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  Table of Contents    
Year : 2011  |  Volume : 54  |  Issue : 3  |  Page : 448-453
Expansion of peripheral and intratumoral regulatory T-cells in hepatocellular carcinoma: A case-control study

1 Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Hepatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Cytology and Gynac Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Radio-diagnosis and Imaging, Postgraduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication20-Sep-2011


Background: Hepatocellular carcinoma (HCC) is notorious for poor prognosis with limited therapeutic options. A better understanding of the role of regulatory T-cells (Tregs) in HCC is important for design of immunotherapy based clinical protocol. The objective of the present study was to evaluate the presence of Tregs in tumor microenvironment in patients with HCC compared to chronic hepatitis (CH). Materials and Methods: The frequency of CD4 + CD25 + Treg cells was evaluated from peripheral blood (PB) of 28 patients of HCC and 30 controls including CH cases and healthy donors using flowcytometry. Intratumoral Treg were also analyzed in tissue samples from 17 HCC cases and 15 CH cases. In addition the expression of FOXP3 and CTLA-4 was also studied by RT-PCR. Results: Frequency of CD4 + CD25 + cells in the PBMCs of HCC cases was significantly higher than in HC (10.8 ± 7.64 vs 3.05 ± 1.30, P < 0.005) and CH patients (2.88 ± 1.92, P < 0.005). Also Treg population was significantly higher in HCC tumor microenvironment compared to CH biopsies (15.8 ± 5.32 vs 5.51 ± 3.40, P < 0.05). Expression of FOXP3 and CTLA-4 was also significantly higher in HCC patients ( P < 0.05) compared to CH group. Conclusions: We provide evidence of an increased population of Treg not only in the PB but also in tumor microenvironment of HCC patients, suggesting association of enhanced Treg activity with poor immune responses to tumor antigens. These findings may in future play a significant role in designing immunotherapeutic approaches in HCC.

Keywords: CTLA-4, FOXP3, hepatocellular carcinoma, immuneregulation, T-regulatory cells

How to cite this article:
Thakur S, Singla A, Chawla Y, Rajwanshi A, Kalra N, Arora SK. Expansion of peripheral and intratumoral regulatory T-cells in hepatocellular carcinoma: A case-control study. Indian J Pathol Microbiol 2011;54:448-53

How to cite this URL:
Thakur S, Singla A, Chawla Y, Rajwanshi A, Kalra N, Arora SK. Expansion of peripheral and intratumoral regulatory T-cells in hepatocellular carcinoma: A case-control study. Indian J Pathol Microbiol [serial online] 2011 [cited 2021 Nov 27];54:448-53. Available from: https://www.ijpmonline.org/text.asp?2011/54/3/448/85073

   Introduction Top

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a global incidence of approximately one million cases a year. [1] The epidemiology of HCC is characterized by marked demographic (age, gender, race/ethnicity) and geographic variations with 80% of cases occurring in those developing countries where viral hepatitis is more prevalent. [2] This tumor is notorious for poor prognosis, due to its invasiveness and frequent association with cirrhosis. Cirrhosis resulting in HCC is also associated with chronic viral hepatitis due to hepatitis B virus (HBV) and hepatitis C virus (HCV) infection besides alcoholism and Aflatoxin from Aspergillus moulds. [3],[4],[5] Surgery and transplantation remain the only curative option for HCC patients. [3] Complications due to underlying cirrhosis are confined to only 5-10% of presenting patients. Remaining 90% of patients, with no gold-standard therapy, are frequently treated with systemic therapies with palliative intent. Since, no drug treatment has, yet, clearly demonstrated a significant beneficial effect on survival or quality of life, there is an urgent need for novel approaches.

Immunotherapeutic approaches using a variety of strategies are in the developmental stage at present. HCC possesses several characteristics that make it an attractive target for immune therapy. [6],[7] These tumors are often infiltrated with lymphocytes and patients with high levels of tumor-infiltrating lymphocytes (TIL) were shown to have a better prognosis after resection. [8]

Recently, many studies have suggested that the tumor microenvironment plays an important role in the establishment and progression of tumors. Lymphocytes contribute to the tumor microenvironment through immunity and inflammation. However, in most patients, tumors continue to progress in spite of a tumor-specific humoral and cellular immune response. Several mechanisms may account for the T-cell dysfunction leading to the evasion of immune response by cancer cells. These include Fas-Fas ligand interaction leading to T-cell apoptosis and reactive oxygen species produced by myelomonocytic cells, [9] which have recently emerged as a potentially important immunosuppressive mechanism for T cells in tumor-bearing individuals. [10],[11]

Treg, characterized by coexpression of CD4 and CD25 (the IL-2-receptor a-chain), are a functionally unique population of T-cells which have been shown to play a critical role in immunologic self-tolerance as well as antitumor immune responses. [12],[13] These cells, generally considered to be the naturally occurring T-regulatory (Treg) cells, represent 5-10% of CD4+ T-cell population in humans. Besides CD25, Tregs constitutively express CTLA4 and GITR on their surface. Another important intracellular marker, forkhead or winged helix family of transcription factor P3 (Foxp3) is now shown to be critical and specific for the development and function of naturally occurring Treg in mice as well as humans. [14]

Although, several groups have reported increased number of CD4 + CD25 + Treg in the peripheral blood (PB) of cancer patients,[15],[16],[17] there are only a few studies that describe an increase in number of Treg among tumor-associated lymphocytes. [18],[19],[20] Little information is available on the number of these cells in the liver of patients with HCC. A few groups have reported an increased proportion of Treg in PB and TIL of patients with HCC, these were not large scale studies and did not estimate the clinicopathological significance of Treg infiltrating HCC. [5]

The principle objective of the present study was to evaluate the existence and expansion of Treg in tumor microenvironment in the HCC patients in comparison to chronic hepatitis (CH) cases using flowcytometry. To the best of our knowledge, ours is the first study to report the enumeration of CD4+CD25+ Tregs using fine needle aspirate cytology (FNAC) samples in HCC.

   Materials and Methods Top

Following an approval by the Institutional Ethics Committee, a baseline study involving 28 HCC patients, 15 CH patients and 15 healthy volunteers (HC) was carried out. Patient characteristics and clinical work up are summarized in [Table 1]. These patients were registered in hepatology clinic of the institute. The cases of HCC were diagnosed on the basis of radiological findings (Ultrasonography, Spiral CT, MRI and angiography) and/or raised a-feto protein (AFP) levels as per European Association for the Study of Liver (EASL) guidelines. Only those cases, in which no radiotherapy, chemotherapy, immunosuppressive therapy or other medical or surgical intervention was given, were included. Cases of multifactorial etiology (viral, drug-induced, metabolic disease, cryptogenic hepatitis) with no evidence of malignancy and no antiviral therapy were included as CH group. Healthy volunteers (HC) were chosen on the basis of normal liver function tests and without history of any recent infections.
Table 1: Patient characteristics and clinical work-up

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After an informed consent, EDTA blood samples were collected from all the patients and healthy volunteers. Ultrasound-guided FNAC of cancer tissue was done in 17 HCC patients in the Department of Radiodiagnosis and X-ray of institute using 5-11 cm long 22-23-gauge needle fitted with 20-ml syringe attached to metallic holder. Specimens of liver biopsy measuring 0.6-0.8 cm were collected from CH patients. FNAC material and the biopsies were divided in two parts. One part collected in PBS was used for flow cytometry and the other part was collected in RNA-holding solution (Sigma-Aldrich, MO, USA) for extraction of RNA and subsequent quantitation of gene expression studies. All the samples were transported under appropriate conditions to the Molecular Immunology laboratory for further analysis.

Peripheral blood mononuclear cells (PBMC) were obtained from venous blood of patients by Ficoll (Lymphoprep, Sigma-Aldrich) density gradient centrifugation. The lymphocyte layer was collected, washed and further stained for phenotyping of Treg. The FNAC sample collected in PBS was passed through a 23-gauge needle, four to five times to obtain a uniform single cell suspension, which was used for the flowcytometric analysis. Biopsies collected from CH patients were minced into 1-mm pieces and digested in a buffer containing 1 mg/ml collagenase (Sigma-Aldrich), 2.5 U/ml hyaluronidase and 0.1 mg/ml DNase (Sigma-Aldrich) at 37°C for 2 hours to obtain a single cell suspension.

The frequency of Treg cells were assessed as percentage of CD4+CD25+ cells among CD4+ cell population in the PB as well as tissue specimen (FNAC or liver biopsy) by immunofluorescene phenotyping using two-color flowcytometry. Cells were stained using anti-CD4-PE and anti-CD25-FITC (BD Biosciences, USA) antibodies. The samples were acquired using a FACScan flowcytometer (Becton Dickinson, USA) and the data analyzed using BD Cell Quest software.

RNA from FNAC specimen from HCC was isolated using Tri-reagent (Sigma-Aldrich) and from liver biopsy tissues of CH cases using RNeasy Micro kit (Qiagen Inc., USA) according to manufacturer's instructions. RNA was reverse transcribed to make cDNA copies by using RevertAid first strand cDNA synthesis kit (MBI Fermentas, Lithuania). The PCR for Foxp3, CTLA-4 and b-actin was done using primers shown in [Table 2]. Semiquantitative estimation of Foxp3 and CTLA-4 relative gene-expression was done by comparing the signal intensities of their PCR products in etihidium bromide-stained agarose gels normalized against that of a house-keeping gene b-actin. The signal intensity of PCR product was determined by densitometric scanning of gels in Image Master VDS gel documentation system using Total Image 1D gel analysis software (Pharmacia Biotech, Sweden).
Table 2: Primers and conditions utilized for semiquantitative RT-PCR

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All values were expressed as mean ± S.D. Statistical analyses were done with SPSS 10.0.1 software (SPSS Inc., Chicago, IL, USA). Associations among the variables were assessed by the Student's t-test, χ2 test, Fisher's exact test. The level of significance was set at P < 0 0.05.

   Results Top

We analyzed the PB from 28 patients with HCC. The gate for CD25+ cells was set using a control tube carrying cells which were stained with CD4-PE and FITC- IgG1k (BD Pharmingen, USA) isotype control for CD25-FITC [Figure 1]. Thus calculated, the frequency of CD4+ CD25+ T cells as a percentage of total CD4+ cells in PB of HCC patients (10.8 ± 7.64) was significantly higher than in the patients of CH (2.88 ± 1.92) and HC (3.05 ± 1.30). The differences were statistically significant between the groups (HCC versus CH P = 0.001, P < 0.005; HCC versus HC P = 0.001, P < 0.005 by unpaired Student's t test). The difference between CH and HC groups was not significant. Representative dot plot of HCC patient is shown in [Figure 2]a and cumulative data for all the patients and normal donors are presented in [Figure 2]b.
Figure 1: A composite fl owcytometry panel showing gating strategy for FITC-CD25: (a) Shows the gate seting using CD4 positive cells stained with CD4-PE and isotype control anti body FITC-IgG1k. (b) Shows the representative plot for CD4+CD25+ cells from peripheral blood of HCC patients. (c) Shows the representative plot for CD4+CD25+ cells from an FNAC sample of HCC patient. (d) Shows representative plot for the CD4+CD25+ cells from the biopsy sample of CH patient

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Figure 2: (a) Representative flowcytogram showing CD4+CD25+lymphocytes in peripheral blood of HCC patients. (b) Scatier plot showing percentage of CD4+CD25+ cells in the peripheral blood of HCC patients (n = 28) signifi cantly higher (P < 0.005) than in CH patients (n = 15) and healthy controls (n = 15)

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The FNAC samples from 17 HCC cases and liver biopsies from 15 CH cases were analyzed for Treg cells. The Flowcytometry data reveals increased frequency of CD4+ CD25+ cells in tumor microenvironment of HCC cases (15.8 ± 5.32) as compared to CH cases (5.51 ± 3.40). The difference was statistically significant (HCC versus CH P = 0.01, P < 0.05 by unpaired Student's t test). The representative dot plots for an HCC patient and a CH patient [Figure 3]a are shown. The cumulative data for all the HCC patients and CH analyzed are shown in [Figure 3]b.
Figure 3: (a) Representative flowcytometric graph of FNAC sample of a HCC patient and biopsy sample of a CH patient. (b) Scatier plot showing percentage of CD4+CD25+ cells in the tumor microenvironment of HCC patients was signifi cantly higher (P < 0.05) than in biopsies from CH patients

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The gene expression for Foxp3 and CTLA-4 in HCC and CH tissue samples was semiquantitatively estimated by reverse transcription of mRNA followed by PCR. The signal intensities of the PCR products were compared to those of b-actin gene from the same RNA samples [Figure 4]a and b. The relative expression of Foxp3 in HCC tumor microenvironment (473 ± 481) was found to be significantly higher than in liver tissue (38.1 ± 33.7) from CH cases. The difference was statistically significant (P = 0.02, P < 0.05). Higher level of Foxp3 expression in HCC strongly indicates the activated state of Treg in tumor microenvironment. The relative expression of CTLA-4 gene in tumor tissue of HCC was 263 ± 184, while no CTLA-4 transcript was detectable in liver tissue of CH patients as analyzed by RT-PCR. This indicates the increased number of Treg cells in tumor microenvironment of HCC patients.
Figure 4: Relative expression of Foxp3 and CTLA4 genes by RT-PCR with respect to actin gene from FNAC samples of HCC patients. (a) and (b) Ethidium bromide-stained gels showing RT-PCR amplifi ed products of FoxP3, CTLA4 and β-actin genes from representative samples. (c) Histogram showing relative gene expression values of FOXP3 and CTLA4 genes in terms of percentage of β-actin gene in HCC samples significantly higher (P < 0.05) than in liver biopsy samples of CH patients which shows no detectable expression of CTLA4

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   Discussion Top

The present study provides an evidence for increased frequency of CD4+ CD25+ Treg lymphocytes in the tumor microenvironment as well as PB of patients with HCC. There are only a few studies wherein the increased prevalence of Treg in PBMCs and TIL of cancer patients has been reported. [19],[21],[22] None of these studies have compared the prevalence of these cells among tissue infiltrating lymphocytes in tumor versus non-tumor tissue environment. We, in the present study, have used FNAC material of HCC patients and liver biopsy material of CH patients to compare the prevalence of CD4+ CD25+ Treg cells among the tissue infiltrating lymphocytes. We observed significantly increased population of CD4+CD25+ cells in PB as well as tumor tissue of HCC patients in comparison with PB and liver tissue of CH patients and PB of HC. Our data shows no significant difference in the number of Treg cells between HC and patients with CH. These results corroborate the data reported previously by others. [23],[24] These findings suggest that expanded population of Treg might be responsible for the suppression of intratumoral effector cell functions and helping the tumor to progress.

Although CD25 is a typical cell surface marker used to identify Treg cells, its specificity is not limited to Treg only but also to other types of activated T cells. Foxp3 represents a more specific marker than currently used cell-surface molecules (such as CD25, CD45RB, CTLA-4 and GITR), which are unable to completely discriminate between Treg cells and activated, effector or memory T cells. [25] We found Foxp3 expression to be significantly increased in tumor microenvironment of HCC in comparison to the tissue environment of CH. Similar results of increased Foxp3 expression have also been reported in TIL in a previous study. [26] Intratumoral Foxp3 expression has earlier been shown to be associated with invasion, size and vascularity in a study on breast carcinoma, indicating its utility as a marker for tumor progression and metastasis. [27] Some recent reports suggest that the expression of Foxp3 mRNA in humans is not limited to CD4+ CD25+ Treg compartment but also detected, although at a very low level, in cell lines and clones with no suppressor activity. [28] These studies suggest that the expression of this gene is possibly a consequence of activation status and the regulatory activity of the cells is exerted only beyond a given Foxp3 expression thresholds.

CTLA-4 has previously been implicated in the functioning of Treg cells. [25] In a previous study cell surface expression of CTLA 4 in cancer patients has been shown to be similar to healthy individuals.[26] However, a detectable expression of CTLA-4 in HCC tissue as compared to non-detectable in liver from CH might have been due to increased number of Treg in HCC rather than an enhanced expression of CTLA4.

So our study provides substantial evidence of increased population of Treg not only in PB but also the intratumoral environment of HCC cases. Studies have demonstrated the negative impact of Treg on the generation of tumor specific CTL as well as on effector mechanism mediated by innate immune response. [29] They showed that antibody-mediated depletion of CD4+ CD25+ cells abrogated immunological unresponsiveness against various syngenic tumors in mice. [30] It has been indicated in several animal models that the efficacy of therapeutic vaccination can be enhanced by depleting Treg and adoptive transfer of Treg impaired tumor specific immunity resulting in tumor progression. [30] In cancer, such strategies that inhibit or deplete Treg and boost antitumor immunity are under investigation. In mice, the removal of CD4+CD25+ T-cells with anti-CD25 depleting antibody, albeit in combination with anti CTLA-4 antibody led to tumor rejection. [31] It has been shown that combination of CTLA-4 blockade and elimination of CD4+CD25+ Treg cells can result in more effective antitumor immunity than in cases when these intervention strategies are applied separately indicating two alternative pathways for suppression of Treg cell activity. [32] Our findings of higher number of Treg cells in patients with HCC have implication both in the pathogenesis of tumor and in design of immunotherapy against it. The abundance of CD4+CD25+ cells among TIL observed in HCC patients is likely to promote a local immunosuppressive effect helping the tumor cells to evade an effector immune response. The elimination of CD4+ CD25+ T-cells may enhance tumor immunity when combined with the current attempts to augment immunogenicity of tumor cells.

   Conclusions Top

On the basis of results of this study we can conclude that there is an expansion of CD4 + CD25 + Treg cells in the PB as well as tumor microenvironment of HCC patients. This suggests that the poor immune responses to tumor antigens may be due to increased Treg cell activity which contributes to the immune dysfunction seen in cancer patients. So, it will not be illogical if the future cancer controlling strategies are directed toward Treg cells.

   Acknowledgment Top

The authors gratefully acknowledge the technical support from histopathology and Immunopathology departments and institute support of residency program to ST. The financial support for reagents and fellowship to AS provided by Department of Biotechnology (Govt. of India).

   References Top

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Correspondence Address:
Sunil K Arora
Department of Immunopathology, PGIMER, Chandigarh - 160 012
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.85073

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  [Table 1], [Table 2]

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13 Increased Frequency of Foxp3+ Regulatory T Cells in Mice with Hepatocellular Carcinoma
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[Pubmed] | [DOI]


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