DNA AND CELL BIOLOGY Volume 33, Number 6, 2014 ª Mary Ann Liebert, Inc. Pp. 355–360 DOI: 10.1089/dna.2013.2332
Dysregulation of Circulating Follicular Helper T Cells in Nonsmall Cell Lung Cancer Weiwei Shi,1 Xiaoyan Li,2 Zhanshan Cha,3 Shengjie Sun,1 Lijie Wang,1 Shunchang Jiao,1 Bo Yang,1 Yan Shi,1 Zhikuan Wang,1 Zhiyong Wu,1 and Guanghai Dai1
Nonsmall cell lung cancer (NSCLC) is greatly affected by the dysregulation of the immune system. Circulating follicular helper T cells (Tfh) play critical roles in inducing B-cell activation and producing various cytokines. In the current study, we investigated levels of circulating Tfh in NSCLC. Circulating Tfh and it subtypes were determined by measuring CD3, CD4, CXCR5, CXCR3, and CCR6 in 62 NSCLC patients and 66 healthy controls using flow cytometry. Data presented that percentage of circulating Tfh in the peripheral CD4 + T cells was significantly increased in NSCLC (14.0%) than in controls (8.7%) ( p < 0.001). Further analysis revealed that the upregulation of Tfh was contributed by the Th2-Tfh subtype and the Th17-Tfh subtype, whereas the percentage of the Th1-Tfh subtype was significantly decreased in patients. Investigating the clinical stages of the patients demonstrated that prevalence of Tfh was significantly elevated in cases with advanced stages (III: 14.2%; IV: 16.4%) than those with primary stages (I: 10.9%; II: 10.8%). In addition, we analyzed Tfh in patients with different histological types. Results showed that the percentage of circulating Tfh was further upregulated in adenocarcima than squamous cell carcinoma or other types. This study suggests the involvement of circulating Tfh in the pathogenesis and progression of NSCLC, and provides a potential pathway for understanding this disease.
Introduction
L
ung cancer is the leading cause of cancer mortality in the world. Millions of people die from this disease each year (Lawrence and Salgia, 2010). Nonsmall cell lung cancer (NSCLC) is the most common type and accounts for up to 80% of all lung cancer cases. Despite the development of antitumor therapy, the prognosis for patients with NSCLC remains poor, with a 5-year survival rate of less than 20% (Lawrence and Salgia, 2010). Recent studies have provided evidence that dysregulation of the immune system plays important roles in the development of NSCLC (Dzionek et al., 2000; Li et al., 2012). Physiologically, CD4 + T cells provide great assistance in antibody responses. These cells are fundamental for the generation of germinal centers (GC), a discrete structure in secondary lymphoid organs to develop B-cell memory (Crotty, 2011). Follicular helper T cells (Tfh) are recently identified CD4 + T helper lineage specialized in the assistance of B cells during GC reactions in secondary lymphoid tissue ( Johnston et al., 2009; Ma et al., 2009). The Tfh cell is generated from activated T cells that express Bcl-6 (Ma
1 2 3
et al., 2009). However, the distinguishing features of these cells lie in the expression of CXCR5, PD1, ICOS, and CD40L. These receptors and ligands promote differentiation, proliferation, and class switching of B cells (Akiba et al., 2005; Rasheed et al., 2006; Bauquet et al., 2009; Pangault et al., 2010). Recent researches have reported that Tfh cells may have a deep impact on the pathogenesis of various cancers such as follicular lymphoma and peripheral T-cell lymphomas (Alizadeh et al., 2000; Marinova et al., 2006; Ito et al., 2008). However, studies of Tfh cells on NSCLC remain very limited. A huge limitation of Tfh research is that most of the cells are located in the GC zone of secondary lymph nodules, which are hard to be accessed (Strauss et al., 2008; de Jong and de Boer, 2009). Several studies have suggested that CD4 + CXCR5 + T cells in blood may act as a counterpart to measure Tfh cell disorders. CD4 + CXCR5 + T cells are known as circulating Tfh, which share functional properties with Tfh cells and appear to represent their circulating memory compartment (Yang et al., 2007). The upregulated percentages of CD4 + CXCR5 + cells in the peripheral blood of patients with autoimmunity and tumors have been
Department of Oncology, PLA General Hospital, Beijing, China. Department of Lung Cancer, Affiliated Hospital of Academy of Military Medical Sciences, PLA, Beijing, China. Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, China.
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reported (Lim and Kim, 2007; Chung et al., 2011; Linterman et al., 2011). However, little is known about the frequency of circulating Tfh cells in NSCLC patients. In the current study, we investigated the CD4 + CXCR5 + circulating Tfh cells and their subtypes in NSCLC patients and examined the potential association of these cells with different clinical characteristics. Materials and Methods Study subjects
The study group included 62 NSCLC cases recruited from the Affiliated Hospital of Academy of Military Medical Sciences and PLA General Hospital. The histological types of lung cancers were identified according to the World Health Organization classifications. The pathologic stage was determined according to the International System for Staging Lung Cancer. The control population was recruited from people who came for general health examinations. People who are relatives or have histories of malignancy and other major diseases (cancers, cardiovascular diseases, hypertension, diabetes, or on constant medications) were excluded from this study. All the control subjects were matched with patient populations in terms of age, sex, and residence area (urban or rural). To exclude the possible effects of ethnicity, only Han Chinese were included in this study. Information of smoking status was gathered from patients and controls. The definition of a nonsmoker in the study is less than 100 cigarettes per year or less than 2 cigarettes per week for a year. Informed consent was obtained from all study participants according to the Helsinki Declaration. This study was approved by the institutional review boards of the Affiliated Hospital of Academy of Military Medical Sciences (200812) and PLA General Hospital (200856). Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque centrifugation of heparinized blood and resuspended in the RPMI tissue culture medium containing 10% fetal calf serum (GIBCO, Grand Island, NY), streptomycin, and penicillin (RPMI-FCS). Surface and intracellular staining and flow cytometry
Antibodies used for staining include FITC-CXCR5, PerCP-CD4, PE/Cy5-CD3, PE-CCR6, APC-CXCR3, or isotype-matched control IgG (Beckton Dickinson, San Diego, CA). Human PBMCs were stained with various antibodies at room temperature for 30 min. Cells were washed twice with phosphate-buffered saline and were subjected to flow cytometry analysis using a FACS Calibur (Beckton Dickinson) and FlowJo software. The CD3 + CD4 + CXCR5 + cells were considered as circulating Tfh. The CD4 + CXCR5 + T cells in blood were subdivided into CXCR3 + Th1-like, CCR6 + Th17-like, and CXCR3 - CCR6 - Th2-like Tfh cells. Statistical analysis
All data were analyzed using SPSS 13.0 software. The Student’s t-test was used for comparison between groups. Value of p < 0.05 is considered as statistical significance.
SHI ET AL. Results Clinical characteristics of the study subjects
Demographic and other selected characteristics of the cases and controls are shown in Table 1. There was no significant difference in age ( p > 0.05) and gender ( p > 0.05) between NSCLC patients and healthy controls. Compared with controls, the NSCLC group presented a higher percentage of smoking status (72.6% vs. 54.5%, p < 0.05), an established risk factor for the disease. As for the histological types of cancer, 48.4% were adenocarcima, 38.7% were squamous cell carcinoma, and 12.9% were other cell types (Table 1). Circulating Tfh cells in NSCLC patients and controls
By flow cytometry, we quantified the CD4 + CXCR5 + T cells (circulating Tfh) in the PBMCs of 62 NSCLC cases and 66 controls. As shown in Figure 1A, an upregulated level of circulating Tfh in CD4 + T cells was detected in NSCLC patients than in controls (mean – SEM 14.0% – 0.6% vs. 8.7% – 0.3%, p < 0.001). We also investigated whether age and gender could affect the level of circulating Tfh. In the 66 controls studied, no difference was observed in the proportion of circulating Tfh between controls older or younger than 50 years of age (Fig. 1B). Similarly, the gender did not show an effect on the prevalence of circulating Tfh (Fig. 1C). We also compared the proportion of Tfh between age, gender, and smoking status in patients, and no significant differences were observed. Since smoking is an established risk factor for NSCLC, we analyzed the potential correlation between smoking and circulating Tfh,
Table 1. Characteristics of Nonsmall Cell Lung Cancer Patients and Controls Characteristics
NSCLC (n = 62) (%)
Controls (n = 66) (%)
Age (mean, range) 53.6 (36–78) 52.9 (25–79) £ 50 28 (45.2) 32 (48.5) > 50 34 (54.8) 34 (51.5) Gender Male 40 (60.6) 41 (62.1) Female 22 (39.4) 25 (37.9) Smoking status Nonsmokers 17 (27.4) 30 (45.5) Smokers 45 (72.6) 36 (54.5) Pack-years value < 30 16 (35.6) 20 (55.6) ‡ 30 29 (64.4) 16 (44.4) Clinical stage I 9 (14.5) II 11 (17.7) III 19 (30.6) IV 23 (37.2) Histological cell type Adenocarcima 30 (48.4) I/II/III/IV 3/5/8/8 Squamous cell 24 (38.7) carcinoma I/II/III/IV 4/4/9/13 Other 8 (12.9) I/II/III/IV 2/2/2/2 NSCLC, nonsmall cell lung cancer.
p-Value > 0.05 > 0.05 > 0.05 < 0.05 < 0.05
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FIG. 1. Proportion of circulating Tfh (CD4 + CXCR5 + T cells) in the CD4 + T cells of nonsmall cell lung cancer (NSCLC) patients and controls (A). Comparison of circulating Tfh in healthy donors with different ages (B), genders (C), and smoking status (D). Tfh, follicular helper T cell.
and no association was identified (Fig. 1D). To further study the correlation between smoking and Tfh, we analyzed the proportion of Tfh and pack-year status in controls, and no significant difference was observed, indicating that Tfh is not affected by smoking. Subsets of circulating Tfh in NSCLC patients and controls
Since circulating Tfh could be further divided into different subtypes such as Th1-Tfh (CXCR3 + CCR6 - ), Th2-
Tfh (CXCR3 - CCR6 - ), and Th17-Tfh (CXCR3 + CCR6 + ), we analyzed levels of these three subtypes in patients and controls (Fig. 2). Data showed that Th1-Tfh, Th2-Tfh, and Th17-Tfh in CD4 + T cells of healthy donors accounted for 3.0%, 2.5%, and 2.4%, respectively (Fig. 2A), whereas these cell subtypes in NSCLC patients accounted for 2.7%, 4.3%, and 5.6% (Fig. 2B). The proportion of Th1-Tfh was significantly decreased in patients than in controls (Fig. 2C). However, Th2-Tfh (Fig. 2D) and Th17-Tfh (Fig. 2E) were significantly elevated in NSCLC patients (1.75-fold high, p < 0.001 and 2.33-fold high, p < 0.001, respectively).
FIG. 2. Proportion of circulating Tfh subtypes, Th1Tfh, Th2-Tfh, and Th17-Tfh in the CD4 + T cells of healthy controls (A) and NSCLC patients (B). Comparison of Th1-Tfh (C), Th2Tfh (D), and Th17-Tfh (E) between controls and patients.
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suggest that circulating Tfh cells may be associated with the progression of NSCLC. Circulating Tfh cells in NSCLC cases with different histological subtypes
FIG. 3. Proportion of circulating Tfh in the CD4 + T cells of NSCLC patients with different tumor stages. p-Values are shown in the table.
NSCLC has several subtypes based on histological examination, in which adenocarcima and squamous cell carcinoma are the two major ones. We investigated the levels of circulating Tfh cells in these NSCLC subtypes. Data showed that the percentage of circulating Tfh cells was significantly increased in adenocarcima than in squamous cell carcinoma or other types (15.9% – 0.8% vs. 12.4% – 0.8% and 11.1% – 1.4%, respectively, Fig. 4A). However, the proportion of Tfh was not significantly different between squamous cell carcinoma and other types ( p = 0.432, Fig. 4A). Stage of the cancer may affect the level of circulating Tfh cells. To exclude this effect, we compared adenocarcima and squamous cell carcinoma with the same stages, and identified that adenocarcima had upregulated levels of circulating Tfh cells than squamous cell carcinoma in advanced stages (stage III and IV), whereas Tfh in stage I/II did not reveal any significant differences between adenocarcima and squamous cell carcinoma ( p = 0.535, Fig. 4B). Discussion
These data suggest a dysregulation of circulating Tfh in NSCLC and indicate the potential involvement of Tfh in this disease. Circulating Tfh cells in NSCLC cases with different stages
The staging system is a standardized way for the cancer care team to summarize information about how far a cancer has spread. Stage I indicates less severity, whereas stage IV indicates high progression of the disease. We analyzed the levels of circulating Tfh cells in patients with different stages (Fig. 3). Results revealed that the percentage of circulating Tfh cells remained similar between stage I (10.9% – 0.8%) and stage II patients (10.8% – 1.4%). However, patients with stage III and IV clearly presented elevated levels of circulating Tfh cells (14.2% – 1.1%, and 16.4% – 0.8%, respectively), in which NSCLC stage IV patients revealed the highest level of Tfh cells. These results
FIG. 4. Proportion of circulating Tfh in CD4 + T cells of NSCLC patients with different histological subtypes (A). Comparison of circulating Tfh between adenocarcima and squamous cell carcinoma with the same stages (B).
Circulating Tfh cell is a novel CD4 + T-cell subset. We investigated circulating Tfh cells in NSCLC and identified the upregulated percentage of these cells in the patients. We also observed a great unbalance of circulating Tfh subsets toward Th2 and Th17 cells in NSCLC. Furthermore, we found that the circulating Tfh cell was correlated with disease progression and was more involved in lung adenocarcima. Studies of human Tfh cells have exploited the fact that CD4 + CXCR5 + T cells comprise a small subset of circulating lymphocytes. However, there are clear differences between CD4 + CXCR5 + T cells in the blood and those in the tonsils. For instance, CD4 + CXCR5 + T cells in the blood do not express BCL-6 and their expression of ICOS and PD1 is substantially lower compared with TFH cells (Ame-Thomas et al., 2007). Despite this, in vitro-cultured blood-derived CD4 + CXCR5 + T cells produce more IL-21, IL-10, and CXCL13 - , which are all features of TFH cells,
CIRCULATING FOLLICULAR HELPER T CELLS IN NSCLC
and are more efficient at inducing B-cell differentiation than CD4 + CXCR5 - T cells. Thus, human circulating CD4 + CXCR5 + T cells have some features of Tfh cells, which indicate that precursors of circulating CD4 + CXCR5 + T cells might have experienced some aspects of a ‘‘Tfh cell differentiation programme’’ in vivo (Clear et al., 2010). Circulating Tfh cells comprise three major subsets: Th1, Th2, and Th17 cells. These subsets can be defined according to the expression of chemokine receptors, expression of transcription factors, and the type of cytokine secretion patterns. Th2 and Th17 cells within the CXCR5 + compartment efficiently induce naive B cells to produce immunoglobulins and to switch isotypes through IL-21 secretion. While CXCR5 + Th2 cells promote IgG and IgE secretion, CXCR5 + Th17 cells are efficient at promoting IgG and, in particular, IgA secretion. In contrast, CXCR5 + Th1 cells do not produce IL-21 and lack the capacity to help naive B cells (Yang et al., 2009). Our data demonstrated that Th2-Tfh and Th17-Tfh cells were significantly increased in NSCLC cases (Fig. 2B), indicating that IL-21-mediated immune responses may play a critical role in the development of NSCLC. Researches have suggested that circulating Tfh cells may ace as diagnostic and/or prognostic biomarkers in human autoimmune diseases and cancers. It has been reported that the proportion of circulating Tfh in CD4 + T cells is correlated with Binet stages in chronic lymphocytic leukemia (Cha et al., 2013). Similarly, our results observed an elevated level of circulating Tfh in NSCLC patients with advanced stages (Fig. 3). In addition, different histological subtypes of NSCLC may have different pathways of tumorigenesis. We identified a further increase of circulating Tfh in adenocarcima than in squamous cell carcinoma or other types (Fig. 4A), suggesting a potentially specific pathway between Tfh and the pathogenesis of lung adenocarcima. In conclusion, it is the first report investigating circulating Tfh cells in NSCLC. The study identified increased levels of these cells in the tumor, especially in lung adenocarcima. In addition, a great unbalance of circulating Tfh subsets toward Th2 and Th17 cells was found in NSCLC patients. These findings provide knowledge in understanding the pathogenesis of NSCLC and identifying potential targets for therapeutic action. Acknowledgments
We thank Dr. Zhanshan Cha and Dr. Hong Xiao for directing the experiments and writing the article. Disclosure Statement
No competing financial interests exist. References
Akiba, H., Takeda, K., Kojima, Y., Usui, Y., Harada, N., Yamazaki, T., and Okumura, K. (2005). The role of ICOS in the CXCR5 + follicular B helper T cell maintenance in vivo. J Immunol 175, 2340–2348. Alizadeh, A.A., Eisen, M.B., Davis, R.E., Ma, C., Lossos, I.S., Rosenwald, A., and Staudt, L.M. (2000). Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503–511.
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Ame-Thomas, P., Maby-El Hajjami, H., Monvoisin, C., Jean, R., Monnier, D., Caulet-Maugendre, S., and Tarte, K. (2007). Human mesenchymal stem cells isolated from bone marrow and lymphoid organs support tumor B-cell growth: role of stromal cells in follicular lymphoma pathogenesis. Blood 109, 693–702. Bauquet, A.T., Jin, H., Paterson, A.M., Mitsdoerffer, M., Ho, I.C., Sharpe, A.H., and Kuchroo, V.K. (2009). The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH17 cells. Nat Immunol 10, 167–175. Cha, Z., Zang, Y., Guo, H., Rechlic, J.R., Olasnova, L.M., Gu, H., and Qian, B. (2013). Association of peripheral CD4 + CXCR5 + T cells with chronic lymphocytic leukemia. Tumour Biol 34, 3579–3585. Chung, Y., Tanaka, S., Chu, F., Nurieva, R.I., Martinez, G.J., Rawal, S., and Dong, C. (2011). Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. Nat Med 17, 983–988. Clear, A.J., Lee, A.M., Calaminici, M., Ramsay, A.G., Morris, K.J., Hallam, S., and Gribben, J.G. (2010). Increased angiogenic sprouting in poor prognosis FL is associated with elevated numbers of CD163 + macrophages within the immediate sprouting microenvironment. Blood 115, 5053– 5056. Crotty, S. (2011). Follicular helper CD4 T cells (TFH). Annu Rev Immunol 29, 621–663. de Jong, D., and de Boer, J.P. (2009). Predicting transformation in follicular lymphoma. Leuk Lymphoma 50, 1406–1411. Dzionek, A., Fuchs, A., Schmidt, P., Cremer, S., Zysk, M., Miltenyi, S., and Schmitz, J. (2000). BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood. J Immunol 165, 6037–6046. Ito, T., Hanabuchi, S., Wang, Y.H., Park, W.R., Arima, K., Bover, L., and Liu, Y.J. (2008). Two functional subsets of FOXP3 + regulatory T cells in human thymus and periphery. Immunity 28, 870–880. Johnston, R.J., Poholek, A.C., DiToro, D., Yusuf, I., Eto, D., Barnett, B., and Crotty, S. (2009). Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. Science 325, 1006–1010. Lawrence, R.E., and Salgia, R. (2010). MET molecular mechanisms and therapies in lung cancer. Cell Adh Migr 4, 146– 152. Li, X., Shi, W., Yu, G., Lin, L., Yang, B., Li, J., and Liu, X. (2012). Interleukin-4-590T/C polymorphism influences the susceptibility to nonsmall cell lung cancer. DNA Cell Biol 31, 797–800. Lim, H.W., and Kim, C.H. (2007). Loss of IL-7 receptor alpha on CD4 + T cells defines terminally differentiated B cellhelping effector T cells in a B cell-rich lymphoid tissue. J Immunol 179, 7448–7456. Linterman, M.A., Pierson, W., Lee, S.K., Kallies, A., Kawamoto, S., Rayner, T.F., and Vinuesa, C.G. (2011). Foxp3 + follicular regulatory T cells control the germinal center response. Nat Med 17, 975–982. Ma, C.S., Suryani, S., Avery, D.T., Chan, A., Nanan, R., Santner-Nanan, B., and Tangye, S.G. (2009). Early commitment of naive human CD4( + ) T cells to the T follicular helper (T(FH)) cell lineage is induced by IL-12. Immunol Cell Biol 87, 590–600. Marinova, E., Han, S., and Zheng, B. (2006). Human germinal center T cells are unique Th cells with high propensity for apoptosis induction. Int Immunol 18, 1337–1345.
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Yang, Z.Z., Novak, A.J., Ziesmer, S.C., Witzig, T.E., and Ansell, S.M. (2009). Malignant B cells skew the balance of regulatory T cells and TH17 cells in B-cell non-Hodgkin’s lymphoma. Cancer Res 69, 5522–5530.
Address correspondence to: Guanghai Dai, MD Department of Oncology PLA General Hospital 28 Fuxing Road Haidian District Beijing 100853 China E-mail: [email protected] Received for publication December 30, 2013; received in revised form January 25, 2014; accepted January 30, 2014.
Dysregulation of Circulating Follicular Helper T Cells in Nonsmall Cell Lung Cancer Weiwei Shi,1 Xiaoyan Li,2 Zhanshan Cha,3 Shengjie Sun,1 Lijie Wang,1 Shunchang Jiao,1 Bo Yang,1 Yan Shi,1 Zhikuan Wang,1 Zhiyong Wu,1 and Guanghai Dai1
Nonsmall cell lung cancer (NSCLC) is greatly affected by the dysregulation of the immune system. Circulating follicular helper T cells (Tfh) play critical roles in inducing B-cell activation and producing various cytokines. In the current study, we investigated levels of circulating Tfh in NSCLC. Circulating Tfh and it subtypes were determined by measuring CD3, CD4, CXCR5, CXCR3, and CCR6 in 62 NSCLC patients and 66 healthy controls using flow cytometry. Data presented that percentage of circulating Tfh in the peripheral CD4 + T cells was significantly increased in NSCLC (14.0%) than in controls (8.7%) ( p < 0.001). Further analysis revealed that the upregulation of Tfh was contributed by the Th2-Tfh subtype and the Th17-Tfh subtype, whereas the percentage of the Th1-Tfh subtype was significantly decreased in patients. Investigating the clinical stages of the patients demonstrated that prevalence of Tfh was significantly elevated in cases with advanced stages (III: 14.2%; IV: 16.4%) than those with primary stages (I: 10.9%; II: 10.8%). In addition, we analyzed Tfh in patients with different histological types. Results showed that the percentage of circulating Tfh was further upregulated in adenocarcima than squamous cell carcinoma or other types. This study suggests the involvement of circulating Tfh in the pathogenesis and progression of NSCLC, and provides a potential pathway for understanding this disease.
Introduction
L
ung cancer is the leading cause of cancer mortality in the world. Millions of people die from this disease each year (Lawrence and Salgia, 2010). Nonsmall cell lung cancer (NSCLC) is the most common type and accounts for up to 80% of all lung cancer cases. Despite the development of antitumor therapy, the prognosis for patients with NSCLC remains poor, with a 5-year survival rate of less than 20% (Lawrence and Salgia, 2010). Recent studies have provided evidence that dysregulation of the immune system plays important roles in the development of NSCLC (Dzionek et al., 2000; Li et al., 2012). Physiologically, CD4 + T cells provide great assistance in antibody responses. These cells are fundamental for the generation of germinal centers (GC), a discrete structure in secondary lymphoid organs to develop B-cell memory (Crotty, 2011). Follicular helper T cells (Tfh) are recently identified CD4 + T helper lineage specialized in the assistance of B cells during GC reactions in secondary lymphoid tissue ( Johnston et al., 2009; Ma et al., 2009). The Tfh cell is generated from activated T cells that express Bcl-6 (Ma
1 2 3
et al., 2009). However, the distinguishing features of these cells lie in the expression of CXCR5, PD1, ICOS, and CD40L. These receptors and ligands promote differentiation, proliferation, and class switching of B cells (Akiba et al., 2005; Rasheed et al., 2006; Bauquet et al., 2009; Pangault et al., 2010). Recent researches have reported that Tfh cells may have a deep impact on the pathogenesis of various cancers such as follicular lymphoma and peripheral T-cell lymphomas (Alizadeh et al., 2000; Marinova et al., 2006; Ito et al., 2008). However, studies of Tfh cells on NSCLC remain very limited. A huge limitation of Tfh research is that most of the cells are located in the GC zone of secondary lymph nodules, which are hard to be accessed (Strauss et al., 2008; de Jong and de Boer, 2009). Several studies have suggested that CD4 + CXCR5 + T cells in blood may act as a counterpart to measure Tfh cell disorders. CD4 + CXCR5 + T cells are known as circulating Tfh, which share functional properties with Tfh cells and appear to represent their circulating memory compartment (Yang et al., 2007). The upregulated percentages of CD4 + CXCR5 + cells in the peripheral blood of patients with autoimmunity and tumors have been
Department of Oncology, PLA General Hospital, Beijing, China. Department of Lung Cancer, Affiliated Hospital of Academy of Military Medical Sciences, PLA, Beijing, China. Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, China.
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reported (Lim and Kim, 2007; Chung et al., 2011; Linterman et al., 2011). However, little is known about the frequency of circulating Tfh cells in NSCLC patients. In the current study, we investigated the CD4 + CXCR5 + circulating Tfh cells and their subtypes in NSCLC patients and examined the potential association of these cells with different clinical characteristics. Materials and Methods Study subjects
The study group included 62 NSCLC cases recruited from the Affiliated Hospital of Academy of Military Medical Sciences and PLA General Hospital. The histological types of lung cancers were identified according to the World Health Organization classifications. The pathologic stage was determined according to the International System for Staging Lung Cancer. The control population was recruited from people who came for general health examinations. People who are relatives or have histories of malignancy and other major diseases (cancers, cardiovascular diseases, hypertension, diabetes, or on constant medications) were excluded from this study. All the control subjects were matched with patient populations in terms of age, sex, and residence area (urban or rural). To exclude the possible effects of ethnicity, only Han Chinese were included in this study. Information of smoking status was gathered from patients and controls. The definition of a nonsmoker in the study is less than 100 cigarettes per year or less than 2 cigarettes per week for a year. Informed consent was obtained from all study participants according to the Helsinki Declaration. This study was approved by the institutional review boards of the Affiliated Hospital of Academy of Military Medical Sciences (200812) and PLA General Hospital (200856). Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque centrifugation of heparinized blood and resuspended in the RPMI tissue culture medium containing 10% fetal calf serum (GIBCO, Grand Island, NY), streptomycin, and penicillin (RPMI-FCS). Surface and intracellular staining and flow cytometry
Antibodies used for staining include FITC-CXCR5, PerCP-CD4, PE/Cy5-CD3, PE-CCR6, APC-CXCR3, or isotype-matched control IgG (Beckton Dickinson, San Diego, CA). Human PBMCs were stained with various antibodies at room temperature for 30 min. Cells were washed twice with phosphate-buffered saline and were subjected to flow cytometry analysis using a FACS Calibur (Beckton Dickinson) and FlowJo software. The CD3 + CD4 + CXCR5 + cells were considered as circulating Tfh. The CD4 + CXCR5 + T cells in blood were subdivided into CXCR3 + Th1-like, CCR6 + Th17-like, and CXCR3 - CCR6 - Th2-like Tfh cells. Statistical analysis
All data were analyzed using SPSS 13.0 software. The Student’s t-test was used for comparison between groups. Value of p < 0.05 is considered as statistical significance.
SHI ET AL. Results Clinical characteristics of the study subjects
Demographic and other selected characteristics of the cases and controls are shown in Table 1. There was no significant difference in age ( p > 0.05) and gender ( p > 0.05) between NSCLC patients and healthy controls. Compared with controls, the NSCLC group presented a higher percentage of smoking status (72.6% vs. 54.5%, p < 0.05), an established risk factor for the disease. As for the histological types of cancer, 48.4% were adenocarcima, 38.7% were squamous cell carcinoma, and 12.9% were other cell types (Table 1). Circulating Tfh cells in NSCLC patients and controls
By flow cytometry, we quantified the CD4 + CXCR5 + T cells (circulating Tfh) in the PBMCs of 62 NSCLC cases and 66 controls. As shown in Figure 1A, an upregulated level of circulating Tfh in CD4 + T cells was detected in NSCLC patients than in controls (mean – SEM 14.0% – 0.6% vs. 8.7% – 0.3%, p < 0.001). We also investigated whether age and gender could affect the level of circulating Tfh. In the 66 controls studied, no difference was observed in the proportion of circulating Tfh between controls older or younger than 50 years of age (Fig. 1B). Similarly, the gender did not show an effect on the prevalence of circulating Tfh (Fig. 1C). We also compared the proportion of Tfh between age, gender, and smoking status in patients, and no significant differences were observed. Since smoking is an established risk factor for NSCLC, we analyzed the potential correlation between smoking and circulating Tfh,
Table 1. Characteristics of Nonsmall Cell Lung Cancer Patients and Controls Characteristics
NSCLC (n = 62) (%)
Controls (n = 66) (%)
Age (mean, range) 53.6 (36–78) 52.9 (25–79) £ 50 28 (45.2) 32 (48.5) > 50 34 (54.8) 34 (51.5) Gender Male 40 (60.6) 41 (62.1) Female 22 (39.4) 25 (37.9) Smoking status Nonsmokers 17 (27.4) 30 (45.5) Smokers 45 (72.6) 36 (54.5) Pack-years value < 30 16 (35.6) 20 (55.6) ‡ 30 29 (64.4) 16 (44.4) Clinical stage I 9 (14.5) II 11 (17.7) III 19 (30.6) IV 23 (37.2) Histological cell type Adenocarcima 30 (48.4) I/II/III/IV 3/5/8/8 Squamous cell 24 (38.7) carcinoma I/II/III/IV 4/4/9/13 Other 8 (12.9) I/II/III/IV 2/2/2/2 NSCLC, nonsmall cell lung cancer.
p-Value > 0.05 > 0.05 > 0.05 < 0.05 < 0.05
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FIG. 1. Proportion of circulating Tfh (CD4 + CXCR5 + T cells) in the CD4 + T cells of nonsmall cell lung cancer (NSCLC) patients and controls (A). Comparison of circulating Tfh in healthy donors with different ages (B), genders (C), and smoking status (D). Tfh, follicular helper T cell.
and no association was identified (Fig. 1D). To further study the correlation between smoking and Tfh, we analyzed the proportion of Tfh and pack-year status in controls, and no significant difference was observed, indicating that Tfh is not affected by smoking. Subsets of circulating Tfh in NSCLC patients and controls
Since circulating Tfh could be further divided into different subtypes such as Th1-Tfh (CXCR3 + CCR6 - ), Th2-
Tfh (CXCR3 - CCR6 - ), and Th17-Tfh (CXCR3 + CCR6 + ), we analyzed levels of these three subtypes in patients and controls (Fig. 2). Data showed that Th1-Tfh, Th2-Tfh, and Th17-Tfh in CD4 + T cells of healthy donors accounted for 3.0%, 2.5%, and 2.4%, respectively (Fig. 2A), whereas these cell subtypes in NSCLC patients accounted for 2.7%, 4.3%, and 5.6% (Fig. 2B). The proportion of Th1-Tfh was significantly decreased in patients than in controls (Fig. 2C). However, Th2-Tfh (Fig. 2D) and Th17-Tfh (Fig. 2E) were significantly elevated in NSCLC patients (1.75-fold high, p < 0.001 and 2.33-fold high, p < 0.001, respectively).
FIG. 2. Proportion of circulating Tfh subtypes, Th1Tfh, Th2-Tfh, and Th17-Tfh in the CD4 + T cells of healthy controls (A) and NSCLC patients (B). Comparison of Th1-Tfh (C), Th2Tfh (D), and Th17-Tfh (E) between controls and patients.
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suggest that circulating Tfh cells may be associated with the progression of NSCLC. Circulating Tfh cells in NSCLC cases with different histological subtypes
FIG. 3. Proportion of circulating Tfh in the CD4 + T cells of NSCLC patients with different tumor stages. p-Values are shown in the table.
NSCLC has several subtypes based on histological examination, in which adenocarcima and squamous cell carcinoma are the two major ones. We investigated the levels of circulating Tfh cells in these NSCLC subtypes. Data showed that the percentage of circulating Tfh cells was significantly increased in adenocarcima than in squamous cell carcinoma or other types (15.9% – 0.8% vs. 12.4% – 0.8% and 11.1% – 1.4%, respectively, Fig. 4A). However, the proportion of Tfh was not significantly different between squamous cell carcinoma and other types ( p = 0.432, Fig. 4A). Stage of the cancer may affect the level of circulating Tfh cells. To exclude this effect, we compared adenocarcima and squamous cell carcinoma with the same stages, and identified that adenocarcima had upregulated levels of circulating Tfh cells than squamous cell carcinoma in advanced stages (stage III and IV), whereas Tfh in stage I/II did not reveal any significant differences between adenocarcima and squamous cell carcinoma ( p = 0.535, Fig. 4B). Discussion
These data suggest a dysregulation of circulating Tfh in NSCLC and indicate the potential involvement of Tfh in this disease. Circulating Tfh cells in NSCLC cases with different stages
The staging system is a standardized way for the cancer care team to summarize information about how far a cancer has spread. Stage I indicates less severity, whereas stage IV indicates high progression of the disease. We analyzed the levels of circulating Tfh cells in patients with different stages (Fig. 3). Results revealed that the percentage of circulating Tfh cells remained similar between stage I (10.9% – 0.8%) and stage II patients (10.8% – 1.4%). However, patients with stage III and IV clearly presented elevated levels of circulating Tfh cells (14.2% – 1.1%, and 16.4% – 0.8%, respectively), in which NSCLC stage IV patients revealed the highest level of Tfh cells. These results
FIG. 4. Proportion of circulating Tfh in CD4 + T cells of NSCLC patients with different histological subtypes (A). Comparison of circulating Tfh between adenocarcima and squamous cell carcinoma with the same stages (B).
Circulating Tfh cell is a novel CD4 + T-cell subset. We investigated circulating Tfh cells in NSCLC and identified the upregulated percentage of these cells in the patients. We also observed a great unbalance of circulating Tfh subsets toward Th2 and Th17 cells in NSCLC. Furthermore, we found that the circulating Tfh cell was correlated with disease progression and was more involved in lung adenocarcima. Studies of human Tfh cells have exploited the fact that CD4 + CXCR5 + T cells comprise a small subset of circulating lymphocytes. However, there are clear differences between CD4 + CXCR5 + T cells in the blood and those in the tonsils. For instance, CD4 + CXCR5 + T cells in the blood do not express BCL-6 and their expression of ICOS and PD1 is substantially lower compared with TFH cells (Ame-Thomas et al., 2007). Despite this, in vitro-cultured blood-derived CD4 + CXCR5 + T cells produce more IL-21, IL-10, and CXCL13 - , which are all features of TFH cells,
CIRCULATING FOLLICULAR HELPER T CELLS IN NSCLC
and are more efficient at inducing B-cell differentiation than CD4 + CXCR5 - T cells. Thus, human circulating CD4 + CXCR5 + T cells have some features of Tfh cells, which indicate that precursors of circulating CD4 + CXCR5 + T cells might have experienced some aspects of a ‘‘Tfh cell differentiation programme’’ in vivo (Clear et al., 2010). Circulating Tfh cells comprise three major subsets: Th1, Th2, and Th17 cells. These subsets can be defined according to the expression of chemokine receptors, expression of transcription factors, and the type of cytokine secretion patterns. Th2 and Th17 cells within the CXCR5 + compartment efficiently induce naive B cells to produce immunoglobulins and to switch isotypes through IL-21 secretion. While CXCR5 + Th2 cells promote IgG and IgE secretion, CXCR5 + Th17 cells are efficient at promoting IgG and, in particular, IgA secretion. In contrast, CXCR5 + Th1 cells do not produce IL-21 and lack the capacity to help naive B cells (Yang et al., 2009). Our data demonstrated that Th2-Tfh and Th17-Tfh cells were significantly increased in NSCLC cases (Fig. 2B), indicating that IL-21-mediated immune responses may play a critical role in the development of NSCLC. Researches have suggested that circulating Tfh cells may ace as diagnostic and/or prognostic biomarkers in human autoimmune diseases and cancers. It has been reported that the proportion of circulating Tfh in CD4 + T cells is correlated with Binet stages in chronic lymphocytic leukemia (Cha et al., 2013). Similarly, our results observed an elevated level of circulating Tfh in NSCLC patients with advanced stages (Fig. 3). In addition, different histological subtypes of NSCLC may have different pathways of tumorigenesis. We identified a further increase of circulating Tfh in adenocarcima than in squamous cell carcinoma or other types (Fig. 4A), suggesting a potentially specific pathway between Tfh and the pathogenesis of lung adenocarcima. In conclusion, it is the first report investigating circulating Tfh cells in NSCLC. The study identified increased levels of these cells in the tumor, especially in lung adenocarcima. In addition, a great unbalance of circulating Tfh subsets toward Th2 and Th17 cells was found in NSCLC patients. These findings provide knowledge in understanding the pathogenesis of NSCLC and identifying potential targets for therapeutic action. Acknowledgments
We thank Dr. Zhanshan Cha and Dr. Hong Xiao for directing the experiments and writing the article. Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Guanghai Dai, MD Department of Oncology PLA General Hospital 28 Fuxing Road Haidian District Beijing 100853 China E-mail: [email protected] Received for publication December 30, 2013; received in revised form January 25, 2014; accepted January 30, 2014.
