observations of the immunogenetic donor–recipient associations.

Taken together, in the present study, we showed significant associations of the MICB genetic variants and sMICB serum levels with the development of post-transplant complications in recipients undergoing allogeneic HSCT. We found that the increased serum sMICB concentration is a negative factor for CMV infection and cGvHD development. We showed that both donor and recipient MICB variants could be potential prognostic markers of CMV and cGvHD. Our results indicate the emerging role of MICB molecule in the context of HSCT outcome.