Differential expression of Tim-3, PD-1, and CCR5 on peripheral T and B lymphocytes in hepatitis C virus-related hepatocellular carcinoma and their impact on treatment outcomes

Abstract

Background and objective

Activation of the immune checkpoints and expression of chemokines and chemokine receptors have been reported to promote HCC progression. This study aimed to assess the differential expression of Tim-3, PD-1, and CCR5 on peripheral blood lymphocytes from patients with HCV-related HCC and correlate their expression with the treatment outcomes.

Patients and methods

The study incorporated 40 patients with chronic HCV-related HCC and 40 healthy controls. Patients were radiologically assessed for hepatic focal lesions and portal vein thrombosis. Response to HCC treatment and overall survival (OS) outcomes were determined. The expression of Tim-3, PD-1, and CCR5 among CD19⁺, CD4⁺, and CD8⁺ lymphocytes was assessed by flow cytometry.

Results

Higher frequencies of CD4⁺ and CD8⁺ cells expressing each of Tim-3 and PD-1 and PD-1⁺CD19⁺ cells were observed in the HCV-related HCC patients in comparison with controls. The highest expression of Tim-3 and PD-1 was by the CD8⁺ cells. Strong relations were detected among PD-1⁺CD19⁺, PD-1⁺CD4⁺ and PD-1⁺CD8⁺ cells. Elevated levels of PD-1⁺ lymphocytes were significantly associated with poor treatment response and shorter OS.

Conclusion

Modulation of the expression of immune checkpoints as Tim-3 and PD-1, and of CCR5 on T cells is somehow related to HCC. CD8⁺ T cells expressing PD-1 were the most relevant to HCC prognosis (OS and treatment response) and could represent a promising target for immune therapy against HCC. Future studies need to focus on exploring PD-1⁺ B cells and Tim-3⁺CD4⁺ cells, which seem to play a significant role in the pathogenesis of HCC.

Electronic supplementary material

The online version of this article (10.1007/s00262-019-02465-y) contains supplementary material, which is available to authorized users.

Introduction

Hepatocellular carcinoma (HCC) is the most common of all primary liver cancers [2]. It is the sixth most prominent cancer globally and the third most common cause of cancer-related deaths. HCC is associated with poor prognosis, because it is usually multifocal at the time of discovery, with metastasis, a high recurrence rate, and few treatment options [3, 4]. The risk for its development is considerably greater in patients with hepatitis B virus (HBV) or hepatitis C virus (HCV)-related cirrhosis [5–7].

The coordinated immune responses of regulatory, helper, and cytotoxic T lymphocytes have a significant impact on the progression of chronic liver disease to HCC [8–10]. Cytotoxic T cells in particular have an essential role in getting rid of infected or malignant liver cells [11]. According to recent research, B cells have shown diverse roles in carcinogenesis and tumor progression [12, 13]. While some have shown that B cells have tumor-promoting functions [14–16], others concluded that B cells have antitumor protective features [17, 18].

Various immune responses, including alterations in immune cell functions, cytokine level, and the expression of immune receptors or ligands, have been reported to promote HCC progression [19–22]. Chemokines and their receptors are critically involved in the pathogenesis of HCC through enhancement of inflammation, angiogenesis, effects on immune cells, and direct effects on tumor invasion, growth and survival [23]. A close relation was observed between the CCL5–CCR5 (chemokine ligand 5—chemokine receptor 5) axis and liver chronic inflammation induced by different pathogens that ultimately led to the development of HCC [24].

Some tumors evade the immune response by expressing inhibitory ligands, which bind co-inhibitory receptors (immune checkpoints) expressed on tumor-specific immune cells and cause immune suppression [25]. PD-1 (programmed cell death protein-1) and Tim-3 (T-cell immunoglobulin and mucin-domain containing-3) are the most studied immune checkpoint receptors [26]. PD-L1 and PD-L2 are the two known ligands for PD-1. Activation of PD-1/PD-L1 signaling is crucial for peripheral T-cell tolerance through down-regulation of effector T-cell-mediated immune responses to guard against immune-mediated tissue damage [27]. Once engaged by its ligands, PD-1 reduces T-cell cytokine responses with substantial effects on IFN-γ, TNF-α, and IL-2 production [27, 28]. PD-1 signaling also affects T-cell differentiation and survival directly by inhibiting the early activation events [29]. It inhibits T-cell activation kinases through SHP2 phosphatase activity and other signaling pathways [30].

Engagement of TIM-3 and PD-1 molecules is associated with exhausted T cells with reduced numbers, functions, and subsequently suppressed immune responses to tumors [25, 31] and chronic viral infections [32]. Their expression by T cells has prognostic implication for a diversity of tumors making them potential targets for cancer immunotherapy [25, 33]. Previous studies have reported higher expression levels of both PD-1 and Tim-3 by CD4⁺ and CD8⁺ T cells in patients with HBV and HCV infection [34–36] and HCC patients [37–39] compared with healthy subjects.

Understanding the complexity of interactions between the different immune cells expressing these immune checkpoints, chemokines, and chemokine receptors is of utmost importance in tailoring newer immune-therapeutics against HCC. Thereby, this study aimed to assess the differential expression of Tim-3, PD-1, and CCR5 by peripheral blood T and B lymphocytes from HCV patients with HCC and correlate their expression with the treatment response and survival outcomes in these patients.

Materials and methods

Forty HCV-related HCC patients and 40 age- and sex-matched healthy controls were incorporated in this case–control study. Patients were recruited from AL-Rajhi Liver Center, South Egypt Cancer Institute, Assiut University Hospital. We excluded from the study patients concurrently infected with HIV, hepatitis A, B, D viruses, or schistosoma. Patients having alcoholic or drug-induced liver disease or treated with immune-modulating drugs were also excluded from this study.

Diagnosis of HCV infection and liver cirrhosis was based on the clinical, biochemical, and ultrasonographic findings. The Child–Pugh score was employed to estimate the severity of cirrhosis; it is also considered a useful marker for global liver function, primarily for the selection of HCC patients for different lines of treatment [40]. Besides, Access 2 (Beckman Coulter, USA) was used to evaluate serum levels of alpha-fetoprotein (AFP).

All patients were radiologically evaluated via triphasic computed tomography (CT) with contrast and magnetic resonance imaging (MRI). HCC diagnosis was based on LI-RADS and AASLD criteria that include early arterial hyperenhancement and delayed venous phase washout appearance and capsule appearance. The number of hepatic focal lesions, portal vein thrombosis, and liver volume, including the Future Liver Remnant (FLR), were also evaluated. The likelihood that the disease is confined to the liver, the size/location of the tumor and the patient’s hepatic function were assessed to determine whether the patient's condition permit resection or not.

Patients with early-stage disease (solitary HCC nodule or up to 3 nodules ≤ 3 cm, Child–Pugh A or B) were identified and evaluated for curative-intent therapies such as resection and radiofrequency ablation (RFA), percutaneous ethanol injection, and trans-arterial chemo-embolization (TACE) by a multidisciplinary team. Systemic treatment was a palliative-intent therapy given for patients with sufficient hepatic reserve and performance status (PS) but no longer eligible for surgical or liver-directed therapies and included:

1- Sorafenib: The first-line standard of care based upon the 3-month median survival benefit demonstrated in the SHARP trial that was used in patients with Child–Pugh A hepatic reserve, with Eastern Cooperative Oncology Group Performance Status (ECOG PS) grades 0–2.

2- Capecitabine (Xeloda) given mainly on a metronomic base that meant to give Xeloda one tablet, 1–3 times daily, continuously without drug-free period according to the tolerance of the patient.

3- Supportive treatments (liver support drugs, platelet transfusion, diuretics, etc.).

All patients were regularly followed-up after treatment and every 3 months with clinical examination, liver function tests, AFP, and triphasic CT abdomen to evaluate their responses, manage toxicities developed, and to determine the overall survival. Our patients were followed up for a range of 2–30 months with a mean follow-up period of 10.7 ± 6.2 months. Clinical and laboratory features of the study subjects are shown in Table 1.

Table 1.

Clinical and laboratory characteristics of the study subjects

Characteristics	HCV–HCC patients (n = 40)	Controls (n = 40)
Age (years)	63.7 ± 1	58 ± 2
Sex
Males	36 (90%)	34 (85%)
Females	4 (10%)	6 (15%)
ALT (U/L)	45.5 ± 7	10.3 ± 0.3
AST (U/L)	94 ± 14	11 ± 0.5
Albumin (gm/L)	30.7 ± 1	40.3 ± 0.1
Total protein (gm/L)	69.9 ± 2	70 ± 3
Total bilirubin (mg/dL)	23.5 ± 2	0.8 ± 0.04
Prothrombin time (seconds)	13.9 ± 0.3	12 ± 0.3
INR	1.2 ± 0.03	1 ± 0.01
Hemoglobin (g/dl)	10.9 ± 0.4	13.2 ± 0.3
WBCs (109/L)	7.2 ± 0.5	8.2 ± 0.3
Platelet count	162.2 ± 14	254.5 ± 11
Liver cirrhosis	40 (100%)	–
Child–Pugh score	7.1 ± 0.3	–
Ascites
No	23 (57.5%)	–
Mild	3 (7.5%)
Moderate	14 (35%)
Hepatic encephalopathy	8 (20%)	–
Splenomegaly	20 (50%)	–
HFL
1	14 (35%)	–
2	8 (20%)
3	18 (45%)
Portal vein thrombosis	31 (77.5%)	–
Alpha-fetoprotein (ng/ml)	12,661.8 ± 11,036.6	–
Patients on Daclatasvir /sofosbuvir treatment regimen	9 (22.5%)	–
Deaths	6 (15%)	–

HCV–HCC hepatitis C related hepatocellular carcinoma, ALT alanine aminotransferase, AST aspartate aminotransferase, INR international normalized ratio, MCV mean corpuscular volume, MCHC mean corpuscular hemoglobin concentration, WBCs white blood cells, HFL hepatic focal lesions

Data expressed as mean ± SE or number (percentage), accordingly

Flow cytometric detection of lymphocytes and their expression of TIM-3, PD-1, and CCR5

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll density gradient centrifugation (Biochrom GmbH, Germany). About 2 × 10⁶ cells were stained with 5 µl fluoroisothiocyanate (FITC) conjugated Tim-3, phycoerythrin (PE) conjugated CD8, peridinium-chlorophyll-protein (Per-CP)-conjugated CD4 and allophycocyanin (APC) conjugated CD19 in one tube. Also, the second tube included 5 µl FITC-conjugated PD-1, PE-conjugated CD8, Per-CP-conjugated CD4, and APC-conjugated CD19. And, the third tube included 5 µl FITC-conjugated CCR5, PE-conjugated CD8, Per-CP-conjugated CD4, and APC-conjugated CD19. All monoclonal antibodies were purchased from Becton Dickinson (BD Bioscience, CA, USA) except Tim-3 and PD-1 from BioLegend (GmbH, Germany). Incubation for 20 min at 4 °C was performed in the dark, and then washing with phosphate buffer saline (PBS) was done. An isotype-matched anti-human IgG negative control was used with each sample. The analysis was accomplished by FACSCalibur flow cytometry with Cell Quest software (BD Biosciences, USA). The lymphocyte population was defined according to their forward and side scatters. The region (R1) was drawn to select the lymphocyte population to assess the expression of CD19, CD8, and CD4. The CD19⁺, CD8⁺, and CD4⁺ lymphocyte subsets were then gated by drawing regions (R2, R3, and R4, respectively). The expression of Tim-3, PD-1, and CCR5 was assessed among the three lymphocyte subsets and was reported as percentages in each cell population (Fig. 1).

Fig. 1.

Flow cytometric detection of lymphocytes and their expression of TIM-3, PD-1, and CCR5. a Forward and side scatter histogram was used to define the lymphocyte population. b, c The expression of CD19, CD8 and CD4 was assessed on the lymphocytes, and then, the CD19⁺, CD8⁺, and CD4⁺ lymphocyte subsets were gated for further analysis of TIM-3, PD-1, and CCR5. d–f The expression of Tim-3, PD-1, and CCR5 on CD19. g–i The expression of Tim-3, PD-1, and CCR5 on CD8. j–l The expression of Tim-3, PD-1, and CCR5 on CD4

Statistics

Descriptive data in this study were in the form of mean, median, range, standard error, and percentages. The Mann–Whitney U test and independent t test were used to compare two groups, while the one-way ANOVA test to compare more than two groups of quantitative variables. A p value < 0.05 was considered significant. The Pearson correlation was employed to assess the relations of immune checkpoints and chemokine receptors with the different treatment outcomes. The overall survival (OS) was defined as the interval from the diagnosis to the date of death, or the last follow up and was estimated by the Kaplan–Meier estimator. All of our results were analyzed using SPSS ver. 24.

Results

Comparison of the mean percentages of CD19⁺, CD4⁺, and CD8⁺ cells expressing Tim-3, PD-1, and CCR5 among groups

The mean percentages of CD4⁺ and CD8⁺cells expressing CCR5 were significantly lower in patients compared with the healthy control group (p < 0.0001). On the contrary, a noticeable higher frequency of CD4⁺ and CD8⁺ cells expressing each of Tim-3 and PD-1 and also of PD-1⁺CD19⁺ cells was observed in patients in comparison with controls (p < 0.0001). The results are summarized in Table 2.

Table 2.

Mean percentages of CD19⁺, CD4⁺ andCD8⁺ cells expressing Tim-3, PD-1, and CCR5 among groups

Cells	HCV–HCC patients (n = 40)	Controls (n = 40)	p value
Tim-3+ CD19+ (%)	1.3 ± 0.01	1.2 ± 0.01	0.2
Tim-3+ CD4+ (%)	8.9 ± 0.5	4 ± 0.3	< 0.0001
Tim-3+ CD8+ (%)	10.4 ± 1	5.5 ± 0.2	< 0.0001
PD-1+CD19+ (%)	23 ± 0.5	17.2 ± 0.5	< 0.0001
PD-1+CD4+ (%)	20.3 ± 0.4	15.2 ± 0.4	< 0.0001
PD-1+CD8+ (%)	30.9 ± 1	21.1 ± 0.4	< 0.0001
CCR5+CD19+ (%)	10.6 ± 0.4	11.5 ± 0.5	0.1
CCR5+CD4+ (%)	10.6 ± 0.6	15.1 ± 0.9	< 0.0001
CCR5+CD8+ (%)	20 ± 0.3	22.5 ± 0.5	< 0.0001

n number, HCV–HCC hepatitis C related hepatocellular carcinoma

Results expressed as mean percentages ± SE

Independent T Test, bold p values indicate significance (< 0.05)

Differences in the expression of Tim-3, PD-1, and CCR5 between the CD19⁺, CD4⁺, and CD8⁺ cells in the study patients

Differences in the expression of Tim-3, PD-1, and CCR5 between the different lymphocytes subsets are illustrated in “Supplementary Fig. 1”. The highest expression of Tim-3, PD-1, and CCR5 was detected among the CD8⁺ cells compared with CD4⁺ T cells and B cells (p < 0.0001). The expression of CCR5 was more or less equal by both CD19⁺ and CD4⁺ cells (p value = 0.96). The expression of Tim-3 by both CD8⁺and CD4⁺cells was markedly higher than that by CD19⁺cells (p < 0.0001). PD-1 expression by CD8⁺cells was higher than that by CD19⁺ cells followed by CD4⁺cells (p < 0.0001).

Correlations among CD19⁺, CD4⁺, and CD8⁺ cells expressing Tim-3, PD-1 and CCR5, and their relations with the other laboratory parameters

Several relations were observed among the studied cells, as shown in Table 3. A perfect correlation was found between CD19⁺ and CD4⁺ cells expressing PD-1. Moreover, CD19⁺ and CD4⁺ cells expressing PD-1 showed moderate positive correlations with Tim-3⁺CD4⁺ and PD-1⁺CD8⁺ cells and a moderate negative correlation with CCR5⁺CD4⁺ cells. A moderate positive correlation was also observed between CD4⁺ and CD8⁺ cells expressing Tim-3.

Table 3.

Correlations among CD19⁺, CD4⁺ and CD8⁺ cells expressing Tim-3, PD-1, and CCR5

Cells	Tim-3+ CD19+ (%)	Tim-3+ CD4+ (%)	Tim-3+ CD8+ (%)	PD-1+CD19+ (%)	PD-1+CD4+ (%)	PD-1+CD8+ (%)	CCR5+CD19+ (%)	CCR5+CD4+ (%)	CCR5+CD8+ (%)
Tim-3+ CD19+ (%)	–	r = 0.17 p value = 0.06	r = 0.06 p value = 0.3	r = 0.2 p value = 0.03	r = 0.2 p value = 0.03	r = − 0.02 p value = 0.4	r = − 0.1 p value = 0.2	r = − 0.2 p value = 0.01	r = 0.2 p value = 0.04
Tim-3+ CD4+ (%)	r = 0.17 p value = 0.06	–	r = 0.7 p value =  < 0.0001	r = 0.6 p value < 0.0001	r = 0.6 p value < 0.0001	r = 0.4 p value < 0.0001	r = − 0.04 p value = 0.4	r = − 0.2 p value = 0.02	r = − 0.4 p value < 0.0001
Tim-3+ CD8+ (%)	r = 0.06 p value = 0.3	r = 0.7 p < 0.0001	–	r = 0.3 p value = 0.004	r = 0.3 p value = 0.004	r = 0.3 p value = 0.01	r = 0.17 p value = 0.07	r = − 0.1 p value 0.2	r = − 0.3 p value = 0.004
PD-1+CD19+ (%)	r = 0.2 p value = 0.03	r = 0.6 p value < 0.0001	r = 0.3 p value = 0.004	–	r = 1 p < 0.0001	r = 0.6 p value < 0.0001	r = − 0.2 p value = 0.04	r = − 0.5 p value < 0.0001	r = − 0.2 p value = 0.02
PD-1+CD4+ (%)	r = 0.2 p value = 0.03	r = 0.6 p value < 0.0001	r = 0.3 p value = 0.004	r = 1 p < 0.0001	–	r = 0.6 p value < 0.0001	r = − 0.2 p value = 0.04	r = − 0.5 p value < 0.0001	r = − 0.2 p value = 0.02
PD-1+CD8+ (%)	r = − 0.02 p value = 0.4	r = 0.4 p value < 0.0001	r = 0.3 p value = 0.01	r = 0.6 p value < 0.0001	r = 0.6 p value < 0.0001	–	r = − 0.3 p value = 0.007	r = − 0.4 p value = 0.001	r = − 0.2 p value = 0.02
CCR5+CD19+ (%)	r = − 0.1 p value = 0.2	r = − 0.04 p value = 0.4	r = 0.17 p value = 0.07	r = − 0.2 p value = 0.04	r = − 0.2 p value = 0.04	r = − 0.3 p value = 0.007	–	r = − 0.06 p value = 0.3	r = − 0.2 p value = 0.02
CCR5+CD4+(%)	r = − 0.2 p value = 0.01	r = − 0.2 p value = 0.02	r = − 0.1 p value 0.2	r = − 0.5 p value < 0.0001	r = − 0.5 p value < 0.0001	r = − 0.4 p value = 0.01	r = − 0.06 p value = 0.3	–	r = − 0.08 p value = 0.2
CCR5+CD8+ (%)	r = 0.2 p value = 0.04	r = − 0.4 p value < 0.0001	r = − 0.3 p value = 0.004	r = − 0.2 p value = 0.02	r = − 0.2 p value = 0.02	r = − 0.2 p value = 0.02	r = − 0.2 p value = 0.02	r = 0.08 p value = 0.2	–

Pearson correlation, r correlation coefficient, bold r and p values indicate significance (p < 0.05)

Moreover, tested cells have only shown a few significant correlations with most of the evaluated laboratory parameters. Tim-3⁺CD4⁺ cells had a positive correlation with the level of total bilirubin (r = 0.3, p value = 0.03) and a negative correlation with albumin (r = − 0.3, p value = 0.049). The level of CCR5⁺CD8⁺ cells was negatively correlated with the platelet count (r = − 0.3, p value = 0.02). None of the cells expressing the tested markers showed significant correlations with AFP or the number of hepatic focal lesions.

Comparison of the levels of CD19⁺, CD4⁺, and CD8⁺ cells expressing Tim-3, PD-1, and CCR5 between patients on Daclatasvir/ sofosbuvir treatment regimen and patients not receiving treatment for HCV

Only the mean percentages of both CD4⁺ and CD8⁺ cells expressing Tim-3 were significantly higher in non-treated patients compared with those who were on a treatment regimen, as shown in Table 4.

Table 4.

Comparison of the levels of CD19⁺, CD4⁺ and CD8⁺ cells expressing Tim-3, PD-1, and CCR5 (A) between HCV-related HCC patients on Daclatasvir/ sofosbuvir treatment regimen and patients not receiving treatment for HCV and (B) between patients with different HCC treatment responses

Response	Tim-3- CD19	Tim-3- CD4	Tim-3- CD8	PD-1- CD19	PD-1- CD4	PD-1- CD8	CCR5- CD19	CCR5- CD4	CCR5- CD8
A) Daclatasvir/ sofosbuvir treatment*
Non HCV treated (n = 31)	1.3 ± 0.01	9.6 ± 0.6	11.4 ± 1.3	23.2 ± 0.6	20.5 ± 0.5	30.7 ± 1	10.5 ± 0.5	10.9 ± 0.6	20 ± 0.3
HCV treated (n = 9)	1.3 ± 0.04	6.6 ± 0.8	7 ± 1	22.4 ± 1	19.7 ± 0.9	31.8 ± 1.9	10.8 ± 0.5	9.4 ± 1	20 ± 0.8
p value	0.6	0.02	0.009	0.5	0.5	0.6	0.8	0.4	0.7
B) HCC treatment response**
CR (n = 13)	1.2 ± 0.1	5 ± 2	6.4 ± 0.5	16.7 ± 0.3	14.7 ± 0.2	20 ± 0.01	12.3 ± 3	14.1 ± 2	21.1 ± 0.4
PR (n = 10)	1.2 ± 0.03	6.6 ± 1	7.6 ± 1	19.9 ± 2	17.5 ± 2	23.3 ± 2	10.4 ± 1	14.3 ± 2	19.97 ± 1
SD (n = 10)	1.2 ± 0.03	6.4 ± 2	8.9 ± 2	20.6 ± 0.3	18.1 ± 0.3	27 ± 1	10.3 ± 1	10.8 ± 1	21.3 ± 0.6
PD (n = 7)	1.2 ± 0.3	7.8 ± 1	8.98 ± 0.8	23.4 ± 1	20.6 ± 3	35.96 ± 1	9.6 ± 0.7	9.95 ± 2	20.5 ± 0.4
p value	0.7	0.4	0.6	0.003	0.003	0.001	0.2	0.1	0.7

HCC hepatocellular carcinoma, CR complete response, PR partial response, SD stable disease, PD progressive disease n number

Results expressed as mean percentages ± SE

*Mann Whitney U test and

**One-way ANOVA test for significance, bold p values indicate significance (< 0.05)

Relations of the levels of CD19⁺, CD4⁺and CD8⁺ cells expressing Tim-3, PD-1 and CCR5 with the different patterns of response to HCC treatment

The percentages of expression of Tim-3 and CCR5 did not have a significant impact on the response to HCC treatment, while there was a considerable increase in the proportions of cells expressing PD-1 with the worsening of the response to treatment as presented in Table 4.

Overall survival in HCV-related HCC patients

The median overall survival of the whole study sample was 12 months ± 0.9, with a confidence interval from 10.2 to 13.8 (Fig. 2). Our results elucidated several associations between OS and the different immune cells. A positive correlation was observed between OS and PD-1⁺ CD4⁺/PD-1⁺CD8⁺ ratio (r = 0.4, p = 0.02). Conversely, OS showed negative correlations with each of CD19⁺, CD4⁺ and CD8⁺ lymphocytes expressing PD1 (r = − 0.6, p < 0.0001, r = − 0.6, p < 0.0001and r = − 0.8, p < 0.0001, respectively) (Fig. 2). Also, OS was inversely associated with Tim-3⁺CD4⁺ cells (r = − 0.4, p = 0.01).

Fig. 2.

Overall survival curve of the whole study population (a) and its correlation with PD-1⁺CD8⁺ cells (b)

Discussion

The significance of T cells in combating tumors has become extensively investigated and well known. Meanwhile, the likely role of B cells in the immune response to tumors is less well studied [41]. Tumors are rich in B cells [42], but the abundant subsets and their biologic role in tumor pathogenesis are poorly understood [43].

The expression and function of CCR5 on T cells are well established [44, 45]. Aberrant CCR5 expression can be detected in different kinds of tumors. Higher CCR5 expression by B cells was found in lymphoma patients [46]. Agreeing with a previous study [47], CCR5 expression by both peripheral CD4⁺ and CD8⁺T lymphocytes was significantly lower in our HCC patients compared with the healthy control; this was more evident in CD4⁺ T lymphocytes. Liu et al. reported an increased T-cell expression of this chemokine receptor in the tumor tissue, proposing that they might be involved in directing the migration of T cells to the tumor area [48] and in optimizing helper-dependent CD8 + T cell priming [49]. In contrast, no difference was observed in the level of CCR5 expression by B cells between our HCC patients and the healthy subjects.

Several studies [34–36] have shown higher expression levels of both PD-1 and Tim-3 by CD4⁺ and CD8⁺ T cells in patients with HBV and HCV infection both in the liver and in peripheral blood compared with healthy subjects. Others have reported the elevated frequency of CD4⁺ and CD8⁺ T cells expressing PD-1 and Tim-3 in HCC patients [37–39]. Even though Li and colleagues [50] stated that the expression levels of Tim-3 by CD8⁺ T cells was related to a poor prognosis for tumor progression, they noted that the frequency of cells expressing Tim-3 did not show significant correlations with AFP or tumor multiplicity.

In agreement with the previous studies, the levels of CD4⁺ and CD8⁺ T cells expressing PD-1 and Tim-3 were higher in our HCC patients compared with the healthy control group and were not showing any correlations with either AFP or the number of hepatic focal lesions. The highest expression of Tim-3 and PD-1 was also detected among the CD8⁺ cells indicating the marked exhaustion of these cells in HCC patients. Moreover, a moderate correlation was found between CD4 and CD8 cells expressing PD-1. Similar to a prior study [34], there was a strong correlation between the expression of Tim-3 by CD4⁺ cells and that by CD8⁺ T cells.

The role offered by PD-1 expression on B cells still was not well studied [51]. A recent study [43] has identified a PD-1 expressing B cell subset whose frequency correlates with the disease stage and is associated with early recurrence of HCC. In the same way, our results indicated that the level of expression of PD-1 by B cells increases significantly in HCC patients having HCV compared with the control. PD-1 expression level by B cells was even more than that by CD4⁺ T cells. It is worth mentioning that B cells expressing PD-1 showed a perfect correlation with CD4⁺PD-1⁺ cells and moderate correlations with CD4⁺TIM-3⁺ and CD8⁺PD-1⁺ cells, which probably proposes that B cells expressing PD-1, CD4 and CD8 cells jointly participate in the immune dysfunction occurring in HCV patients with HCC. Furthermore, no abnormal expression of Tim-3 was detected among B cells.

In our study, CD4⁺ cells expressing CCR5 were inversely related to each of CD4⁺PD-1⁺ and CD19⁺PD-1⁺ cells. This was somewhat in line with an earlier study [52], which reported a down-regulation of some molecules affecting T-cell trafficking to and/or retention in the tumor following the co-inhibition of the PD-1 and Tim-3 signaling pathways.

Sofosbuvir/daclatasvir combination of direct-acting antiviral drugs (DAA) is one of the favored regimens in the WHO guidelines for HCV treatment with cure rates above 95% [53–55]. It is of utmost importance to understand how DAA treatment restores the immune fitness in the liver, the number, and function of exhausted lymphocytes and the expression of inhibitory molecules on liver cells [56, 57]. In our patients, a marked reduction of the levels of CD4 and CD8 T cells expressing Tim-3 was found in the patients on Daclatasvir/sofosbuvir treatment regimen compared with those not receiving treatment for HCV. The previous finding suggests a means by which these drugs can enhance the dampened immune response to HCC.

Previous studies have elicited that a higher expression of Tim-3 [37, 58, 59] and PD-1 [38] was significantly correlated with shorter OS and a more advanced tumor stage in cancer patients. Tim-3 and PD-1 cause T-cell exhaustion which enhances HCC growth [60] with a more advanced tumor stage and shorter survival. Similarly, our results showed that elevated levels of Tim-3 CD4⁺ cells and PD-1⁺ lymphocytes were correlated significantly with a shorter OS. In addition, lower proportions of PD-1⁺ lymphocytes were associated with a better response to treatment in our patients.

In the current study, we analyzed the differential expression of Tim-3, PD-1, and CCR5 by peripheral blood T and B lymphocytes from HCV patients with HCC and correlated their levels with the treatment response and survival outcomes. Our results suggest that, of all the tested markers, PD-1 was the most relevant to HCC prognosis. This was verified by the better OS and improved response to HCC treatments associated with the lower levels of cells expressing PD-1, especially CD8⁺ cells. No doubt that the significantly higher levels of T cells expressing Tim-3 point to their likely importance in HCC pathogenesis. However, it seems that they may be indirectly affecting tumor progression. Even though a better OS was somewhat related to the lower levels of CD4⁺ expressing Tim-3, the lower levels of these cells were not significantly associated with improved response to treatment. Tim-3⁺CD4⁺ cells were the only cells showing a significant positive correlation with bilirubin and a negative correlation with albumin. Together with Tim-3⁺CD8⁺ cells, they were the only cells displaying decreased levels in patients receiving HCV treatment. B cells expressing PD-1 also seem to have an essential role in HCC. This is supported by the strong relations detected between the percentage of these cells and PD-1⁺CD4⁺ and PD-1⁺CD8⁺ cells. Thus, future studies should focus on a more thorough exploration of this subset of B cells.

The study was limited by the small sample size and lack of gene expression work. Thereby, reliable conclusions regarding the interrelationship between the expression levels of these molecules and the relation between their levels and achieving sustained treatment response were challenging to be drawn. Further studies with more detailed immunological characterization are needed to confirm the impact of HCC treatments on different B- and T-cell subsets expressing these molecules.

Conclusion

Modulation of the expression of immune checkpoints as Tim-3 and PD-1, and of CCR5 on T cells is somehow related to HCC. CD8⁺ T cells expressing PD-1 were the most relevant to HCC prognosis (OS and treatment response) and could represent a promising target for immune therapy against HCC. Future studies need to focus on exploring PD-1⁺ B cells and Tim-3⁺CD4⁺ cells, which seem to play a significant role in the pathogenesis of HCC.

Electronic supplementary material

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Abbreviations

APC

Allophycocyanin

CCL5-CCR5

Chemokine ligand 5-chemokine receptor 5

DAA

Direct-acting antiviral drugs

FITC

Fluoroisothiocyanate

FLR

Future liver remnant

HCC

Hepatocellular carcinoma

OS

Overall survival

PBMCs

Peripheral blood mononuclear cells

PBS

Phosphate buffer saline

PD-1

Programmed cell death protein-1

PE

Phycoerythrin

Per-CP

Peridinium-chlorophyll-protein

PS

Performance status

RFA

Radiofrequency ablation

TACE

Trans-arterial chemo-embolization

Tim-3

T-cell immunoglobulin and mucin-domain containing-3

Author contributions

AMZ, HFH, AR and OE-B conceived and designed the experiments. AR, ASE, EAH, HF and AS recruited patients, carried out the clinical investigations and collected patients’ clinical data. AMZ performed the experiments. AMZ, HFH and OE-B shared in the analysis of the flow cytometry data. OE-B. performed the statistical analysis. OE-B and AMZ accomplished the interpretation of results and wrote the initial draft. All authors participated in critical review and revision of the final manuscript, and HFH managed the submission of the manuscript.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interests.

Ethical approval

The research was done according to the ethical guidelines of the Declaration of Helsinki 1975 and was accepted by the local ethics committee of the Faculty of Medicine, Assiut University, Assiut, Egypt (IRB no: 17300300).

Informed consent

Written informed consent was obtained from all participants before inclusion in the study. Participants consented for the use of their specimens and data for research and publication.