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

Z-Z Yang1, DM Grote1, B Xiu2, SC Ziesmer1, TL Price-Troska1, LS Hodge1, DM Yates1, AJ Novak1 and SM Ansell1

Transforming 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 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 (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 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 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 (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 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 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. instructions (BD Biosciences, San Jose, CA, USA). Briefly, fresh isolated It is well known that TGF-b induces the expression of Foxp3, a mononuclear cells from biopsy specimens of patients with B-cell NHL were transcriptional factor critically important for regulatory T cells. incubated with IL-2 (50 ng/ml) for 30 min, then fixed and permeabilized Thus, we examined the induction of Foxp3 and CD70 in activated using a phosflow kit (BD Biosciences). Cells were stained with anti-pStat5- CD4 þ T cells by TGF-b. As shown in Figure 1f, treatment with TGF- Alexa647 Ab plus anti-CD3-FITC and CD70-PE antibodies for 30 min and þ analyzed by flow cytometry. b upregulated expression of both Foxp3 and CD70 on CD4 T cells. The numbers of CD70 þ Foxp3 À , CD70 þ Foxp3 þ or CD70 À Foxp3 þ T cells were significantly increased in response to TGF-b. Small interfering RNA (siRNA) transfection Transfection of CD4 þ T cells with siRNA was performed according to the manufacturer’s instruction (Qiagen, Cambridge, MA, USA). Genesolution TGF-b preferentially upregulates CD70 expression on Tm cells siRNA and Hiperfect Transfection Reagent were purchased from Qiagen. We then measured CD70 induction in activated CD4 þ CD45RA þ þ þ In all, 100 nM siRNA for CD70 or Stat5 or a scrambled siRNA was transfected naive (Tn) and CD4 CD45RO Tm cells. TGF-b was able to in a 24-well tissue culture plate for 24 h. After washing, cells were cultured upregulate CD70 expression on both Tn and Tm cells. However, a in OKT3-coated plate in the presence or absence of TGF-b 50 ng/ml for 3 more substantial induction of CD70 was predominantly observed days, and CD70 expression was measured by flow cytometry. in Tm cells as compared with Tn cells (Figure 2a). When we extended the culture time with TGF-b, CD70 induction was Statistical analysis diminished and lasted for only a short time in Tn cells. However, Statistical analysis was performed using the Student’s t-test. Significance CD70 induction was much greater and maintained for a relatively was determined at Po0.05. Overall survival was measured from the date of longer period of time in Tm cells (Figure 2b). Supporting this diagnosis until death from any cause. Patients alive and still at risk of death þ À finding, we noted that CD4 CD62L effector Tm cells accounted at last follow-up evaluation were censored for the analysis of overall for most of the observed TGF-b-mediated CD70 induction (Figures survival. Survival of all patients was estimated using the Kaplan-Meier method. The univariate association between CD70 expression and survival 2c and d). This result indicated that CD70 induction by TGF-b is was determined with the log- test. preferential in Tm cells. Next, we examined the expression pattern of Foxp3 and CD70 induced by TGF-b in purified activated Tn and Tm cells. Although RESULTS TGF-b was able to induce the expression of Foxp3 or CD70 in both TGF-b induces CD70 expression on T cells Tn and Tm cells, we observed a preferential induction pattern We have recently shown that TGF-b has a critical role in mediating between Foxp3 and CD70 in Tn and Tm cells (Figures 2e and f; b T-cell differentiation in the tumor microenvironment of B-cell Supplementary Figure S4). Namely, in Tn cells, TGF- had a greater 24,28 effect on Foxp3 induction than CD70. In contrast, TGF-b-mediated NHL. To further evaluate the role of TGF-b on immune responses, we determined the effect of TGF-b on the expression of CD70 upregulation was more profound than that of Foxp3 in Tm b various cell surface markers, cytokines, transcription factors and cells (Figure 2g). These results suggest that TGF- differentially signaling molecules in intratumoral T cells derived from patients exerts its function on Tn and Tm cells. with B-cell NHL using flow cytometry (Figure 1a). As expected, TGF-b inhibited the expression of most of the cell surface and IL-2/Stat5 signaling is involved in TGF-b-mediated CD70 induction signaling molecules tested. However, TGF-b increased expression in T cells of three molecules, one of which was Foxp3, which is well known As Smad3 activation is a key component of the TGF-b signaling to be upregulated by TGF-b. Consistent with previous reports, pathway, we first determined the role of Smad3 in TGF-b- TGF-b also increased IL-2 expression.29,30 Surprisingly, the mediated CD70 induction using a specific Smad3 inhibitor SIS3.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1872 – 1884 TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1874

Figure 1. TGF-b induces CD70 expression on T cells. (a) Expression of a variety of molecules in activated intratumoral T cells from lymphoma patients treated with or without TGF-b. The expression levels were expressed as fold change and converted to logarithmic number. *Po0.05, compared with untreated cells. (b) CD70 expression on peripheral blood CD4 þ T cells from healthy donors treated with or without TGF-b detected by flow cytometry (upper panel) or cytofluorescence (lower panel). Isotype control for CD70 was included. (c) A summary of CD70 induction on CD4 þ T cells treated without (NIL) or with TGF-b. CD70 expression on CD4 þ T cells was measured by flow cytometry and calculated as the percentage or mean fluorescence intensity (MFI). Each line represents an individual sample of peripheral blood from healthy donors (n ¼ 15). (d, e) CD70 expression on CD4 þ T cells from healthy donors treated with TGF-b at escalating doses (d)(n ¼ 3) or at different time points (e), (n ¼ 3). (f) CD70 and Foxp3 co-expression on CD4 þ T cells from peripheral blood of healthy donors treated with or without TGF-b. Intracellular staining was performed using a Foxp3 staining kit (Biolegend). The numbers of CD70 þ Foxp3 À , CD70 þ Foxp3 þ or CD70 À Foxp3 þ T cells induced by TGF-b were summarized graphically (right, n ¼ 5).

Activated CD4 þ T cells treated with SIS3 alone showed no effect CD70 induction, we observed that IL-2 depletion by an anti-IL-2 on CD70 expression (Figure 3a). However, CD70 induction by Ab led to complete inhibition of CD70 induction on T cells TGF-b was completely blocked when T cells were treated with activated with T-cell receptor stimulation (Figure 3b). This 5 mg/ml SIS3. This result suggests that TGF-b-induced CD70 depletion also attenuated TGF-b-mediated CD70 induction expression is Smad3 dependent. on T cells although CD70 upregulation was not completely Given that TGF-b induces IL-2 production, we next tested blocked (Figure 3b). We observed that this attenuation whether IL-2 signaling is involved in TGF-b-mediated CD70 was more pronounced in Tn than in Tm cells and affected induction. Although addition of exogenous IL-2 had no effect on both CD70 and Foxp3 upregulation by TGF-b (Figure 3c,

Leukemia (2014) 1872 – 1884 & 2014 Macmillan Publishers Limited TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1875

Figure 2. TGF-b preferentially upregulates CD70 expression on Tem cells. (a) Co-expression of CD70 and CD25 on day 3 on naive or Tm cells þ þ from peripheral blood of healthy donors treated with or without TGF-b (n ¼ 5). Naive and Tm cells were isolated using CD4 CD45RA naive þ cell isolation kit (Miltenyi Biotec). (b) CD70 induction on CD4 naive or Tm cells from peripheral blood of healthy donors treated with or without TGF-b on different days (n ¼ 3). CD70 induction was calculated by percentage of CD70 þ T cells treated with TGF-b.(c) CD70 expression on CD4 þ CD62L þ or CD62L À T cells from peripheral blood of healthy donors treated with or without TGF-b (n ¼ 5). þ (d) Co-expression of CD70 and CD62L on CD4 Tm cells from peripheral blood of healthy donors treated with or without TGF-b in the þ presence or absence of anti-IL-2 Ab (n ¼ 5). (e, f) Dot plots (e) or graphs (f) summarizing Foxp3 and CD70 expression in CD4 naive or Tm cells from peripheral blood of healthy donors treated with or without TGF-b (n ¼ 6). (g) A graph showing fold change of Foxp3 or CD70 expression þ in CD4 naive or Tm cells from peripheral blood of healthy donors (n ¼ 6). The fold change was expressed as TGF-b-treated vs untreated cells.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1872 – 1884 TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1876 Supplementary Figure S4). These results suggest that IL-2 Although activation is required, TGF-b-induced CD70 expression signaling is essential for activation-induced CD70 expression may occur through a different pathway. Although the vast and also has a role in TGF-b-mediated CD70 induction. majority of CD70-expressing cells are CD25 þ or Ki-67 þ activated

Leukemia (2014) 1872 – 1884 & 2014 Macmillan Publishers Limited TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1877 T cells, only a subset of CD25 þ or Ki-67 þ T cells were able to CD70 þ T cells account for TGF-b-mediated effects and are respond to TGF-b and upregulate CD70 expression (Figure 3d). functionally exhausted However, the expression of CD25 or Ki-67 was inhibited in CD4 þ We found that CD4 þ T cells treated with TGF-b displayed T cells treated with TGF-b (Figures 3d and 4a). The association decreased expression of the activation markers CD25 and Ki-67 as between increased CD70 expression and downregulated CD25 or well as reduced proliferation as indicated by decreased numbers Ki-67 suggests that TGF-b-mediated CD70 enhancement is not of CFSEdim cells (Figure 5a). TGF-b also inhibited (IFN)-g simply due to T-cell activation. production but enhanced IL-2 expression by CD4 þ T cells It has been shown that Stat5 activation has a crucial role in TGF- (Figure 5b). Given that TGF-b upregulates CD70 expression, we b-mediated induction of Foxp3 in T cells.31 Given the similar effect tested whether CD70 induction has a role in mediating the effects of TGF-b on CD70 expression, we wondered whether Stat5 was of TGF-b on T cells. As shown in Figure 5c, CD4 þ CD70 þ T cells also involved in CD70 induction by TGF-b. To test this, we were the major population responsible for IFN-g production, and transfected Stat5 siRNA into CD4 þ T cells and monitored CD70 TGF-b treatment significantly reduced IFN-g expression by CD70 þ expression. We observed that TGF-b-mediated CD70 induction T cells. Although both CD70 þ and CD70 À T cells were able to was attenuated when activated T cells were transfected with Stat5 produce IL-2, IL-2 production was more profoundly enhanced in siRNA (Figure 3e). To further confirm the role of IL-2/Stat5 TGF-b-induced CD70 þ cells than in CD70 À T cells. These results signaling in TGF-b-mediated CD70 induction, we treated T cells suggest that CD70 induction accounts for TGF-b-mediated effects with the calcineurin inhibitor cyclosporin A (CsA) and mammalian on CD4 þ T cells. target of rapamycin inhibitor Rapamycin (Rapa), which block To further functionally characterize CD70 þ T cells, we measured nuclear factor of activated T-cell activation and suppress IL-2/Stat5 cytokine production as well as Stat phosphorylation in CD70 þ signaling. As shown in Figures 3f and g, both CsA and Rapa were T cells isolated from biopsy specimens of lymphoma patients. able to inhibit activation-induced CD70 expression on activated As shown in Figure 5d, intratumoral CD70 À T cells were T cells in a dose-dependent manner. Furthermore, in the presence predominantly the source of cytokine production, whereas of either CsA or Rapa, TGF-b mediated induction of CD70 on CD70 þ T cells failed to produce cytokines when stimulated with activated T cells was no longer observed. PMA/Ion. Next, we determined whether signal transduction Supporting the above findings, we also observed that IL-2/Stat5 was also inhibited in CD70 þ T cells. Intratumoral T cells were signaling was positively associated with CD70 expression in two T-cell treated with or without IL-2, and Stat5 phosphorylation was lines, Jurkat and Karpas 299 (Supplementary Table S1). Karpas 299 measured in CD70 À or CD70 þ T cells. We found that a cells, which constitutively express CD70 on the cell surface, respond to subpopulation of intratumoral T cells were able to respond to IL-2 with subsequent activation of Stat5. In contrast, Jurkat cells do not IL-2 with subsequent activation of Stat5. However, CD70 À T cells express surface CD70 nor respond to IL-2. Similarly, IL-2 receptor a is accounted for this response, whereas CD70 þ T cells completely expressed on Karpas 299 cells and is absent on Jurkat cells. Taken lacked the capacity (Figure 5e). together, these data indicate that IL-2/Stat5 signaling has a role in TGF-b-mediated CD70 expression on T cells. Exhausted CD70 þ T cells are highly susceptible to apoptosis TGF-b is known to induce apoptosis in a variety of cell types. We þ CD70 T cells express exhaustion markers PD-1 and TIM-3 observed that treatment with TGF-b induces apoptosis of B cells To phenotypically characterize CD70 þ T cells, we measured the and results in a significant decrease in cell number of both resting expression of an array of surface markers on resting CD70 þ T cells and lipopolysaccharide- or CD40L-activated B cells during short- isolated from specimens of FL patients (Figure 4a). Intratumoral term culture (3 days) (Supplementary Figure S5A). However, we CD70 þ T cells expressed high levels of CD45RO, indicating that failed to see a decrease in cell number of either resting or þ the cells were Tm cells. Although the majority of CD70 T cells are activated T cells during the same period of culture (Supplementary CD27 þ and CD28 þ , the loss of CD27 and CD28 was substantial. Figure S5B). When we measured apoptosis, TGF-b treatment had Most CD8 þ CD70 þ and some CD4 þ CD70 þ T cells expressed PD-1 no effect on CD4 þ T-cell apoptosis for the first several days and TIM-3. Therefore we compared the expression of PD-1 and (Supplementary Figure S5C), which is consistent with previous TIM-3 between CD4 þ CD70 À and CD4 þ CD70 þ T cells. We found studies. However, upon extended culture, TGF-b eventually led to that both TGF-b-induced and preexisting CD70 þ T cells express a decreased viability of T cells (Figure 6a). This late-onset effect significantly higher levels of PD-1 and TIM-3 compared with suggests that other earlier changes in T cells by TGF-b are CD70 À T cells (Figure 4b), Supporting this finding, TGF-b necessary for the cells to become susceptible to apoptotic treatment upregulated PD-1 expression on T cells while no similar induction. Therefore we tested whether CD70 induction effects can be seen by other cytokines (Figure 4c). TGF-b-mediated contributes to the late-onset TGF-b-induced apoptosis in CD4 þ PD-1 upregulation was mainly seen on CD70 þ T cells (Figure 4d). T cells. Indeed, we found that CD70 þ T cells displayed a Although TGF-b treatment caused downregulation of TIM-3 significantly higher rate of apoptosis than CD70 À T cells. (Figure 4c), CD70 À T cells accounted for the majority of this Treatment with TGF-b did not induce apoptosis in activated downregulation (Figure 4d). The majority of CD70 þ T cells CD70 À T cells but increased apoptosis in activated CD70 þ T cells maintained TIM-3 expression on the cell surface. These results (Figure 6b). This finding was further supported by the observation implied that CD70 þ T cells are exhausted cells. that the increased number of CD70 þ T cells, induced by

Figure 3. IL-2/Stat5 signaling is involved in TGF-b-mediated CD70 induction on T cells. (a) CD70 expression on CD4 þ T cells from peripheral blood of healthy donors treated with or without SIS3 at 1 (SIS3 (1)) or 5 (SIS3 (5)) mg/ml in the presence or absence of TGF-b. The induction of CD70 on T cells induced by TGF-b is summarized in graphic form (right, n ¼ 3). (b) CD70 expression on CD4 þ T cells from peripheral blood of healthy donors treated with or without anti-IL-2 Ab (1 mg/ml) in the presence or absence of TGF-b (n ¼ 5). (c) Co-expression of CD70 and Foxp3 on CD4 þ CD45RA þ (naive) or CD4 þ CD45RO þ (memory) T cells from peripheral blood of healthy donors treated with or without anti-IL-2 Ab in the presence or absence of TGF-b (n ¼ 3). (d) CD70 and CD25 or Ki-67 co-expression on CD4 þ T cells from peripheral blood of healthy donors cultured in anti-CD3-coated plates with or without anti-CD28 Ab in the presence or absence of TGF-b. Ki-67 expression was measured using intracellular staining (n ¼ 3). (e) Co-expression of CD70 and CD25 on CD4 þ T cells from peripheral blood of healthy donors transfected with no siRNA, control siRNA or Stat5 siRNA in the presence or absence of TGF-b. MFI was calculated for CD70 þ population (n ¼ 3). (f, g) CD70 expression on CD4 þ T cells from peripheral blood of healthy donors treated with escalated doses of cyclosporin A (CsA) (f) or rapamycin (Rapa) (g) in the presence or absence of TGF-b (n ¼ 2).

& 2014 Macmillan Publishers Limited Leukemia (2014) 1872 – 1884 TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1878

Figure 4. CD70 þ T cells express exhaustion markers PD-1 and TIM-3. (a) Histograms showing CD45RO, CD27, CD28, 2B4, LAG-3, CD25, TIM-3, PD-1 and CD57 on intratumoral preexisting CD4 þ CD70 þ or CD8 þ CD70 þ T cells from B-cell NHL. (b) Representative histograms showing PD-1 and TIM-3 expression on CD70 À and CD70 þ T cells. The expression levels of PD-1 and TIM-3 on CD70 À or CD70 þ T cells from either TGF-b-induced or intratumoral preexisting T-cells are summarized in graphic form (right, n ¼ 5). (c) Graphs showing CD70, PD-1 or TIM-3 expression on CD4 þ T cells treated with or without TGF-b, IL-2 and IL-21 at the indicated time points (n ¼ 2). (d) Graphs showing PD-1 or TIM-3 expression on CD4 þ CD70 À or CD70 þ T cells treated with or without TGF-b (n ¼ 5).

escalating doses of TGF-b, were annexin V (AnV) þ or propidium active form of caspase-3 in activated CD4 þ T cells treated with or iodide (PI) þ (Figure 6c). Using untreated, viable (PI negative) cells without TGF-b. As shown in Figure 6e, TGF-b treatment from a representative sample, a subset of CD70 þ T cells and a small upregulated the active form of caspase-3 expression in CD4 þ number of CD70 À T cells were positive for AnV (Figure 6d). TGF-b T cells. Among viable cells, a portion of CD70 þ T cells and a treatment resulted in positive AnV staining in the vast majority of smaller percentage of CD70 À T cells stained positively for CD70 þ T cells while no significant change was seen in CD70 À caspase-3 in the untreated population. TGF-b treatment signifi- T cells (Figure 6d). These results strongly indicate that TGF-b cantly increased the number of CD70 þ T cells expressing caspase-3 treatment affects the susceptibility of CD70 þ T cells to apoptosis. while only a modest change was detected in CD70 À T cells Caspase-3 activation serves as a marker of apoptosis and causes (Figure 6e). Caspase-3 induction in TGF-b-treated CD4 þ T cells cell death. To confirm whether CD70 þ T cells are the major subset was time course dependent and was not observed at early time of TGF-b-mediated apoptosis, we measured the expression of the points (Supplementary Figure S6). This is in line with the

Leukemia (2014) 1872 – 1884 & 2014 Macmillan Publishers Limited TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1879

Figure 5. CD70 þ T cells account for TGF-b-mediated effects and are functionally exhausted. (a) Expression of CD25 (n ¼ 6), Ki-67 (n ¼ 4) or CFSEdim (n ¼ 6) on CD4 þ T cells from peripheral blood of healthy donors treated with or without TGF-b determined by flow cytometry. (b, c) Expression of IFN-g or IL-2 in CD4 þ T cells (b)(n ¼ 6) or CD4 þ CD70 À / þ T cells (c)(n ¼ 5) from peripheral blood of healthy donors treated with or without TGF-b determined by intracellular staining. (d) Mononuclear cells freshly isolated from biopsy specimens of B-cell NHL were stimulated with PMA/Ion plus Brefeldin A for 4 h and analyzed by intracellular staining. Plots were generated gating on CD3 þ cells (n ¼ 4). (e) Mononuclear cells freshly isolated from biopsy specimens of B-cell NHL were stimulated with IL-2 for 30 min, and phosphorylation of Stat5 in CD70 À or CD70 þ T cells was measured by phosflow staining. Plots were generated by gating on CD3 þ cells (n ¼ 2). observation that TGF-b-induced apoptosis only occurs after manner. Treatment with TGF-b resulted in a significant increase in prolonged incubation periods. soluble CD27 (Figure 7c). In the presence of an anti-CD70 Ab, To test whether CD27/CD70 interaction is involved in TGF-b- the TGF-b-induced increase in soluble CD27 level was attenuated mediated apoptosis of activated CD4 þ T cells, we used an anti- (Figure 7d). Concomitant with soluble CD27 levels, the TGF-b- CD70 Ab to block the interaction between CD27 and CD70 and mediated decrease in surface CD27 expression was inhibited by determined the apoptosis of CD4 þ T cells. As shown in Figures 6f treatment with the anti-CD70 Ab (Figure 7e). These results and g, treatment of cells with an anti-CD70 Ab reduced the suggested that while cleavage contributes to surface CD27 number of PI þ cells induced by TGF-b on day 7. This was downregulation, CD70 upregulation might be responsible for confirmed in repeat experiments that consistently showed that TGF-b-mediated shedding of CD27 from T cells. the viability of CD4 þ T cells treated with TGF-b was improved by Next, we determined whether CD27/CD70 signaling has a role exposure to an anti-CD70 blocking Ab (Figure 6g). in TGF-b-mediated T-cell exhaustion by blocking the CD27/CD70 interaction. As shown in Figure 7f, while TGF-b treatment showed a modest inhibition of IFN-g production on day 1, prolonged CD27-CD70 interaction contributes to TGF-b-induced T-cell incubation with TGF-b significantly suppressed IFN-g production exhaustion by CD4 þ T cells on day 7. Treatment with a blocking anti-CD70 Ab To investigate whether CD27-CD70 interaction has a role in TGF- alone slightly inhibited IFN-g expression by CD4 þ T cells. b-induced T-cell exhaustion, we first determined the expression of However, in the presence of the CD70 Ab, TGF-b-mediated CD27 on CD4 þ T cells treated with or without TGF-b, as it has inhibition was attenuated on day 7 (Figure 7f). Similarly, while been shown that CD70 expression is associated with CD27 TGF-b alone inhibited Ki-67 expression in CD4 þ T cells, this downregulation on T cells.18 We observed that TGF-b inhibition was reduced in the presence of the blocking anti-CD70 downregulated CD27 expression on the surface CD4 þ T cells Ab on day 7 (Figure 7g). These results suggest that CD27/CD70 and resulted in an increase in the number of CD70 þ CD27 À T cells interaction is involved in TGF-b-mediated T-cell exhaustion. (Figures 7a and b). Because it has been well known that CD27 can be cleaved from the cell surface, we next tested whether TGF- b-induced CD27 downregulation is due to shedding of surface The frequency of CD70 þ T cells is associated with a poor outcome CD27, resulting in elevated levels of soluble CD27. As shown in in FL patients Figure 6c, although culture medium had no soluble CD27, activated Because lymphoma-associated CD70-expressing T cells were T cells shed CD27 into culture medium at a time-dependent functionally incompetent, we predicted that intratumoral CD70 þ

& 2014 Macmillan Publishers Limited Leukemia (2014) 1872 – 1884 TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1880

Figure 6. Exhausted CD70 þ T cells are highly susceptible to apoptosis. (a) Graph showing viability of activated CD4 þ T cells from peripheral blood of healthy donors treated with TGF-b at different time points. Cell viability was measured using annexin V (AnV) and propidium iodide (PI) staining and reported as the numbers of cells lacking AnV and PI staining (n ¼ 6). *Po0.05; **Po0.01. (b) Graph showing apoptosis in activated CD4 þ CD70 À or CD70 þ T cells from peripheral blood of healthy donors treated with or without TGF-b on day 7. Apoptosis was measured by AnV and PI staining and calculated by the numbers of cells staining positive for AnV and PI (n ¼ 6). (c) Dot plots showing co- staining of AnV or PI and CD70 on activated CD4 þ T cells from peripheral blood of healthy donors treated with or without TGF-b at different doses for 7 days (n ¼ 4). (d) Dot plots showing co-staining of AnV and CD70 on CD4 þ T cells from peripheral blood of healthy donors treated with or without TGF-b for 7 days. The plots were generated gating on viable (PI À ) cells (left panel). (e) Left: Graph showing expression of the active form of caspase-3 in CD4 þ T cells treated with or without TGF-b for 7 days. Caspase-3 expression was determined by intracellular staining (n ¼ 5). Right: Plots showing co-expression of caspase-3 and CD70 on CD4 þ T cells treated with or without TGF-b for 7 days. (f, g) Representative dot plots showing co-staining of AnV and PI on CD4 þ T cells treated with or without TGF-b in the presence of immunoglobulin G (IgG) or anti-CD70 Ab for 7 days. Three individual experiments are graphed in Figure 5g. The percentage of viable cells is calculated as the number of AnV/PI À cells.

Leukemia (2014) 1872 – 1884 & 2014 Macmillan Publishers Limited TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1881

Figure 7. CD27-CD70 interaction contributes to TGF-b-induced T-cell exhaustion. (a) Representative dot plots showing co-expression of CD27 and CD70 on CD4 þ T cells treated with or without TGF-b (n ¼ 6). (b) A summary of CD27 expression on CD4 þ or on CD70 þ T cells treated with or without TGF-b (n ¼ 6). (c, d) A summary of soluble CD27 concentration in culture medium of CD4 þ T cells treated with or without TGF-b in the presence of immunoglobulin G (IgG) or anti-CD70 Ab for the indicated time points. Soluble CD27 concentration was measured by CD27 enzyme-linked immunosorbent assay kit (n ¼ 3). (e) Representative histograms showing expression of CD27 on CD4 þ T cells treated with or without TGF-b in the presence of IgG or anti-CD70 Ab for 5 days (n ¼ 3). (f, g) Representative dot plots showing production of IFN-g and IL-2 (f) or Ki-67 (g)byCD4þ T cells treated with or without TGF-b in the presence of IgG or anti-CD70 Ab for the indicated time points (n ¼ 3).

T cells would adversely affect patient outcomes in B-cell NHL. We but all patients subsequently received chemotherapy in combina- first measured the numbers of CD70 þ T cells in biopsy specimens tion with rituximab as therapy for the disease. CD70 was highly from a cohort of 32 untreated FL patients. The specimens from expressed on a subset of T cells from LNs from FL patients, LNs of patients with follicular hyperplasia were used as a control. whereas its expression was negligible or low on T cells from The biopsy specimens from FL patients were collected at benign LNs (Figure 8a). On average, the number of CD70 þ T cells diagnosis with variable histological grades (g1: 17 patients; g2: 7 accounted for 11.32±1.248% of CD3 þ T cells in LNs of FL patients patients; g3a: 8 patients). Many patients were initially observed, (range: 1.9–28.5%, n ¼ 32) as compared with 5.1±1.9 in healthy

& 2014 Macmillan Publishers Limited Leukemia (2014) 1872 – 1884 TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1882 sequentially stained the same tissue section for CD70, CD20 and CD3 and visualized co-expression of these using a novel method devised by Glass et al.32 The resulting images captured were overlaid, and each assigned a color using Adobe Photoshop CS2 (Adobe Systems, Inc., San Jose, CA, USA). As shown in Figure 8c (bottom), CD70 staining (red) was seen in both the intra- and extra-follicular regions and was co-expressed with CD20 (yellow, mostly intra-follicular) and CD3 (blue, mostly extra- follicular). These results were consistent with the flow cytometry results. To test whether elevated numbers of CD70 þ T cells had an impact on patient outcome, we performed a Kaplan-Meier analysis to correlate the number of intratumoral CD70 þ T cells at diagnosis with overall survival in a cohort of FL patients who all received chemotherapy and rituximab during their disease course. Using the median of 11% of CD70 þ T cells as a cutoff, we found that higher numbers of CD70 þ T cells were significantly associated with a shorter overall survival in FL patients (Figure 8d).

DISCUSSION The mechanism by which CD70 expression is regulated remains largely unstudied, and little is known about cytokine regulation of CD70 expression on T cells. Non-specific activation of T cells is able to upregulate CD70 expression, but the extent of the upregulation is modest. In the presence of TGF-b, however, we find that CD70 expression levels are significantly increased. This induction was specific; none of other cytokines tested was able to regulate CD70 expression on T cells, including IL-2, which has been shown by others to upregulate CD70 expression on CD8 þ T cells in patients with metastatic melanoma.33 We have shown that the Smad signaling pathway is required and that Stat5 signaling also has an important role although the predominant effect of IL-2/Stat5 signaling was seen in naive T cells. This is in agreement with the finding that Stat5 signaling is essential for TGF-b-mediated Foxp3 induction in T cells.31 The finding that CD70 and Foxp3 can be induced in either the same cells or in separate cells indicates that they may use unique signaling pathways when responding to TGF-b. Supporting this, we found that TGF-b predominantly upregulates CD70 expression on Tm cells while Foxp3 expression is preferentially upregulated in Tn cells. This finding is consistent with the result that IL-2/Stat5 signaling has a greater effect on TGF-b-mediated CD70 expression Figure 8. The frequency of CD70 þ T cells is associated with a poor in naive T cells. outcome in FL patients. (a) Representative dot plots showing CD70 It was previously unclear how CD70 acquisition affects T-cell expression on CD3 þ cells from biopsy specimens of individuals with function. In this study, we observed that both TGF-b-induced and follicular hyperplasia (NM) or FL patients. (b) A summary of preexisting intratumoral CD70 þ T cells displayed phenotypic and frequency of CD70-expressing T cells from biopsy specimens of functional changes similar to that in T-cell exhaustion. For individuals with reactive follicular hyperplasia (NM) or FL patients. þ þ þ example, CD70 T cells had higher PD-1 and TIM-3 expression CD70 CD3 T cells were measured by flow cytometry. (c)Top compared with CD70 À T cells. The function of these cells was panel, a representative image from one of five FL patient samples showing CD70 expression measured by immunohistochemistry. suppressed, and they exhibited reduced cytokine production and Tonsil tissue was used as a control. Bottom, a representative image impaired signal transduction. Previous studies have suggested from a FL patient sample showing co-expression of CD70 (red), that TGF-b may induce T-cell exhaustion that leads to decreased CD20 (yellow) and CD3 (blue) measured by immunohistochemistry. T-cell proliferation and function.25–27 However, the cell population The method involves sequential staining of the same tissue section, responsible for this effect was unknown. The results we present in decolorizing and antigen-antibody dissociation of each antibody this study clearly show that CD70 upregulation is associated with used. Slides initially positive for CD70 were co-stained stained with TGF-b-induced exhaustion of effector Tm cells. CD3 and then CD20. (d) Kaplan-Meier curve for overall survival of FL Although TGF-b is known to induce apoptosis in a variety of patients treated with chemoimmunotherapy (n ¼ 32) by the number types of cells, TGF-b-mediated apoptosis in T cells is only observed of CD3 þ CD70 þ T cells with a cutoff of 11%. after extended culture. Studies have suggested that co-stimulation via CD28 has a crucial role in inhibiting TGF-b-induced apoptosis tissue (range: 1.3–21.0%, n ¼ 10, P ¼ 0.016) (Figure 8b). In addition in T cells34,35 and may contribute to this late-onset apoptosis. We to flow cytometry, we did immunohistochemistry to determine provide evidence that CD70 induction is responsible for this CD70 expression in FL specimens (Figure 8c, top panel). Tonsil delayed TGF-b-mediated apoptosis. These exhausted CD70 þ tissues were stained as a control. We found that CD70 was brightly T cells acquire more pro-apoptotic markers such as caspase-3 stained in cells within follicles (B-cell region) as well as cells from than CD70 À T cells and are more susceptible to activation- interfollicle areas (T-cell region). In contrast, CD70 staining in tonsil induced cell death. was modest with dim staining of a few cells within follicles. To Although we show that CD70 þ T cells are the major cell further topographically identify CD70 þ T cells in FL tissue, we population contributing to TGF-b-mediated effects, it is unknown

Leukemia (2014) 1872 – 1884 & 2014 Macmillan Publishers Limited TGF-b induces CD70 upregulation and T-cell exhaustion Z-Z Yang et al 1883 whether CD27/CD70 interaction has a role in TGF-b-induced T-cell 5 Ulsenheimer A, Gerlach JT, Gruener NH, Jung MC, Schirren CA, Schraut W et al. exhaustion. Previous studies have shown that excessive or chronic Detection of functionally altered hepatitis C virus-specific CD4 T cells in acute and CD27-CD70 signaling can lead to exhaustion in a murine model chronic hepatitis C. Hepatology 2003; 37: 1189–1198. and results in impaired function of CD8 þ T cells.15,36,37 Our finding 6 Urbani S, Amadei B, Tola D, Massari M, Schivazappa S, Missale G et al. that TGF-b-mediated downregulation of surface CD27 on CD70 þ PD-1 expression in acute hepatitis C virus (HCV) infection is associated with T cells is due to shedding of CD27, creates a model whereby CD70 HCV-specific CD8 exhaustion. J Virol 2006; 80: 11398–11403. expression on T cells leads to stimulation (or overstimulation) of 7 Kasprowicz V, Schulze Zur Wiesch J, Kuntzen T, Nolan BE, Longworth S, Berical A neighboring T cells (or auto-stimulation) through CD27 at which et al. High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8 þ and CD4 þ T cells during acute HCV infection, irrespective of clinical outcome. J Virol point CD27 is cleaved from the cell surface. We observed that 2008; 82: 3154–3160. blocking CD70 reverses CD27 loss on the cell surface and restores 8 Mumprecht S, Schurch C, Schwaller J, Solenthaler M, Ochsenbein AF. cell function, including IFN-g production and cell viability, Programmed death 1 signaling on chronic myeloid leukemia-specific T cells confirming the role of CD27/CD70 interaction in TGF-b-mediated results in T-cell exhaustion and disease progression. Blood 2009; 114: 1528–1536. T-cell exhaustion. 9 Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE The role of CD70-CD27 interaction in tumors is controversial. et al. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels Although numerous studies suggest that CD70-CD27 interaction of PD-1 and are functionally impaired. Blood 2009; 114: 1537–1544. improves antitumor immunity, many other reports find the 10 Klein L, Trautman L, Psarras S, Schnell S, Siermann A, Liblau R et al. Visualizing the opposite. It has been shown that CD27 ligation by CD70- course of antigen-specific CD8 and CD4 T cell responses to a growing tumor. Eur J Immunol 2003; 33: 806–814. expressing tumor cells,38,39 genetically modified tumor cells 40–43 44–46 11 Fourcade J, Sun Z, Benallaoua M, Guillaume P, Luescher IF, Sander C et al. expressing CD70, soluble CD70 or agonistic anti-CD27 Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen- 47–49 antibodies induces an antitumor response by activating specific CD8 þ T cell dysfunction in melanoma patients. J Exp Med 2010; 207: cytotoxic T and enhancing tumor-specific 2175–2186. cytotoxicity. In contrast, it has been suggested that the CD70- 12 Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting CD27 interaction may favor immune escape through the Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor development of intratumoral regulatory T cells,50,51 the immunity. J Exp Med 2010; 207: 2187–2194. promotion of apoptosis48,52,53 or the depletion of 13 Yang ZZ, Grote DM, Ziesmer SC, Niki T, Hirashima M, Novak AJ et al. IL-12 upre- natural killer cell.36,54 In B-cell malignancies, this discrepancy gulates TIM-3 expression and induces T cell exhaustion in patients with follicular non-Hodgkin lymphoma. J Clin Invest 2012; 122: 1271–1282. remains. Although CD70-CD27 interaction prevents T-cell 14 Tesselaar K, Xiao Y, Arens R, van Schijndel GM, Schuurhuis DH, Mebius RE et al. anergy and enhances cytotoxic activity of tumor-specific 39 Expression of the murine CD27 ligand CD70 in vitro and in vivo. J Immunol 2003; cytotoxic T lymphocytes in acute lymphoblastic leukemia, 170:33–40. high CD70 expression levels correlate to shorter overall survival 15 van Gisbergen KP, van Olffen RW, van Beek J, van der Sluijs KF, Arens R, Nolte MA in both the GCB and ABC subtypes in diffuse large B-cell et al. Protective CD8 T cell memory is impaired during chronic CD70-driven lymphoma.55 Our data suggest that when T cells acquire costimulation. J Immunol 2009; 182: 5352–5362. CD70 expression, they become exhausted cells and their 16 Oelke K, Lu Q, Richardson D, Wu A, Deng C, Hanash S et al. Overexpression of function is impaired, which correlates with an inferior clinical CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated outcome in FL patients. with DNA methylation inhibitors. Arthritis Rheum 2004; 50: 1850–1860. In summary, CD70 þ T cells represent a subpopulation of 17 Han BK, White AM, Dao KH, Karp DR, Wakeland EK, Davis LS. Increased prevalence of activated CD70 þ CD4 þ T cells in the periphery of patients with systemic immune cells that is functionally exhausted, and the presence of þ lupus erythematosus. Lupus 2005; 14: 598–606. intratumoral CD70 T cells in FL impacts patient outcome. We, for 18 Lee WW, Yang ZZ, Li G, Weyand CM, Goronzy JJ. 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