Glioma is the most common malignant tumor type of the central nervous system and the 5-year overall survival (OS) is <10%. According to the biological behavior and malignancy of the tumor, glioma may be divided into four grades from World Health Organization (WHO) grade I to IV. Low-grade glioma (LGG) includes WHO grade-I and -II tumors, while the other two grades III and IV are classified as high-grade glioma (HGG). Of note, glioblastoma multiforme (GBM), the most malignant glioma type with WHO grade IV, accounts for ~50% of glioma cases, and has a median survival time of 14.2 months and a 5-year survival rate of <5% (1). In the past decades, despite improvements in surgical, radio- and chemotherapies, the treatment of glioma has remained a tremendous challenge (2,3). However, with the development of novel emerging immunotherapies, which aim to reinvigorate antitumor immune responses, outcomes have been significantly improved in a variety of advanced hematologic and solid malignancies (4–8). This points out a new direction in terms of treatment strategies for glioma. Thus, novel therapeutic approaches targeting the interaction between the tumor microenvironment and immune response are urgently required in this field.

Various preclinical studies have demonstrated the success of immunotherapy-based approaches in animal models and numerous phase I and II clinical trials suggested immunotherapy to be safe and, in certain cases, improve progression-free survival (PFS) and OS (9–13). Preclinical studies using murine models with orthotopic-transplanted gliomas have provided a marked benefit of checkpoint inhibitors used individually or in combination with other immunotherapeutic strategies (42). Numerous glioma-associated antigens, including interleukin (IL)-13 receptor subunit α2, human epidermal growth factor receptor 2, EPH receptor A2, gp100 and AIM-2 are being targeted in glioma (14–16). In addition, tumor-specific neoantigens, including epidermal growth factor receptor variant III, are being used to target tumor cells (16,17). The successful preclinical studies have prompted a number of clinical studies using dendritic cell vaccines (11). Furthermore, considerable progress has been achieved in immunotherapy with antibodies, adoptive T-cell transfer and chimeric antigen receptor T cells in their respective fields (18–20). Among the aforementioned therapeutic strategies, immune checkpoint blockade appears to be an exciting avenue that warrants further development based on the preclinical studies.

Immune checkpoint proteins are surface molecules on certain immune cell populations that activate or inhibit immune function when engaged to their ligands. Numerous studies have indicated an interaction between the expression of the co-inhibitory protein and tumor immune escape (21). Therefore, immunotherapy based on blocking the interaction between an immune checkpoint protein and its ligands has the potential to restore functional immune cells and inhibit tumor progression.

The B7 family, an important class of the immune checkpoint superfamily, has exhibited great potential for regulating T-cell function and participating in the immune response. The growing B7 family is now comprised of 10 members, including CD80 (B7-1), CD86 (B7-2), programmed cell death 1 ligand 1 (PD-L1 or B7-H1), PD-L2 (B7-DC), inducible T cell co-stimulator ligand (B7-H2), CD276 (B7-H3), B7-H4, V-set immunoregulatory receptor (B7-H5), B7-H6 and human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2 or B7-H7) (22). Among these ligands, PD-L1 and PD-L2 represent two ligands for the PD-1 receptor. Recent studies have indicated that upregulation of PD-1 and PD-L1 in tumor tissue was associated with poor prognosis in certain cancer types, demonstrating that PD-1 and PD-L1 may inhibit the function of T-cells and promote the immune escape of tumor cells (23–25). The clinical application of a specific antibody which inhibits the PD-1/PD-L1 pathway has achieved satisfactory curative effects (26,27). A recent study reported that upregulation of PD-1 in glioma predicted a poor prognosis (28), indicating the potential value of this immune checkpoint protein as a therapeutic target in glioma.

HHLA2 is the most recently discovered member of the B7 family. Transmembrane and immunoglobulin domain-containing 2 (TMIGD2, also known as IGPR-1 or CD28H) is the only known receptor identified for HHLA2 (29). While its exact function remains elusive, it has been reported to have co-stimulatory as well as co-inhibitory properties (30,31). Zhu et al (31) indicated that the interaction between CD28H and B7-H7 on antigen-presenting cells (APCs) co-stimulated human T-cell proliferation and cytokine production via a pathway involving AKT phosphorylation. By contrast, Zhao et al (30) proposed the opposite function for B7H7: In the presence of the T-cell antigen receptor (TCR) signaling pathway, B7-H7 inhibits the proliferation of CD4⁺ and CD8⁺ T cells. In addition, B7-H7 significantly reduces cytokine production by T cells, including interferon-γ, tumor necrosis factor-α, IL-5, IL-10, IL-13, IL-17α and IL-22. Thus, the ligation of B7-H7 to T cells suppresses T-cell responses. As with B7-H3, a T-cell co-inhibitory role and a co-stimulatory role have been reported for this ligand (22). One explanation is that HHLA2 has two ligands with opposite functions-TMIGD has a co-stimulatory role, while the other remains elusive. HHLA2 on APCs or tumor cells may interact with unknown ligands and exert a co-inhibitory function in the microenvironment of certain cancers. Furthermore, it may promote angiogenesis within the tumor microenvironment via its interaction with TMIGD2 expressed in the endothelium.

The expression of HHLA2 has been reported in a large proportion of tumor specimens, including breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney and esophageal, but not in endometrial, gallbladder, laryngeal, stomach and uterine cancer or in lymphoma (29). To date, no systematic study on the expression status and biological function of HHLA2 in patients with glioma has been performed, to the best of our knowledge. The present study aimed to examine the expression of HHLA2 in normal brain specimens and tumor specimens obtained from patients with glioma. Furthermore, the potential mechanistic role of HHLA2 in glioma and the association between HHLA2 expression and tumor behavior were investigated, and its clinical utility as a prognostic predictor was assessed.

The present study first focused on detecting the expression level of HHLA2 in normal brain tissue and tumor tissue obtained from patients with glioma by IHC labeling in the Human Protein Atlas dataset. By individually analyzing the normal brain tissue and tumor tissue, it was revealed that HHLA2 was absent in normal brain cells, including glial cells and neurons, but abundant in endothelial cells. Furthermore, it was observed that tumoral HHLA2 expression was absent in HGG, particularly in GBM. Of note, in contrast to the aforementioned, weak expression of HHLA2 in the nuclei of tumor cells was observed in part of the patients with LGG. This result suggested a downward trend in HHLA2 expression with the increase in the degree of malignancy of the tumor, which is opposite to previous results according to which HHLA2 expression was not detected in most organs, but was widely expressed in human cancers of the breast, lung, thyroid, skin, pancreas, ovary, liver, bladder, colon, prostate, kidney and esophagus (29). However, Yan et al (37) also reported that HHLA2 was widely overexpressed in early pancreatic precancerous lesions compared with pancreatic cancer, although it was not expressed in normal acinar, islet and ductal cells. Furthermore, overexpression of HHLA2 was indicated to be significantly associated with a better outcome. In addition, Zhu et al (31) indicated that HHLA2 engaged with CD28H to co-stimulate human T-cell proliferation and cytokine production via a pathway involving AKT phosphorylation. Based on the aforementioned results, it is reasonable to assume that higher HHLA2 expression in the early stage of glioma co-stimulated the immune response, while the expression decreased with the increasing malignancy of the tumor. It may be speculated that the expression model of HHLA2 in glioma is similar to that in the pancreas. However, most patients with glioma only present at the hospital after evident clinical symptoms have occurred. On this account, it is difficult to obtain tumor tissue in the early stage of glioma for IHC labeling and detection of HHLA2 expression. Furthermore, the present cancer model was validated in the TCGA dataset at the mRNA level at the same time.

Through analysis of TCGA, the largest cancer dataset, HHLA2 expression in malignant glioma was assessed at the transcriptional level. It was revealed that HHLA2 expression in GBM was significantly lower than that in glioma of other grades. According to a study by Eckel-Passow et al (32), a single TERT mutation predicted poor prognosis in glioma, while IDH mutation and 1p/19q co-deletion predicted a favorable outcome. In the present study, tumors with IDH mutation, 1p/19q co-deletion and wild-type TERT expressed higher levels of HHLA2, which was in line with our previous assumption. Furthermore, the expression levels of HHLA2 were detected in four different molecular subtypes of glioma defined by TCGA: PN, NE, CL and ME (38,39). Studies have indicated that PN and NE subtypes mostly occurred among LGG and were associated with a favorable prognosis, while the CL and ME subtypes were associated with a worse outcome (38,40). Of note, in the NE subtype, a significantly higher expression of HHLA2 compared with that in the other subtypes was observed, and the HHLA2 expression status was a good predictor for NE-subtype glioma. These results were in line with a previous study and indicated that higher expression of HHLA2 may predict a favorable outcome (37).

The present study further explored the expression model of TIICs in glioma, and it was indicated that macrophages were markedly increased in GBM vs. LGG. TAMs, developed from monocytes, have been confirmed to be the most important type of immune cell in the stroma of tumors, accounting for 50% of all immune cells, and to have an important role in neoplasia, metastasis, immune escape and tumoral angiogenesis (33,34). Immature monocytes migrate to tumor tissues and develop into TAMs through several cytokines, including VEGF, PDGF, colony-stimulating factor-1 (CSF-1) and C-C motif chemokine ligand 2. Notably, a previous study confirmed that HHLA2 was constitutively expressed on human monocytes (22). Thus, the association between TAMs and the expression levels of HHLA2 was explored in the present study. Lower TAMs were observed in the HHLA2 high-expression group and predicted a better outcome. To further explore the mechanism of HHLA2 acting on TAMs, several cytokines linked to angiogenesis and processes that develop monocytes into TAMs were selected for assessment, revealing that HHLA2 was negatively correlated with VEGF and PDGF. These results demonstrated that HHLA2 may inhibit TAM development and tumor angiogenesis via anti-VEGF and anti-PDGF processes. Kumar et al (41) reported that anti-CSF1 receptor, specifically targeting TAMs, plus anti-PD1 treatment significantly improved therapies, compared with anti-PD1 treatment alone. HHLA2, which may not only inhibit TAM development but also co-stimulate immune function, has promising potential in immune therapy for patients with glioma.

The function of HHLA2 in the immune microenvironment of glioma was then assessed. Taking its role as an immune stimulator into account, its correlation with several common immune inhibitors was explored. According to Kamran et al (42), TGF-β and IL-10 are central to maintaining the immunosuppressive microenvironment of glioma. Thus, TGF-β and IL-10 were selected as representative immune inhibitors and included in the analysis. It was observed that IL-10 was significantly increased in the low HHLA2 expression group, which was also the case for TGF-β. The function of HHLA2 was then explored in-depth via GO and KEGG enrichment analysis. The results demonstrated that HHLA2 was positively associated with response to toxic substances, regulation of neuronal death, the glutamate receptor signaling pathway and regulation of protein processes. This indicated that HHLA2 is able to prevent normal neurons from damage by negatively regulating the glutamate receptor signaling pathway. Furthermore, genes which were negatively correlated with HHLA2 were enriched in cell cycle, DNA replication, DNA repair, activation of E2F1 target genes at G1/S, the FOXM1 pathway and the ATR pathway. Activation of E2F1 target genes at G1/S, the FOXM1 pathway and the ATR pathway have been previously confirmed to promote neoplastic proliferation and tumor progression (43–45). These results indicated that HHLA2 inhibits tumor progression in the early stage of glioma by participating in early immune response and inhibiting neoplastic proliferation and angiogenesis. Hence, exploring the mechanism of HHLA2 acting on tumor cells may facilitate the discovery of a cure for this disease.

Notably, the present study was the first to report on the prognostic significance of HHLA2 in glioma. Kaplan-Meier survival analysis revealed that higher HHLA2 expression was consistently and significantly associated with better survival in glioma and GBM. Furthermore, a subsequent Kaplan-Meier survival curve analysis for each subtype yielded a similar result for the MΕ subtype of glioma. Lin et al (40) reported that the CL subtype had the worst outcome and accounted for a large part of GBM, while the NE and PN subtypes had a high proportion of LGG with a better outcome. These results may explain for the absence of a significant correlation between higher HHLA2 expression and better prognosis in the CL, NE and PN subtypes of glioma. Furthermore, the univariate analysis indicated that HHLA2 expression was a significant prognostic factor, while multivariate analysis demonstrated that the expression of HHLA2 was not an independent prognostic factor. Regarding the limitations of the present study, part of the patients included were lost to follow-up. Thus, the potential of HHLA2 to be an independent prognostic predictor for glioma should be further investigated in a larger and more comprehensive dataset.

However, in other types of human cancer, HHLA2 expression is not always a favourable predictor for patient survival. The prognostic significance of HHLA2 expression in osteosarcoma and colorectal carcinoma was identified to be correlated with metastasis and poor survival (46,47). Similarly, in triple-negative breast cancer, overexpression of HHLA2 was associated with lymph node positivity and advanced stage of the disease at the time of diagnosis, and also with an increased risk of recurrence (29). HHLA2 has also been reported to predict a favorable outcome in pancreatic ductal adenocarcinoma and gastric cancer (37,48). One explanation is that HHLA2 has two opposite ligands (22), including TMIGD that has a co-stimulatory role, while the other ligand remains elusive. TMIGD was indicated to be the major receptor in certain cancer types due to the tumoral immune microenvironment. It is not uncommon for members of the B7 family to have a dual function depending on the immune environment, tumor microenvironment or interaction with different receptors (22). HHLA2 belongs to group III of the B7 family, which also includes B7-H3 (also known as CD276) and B7×. As with HHLA2, the prognostic significance of B7-H3 and B7× also remains to be further delineated (49,50). Wang et al (51) reported that B7-H3 was positively associated with the Toll-like receptor signaling pathway and predicted poor survival for glioma patients. Zhou et al (52) also identified that overexpression of B7-H3 was associated with the malignancy grade of brainstem gliomas. However, results demonstrating B7-H3 as a co-stimulatory molecule associated with prolonged survival in pancreatic cancer (53) and gastric cancer (54) have also been reported. Therefore, the association between HHLA2 and other immune checkpoint molecules was then analyzed, revealing a negative correlation with checkpoint inhibitors, including PD-1, LAG3 and B7-H3. These results indicated that anti-PD-1 plus anti-B7-H3 treatment may be a strategy for glioma treatment, as it may lead to upregulation of HHLA2. Notably, the present study was the first to indicate that HHLA2 may act as a co-stimulatory molecule in glioma, in contrast to other B7 family members, which are commonly characterized as immune checkpoint inhibitors.

To date, observable progress has been made in the area of immunotherapy. However, blocking the PD-1/PD-L1 pathway was only effective in a small number of cases and most patients with glioma still suffered from disease. Thus, novel therapeutic strategies targeting other immune checkpoint molecules are in urgent demand. HHLA2, acting as an immune stimulator and inhibiting the formation of TAMs, may be a potential therapeutic target.

To the best of our knowledge, the present study was the first to explore the biological function and clinical roles of HHLA2 in glioma. The results indicated that HHLA2 acts as an immune stimulator and inhibits the formation of TAMs. Furthermore, HHLA2 expression was significantly correlated with a favorable outcome. The present study indicated its potential as a prognostic predictor and novel therapeutic target.