Role of soluble human leukocyte antigen-G in virus-associated cancers: A focused minireview

Abstract

Soluble human leukocyte antigen-G (sHLA-G), a non-classical major histocompatibility complex class I molecule, arises either through proteolytic cleavage of membrane-bound isoforms or via secretion of soluble isoforms. sHLA-G exerts potent immunosuppressive effects, facilitating immune escape of cancer cells within the tumor microenvironment by inhibiting the cytotoxic activity of natural killer cells, cytotoxic T lymphocytes, and other immune effectors. Emerging evidence indicates that aberrant expression of sHLA-G contributes to tumor progression in virus-associated malignancies, including human papillomavirus-driven cervical cancer and hepatitis B virus/hepatitis C virus-related hepatocellular carcinoma. sHLA-G is detectable in various body fluids, including plasma and serum, highlighting its potential as both a diagnostic and prognostic biomarker as well as a therapeutic target.

Key Words: Soluble human leukocyte antigen-G; Virus-associated cancers; Immune escape; Diagnostic biomarker; Prognostic biomarker; Immune evasion

Core Tip: Increased soluble human leukocyte antigen-G (sHLA-G) levels are consistently associated with poor prognosis in hepatitis C virus/hepatitis B virus -related hepatocellular carcinoma and human papillomavirus-driven cervical cancer, underscoring its potential as a diagnostic and prognostic biomarker. High-risk human papillomavirus oncoproteins (E6 and E7) and hepatitis C virus proteins (core protein and nonstructural protein 3) may contribute to increased sHLA-G levels through cytokine-mediated immunosuppressive signalling pathways. The mechanisms by which sHLA-G-immunoglobulin-like transcript 2/immunoglobulin-like transcript 4 interactions modulate immune suppression in these virus-driven cancers remain incompletely understood. sHLA-G isoforms are primarily detected in plasma and other biological fluids using sandwich enzyme-linked immunosorbent assays, providing a reliable method for both clinical and research applications.

INTRODUCTION

Several human oncogenic viruses have been identified and collectively account for approximately 10%-15% of cancers worldwide[1]. These include human papillomavirus (HPV), hepatitis B virus (HBV) and hepatitis C virus (HCV), Epstein-Barr virus, Kaposi’s sarcoma-associated herpesvirus, human T-cell lymphotropic virus type 1, and Merkel cell polyomavirus[2]. HPV plays a central role in the pathogenesis of cervical carcinoma[3], whereas chronic HBV and HCV infections are major etiological drivers of hepatocellular carcinoma (HCC)[4]. Epstein-Barr virus is associated with nasopharyngeal carcinoma, gastric carcinoma, Burkitt lymphoma, and Hodgkin lymphoma[5,6], whereas Kaposi’s sarcoma-associated herpesvirus is the causative agent of Kaposi’s sarcoma[7]. These oncogenic viruses employ immune-evasion strategies to circumvent host antiviral defenses, thereby promoting viral persistence and malignant transformation[8-10]. One such mechanism may involve the upregulation of human leukocyte antigen-G (HLA-G) expression in virus-infected cells or increased levels of soluble HLA-G (sHLA-G) within the tumor microenvironment (TME)[11,12].

sHLA-G isoforms strongly suppress immune responses and play a pivotal role in tumor immune escape by inhibiting the cytotoxic activity of natural killer (NK) cells, cytotoxic T lymphocytes, and other immune effector populations[13]. Emerging evidence suggests that aberrant sHLA-G expression contributes to tumor progression in virus-associated malignancies[12,14,15].

This article summarizes the mechanisms underlying the immunomodulatory role of sHLA-G in virus-induced cancers, discusses its potential as a diagnostic and prognostic biomarker, and highlights current approaches for its detection and quantification.

STRUCTURE OF SHLA-G ISOFORMS

HLA-G, a non-classical human leukocyte antigen class I (HLA-I) molecule, is predominantly expressed in extravillous cytotrophoblasts during pregnancy and, under homeostatic conditions, is otherwise restricted to a limited number of immunologically privileged sites[16]. The HLA-G gene generates seven protein isoforms: Four membrane-bound (HLA-G1, -G2, -G3, and -G4) and three soluble (HLA-G5, -G6, and -G7)[17,18]. HLA-G1 represents the full-length isoform and comprises three extracellular domains (alpha 1, alpha 2, and alpha 3), a transmembrane segment, and a short cytoplasmic tail (Figure 1). It assembles with β₂-microglobulin to form a heterotrimeric complex (Figure 1). sHLA-G1 can arise through proteolytic shedding of membrane-bound HLA-G1[18]. In contrast, the secreted isoforms HLA-G5, -G6, and -G7 correspond structurally to HLA-G1, -G2, and -G3, respectively, but lack the transmembrane and cytoplasmic domains, which are replaced by a short hydrophilic tail encoded by intron retention (Figure 1)[19]. Owing to this structural distinction, secreted HLA-G isoforms can be differentiated from shed HLA-G molecules using isoform-specific monoclonal or polyclonal antibodies[20].

Figure 1 Soluble human leukocyte antigen-G-mediated immune evasion in virus-associated cancers. Solid lines indicate experimentally demonstrated mechanisms, whereas dashed lines represent putative or hypothesized pathways. sHLA-G: Soluble human leukocyte antigen-G; HPV: Human papillomavirus; HCV: Hepatitis C virus; HBV: Hepatitis B virus; ILT2: Immunoglobulin-like transcript 2; ILT4: Immunoglobulin-like transcript 4; NK cell: Natural killer cell; CD8⁺ T cell: Cytotoxic T lymphocyte; IL-10: Interleukin-10; IFN-α: Interferon alpha; IFN-β: Interferon beta; α1, α2, α3: Alpha-1, alpha-2, and alpha-3 extracellular domains of human leukocyte antigen-G; β2m: Β₂-microglobulin; TME: Tumor microenvironment.

DETECTION AND QUANTIFICATION OF SHLA-G ISOFORMS

Methods of measurements

Elevated circulating sHLA-G levels have been reported in multiple malignancies[20,21], including virus-associated cancers, underscoring their potential utility as diagnostic and prognostic biomarkers[22]. sHLA-G isoforms, particularly sHLA-G1 and HLA-G5, are detectable in plasma, serum, and other body fluids using enzyme-linked immunosorbent assay (ELISA). Sandwich and competitive ELISA formats are among the most widely used and validated methods for quantifying sHLA-G1 and HLA-G5 in biological fluids from patients and healthy controls, demonstrating acceptable sensitivity, specificity, and analytical reproducibility across studies (Table 1)[12,19,23-26]. At a cut-off of 49 U/mL, sHLA-G achieved 100% specificity[24]. Receiver-operating characteristic curve areas were 0.97, 0.91, 0.98, and 0.80 for colorectal, gastric, esophageal, and non-small cell lung cancers, respectively[24]. Further studies are needed to confirm the diagnostic accuracy of sHLA-G in predicting virus-associated cancers. In addition, plasma-derived sHLA-G can be enriched by immunoprecipitation and subsequently characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie staining to distinguish monomeric and dimeric forms[23].

Table 1 Comparative profile of soluble human leukocyte antigen-G in human papillomavirus-driven cervical cancer vs hepatitis B virus/hepatitis C virus-driven hepatocellular carcinoma.

Cancer type	Cervical cancer	Hepatocellular carcinoma
Oncogenic virus	HPV16/18 (high-risk types)	HBV and/or HCV (approximately 80% of cases)
sHLA-G detection	Serum/plasma by sandwich ELISA	Serum/plasma by sandwich ELISA
sHLA-G alterations	↑ sHLA-G in cervical cancer patients	↑ sHLA-G in HCC
	HLA-G5 low in cervical tumor tissue - ↑ sHLA-G and IL6/8/10/17 → Th2-biased immunity → immune suppression and HPV persistence
Clinical implications	Progressive sHLA-G increase from HPV + cervix → cancer	↑ sHLA-G in HCC vs liver cirrhosis, chronic hepatitis, or asymptomatic infection

HPV: Human papillomavirus; HCV: Hepatitis C virus; HBV: Hepatitis B virus; ELISA: Enzyme-linked immunosorbent assays; IL: Interleukin; Th2: T helper 2; sHLA: Soluble human leukocyte antigen; HLA-G: Human leukocyte antigen; HCC: Hepatocellular carcinoma.

Analytical challenges and limitations

The quantification of circulating sHLA-G by ELISA remains technically challenging due to issues related to antibody specificity, isoform heterogeneity, and assay standardization. HLA-G monoclonal antibodies recognize distinct epitopes and may differ in isoform specificity[27]. Moreover, some anti-HLA-I antibodies may cross-react with classical HLA-I molecules, potentially compromising assay specificity[27]. ELISA methods differ in their capacity to detect sHLA-G isoforms, particularly secreted HLA-G5 and shed HLA-G1, depending on the antibody pairs employed[19]. Furthermore, despite efforts toward harmonization, the lack of universally accepted reference standards and calibrators contributes to interlaboratory variability[28].

Serum sHLA-G levels can also be influenced by biological factors, including the cytokine milieu[29] and HLA-G polymorphisms[30,31]. Certain ELISA formats may preferentially detect monomeric forms while underrepresenting biologically active disulfide-linked HLA-G dimers, which exhibit stronger immunosuppressive properties[32-34]. sHLA-G has not been associated with conventional biomarkers, including HBV DNA levels and alpha-fetoprotein, in patients with HCC, which limits its utility as a surrogate biomarker of disease severity[15].

Taken together, these factors should be carefully considered when interpreting the value of sHLA-G as a biomarker.

SHLA-G AND HBV/HCV-INDUCED HCC

Chronic HCV and HBV infections account for approximately 80% of HCC cases worldwide[35]. HCV core and nonstructural protein 3 proteins have been reported to stimulate interleukin (IL)-10 production in monocytes and dendritic cells, creating a cytokine milieu that promotes an immunoregulatory microenvironment[36]. This environment may upregulate HLA-G expression on monocytes and facilitate immune evasion[29]. Amiot et al[37] further demonstrated that sHLA-G production by human mast cells from HCV-infected livers is significantly upregulated after IL-10 stimulation in vitro, suggesting that virus-related cytokine environments contribute to local immunosuppressive signaling via cytokine-mediated induction of HLA-G.

Genetic background can also modulate HLA-G expression. HCC tissues carrying the 14-bp deletion/deletion (del/del) genotype exhibit significantly higher HLA-G levels compared with heterozygous (ins/del) or insertion/insertion (ins/ins) genotypes, indicating that post-transcriptional regulatory polymorphisms influence tumor-associated HLA-G expression[38].

Multiple NK cell receptors regulate NK-cell responsiveness by directly or indirectly recognizing HLA class I molecules. Among these, immunoglobulin-like transcript (ILT) 2 and ILT4 use sHLA-G as their ligands[39,40]. Chronic infection with HBV or HCV promotes persistent inflammatory signaling and increased production of IL-10 by monocytes and dendritic cells[41,42]. This immunosuppressive cytokine may contribute to the development of an exhausted NK-cell phenotype[43]. Consistent with this model, Sakata et al[44] showed that NK cells frequently exhibit increased ILT2 expression in HCC, which is associated with reduced cytotoxic capacity and impaired antitumor activity (Figure 1). Furthermore, in vitro studies demonstrate that HLA-G binding to ILT2/ILT4 inhibits NK- and CD8⁺ T-cell cytotoxicity[45]. Nevertheless, the precise molecular mechanisms governing sHLA-G-ILT2/ILT4 interactions within the HCC TME and in patient-derived immune subsets remain incompletely understood.

Aberrant upregulation of sHLA-G has been consistently observed in HCC and is associated with tumor metastasis and poor clinical outcomes (Table 1). Serum sHLA-G levels are elevated in patients with active hepatitis B and HCC, enabling discrimination between HCC and cirrhosis and supporting its potential utility as a diagnostic biomarker[15]. Mocci et al[12] further demonstrated that elevated circulating sHLA-G independently predicts poor prognosis in patients with virus-related HCC. Moreover, patients with HCC exhibit significantly higher sHLA-G levels than individuals with liver cirrhosis, chronic hepatitis, or asymptomatic viral infection[46].

SHLA-G AND HPV-INDUCED CERVICAL CANCER

Gynecological malignancies, particularly cervical cancer, are multifactorial diseases in which immunogenetic factors play a critical role. Persistent infection with high-risk HPV genotypes (HPV16/18) is the principal etiological factor in cervical carcinogenesis. Circulating sHLA-G levels are frequently elevated in patients with cervical cancer (Table 1). A meta-analysis by Tizaoui et al[47] reported significantly higher serum sHLA-G concentrations in Asian women with cervical cancer compared with controls. An Indian case-control study further showed a progressive increase in plasma sHLA-G levels from HPV-infected cervical lesions to invasive carcinoma, supporting its potential role in immune evasion, viral persistence, and tumor progression[11].

HPV oncoproteins may indirectly induce HLA-G expression through cytokine-mediated and transcriptional mechanisms. A case-control study demonstrated concurrent elevation of plasma sHLA-G and cytokines, including IL-6, IL-8, IL-10, and IL-17, reflecting a T helper 2-skewed immune profile that may favor immune suppression and persistent HPV infection[26]. HPV E6 and E7 enhance transforming growth factor-beta1 (TGF-β1) promoter activity[48], and increased TGF-β1 promotes immune evasion and tumor progression[49]. Additionally, E6 and E7 also upregulate IL-6 expression in keratinocytes, particularly in cells expressing E6* isoforms of HPV16 and HPV18[50], while IL-10 expression is partially driven by the transcriptional activity of E2, E6, and E7[51]. Because IL-10 and TGF-β1 are known inducers of HLA-G through signal transducer and activator of transcription 3 dependent pathways, HPV-driven cytokine remodeling of the TME may create permissive conditions for HLA-G transcription and secretion.

NK cells in HPV-associated cervical cancer exhibit an impaired cytotoxic phenotype (Figure 1)[52]. HPV-driven alterations of the local cytokine milieu can inhibit NK-cell cytotoxicity and facilitate immune evasion in cervical neoplasia[49,53]. However, direct evidence linking sHLA-G-ILT2/ILT4 interactions to NK-cell functional exhaustion in HPV-related malignancies remains limited, highlighting the need for further mechanistic investigation.

Interestingly, HLA-G5 expression is downregulated in invasive cervical tumor tissues, regardless of metastatic status[14], in contrast to the elevated circulating sHLA-G levels observed in patient plasma[26,47]. This discrepancy may arise from biological compartmentalization, wherein membrane-bound and soluble HLA-G isoforms exhibit distinct tissue distribution, as well as methodological differences in isoform-specific detection. Elevated serum sHLA-G may also result from secretion by immune cells, local production within TME, or release via extracellular vesicles. Although HPV oncoproteins E5 and E7 are known to downregulate classical HLA-I molecules by interfering with antigen processing and transport pathways[54-56], their specific effects on HLA-G isoforms, particularly HLA-G5, remain incompletely defined.

TREATMENT

Standard-of-care treatments

Management of HPV-associated cervical cancer and HCV/HBV-induced HCC is primarily guided by disease stage and underlying viral etiology. In early-stage HCC, curative strategies including surgical resection, liver transplantation, or ablative therapies are preferred[57]. Intermediate-stage tumors are typically managed with locoregional therapies such as transarterial chemoembolization[58], whereas advanced HCC is now treated as first-line therapy with the combination of atezolizumab and bevacizumab[59]. Tyrosine kinase inhibitors, including sorafenib or lenvatinib, are reserved for second-line therapy or for patients in whom immunotherapy is contraindicated[59]. Concurrent antiviral therapy targeting HBV or HCV replication is recommended to reduce the risk of tumor recurrence[60]. HBV vaccination remains a key strategy for reducing HCC incidence[61].

For HPV-associated cervical cancer, early-stage disease is usually treated with radical hysterectomy and pelvic lymphadenectomy[62], whereas locally advanced tumors require concurrent chemoradiotherapy[63]. Recurrent or metastatic disease is managed with platinum- or taxane-based chemotherapy, with or without bevacizumab, and programmed death-ligand 1 (PD-L1)-positive tumors may be treated with pembrolizumab[64]. Vaccination against HPV remains a critical approach for preventing invasive cervical cancer[65,66].

Immune checkpoints sHLA-G/ILT-2/4

Emerging evidence highlights the sHLA-G–ILT2/ILT4 axis as a promising immunotherapeutic target in both HPV-driven cervical cancer and HBV/HCV-related HCC[67,68]. Although immune checkpoint inhibitors targeting the programmed death 1 (PD-1)/PD-L1 pathway have improved outcomes, resistance remains a significant challenge[59]. sHLA-G mediates immune suppression via mechanisms distinct from PD-1/PD-L1, including NK-cell inhibition and alternative T-cell suppressive pathways[45].

Blockade of the sHLA-G–ILT2/ILT4 axis could potentially enhance the efficacy of PD-1/PD-L1 inhibitors. ILT2/ILT4 antagonists and HLA-G-targeting antibodies are currently under early clinical investigation for solid tumors and have been shown to restore NK- and CD8⁺ T-cell function in preclinical models[45]. However, these approaches have not yet been evaluated specifically in HPV-associated cervical cancer or HBV/HCV-induced HCC. Future studies should assess whether combination strategies targeting both PD-1/PD-L1 and sHLA-G/ILT2 pathways can overcome immune evasion and restore antitumor immunity in virus-associated malignancies.

CONCLUSION

Human oncogenic viruses, including HPV, HBV, and HCV, exploit immune-evasion mechanisms to sustain persistent infection and promote malignant transformation. Among these, sHLA-G-mediated immunosuppression within TME plays a pivotal role by impairing NK-cell and CD8⁺ T-cell cytotoxicity. Elevated circulating sHLA-G levels are consistently associated with poor prognosis in HPV-driven cervical cancer and HBV/HCV-related HCC, supporting its potential as a biomarker. Nevertheless, technical limitations, biological variability, and incomplete functional correlation currently restrict its clinical applicability. Targeting the sHLA-G–ILT2/ILT4 axis represents a promising therapeutic strategy. Future studies should clarify its mechanistic contribution to viral oncogenesis and determine whether HLA-G-directed interventions can meaningfully enhance antitumor immunity in virus-associated malignancies.