Leukemia (2014) 28, 1872–1884 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE TGF-b upregulates CD70 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin’s lymphoma Z-Z Yang1, DM Grote1, B Xiu2, SC Ziesmer1, TL Price-Troska1, LS Hodge1, DM Yates1, AJ Novak1 and SM Ansell1 Transforming growth factor beta (TGF-b) has an important role in mediating T-cell suppression in B-cell non-Hodgkin lymphoma (NHL). However, the underlying mechanism responsible for TGF-b-mediated inhibition of effector memory T (Tm) cells is largely unknown. As reported here, we show that exhaustion is a major mechanism by which TGF-b inhibits Tm cells, and TGF-b mediated exhaustion is associated with upregulation of CD70. We found that TGF-b upregulates CD70 expression on effector Tm cells while it preferentially induces Foxp3 expression in naive T cells. CD70 induction by TGF-b is Smad3-dependent and involves IL-2/Stat5 þ signaling. CD70 T cells account for TGF-b-induced exhaustion of effector Tm cells. Both TGF-b-induced and preexisting intratumoral þ CD70 effector Tm cells from B-cell NHL have an exhausted phenotype and express higher levels of PD-1 and TIM-3 compared with CD70 À T cells. Signaling transduction, proliferation and cytokine production are profoundly decreased in these cells, and they are highly susceptible to apoptosis. Clinically, intratumoral CD70-expressing T cells are prevalent in follicular B-cell lymphoma (FL) biopsy specimens, and increased numbers of intratumoral CD70 þ T cells correlate with an inferior patient outcome. These findings confirm TGF-b-mediated effector Tm cell exhaustion as an important mechanism of immune suppression in B-cell NHL. Leukemia (2014) 28, 1872–1884; doi:10.1038/leu.2014.84 INTRODUCTION especially which subpopulation contributes to TGF-b-mediated T-cell exhaustion is a type of immune response describing the T-cell inhibition possibly by T-cell exhaustion, is unknown. condition in which T cells exhibit reduced differentiation, In the present study, we have identified TGF-b to be a key proliferation and effector function. T-cell exhaustion is initially regulator of CD70 expression on T cells. We then determined the recognized and characterized in chronic viral infections.1–7 In phenotypical and functional changes of TGF-b-induced or þ tumors, it has been observed that intratumoral T cells display a intratumoral preexisting CD70 T cells as well as the clinical phenotypic and functional profile similar to that of exhausted impact of CD70-expressing T cells on patient outcome in FL. The T cells from chronic viral infection.8–10 Phenotypically, PD-1 data we present in this study demonstrate the biological and expression has been demonstrated to be a marker to identify clinical significance of TGF-b-mediated CD70 induction and the 3,4 11,12 exhausted T cells in viral infection and tumors. Recently, we subsequent inhibition of Tm cell function. found that interleukin (IL)-12 induces T-cell exhaustion through upregulating TIM-3 in patients with follicular lymphoma (FL).13 Co-stimulatory molecule CD70 can be expressed on T cells MATERIALS AND METHODS upon T-cell receptor stimulation.14 CD70 expression causes a Patient samples 15 change in T-cell function, and high levels of CD70 have been Patients providing written informed consent were eligible for this study if shown to be involved in the pathophysiology of several they had a tissue biopsy that on pathological review showed follicular diseases.16–18 Over the recent decades, efforts to explore the B-cell NHL and adequate tissue or peripheral blood to perform the underlying mechanism of CD70 upregulation on T cells have experiments. Peripheral blood mononuclear cells from healthy donors and proved difficult.19 Studies have suggested that DNA methylation normal specimens from patients with follicular hyperplasia were used as of the CD70 promoter gene has an important role in CD70 controls. The use of human tissue samples for this study was approved by the Institutional Review Board of the Mayo Clinic/Mayo Foundation. upregulation on T cells in various autoimmune diseases.20,21 However, it is not known which cytokine can upregulate CD70 expression on T cells. Cell isolation and culture Cytokine transforming growth factor beta (TGF-b) exerts the Fresh tumor biopsy specimens from patients with FL and control lymph greatest impact on T cells by inhibiting their activation, prolifera- nodes (LNs) were gently minced over a wire mesh screen to obtain a cell tion, differentiation and survival.22,23 B cells, including malignant suspension. The cell suspension or peripheral blood from patients or healthy donors was centrifuged over Ficoll-Hypaque at 500 g for 15 min to B cells, are a source of inhibitory cytokines such as IL-10 and þ þ þ þ TGF-b, suggesting a role for TGF-b in B-cell NHL.24 An important isolate mononuclear cells. CD3 , CD4 , CD8 T cells and CD19 B cells question arises about which type of response is responsible for TGF- were isolated using positive selection with CD3, CD4, CD8 or CD19 microbeads (Miltenyi Biotec, Auburn, CA, USA). CD4 þ CD45RA þ or CD4 þ b-mediated suppression of effector memory T (Tm)cells.Several CD45RO þ T-cell subsets were purified by using EasySep Human Naive studies have implied that TGF-b may induce T-cell exhaustion that CD4 þ T Cell Enrichment Kit (StemCell Technologies, Vancouver, BC, leads to a declined T-cell proliferation and function as well as Canada). T cells were cultured in anti-CD3 antibody (Ab)-coated plates in enhanced cell death.25–27 However, the underlying mechanism, the presence of anti-CD28. All experiments have been done on anti-CD3 1Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN, USA and 2Department of Hematology, Tongji Hospital, Tongji University, Shanghai, China. Correspondence: Dr Z-Z Yang or Dr SM Ansell, Division of Hematology and Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. E-mail: [email protected] or [email protected] Received 30 October 2013; revised 17 January 2014; accepted 3 February 2014; accepted article preview online 26 February 2014; advance online publication, 21 March 2014 TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1873 Ab-activated T cells unless otherwise noted as resting cells. CD4 þ T cells expression of CD70 was dramatically enhanced on T cells were used in the majority of experiments. following treatment with TGF-b (Figure 1a). CD70 is normally absent on resting T cells and is induced upon Intracellular staining activation (Supplementary Figure S1A). With the addition of TGF-b, Cells were washed and subjected to fixation, permeabilization, stained with CD70 expression on activated T cells is significantly upregulated fluorochrome-conjugated antibodies and analyzed by flow cytometry. For (Figure 1b). TGF-b not only increased the number of T cells cytokine induction, we cultured CD4 þ T cells in anti-CD3-coated plates expressing CD70 but also enhanced the magnitude of CD70 and treated with or without stimuli for 3 days. Cells were restimulated with expression on T cells in a dose- and time-dependent manner phorbol myristate acetate (PMA)/Ion plus Brefeldin A for 4 h and analyzed (Figures 1b–e). by flow cytometry. For cytokine production by intratumoral T cells, we To determine whether CD70 upregulation was specific to TGF-b, stimulated cells with PMA/Ion plus Brefeldin A for 4 h and analyze by flow we tested a panel of cytokines for their capacity to upregulate cytometry. Foxp3 expression was determined using Foxp3 detection kit CD70 expression. Among the 12 tested, TGF-b remained the only (Biolegend, San Diego, CA, USA) following the manufacturer’s instructions. For Ki-67 expression, cells were incubated with 70% ethanol for 1 h, stained cytokine that strongly induced CD70 expression (Supplementary with Ki-67-APC Ab and analyzed by flow cytometry. Figure S1B). Although a previous study has reported that IL-2 upregulates CD70 expression on CD8 þ T cells in mice,15 we found that CD70 expression was not upregulated on human T cells Enzyme-linked immunosorbent assay treated with IL-2 alone, and the addition of IL-2 had no further Concentration of soluble CD27 in experimental supernatants was impact on TGF-b-mediated induction of CD70 on activated T cells measured using an Instant enzyme-linked immunosorbent assay kit from eBioscience (San Diego, CA, USA; no. BMS286INST). For each sample, 50 ml (Supplementary Figure S2). were done in duplicate and incubated for 3 h at room temperature on We next determine whether TGF-b was able to upregulate CD70 rotary shaker. After washing the micro wells, 100 ml TMB Substrate was expression on activated B cells or monocytes. As shown in added to each well and color was allowed to develop for 10 min. Reaction supplementary Figure S3, although activation through lipopoly- was stopped by adding 100 ml of Stop Solution, and the plate was read saccharide, but not CD40L, enhanced CD70 expression, TGF-b was immediately on a SpectraMax 190 plate reader (Sunnyvale, CA, USA) at unable to upregulate CD70 expression on either CD19 þ or 450 nm using the SoftMax Pro software program (Sunnyvale, CA, USA). CD11c þ cells. In fact, treatment with TGF-b was associated with a slight decrease in CD70 expression on both resting and activated Signal transducer and activator of transcription factor 5 (Stat5) CD19 þ or CD11c þ cells (Supplementary Figure S3). These phosphorylation assay results indicate that TGF-b-induced CD70 expression is specific The phosphorylation of Stat5 was detected following the manufacturer’s to T cells.