Characterization of γδ regulatory T cells from peripheral blood in patients with multiple myeloma

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Abstract

γδ regulatory T cells are able to inhibit the activation and function of T cells involved in antigen-specific immune responses. This study aimed to investigate the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in patients diagnosed as multiple myeloma (MM). We measured the levels of γδ T cells, the distribution and clonally amplified TCR Vγ and VδT cells in peripheral blood of healthy donors, patients recently diagnosed with MM, and MM patients in remission cohorts. In addition, we evaluated the ability of γδ regulatory T cells to inhibit the proliferation of CD4+CD25- T cells and detected the expression of immunoregulatory-associated molecules. We found that the levels of γδ regulatory T cells from the peripheral blood in patients of MM were significantly higher than those in healthy donors. Comparison of γδT regulatory cells function in MM and healthy donors showed similarly inhibitory effects on the proliferation of T cells. Additionally, TLR8 expression level increased significantly in MM patients compared to healthy donors, while the expression levels of Foxp3, CD25, CTLA4, GITR, GATA3 and Tbet in MM patients and healthy donors showed no significant difference. Taken together, our study reveals the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in MM patients.

Introduction

Multiple myeloma (MM) is one kind of malignant tumor of plasma cell disorder [1], [2]. MM patients are often complicated with anemia, higher M-protein, bone lesions and organ injury [3]. γδT cells can promote immune response against myeloma through their direct cytotoxic activity and through stimulating or regulating biological functions of other types of cells such as cytotoxic CD8+ T cells [4], [5]. However, recent studies suggest that tumor infiltrating γδ T cells may be a good prognostic factor of cancer because the presence of these cells in tumor microenvironment was correlated with poor prognosis for breast and colon cancer patients [6], [7]. The expansion of regulatory γδT cells (γδTreg cells) in patients with nasopharyngeal, renal and gastric cancer has been confirmed [8]. However, the number and the potential role of γδTreg cells in supressing anti-tumor immune responses in MM patients remain largely unclear.

In this study, we measured the levels of γδTreg cells in recently diagnosed MM patients, relapsed MM patients and healthy control volunteers. Additionally, we tried to determined the relationship between clinical features of MM and γδTreg cells. Furthermore, we detected the expression of immunoregulatorory-associated molecules in peripheral blood from patients with MM and healthy donors.

Section snippets

Subjects

Total 10 normal volunteers (Table 1), 11 remission (CR, VGPR and PR) patients with myeloma (Table 2) and 7 new-onset or relapse patients with myeloma (Table 3) were enrolled and their peripheral blood cells were applied for the assay in vitro and the isolation of γδT cells. Obtained written informed consent was necessary from each subject before being enrolled in our study, and approved protocol by Ethics Committee of Nanfang Medical University was indicated.

Isolation and culture of γδT cells

γδT cells were purified from PBMCs

Quantitative RT-PCR

Total RNA was isolated using RNA isolation kit following the manufacturer's instructions (Qiagen, Germantown, MD, USA). RNA was reverse transcribed using the SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). cDNA was analyzed using a quantitative PCR (qPCR) with aCFX Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The sequences of primers for immunoregulatory-associated molecules were shown in Table 5.The relative RNA levels were measured by normalizing with β2

The γδT cells numbers and percentages of lymphocytes among healthy control, remission myeloma patients and new onset/relapse myeloma patients

The percentages of γδ T cells among lymphocytes was 3.09± 0.11% (n = 10) and the numbers of γδ T cells were 43.1 ± 2.86 per 1 μl of PB in healthy controls. The percentages of γδ T cells among lymphocytes was 2.66± 0.15% (n = 11) and numbers of γδ T cells were 36.30 ± 2.34 per 1 μl of PB in remission myeloma patients, significantly reduced compared to healthy controls (P < 0.05). The percentages of γδ T cells among lymphocytes were 1.58± 0.12% (n = 7) of lymphocytes and numbers ofγδ T cells were

Discussion

Human γδT cells are immune cells that harbor specialized T-cell receptors (TCRs) which can recognize nonpeptidic phosphorylated metabolites of isoprenoid biosynthesis produced by stressed cells and microorganisms. γδT cells represent only a fraction of T cells found in peripheral blood and play important role in immuno response to infections and tumors [4], [5].

γδ T cells promote immune response against various tumors such as myeloma, colon, breast, lymphoma, and prostate cancer directly by

Conflict of interest statement

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript entitled “Characterization of γδ regulatory T cells from peripheral blood in patients with multiple myeloma”.

Acknowledgement

This study was supported by grants from Medical Science and Technology Project of Zhejiang Province (No. 2014KYA132); Wenzhou City Science and Technology Projects (No. Y20140072); Natural Science Foundation of Zhejiang Province (No. Q16H080007)and Grant of National Natural Science Foundation (No.81600167).

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