Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia

Abstract

Herpesvirus entry mediator (HVEM) is a molecular switch that can modulate immune responses against cancer. The significance of HVEM as an immune checkpoint target and a potential prognostic biomarker in malignancies is still controversial. This study aims to determine whether HVEM is an immune checkpoint target with inhibitory effects on anti-tumor CD4⁺ T cell responses in vitro and whether HVEM gene expression is dysregulated in patients with acute lymphocytic leukemia (ALL). HVEM gene expression in tumor cell lines and peripheral blood mononuclear cells (PBMCs) from ALL patients and healthy controls was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Tumor cells were left untreated (control) or were treated with an HVEM blocker before co-culturing with CD4⁺ T cells in vitro in a carboxyfluorescein succinimidyl ester (CFSE)-dependent proliferation assay. HVEM expression was upregulated in the chronic myelogenous leukemia cell line (K562) (FC = 376.3, p = 0.086) compared with normal embryonic kidney cells (Hek293). CD4⁺ T cell proliferation was significantly increased in the HVEM blocker-treated K562 cells (p = 0.0033). Significant HVEM differences were detected in ALL PBMCs compared with the controls, and these were associated with newly diagnosed ALL (p = 0.0011) and relapsed/refractory (p = 0.0051) B cell ALL (p = 0.0039) patients. A significant differentiation between malignant ALL and the controls was observed in a receiver operating characteristic (ROC) curve analysis with AUC = 0.78 ± 0.092 (p = 0.014). These results indicate that HVEM is an inhibitory molecule that may serve as a target for immunotherapy and a potential ALL biomarker.

Keywords: CD4+ T cells; acute lymphocytic leukemia; co-inhibitory molecules; herpesvirus entry mediator; immune checkpoint blockade; immune checkpoint receptor.

Figure 1

Light microscopy images and the expression of herpesvirus entry mediator (HVEM) surface protein on breast cancer (MCF-7), hepatocellular carcinomas (HepG2), chronic myelogenous leukemia (K562), and human embryonic kidney (Hek293) cells. The left column represents the microscope and a magnification of 100 pt using NIS-Elements F 4.00.00 software (Nikon Instruments, USA) under a light microscope (Nikon Eclipse, Fujisawa, Japan). Data collected from four separate experiments represents cell proliferation, morphology, and attachment nature. The right column shows histogram graphs representing the measurements of HVEM expression after staining with anti-HVEM-PE. The dotted-line histograms show the unstained control cells, whereas the line histograms represent the HVEM-stained cells. The numbers at the top of each histogram indicate the MFI of the unstained (left) and HVEM-PE (right). The number in the middle represents the percentage of HVEM-positive cells. The results were collected from two wells in three separate experiments. Magnification: 100 pt.

Figure 2

Light microscopic images of K562 (left) and Hek293 (right): (A) alone, (B) HVEM untreated cells with CFSE CD4⁺ T cells at one tumor or normal to ten T cells, or (C) HVEM blocker treated cells with CFSE CD4⁺ T cells after K562 and Hek293 for 72 h of incubation. Data are representative of three separate experiments. Magnification: 100 μm.

Figure 3

Proliferation of CFSE-labeled CD4⁺ T cells in response to (A) K562 tumor cells or (B) Hek293 healthy control cells without HVEM blocker treatment (left column), or after K562 and Hek293 treatment with HVEM blocker (middle column); and non-proliferated CFSE CD4⁺ T cells (right column) after 72 h of incubation at 37 °C. Data are representative of two separate experiments.

Figure 4

Mean ± SD of fluorescence intensity of CFSE-labeled CD4⁺ T cells before proliferation on day zero and after 72 h of incubation with either K562 tumor cells or with Hek293 normal cells that were left untreated or treated with 20 ng HVEM blocker. Cells were cultured at a ratio of one tumor or normal cell to ten CFSE CD4⁺ T cells. ** indicates strong significant differences between groups with p = 0.0033, whereas “ns” indicates no significant differences with p = 0.5918. The results were collected from three separate experiments.

Figure 5

Relative expression of HVEM gene in patients with malignant ALL compared with non-malignant healthy controls. RNA from PBMCs were isolated, RT-qPCR determined the gene expression of HVEM, and the expression of GAPDH was normalized. (A) represents HVEM expression in all study subjects, 10 non-malignant controls and 23 malignant ALL patients, and ** shows significant differences between groups with p = 0.0064 (using the Mann–Whitney multiple comparisons test). (B) shows the expression of HVEM in three ALL categories (newly diagnosed, remission, and relapse/refractory) compared with the non-malignant control, and ** shows significant differences between non-malignant and either freshly diagnosed groups with p = 0.0011 or relapse/refractory with p = 0.0051 (using a one-way ANOVA multiple comparison test). (C) represents HVEM relative expression in three types of ALL (pre-B-ALL, B-ALL, and T-ALL) compared with the non-malignant controls, and ** shows significant differences between groups with p = 0.0039 (using a one-way ANOVA multiple comparison test). In addition, “ns” represents non-significant between groups. All statistical analyses were conducted using GraphPad Prism 10.0.3.

Figure 6

Receiver operating characteristic curve for HVEM gene expression in patients with malignant ALL compared with non-malignant healthy controls: (A) in all samples, (B) in newly diagnosed ALL, (C) in remission ALL, and (D) in relapse/refractory. AUC, the area under the curve, values are 0.7762 (p = 0.0142), 0.8889 (p = 0.0133), 0.6543 (p = 0.1985), and 0.7472 (p = 0.0359), respectively, indicating that HVEM is a potential biomarker in ALL patients, especially in newly diagnosed patients and those in the relapse/refractory phase.