Increased frequencies of Th17 in the peripheral blood of patients with chronic lymphocytic leukemia: A one year follow-up

Objective : In this study, we aimed to investigate changes of peripheral Th17 and Treg cells frequencies in the newly-diagnosed Chronic Lymphocytic Leukemia (CLL) patients for 12 months. Methods : In this research, 50 CLL patients were enrolled. Circulating Th1, Th17 cells and CD4+CD25+Foxp3+Treg cells were analyzed by flow cytometry. Plasma levels of related cytokines were detected by enzyme-linked immuno sorbent assay (ELISA). The study was carried out from January 2012 to October 2013 at Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, P.R. China. Results: Compared with healthy controls, Th17 cells related cytokines were significantly increased in CLL patients, while Treg cells related cytokines were significantly lowered. In the follow-up, we found that the frequency of Treg cells was irregular, while the frequency of Th17 cells was gradually decreased. Conclusion: Our study suggested that Th17 cells may play important role in the immune regulation of CLL, and may become a new target in CLL therapy.


INTRODUCTION
Chronic Lymphocytic Leukemia (CLL) is a low-grade lymphoproliferative tumor, which is characterized by monoclonal B lymphocytes accumulation, apoptosis inhibition as well as infiltration of peripheral blood, bone marrow and lymph nodes. 1 A large number of evidences suggested that the CD4 + T cell-mediated autoimmune regulator imbalance may play a key role in the pathogenesis and development of CLL. 2,3 CD4 + T helper cells (Th17 cell) and CD4 + CD25 + Foxp3 + regulatory T cells (Treg cell) are two novel subsets of CD4 + T cell. Th17 cell is an important mediator of cancer, chronic inflammation and autoimmune diseases through secretion of proinflammation cytokines, such as IL-17A, IL-17F, IL-22, IL-21 and IFN-γ. 4,5 Treg cell plays a role in antiinflammatory and maintain autoimmune tolerance, which also shows negative immuno-regulatory function via cell-contact inhibition and secretion of inhibitory cytokines (such as IL-10, TGF-β). 6 Both of Th17 and Treg cells can be activated by autoimmune or inflammation-mediated immune response. 7 Lots of studies have shown that Th17/Treg imbalance played an important role in autoimmune diseases and tumorigenesis. 8,9 However, until now, there are no follow-up on cell level of CLL in China. Our primary results showed that the frequency of Th17 cells was elevated and the frequency of Treg cells was reduced in the peripheral blood of CLL patients. 10 In this study, we evaluated the cytokines secreted by Th17 and Treg cells in the peripheral blood of CLL patients. Furthermore, we followed up the frequencies of Treg and Th17 cells in patients with CLL (Rai stage III and IV, Rai stage: cytologic staging system for chronic lymphocytic leukemia, which divides it into low (0), intermediate (I and II), and high-risk stages (III and IV).

Patients:
From January 2012 to October 2013, Peripheral blood from 20 healthy individuals (16 males; 4 females; mean age 59.4±15.8 years) and 50 patients with CLL (38 males; 12 females; mean age 61.7±12.3 years) were obtained following approval by the Ethics Committee of the Chinese Human Genome and the Ethics Committee of West China Hospital, and informed consents were obtained from all participants. All peripheral blood samples from healthy donors (controls) and patients were anti-coagulated with heparin. Diagnostic criteria for CLL were based on WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, and CLL remission criteria were based on the National Cancer Institute-sponsored Working Group (NCI-WG) on Chronic Lymphocytic Leukemia (including PR and CR). Staging was performed according to the Rai classification for CLL. Patients' characteristics of all patients are summarized in Table-I. Twenty newly-diagnosed patients (Rai stage III-IV) were followed up at the time of 3 months (M3), 6 months (M6) and 12 months (M12) after the treatment with Chlorambucil (Gloxo SmithKline, UK), and 10 newly-diagnosed Rai stage I-II and 20 remission patients were excluded in the follow-up. Antibodies: Cell phenotype of T cells was defined by multicolor flow cytometry. All the antibodies, including peridinin chlorophyll protein (PerCP)conjugated CD3; fluorescein isothiocyanate (FITC)conjugated CD8; phycoerythrin (PE) -conjugated IL-17A; allophycocyanin (APC) -conjugated IFNγ; PerCP-CD3/FITC-CD4/PE-CD8 and the corresponding isotype control antibodies were from Becton Dickinson Biosciences (San Diego, USA). Human Treg Staining Kit (including Fixation / Permeabilization) was from eBioscience (San Diego, California, USA). Cells were stained according to the manufacturer's recommendations. Cell preparation: For analysis of Th17, 500 μl of whole blood sample was cultured in complete culture medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum) for 4 h, in the presence of phorbol myristate acetate (PMA, PMA is used to stimulate the intracellular cytokine production together with ionomycin, 50 ng/ml, Sigma, USA), ionomycin (1 μg/ml, Sigma, USA) and monensin (a polyether antibiotic, 1 μg/ml, BD, USA). The incubators were set at 37°C, 5% CO 2 . For analysis of Treg, 50 μl of whole blood sample was aliquoted into a tube for further staining. Surface and intracellular staining: For Th17 analysis, 70 μl of stimulated whole blood was

Enzyme-linked immunosorbent assay (ELISA):
For all patients and control subjects, a 3-mL fasting blood sample was drawn into a BD Vacutainer tube containing heparin, plasma was obtained after centrifugation and stored at −20°C for the measurement of the cytokines. Student t tests and further verified by the Wilcoxon tests based on ranks. When multiple groups are present, the analysis of variance models and the analysis of variance F tests were used to evaluate the overall differences among these groups. Statistical significance was defined as p <0.05. Data were analyzed using SPSS 16.0 software.

Change in Treg and Th17 cell numbers in Rai III and IV CLL patients at 12 month follow-up:
Twenty CLL patients at Rai stage III and IV were followed for one year, at which time 14 patients continued in the study. Five patients dropped out of the study to receive treatment in other hospitals, and one patient could no longer be contacted. We tested lymphocyte numbers, CD4+ and CD8+ T cells ratios (Fig.1), and Treg and Th17 cell frequencies (Fig.2)

DISCUSSION
CLL is uncommon in China, with an incidence of only 1/20-1/30. In recent years, an increasing number of patients have been diagnosed with CLL, most of whom were evaluated at middle or late stage upon first visit. Several studies have shown significant degradation of T lymphocyte function and ratios in patients with B-cell CLL, 11 but the underlying cellular and molecular mechanisms for these changes are still unclear.
In our primary study, we found that the number of CD4+ T cells in the peripheral blood of CLL patients was elevated, and that therapy reduced the aberrant ratio of these cells. 10 However, our one year follow-up of Rai stage III and IV CLL patients showed no significant change in the number of peripheral CD4+ and CD8+ T cells. One possible reason is that only a partial number of patients were in complete remission after one year treatment, or alternatively, that the notable reduction in peripheral lymphocytes may have altered CD4+ T cell proportions.
This study also showed that the concentrations of IL-6, IL-17 and IL-23 were all significantly higher in CLL patients, while concentrations of Th17 in patients with CLL  14 The disagreement between our results and other studies may be due to several reasons including statistical methods (absolute count vs. relative proportion), race, and unbalanced autoimmune function after short term treatment. We plan to continue our follow-up study utilizing a larger population and a longer treatment term. Reports of Th17 cells have also been contradictory. Jadidi-Niaragh et al. showed a lower frequency of Th17 cells in CLL patients, 15 but Jain et al. reported that the frequency of Th17 cells in peripheral blood and spleen cell suspensions was higher in patients with CLL. 16 In our study, there was no significant change in Th17 cells after three months treatment, but Th17 cell frequency gradually declined as patients continued to receive treatment. This result is in accordance with our primary result, and further suggested that the frequency of Th17 cells in patients with CLL was initially upregulated, followed by down regulation after treatment.