Reciprocal CD4 T-Cell Balance of Effector CD62L CD4 and CD62LCD25 CD4 Regulatory TCells in Small Cell Lung Cancer Reflects Disease Stage

Purpose: Small cell lung cancer (SCLC) possesses high tendency to disseminate. However, SCLC patients with paraneoplastic syndromemediated by immunity against onconeural antigens remain in limited-stage disease (LD) without distant metastases. Cumulative evidence regulates that a balance between immune and regulatory T (Treg) cells determines the magnitude of immune responses to not only self-antigens but also tumor-associated antigens.The purpose of this study was to elucidate the immunologic balance induced in SCLC patients. Experimental Design:We analyzed Tcells in the peripheral blood of 35 consecutive SCLC patients, 8 long-term survivors, and19 healthy volunteers. Results: Purified CD4 Tcells with down-regulated expressionof CD62L (CD62L) produced IFN-g, interleukin (IL)-4, and IL-17, thus considered to be immune effectorTcells (Teff). Significantly moreTeff cell numbers were detected in LD-SCLC patients than that of extended-stage SCLC (ED-SCLC). By contrast, induction of CD62LCD25 CD4 Treg cells was significantly higher in ED-SCLC patients. Long-term survivors of SCLCmaintained a highTeff toTreg cell ratio, whereas patients with recurrent disease exhibited a lowTeff toTreg cell ratio.Teff cells in LD-SCLC patients included more IL-17^ producing CD4 Tcells (Th17). Moreover, dendritic cells derived from CD14 cells of LD-SCLC patients secreted more IL-23. Conclusion:These results show that CD4 T-cell balancemay be a biomarker that distinguishes ED-SCLC from LD-SCLC and predicts recurrence. This study also suggests the importance of inducingTeff cells, particularlyTh17 cells, while eliminatingTreg cells to control systemic dissemination of SCLC. Small cell lung cancer (SCLC) is an aggressive disease with a strong tendency to disseminate. Approximately 15% to 20% of SCLC patients whose tumors are confined to the hemithorax and mediastinum and lack detectable distant metastases are considered to be limited-stage disease (LD). These LD-SCLC patients are often cured by treatment management. However, although no distant metastases are detected in LD-SCLC patients, regional treatments such as surgical resection or thoracic radiation therapy alone seldom result in a cure suggesting systemic micrometastases. Thus, repeated invasion by SCLC cells into the peripheral blood, some of which remain in the blood as circulating tumor cells, is considered to occur not only in extended-stage disease (ED) patients possessing prominent distant metastases but also in LD patients (1, 2). The reason why circulating tumor cells are unable to establish visible distant metastases in LD patients is unclear because no biological differences have been detected between tumor cells of LD-SCLC and ED-SCLC. SCLC is considered to be a relatively immunogenic tumor because it occasionally causes paraneoplastic syndromes such as the Lambert-Eaton myasthenic syndrome (LEMS) mediated by an immunologic mechanism that recognizes shared onconeural antigens. Interestingly, SCLC patients suffering from LEMS tend to remain in long-term LD state and have favorable prognoses (3). Thus, it is postulated that the immune response, which attacks the neuromuscular system in LEMS patients, also fights the SCLC cells to constrain tumor progression. In the immune system, regulatory CD4 T (Treg) cells with constitutive expression of the interleukin (IL)-2 receptor a chain (CD25) and the transcription factor forkhead box P3 (FOXP3) play a pivotal role in peripheral tolerance to self and Human Cancer Biology Authors’ Affiliations: Division of Respiratory Medicine, Department of Homeostatic Regulation and Development, Course for Biological Functions and Medical Control, Graduate School of Medical and Dental Sciences, Niigata University; Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, Niigata, Japan Received 5/2/08; revised 7/16/08; accepted 7/21/08. Grant support: Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture ofJapan; Niigata University Grant for Promotion of Project; and Niigata University Grant for Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: K. Koyama and H. Kagamu contributed equally to this work. Requests for reprints: Hiroshi Kagamu, Division of Respiratory Medicine, Department of Homeostatic Regulation and Development, Course for Biological Functions and Medical Control, Graduate School of Medical and Dental Sciences, NiigataUniversity,1-757Asahimachi-dori, Niigata 951-8520, Japan. Phone: 81-25227-2200; Fax: 81-25-227-0931; E-mail: [email protected]. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-1156 www.aacrjournals.org Clin Cancer Res 2008;14(21) November1, 2008 6770 Research. on March 31, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from non-self antigens, including tumor-associated antigens (4). It has been shown that Treg cell numbers increase in cancerbearing patients and that the number of Treg cells correlates with the prognosis (5–10). We have reported that in addition to tumor-specific Treg cells, antitumor effector T (Teff) cells were generated in the same tumor-draining lymph nodes during tumor progression (11). T cells with down-regulated CD62L expression (CD62L) that were isolated from the tumor-draining lymph nodes mediated antitumor reactivity when infused i.v., resulting in the regression of established tumors (12, 13). When coinfused, Treg cells purified as CD62LCD25 CD4 from the same tumor-draining lymph nodes could inhibit the antitumor therapeutic efficacy of Teff cells. Importantly, the suppression of antitumor reactivity depended on the ratio of Teff to Treg cells (11). Taken together, it seems that the balance between induced CD4 Teff and Treg cells determines the immune responses against tumors. However, the CD4 T-cell balance in human malignancies has not been elucidated. Here, we examined the peripheral blood mononuclear cells (PBMC) in 35 consecutive SCLC patients, 8 long-term survivors who had been disease-free for >3 years after treatment, and 19 healthy volunteers after obtaining a written informed consent. CD62L CD4 T cells isolated from the peripheral blood exhibited primed effector T-cell function to secrete types 1 (Th1), 2 (Th2), and 17 (Th17) helper T-cell cytokines. On the other hand, the CD62LCD25 CD4 T-cell subpopulation showed cytosolic expression of FOXP3 and regulatory function to suppress cytokine production and inhibit the proliferation of Teff cells. A reciprocal balance was detected between the CD62LCD25 CD4 Treg cells and the effector CD62L CD4 T cells. The former increased in ED-SCLC patients, and in contrast, the latter significantly increased without induction of Treg cells in LD-SCLC patients. Moreover, cytokine analyses revealed that the CD62L CD4 T cells purified from LDSCLC patients included more Th17 cells that produce preferentially IL-17 and that dendritic cells (DC) derived from CD14 cells of LD-SCLC patients produced more IL-23. Materials andMethods Patients. The present study comprised 35 consecutive SCLC patients, 8 long-term survivors, and 19 healthy volunteers from a single institution (Niigata University Medical and Dental Hospital, Niigata, Japan; Table 1). LD-SCLC patients who had been disease-free for >3 y after treatment were considered to be long-term survivors. Specimens were collected after obtaining written informed consent approved by the Niigata University Ethical Committee. Cell purification. PBMCs were obtained by centrifugation over Ficoll-Hypaque gradients. CD4 and CD8 T cells were purified by positive selection using CD4 or CD8 T-cell isolation kits (Dynal Table 1. Patient characteristics