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. Author manuscript; available in PMC: 2014 Sep 4.
Published in final edited form as: Leukemia. 2014 Feb 26;28(9):1872–1884. doi: 10.1038/leu.2014.84

TGF-β upregulates CD70 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin’s lymphoma

Zhi-Zhang Yang 1, Deanna M Grote 1, Bing Xiu 2, Steven C Ziesmer 1, Tammy L Price-Troska 1, Lucy S Hodge 1, Danielle M Yates 1, Anne J Novak 1, Stephen M Ansell 1
PMCID: PMC4145058  NIHMSID: NIHMS574897  PMID: 24569779

Abstract

Transforming growth factor beta (TGF-β) plays an important role in mediating T-cell suppression in B-cell non-Hodgkin lymphoma (NHL). However, the underlying mechanism responsible for TGF-β-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-β inhibits Tm cells, and TGF-β mediated exhaustion is associated with upregulation of CD70. We found that TGF-β upregulates CD70 expression on effector Tm cells while it preferentially induces Foxp3 expression in naïve T cells. CD70 induction by TGF-β is Smad3-dependent and involves IL-2/Stat5 signaling. CD70+ T cells account for TGF-β-induced exhaustion of effector Tm cells. Both TGF-β-induced and preexisting intratumoral CD70+ effector Tm cells from B-cell NHL have an exhausted phenotype and express higher levels of PD-1 and TIM-3 compared to CD70− 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+ T cells correlate with an inferior patient outcome. These findings confirm TGF-β-mediated effector Tm cell exhaustion as an important mechanism of immune suppression in B-cell NHL.

Keywords: TGF-β, CD70, T-cell exhaustion, B-cell non-Hodgkin lymphoma

Introduction

T-cell exhaustion is a type of immune response describing the condition in which T cells exhibit reduced differentiation, proliferation and effector function. T-cell exhaustion is initially recognized and characterized in chronic viral infections(1-7). In tumors, it has been observed that intratumoral T cells display a phenotypic and functional profile similar to that of exhausted T cells from chronic viral infection (8-10). Phenotypically, PD-1 expression has been demonstrated to be a marker to identify exhausted T cells in viral infection(3, 4) and tumors (11, 12). Recently, we found that IL-12 induces T-cell exhaustion through up-regulating TIM-3 in patients with follicular lymphoma(13).

Co-stimulatory molecule CD70 can be expressed on T cells upon TCR stimulation(14). CD70 expression causes a change in T cell function(15), and high levels of CD70 have been shown to be involved in the pathophysiology of several diseases(16-18). Over recent decades, efforts to explore the underlying mechanism of CD70 upregulation on T cells have proved difficult(19). Studies have suggested that DNA methylation of the CD70 promoter gene plays an important role in CD70 upregulation on T cells in various autoimmune diseases(20, 21). However it is not known which cytokine can up-regulate CD70 expression on T cells.

Cytokine TGF-β exerts the greatest impact on T cells by inhibiting their activation, proliferation, differentiation and survival(22, 23). B cells including malignant B cells are a source of inhibitory cytokines such as IL-10 and TGF-β, suggesting a role of TGF- β in B-cell NHL (24). An important question arises about which type of response is responsible for TGF-β-mediated suppression of effector Tm cells. Several studies have implied that TGF-β may induce T-cell exhaustion that leads to a declined T-cell proliferation and function as well as enhanced cell death(25-27). However, the underlying mechanism, especially which subpopulation contributes to TGF-β-mediated T cell inhibition possibly by T-cell exhaustion, is unknown.

In the present study, we have identified TGF-β to be a key regulator of CD70 expression on T cells. We then determined the phenotypical and functional changes of TGF-β-induced or intratumoral preexisting CD70+ T cells as well as the clinical impact of CD70-expressing T cells on patient outcome in FL. The data we present in this study demonstrate the biological and clinical significance of TGF-β-mediated CD70 induction and the subsequent inhibition of Tm cell function.

Materials and methods

Patient samples

Patients providing written informed consent were eligible for this study if they had a tissue biopsy that on pathologic review showed follicular B-cell NHL and adequate tissue or peripheral blood to perform the experiments. Peripheral blood mononuclear cells from healthy donors and normal specimens from patients with follicular hyperplasia were used as controls. The use of human tissue samples for this study was approved by the Institutional Review Board of the Mayo Clinic/Mayo Foundation.

Cell isolation and culture

Fresh tumor biopsy specimens from patients with FL and control lymph nodes (LNs) were gently minced over a wire mesh screen to obtain a cell suspension. The cell suspension or peripheral blood from patients or healthy donors was centrifuged over Ficoll-Hypaque at 500 g for 15 minutes to isolate mononuclear cells. CD3+, CD4+, CD8+ T cells and CD19+ B cells were isolated using positive selection with CD3, CD4, CD8 or CD19 microbeads (Miltenyi Biotec). CD4+CD45RA+ or CD4+CD45RO+ T-cell subsets were purified by using EasySep® Human Naïve CD4+ T Cell Enrichment Kit (StemCell Technologies, Vancouver, Canada). T cells were cultured in anti-CD3 Ab-coated plates in the present of anti-CD28. All experiments have been done on anti-CD3 Ab-activated T cells unless otherwise noted as resting cells. CD4+ T cells were used in the majority of experiments.

Intracellular staining

Cells were washed and subjected to fixation, permeabilization, stained with fluorochrome-conjugated antibodies and analyzed by flow cytometry. For cytokine induction, we cultured CD4+ T cells in anti-CD3-coated plates and treated with or without stimuli for 3 days. Cells were restimulated with PMA/Ion plus Brefeldin A for 4 hrs and analyzed by flow cytometry. For cytokine production by intratumoral T cells, we stimulated cells with PMA/Ion plus Brefeldin A for 4 h and analyze by flow cytometry. Foxp3 expression was determined using Foxp3 detection kit (Biolegend, San Diego, CA) following the manufacturer’s instructions. For Ki-67 expression, cells were incubated with 70% ethanol for 1 hr, stained with Ki-67-APC Ab and analyzed by flow cytometry.

ELISA assay

Concentration of soluble CD27 in experimental supernatants was measured using an Instant ELISA kit from eBioscience (# BMS286INST). For each sample, 50μl were done in duplicate and incubated 3 hours at room temperature on rotary shaker. After washing the micro wells, 100μl TMB Substrate was added to each well and color allowed to develop for 10 minutes. Reaction was stopped by adding 100μl of Stop Solution and plate was read immediately on a SpectraMax 190 plate reader at 450nm using SoftMax Pro software program.

Stat5 phosphorylation assay

The phosphorylation of Stat5 was detected following the manufacturer’s instructions (BD Biosciences, San Jose, CA). Briefly, fresh-isolated mononuclear cells from biopsy specimens of patients with B-cell NHL were incubated with IL-2 (50ng/ml) for 30 min, then fixed and permeabilized using a phosflow kit (BD Biosciences, San Jose, CA). Cells were stained with anti-pStat5-Alexa647 antibody plus anti-CD3-FITC and CD70-PE antibodies for 30 minutes and analyzed by flow cytometry.

siRNA transfection

Transfection of CD4+ T cells with siRNA was performed according to the manufacturer’s instruction (Qiagen, Cambridge, MA). Genesolution siRNA and Hiperfect Transfection Reagent were purchased from Qiagen. 100nM siRNA for CD70 or Stat5 or a scrambled siRNA was transfected in a 24 well tissue culture plate for 24 hrs. After washing, cells were cultured in OKT3-coated plate in the presence or absence of TGF-β 50ng/ml for 3 days and CD70 expression was measured by flow cytometry.

Statistical analysis

Statistical analysis was performed using Student’s t test. Significance was determined at p < 0.05. Overall survival was measured from the date of diagnosis until death from any cause. Patients alive and still at risk of death at last follow-up evaluation were censored for the analysis of overall survival. Survival of all patients was estimated using the Kaplan-Meier method. The univariate association between CD70 expression and survival was determined with the log-rank test.

Results

TGF-β induces CD70 expression on T cells

We have recently shown that TGF-β plays a critical role in mediating T cell differentiation in the tumor microenvironment of B-cell NHL(24, 28). To further evaluate the role of TGF-β on immune responses, we determined the effect of TGF-β on the expression of various cell surface markers, cytokines, transcription factors and signaling molecules in intratumoral T cells derived from patients with B-cell NHL using flow cytometry (Figure 1a). As expected, TGF-β inhibited the expression of most of the cell surface and signaling molecules tested. However, TGF-β increased expression of three molecules, one of which was Foxp3, which is well known to be upregulated by TGF-β. Consistent with previous reports, TGF-β also increased IL-2 expression(29, 30). Surprisingly, the expression of CD70 was dramatically enhanced on T cells following treatment with TGF-β (Figure 1a).

Figure 1. TGF-β induces CD70 expression on T cells.

Figure 1

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(a) Expression of a variety of molecules in activated intratumoral T cells from lymphoma patients treated with or without TGF-β. The expression levels were expressed as fold change and converted to logarithmic number. *: p<0.05, compared to . (b) CD70 expression on peripheral blood CD4+ T cells from healthy donors treated with or without TGF-β detected by flow cytometry (upper panel) or cytofluorescence (lower panel). Isotype control for CD70 was included. (c) A summary of CD70 induction on CD4+ T cells treated without (NIL) or with TGF-β. CD70 expression on CD4+ T cells was measured by flow cytometry and calculated as percentage or mean fluorescence intensity (MFI). Each line represents an individual sample of peripheral blood from healthy donors (n=15). (d, e) CD70 expression on CD4+ T cells from healthy donors treated with TGF-β at escalating doses (d, n=3) or at different time points (e, n=3). (f) CD70 and Foxp3 co-expression on CD4+ T cells from peripheral blood of healthy donors treated with or without TGF-β. Intracellular staining was performed using a Foxp3 staining kit (Biolegend, San Diego, CA). The numbers of CD70+Foxp3−, CD70+Foxp3+ or CD70−Foxp3+ T cells induced by TGF-β were summarized graphically (right, n=5).

CD70 is normally absent on resting T cells and is induced upon activation (Supplementary Figure 1A). With the addition of TGF-β, CD70 expression on activated T cells is significantly upregulated (Figure 1b). TGF-β not only increased the number of T cells expressing CD70 but also enhanced the magnitude of CD70 expression on T cells in a dose- and time-dependent manner (Figure 1b-e).

To determine whether CD70 upregulation was specific to TGF-β, we tested a panel of cytokines for their capacity to upregulate CD70 expression. Among the twelve tested, TGF-β remained the only cytokine that strongly induced CD70 expression (Supplementary Figure 1B). While a previous study has reported that IL-2 upregulates CD70 expression on CD8+ T cells in mice(15), we found that CD70 expression was not upregulated on human T cells treated with IL-2 alone, and the addition of IL-2 had no further impact on TGF-β-mediated induction of CD70 on activated T cells (Supplementary Figure 2).

We next determine whether TGF-β was able to upregulate CD70 expression on activated B cells or monocytes. As shown in supplementary Figure 3, while activation through LPS, but not CD40L, enhanced CD70 expression, TGF-β was unable to upregulate CD70 expression on either CD19+ or CD11c+ cells. In fact, treatment with TGF-β was associated with a slight decrease in CD70 expression on both resting and activated CD19+ or CD11c+ cells (Supplementary Figure 3). These results indicate that TGF-β-induced CD70 expression is specific to T cells.

It is well known that TGF-β induces the expression of Foxp3, a transcriptional factor critically important for Treg cells. Thus, we examined the induction of Foxp3 and CD70 in activated CD4+ T cells by TGF-β. As shown in Figure 1f, treatment with TGF-β upregulated expression of both Foxp3 and CD70 on CD4+ T cells. The numbers of CD70+Foxp3−, CD70+Foxp3+ or CD70−Foxp3+ T cells were significantly increased in response to TGF-β.

TGF-β preferentially upregulates CD70 expression on Tm cells

We then measured CD70 induction in activated CD4+CD45RA+ naïve (Tn) and CD4+CD45RO+ memory (Tm) T cells. TGF-β was able to upregulate CD70 expression on both Tn and Tm cells. However, a more substantial induction of CD70 was predominantly observed in Tm cells as compared to Tn cells (Figure 2a). When we extended the culture time with TGF-β, CD70 induction was diminished and lasted for only a short time in Tn cells. However, CD70 induction was much greater and maintained for a relatively longer period of time in Tm cells (Figure 2b). Supporting this finding, we noted that CD4+CD62L− effector Tm cells accounted for most of the observed TGF-β-mediated CD70 induction (Figure 2c, d). This result indicated CD70 induction by TGF-β is preferential in Tm cells.

Figure 2. TGF-β preferentially upregulates CD70 expression on Tem cells.

Figure 2

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

Next, we examined the expression pattern of Foxp3 and CD70 induced by TGF-β in purified activated Tn and Tm cells. Although TGF-β was able to induce the expression of Foxp3 or CD70 in both Tn and Tm cells, we observed a preferential induction pattern between Foxp3 and CD70 in Tn and Tm cells (Figure 2e,f; Supplementary Figure 4). Namely, in Tn cells, TGF-β had a greater effect on Foxp3 induction than CD70. In contrast, TGF-β-mediated CD70 upregulation was more profound than that of Foxp3 in Tm cells (Figure 2g). These results suggest that TGF-β differentially exerts its function on Tn and Tm cells.

IL-2/Stat5 signaling is involved in TGF-β-mediated CD70 induction in T cells

As Smad3 activation is a key component of the TGF-β signaling pathway, we first determined the role of Smad3 in TGF-β-mediated CD70 induction using a specific Smad3 Inhibitor SIS3. Activated CD4+ T cells treated with SIS3 alone showed no effect on CD70 expression (Figure 3a). However, CD70 induction by TGF-β was completely blocked when T cells were treated with 5μg/ml SIS3. This result suggests that TGF-β-induced CD70 expression is Smad3-dependent.

Figure 3. IL-2/Sta5 signaling is involved in TGF-β-mediated CD70 induction on T cells.

Figure 3

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

Given that TGF-β induces IL-2 production, we next tested whether IL-2 signaling is involved in TGF-β-mediated CD70 induction. Although addition of exogenous IL-2 had no effect on CD70 induction, we observed that IL-2 depletion by an anti-IL-2 Ab led to complete inhibition of CD70 induction on T cells activated with TCR stimulation (Figure 3b). This depletion also attenuated TGF-β-mediated CD70 induction on T cells although CD70 upregulation was not completely blocked (Figure 3b). We observed that this attenuation was more pronounced in Tn than Tm cells and affected both CD70 and Foxp3 upregulation by TGF-β (Figure 3c, Supplementary Figure 4). These results suggest that IL-2 signaling is essential for activation-induced CD70 expression and also plays a role in TGF-β-mediated CD70 induction.

Although activation is required, TGF-β-induced CD70 expression may occur through a different pathway. While the vast majority of CD70-expressing cells are CD25+ or Ki-67+ activated T cells, only a subset of CD25+ or Ki-67+ T cells were able to respond to TGF-β and upregulate CD70 expression (Figure 3d). However, the expression of CD25 or Ki-67 was inhibited in CD4+ T cells treated with TGF-β (Figure 3d and 4a). The association between increased CD70 expression and down-regulated CD25 or Ki-67 suggests that TGF-β-mediated CD70 enhancement is not simply due to T cell activation.

Figure 4. CD70+ T cells express exhaustion markers PD-1 and TIM-3.

Figure 4

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

It has been shown that Stat5 activation plays a crucial role in TGF-β-mediated induction of Foxp3 in T cells(31). Given the similar effect of TGF-β on CD70 expression, we wondered whether Stat5 was also involved in CD70 induction by TGF-β. To test this, we transfected Stat5 siRNA into CD4+ T cells and monitored CD70 expression. We observed that TGF-β-mediated CD70 induction was attenuated when activated T cells were transfected with Stat5 siRNA (Figure 3e). To further confirm the role of IL-2/Stat5 signaling in TGF-β-mediated CD70 induction, we treated T cells with the calcineurin inhibitor Cyclosporin A (CsA) and mTOR inhibitor Rapamycin (Rapa), which block NFAT activation and suppress IL-2/Stat5 signaling. As shown in Figure 3f,g, both CsA and Rapa were able to inhibit activation-induced CD70 expression on activated T cells in a dose-dependent manner. Furthermore, in the presence of either CsA or Rapa, TGF-β mediated induction of CD70 on activated T cells was no longer observed.

Supporting the above findings, we also observed that IL-2/Stat5 signaling was positively associated with CD70 expression in two T cell lines, Jurkat and Karpas 299 (Supplementary Table 1). Karpas 299 cells, which constitutively express CD70 on the cell surface, respond to IL-2 with subsequent activation of Stat5. In contrast, Jurkat cells do not express surface CD70 nor respond to IL-2. Similarly, IL-2 receptor α is expressed on Karpas 299 cells and is absent on Jurkat cells. Taken together, these data indicate that IL-2/Stat5 signaling plays a role in TGF-β-mediated CD70 expression on T cells.

CD70+ T cells express exhaustion markers PD-1 and TIM-3

To phenotypically characterize CD70+ T cells, we measured the expression of an array of surface markers on resting CD70+ T cells isolated from specimens of FL patients (Figure 4a). Intratumoral CD70+ T cells expressed high levels of CD45RO, indicating that the cells were memory T cells. While the majority of CD70+ T cells are CD27+ and CD28+, the loss of CD27 and CD28 was substantial. Most CD8+CD70+ and some CD4+CD70+ T cells expressed PD-1 and TIM-3. Therefore, we compared the expression of PD-1 and TIM-3 between CD4+CD70− and CD4+CD70+ T cells. We found that both TGF-β-induced and preexisting CD70+ T cells express significantly higher levels of PD-1 and TIM-3 compared to CD70− T cells (Figure 4b), Supporting this finding, TGF-β treatment upregulated PD-1 expression on T cells while no similar effects can be seen by other cytokines (Figure 4c). TGF-β-mediated PD-1 upregulation was mainly seen on CD70+ T cells (Figure 4d). Although TGF-β treatment caused downregulation of TIM-3 (Figure 4c), CD70− T cells accounted for the majority of this downregulation (Figure 4d). The majority of CD70+ T cells maintained TIM-3 expression on the cell surface. These results implied that CD70+ T cells are exhausted cells.

CD70+ T cells account for TGF-β-mediated effects and are functionally exhausted

We found that CD4+ T cells treated with TGF-β displayed decreased expression of the activation markers CD25 and Ki-67 as well as reduced proliferation as indicated by decreased numbers of CFSEdim cells (Figure 5a). TGF-β also inhibited IFN-γ production but enhanced IL-2 expression by CD4+ T cells (Figure 5b). Given that TGF-β upregulates CD70 expression, we tested whether CD70 induction plays a role in mediating the effects of TGF-β on T cells. As shown in Figure 5c, CD4+CD70+ T cells were the major population responsible for IFN-γ production, and TGF-β treatment significantly reduced IFN-γ expression by CD70+ T cells. Although both CD70+ and CD70− T cells were able to produce IL-2, IL-2 production was more profoundly enhanced in TGF-β-induced CD70+ cells than in CD70− T cells. These results suggest that CD70 induction accounts for TGF-β-mediated effects on CD4+ T cells.

Figure 5. CD70+ T cells account for TGF-β-mediated effects and are functionally exhausted.

Figure 5

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(a) Expression of CD25 (n=6), Ki-67 (n=4) or CFSEdim (n=6) on CD4+ T cells from peripheral blood of healthy donors treated with or without TGF-β determined by flow cytometry. (b. c) Expression of IFN-γ or IL-2 in CD4+ T cells (b, n=6) or CD4+CD70−/+ T cells (c, n=5) from peripheral blood of healthy donors treated with or without TGF-β 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+ cells (n=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− or CD70+ T cells was measured by phosflow staining. Plots were generated by gating on CD3+ cells (n=2).

To further functionally characterize CD70+ T cells, we measured cytokine production as well as Stat phosphorylation in CD70+ T cells isolated from biopsy specimens of lymphoma patients. As shown in Figure 5d, intratumoral CD70− T cells were predominantly the source of cytokine production whereas CD70+ T cells failed to produce cytokines when stimulated with PMA/Ion. Next, we determined whether signal transduction was also inhibited in CD70+ T cells. Intratumoral T cells were treated with or without IL-2, and Stat5 phosphorylation was measured in CD70− or CD70+ T cells. We found that a subpopulation of intratumoral T cells were able to respond to IL-2 with subsequent activation of Stat5. However, CD70− T cells accounted for this response while CD70+ T cells completely lacked the capacity (Figure 5e).

Exhausted CD70+ T cells are highly susceptible to apoptosis

TGF-β is known to induce apoptosis in a variety of cell types. We observed that treatment with TGF-β induces apoptosis of B cells and results in a significant decrease in cell number of both resting and LPS- or CD40L-activated B cells during short-term culture (three days) (Supplementary Figure 5A). However, we failed to see a decrease in cell number of either resting or activated T cells during the same period of culture (Supplementary Figure 5B). When we measured apoptosis, TGF-β treatment had no effect on CD4+ T cell apoptosis for the first several days (Supplementary Figure 5C), which is consistent with previous studies. However, upon extended culture, TGF-β eventually led to a decreased viability of T cells (Figure 6a). This late-onset effect suggests that other earlier changes in T cells by TGF-β are necessary for the cells to become susceptible to apoptotic induction. Therefore, we tested whether CD70 induction contributes to the late-onset TGF-β-induced apoptosis in CD4+ T cells. Indeed, we found that CD70+ T cells displayed a significantly higher rate of apoptosis than CD70− T cells. Treatment with TGF-β did not induce apoptosis in activated CD70− T cells but increased apoptosis in activated CD70+ T cells (Figure 6b). This finding was further supported by the observation that the increased number of CD70+ T cells, induced by escalating doses of TGF-β, were annexin V (AnV)+ or PI+ (Figure 6c). Using untreated, viable (PI negative) cells from a representative sample, a subset of CD70+ T cells and a small number of CD70− T cells were positive for AnV (Figure 6d). TGF-β treatment resulted in positive AnV staining in the vast majority of CD70+ T cells while no significant change was seen in CD70− T cells (Figure 6d). These results strongly indicate that TGF-β treatment affects the susceptibility of CD70+ T cells to apoptosis.

Figure 6. Exhausted CD70+ T cells are highly susceptible to apoptosis.

Figure 6

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(a) Graph showing viability of activated CD4+ T cells from peripheral blood of healthy donors treated with TGF-β 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=6). *: p<0.05; **: p<0.01. (b) Graph showing apoptosis in activated CD4+CD70− or CD70+ T cells from peripheral blood of healthy donors treated with or without TGF-β 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=6). (c) Dot plots showing co-staining of AnV or PI and CD70 on activated CD4+ T cells from peripheral blood of healthy donors treated with or without TGF-β at different doses for 7 days (n=4). (d) Dot plots showing co-staining of AnV and CD70 on CD4+ T cells from peripheral blood of healthy donors treated with or without TGF-β for 7 days. The plots were generated gating on viable (PI−) cells (left panel). (e) Left: Graph showing expression of the active form of caspase-3 in CD4+ T cells treated with or without TGF-β for 7 days. Caspase-3 expression was determined by intracellular staining (n=5). Right: Plots showing co-expression of caspase-3 and CD70 on CD4+ T cells treated with or without TGF-β for 7 days. (f, g) Representative dot plots showing co-staining of AnV and PI on CD4+ T cells treated with or without TGF-β in the presence of 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− cells.

Caspase-3 activation serves as a marker of apoptosis and causes cell death. To confirm whether CD70+ T cells are the major subset of TGF-β-mediated apoptosis, we measured the expression of the active form of caspase-3 in activated CD4+ T cells treated with or without TGF-β. As shown in Figure 6e, TGF-β treatment upregulated the active form of caspase-3 expression in CD4+ T cells. Among viable cells, a portion of CD70+ T cells and a smaller percentage of CD70− T cells stained positively for caspase-3 in the untreated population. TGF-β treatment significantly increased the number of CD70+ T cells expressing caspase-3 while only a modest change was detected in CD70− T cells (Figure 6e). Caspase-3 induction in TGF-β-treated CD4+ T cells was time-course dependent and was not observed at early time points (Supplementary Figure 6). This is in line with the observation that TGF-β-induced apoptosis only occurs after prolonged incubation periods.

To test whether CD27/CD70 interaction is involved in TGF-β-mediated apoptosis of activated CD4+ T cells, we employed an anti-CD70 Ab to block the interaction between CD27 and CD70 and determined apoptosis of CD4+ T cells. As shown in Figure 6f,g, treatment of cells with an anti-CD70 Ab reduced the number of PI+ cells induced by TGF-β on day 7. This was confirmed in repeat experiments that consistently showed that the viability of CD4+ T cells treated with TGF-β was improved by exposure to an anti-CD70 blocking Ab (Figure 6g).

CD27-CD70 interaction contributes to TGF-β-induced T-cell exhaustion

To investigate whether CD27-CD70 interaction plays a role in TGF-β-induced T-cell exhaustion, we first determined the expression of CD27 on CD4+ T cells treated with or without TGF-β, since it has been shown that CD70 expression is associated with CD27 downregulation on T cells(18). We observed that TGF-β downregulated CD27 expression on the surface CD4+ T cells and resulted in an increase in the number of CD70+CD27− T cells (Figure 7a,b). Because it has been well known that CD27 can be cleaved from the cell surface, we next tested whether TGF-β-induced CD27 downregulation is due to shedding of surface CD27 resulting in elevated levels of soluble CD27. As shown in Figure 6c, while culture medium had no soluble CD27, activated T cells shed CD27 into culture medium at a time-dependent manner. Treatment with TGF-β resulted in a significant increase in soluble CD27 (Figure 7c). In the presence of an anti-CD70 Ab, the TGF-β-induced increase in soluble CD27 level was attenuated (Figure 7d). Concomitant with soluble CD27 levels, the TGF-β-mediated decrease in surface CD27 expression was inhibited by treatment with the anti-CD70 Ab (Figure 7e). These results suggested that while cleavage contributes to surface CD27 downregulation, CD70 upregulation might be responsible for TGF-β-mediated shedding of CD27 from T cells.

Figure 7. CD27-CD70 interaction contributes to TGF-β-induced T-cell exhaustion.

Figure 7

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(a) Representative dot plots showing co-expression of CD27 and CD70 on CD4+ T cells treated with or without TGF-β (n=6). (b) A summary of CD27 expression on CD4+ or on CD70+ T cells treated with or without TGF-β (n=6). (c, d) A summary of soluble CD27 concentration in culture medium of CD4+ T cells treated with or without TGF-β in the presence of IgG or anti-CD70 Ab for indicated time points. Soluble CD27 concentration was measured by CD27 ELISA kit (n=3). (e) Representative histograms showing expression of CD27 on CD4+ T cells treated with or without TGF-β in the presence of IgG or anti-CD70 Ab for 5 days (n=3). (f, g) Representative dot plots showing production of IFN-γ and IL-2 (f) or Ki-67 (g) by CD4+ T cells treated with or without TGF-β in the presence of IgG or anti-CD70 Ab for indicated time points (n=3).

Next, we determined whether CD27/CD70 signaling plays a role in TGF-β-mediated T-cell exhaustion by blocking the CD27/CD70 interaction. As shown in Figure 7f, while TGF-β treatment showed a modest inhibition of IFN-γ production on day 1, prolonged incubation with TGF-β significantly suppressed IFN-γ production by CD4+ T cells on day 7. Treatment with a blocking anti-CD70 Ab alone slightly inhibited IFN-γ expression by CD4+ T cells. However, in the presence of the CD70 Ab, TGF-β-mediated inhibition was attenuated on day 7 (Figure 7f). Similarly, while TGF-β alone inhibited Ki-67 expression in CD4+ T cells, this inhibition was reduced in the presence of the blocking anti-CD70 Ab on day 7 (Figure 7g). These results suggest that CD27/CD70 interaction is involved in TGF-β-mediated T-cell exhaustion.

The frequency of CD70+ T cells is associated with a poor outcome in FL patients

Because lymphoma-associated CD70-expressing T cells were functionally incompetent, we predicted that intratumoral CD70+ T cells would adversely affect patient outcomes in B-cell NHL. We first measured the numbers of CD70+ T cells in biopsy specimens from a cohort of 32 untreated follicular lymphoma (FL) patients. The specimens from lymph nodes of patients with follicular hyperplasia were used as a control. The biopsy specimens from FL patients were collected at diagnosis with variable histological grades (g1: 17 patients; g2: 7 patients; g3a: 8 patients). Many patients were initially observed but all patients subsequently received chemotherapy in combination with rituximab as therapy for the disease. CD70 was highly expressed on a subset of T cells from lymph nodes (LNs) from FL patients, while its expression was negligible or low on T cells from benign LNs (Figure 8a). On average, the number of CD70+ T cells accounted for 11.32% ± 1.248% of CD3+ T cells in LNs of FL patients (range: 1.9%-28.5%, n=32) as compared to 5.1 ± 1.9 in healthy tissue (range: 1.3%-21.0%, n=10, p=0.016) (Figure 8b). In addition to flow cytometry, we did immunohistochemistry to determine CD70 expression in FL specimens (Figure 8c, top panel). Tonsil tissues were stained as a control. We found that CD70 was brightly stained in cells within follicles (B cell region) as well as cells from interfollicle areas (T cell region). In contrast, CD70 staining in tonsil was modest with dim staining of a few cells within follicles. To further topographically identify CD70+ T cells in FL tissue, we sequentially stained the same tissue section for CD70, CD20 and CD3 and visualized co-expression of these antigens using a novel method devised by Glass and colleagues (32). The resulting images captured were overlaid and each antigen assigned a color using Adobe Photoshop CS2 (Adobe Systems, Inc.; San Jose, CA). 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+ T cells had an impact on patient outcome, we performed a Kaplan-Meier analysis to correlate the number of intratumoral CD70+ T cells at diagnosis with overall survival in a cohort of follicular lymphoma patients who all received chemotherapy and rituximab during their disease course. Using the median of 11% of CD70+ T cells as a cutoff, we found that higher numbers of CD70+ T cells were significantly associated with a shorter overall survival in FL patients (Figure 8d).

Figure 8. The frequency of CD70+ T cells is associated with a poor outcome in FL patients.

Figure 8

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(a) Representative dot plots showing CD70 expression on CD3+ cells from biopsy specimens of individuals with follicular hyperplasia (NM) or FL patients. (b) A summary of frequency of CD70-expressing T cells from biopsy specimens of individuals with reactive follicular hyperplasia (NM) or FL patients. CD70+CD3+ T cells were measured by flow cytometry. (c) Top panel, a representative image from one of five FL patient samples showing CD70 expression measured by immunohistochemistry. Tonsil tissue was used as a control. Bottom, a representative image from a FL patient sample showing co-expression of CD70 (red), CD20 (yellow) and CD3 (blue) measured by immunohistochemistry. The method involves sequential staining of the same tissue section, decolorizing and antigen-antibody dissociation of each antibody used. Slides initially positive for CD70 were co-stained stained with CD3 and then CD20. (d) Kaplan-Meier curve for overall survival of FL patients treated with chemoimmunotherapy (n=32) by the number of CD3+CD70+ T cells with a cutoff of 11%.

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-β, 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 up-regulate CD70 expression on CD8+ T cells in patients with metastatic melanoma(33).

We have shown that the Smad signaling pathway is required, and that Stat5 signaling also plays an important role although the predominant effect of IL-2/Stat5 signaling was seen in naïve T cells. This is in agreement with the finding that Stat5 signaling is essential for TGF-β-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 employ unique signaling pathways when responding to TGF-β. Supporting this, we found that TGF-β 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-β-mediated CD70 expression on naïve T cells.

It was previously unclear how CD70 acquisition affects T cell function. In this study, we observed that both TGF-β-induced and preexisting intratumoral CD70+ T cells displayed phenotypic and functional changes similar to that in T-cell exhaustion. For example, CD70+ T cells had higher PD-1 and TIM-3 expression comparing to CD70− T cells. The function of these cells was suppressed and they exhibited reduced cytokine production and impaired signal transduction. Previous studies have suggested that TGF-β may induce T-cell exhaustion that leads to decreased T-cell proliferation and function (25-27). However, the cell population responsible for this effect was unknown. The results we present in this study clearly show that CD70 upregulation is associated with TGF-β-induced exhaustion of Tem cells.

While TGF-β is known to induce apoptosis in a variety of types of cells, TGF-β-mediated apoptosis in T cells is only observed after extended culture. Studies have suggested that co-stimulation via CD28 plays a crucial role in inhibiting TGF-β-induced apoptosis in T cells(34, 35) and may contribute to this late-onset apoptosis. We provide evidence that CD70 induction is responsible for this delayed TGF-β-mediated apoptosis. These exhausted CD70+ T cells acquire more pro-apoptotic markers such as caspase-3 than CD70− T cells and are more susceptible to activation-induced cell death.

Although we show that CD70+ T cells are the major cell population contributing to TGF-β-mediated effects, it is unknown whether CD27/CD70 interaction plays a role in TGF-β-induced T-cell exhaustion. Previous studies have shown that excessive or chronic CD27-CD70 signaling can lead to exhaustion in a murine model and results in impaired function of CD8+ T cells (15, 36, 37). Our finding that TGF-β-mediated downregulation of surface CD27 on CD70+ T cells is due to shedding of CD27, creates a model whereby CD70 expression on T cells leads to stimulation (or overstimulation) of neighboring T cells (or auto-stimulation) through CD27 at which point CD27 is cleaved from the cell surface. We observed that blocking CD70 reverses CD27 loss on the cell surface and restores cell function, including IFN-γ production and cell viability, confirming the role of CD27/CD70 interaction in TGF-β-mediated T-cell exhaustion.

The role of CD70-CD27 interaction in tumors is controversial. While numerous studies suggest that CD70-CD27 interaction improves antitumor immunity, many other reports find the opposite. It has been shown that CD27 ligation by CD70-expressing tumor cells (38, 39), genetically modified tumor cells expressing CD70 (40-43), soluble CD70 (44-46) or agonistic anti-CD27 antibodies (47-49) induces an antitumor response by activating CTLs and enhancing tumor specific cytotoxicity. In contrast, it has been suggested that the CD70-CD27 interaction may favor immune escape through the development of intratumoral Treg cells (50, 51), the promotion of lymphocyte apoptosis (48, 52, 53) or the depletion of NK cell (36, 54). In B-cell malignancies, this discrepancy remains. While CD70-CD27 interaction prevents T-cell anergy and enhances cytotoxic activity of tumor-specific CTL in acute lymphoblastic leukemia (39), high CD70 expression levels correlate to shorter overall survival in both the GCB and ABC subtypes in DLBCL (55). Our data suggest that when T cells acquire CD70 expression, they become exhausted cells and their function is impaired, which correlates with an inferior clinical outcome in follicular lymphoma patients.

In summary, CD70+ T cells represent a subpopulation of immune cells that is functionally exhausted, and the presence of intratumoral CD70+ T cells in follicular lymphoma impacts patient outcome. We, for the first time, identify TGF-β as the cytokine responsible for upregulation of CD70 expression on T cells thereby inducing T-cell exhaustion via CD27-CD70 signaling. These findings describe a novel mechanism to account for TGF-β-mediated suppressive effects on T cells in follicular lymphoma patients.

Supplementary Material

Supplemental Fig 1
Supplemental Fig 2
Supplemental Fig 3
Supplemental Fig 4
Supplemental Fig 5
Supplemental Fig 6
Supplemental Table 1

Acknowledgments

Supported in part by grants from the National Institutes of Health (P50 CA97274), the Lymphoma Research Foundation, the Leukemia & Lymphoma Society and the Predolin Foundation.

Footnotes

Conflict-of-interest disclosure: The authors declare no conflict of interest.

Supplementary information is available at Leukemia’s website.

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Associated Data

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Supplementary Materials

Supplemental Fig 1
NIHMS574897-supplement-Supplemental_Fig_1.tif (207.6KB, tif)
Supplemental Fig 2
NIHMS574897-supplement-Supplemental_Fig_2.tif (331.4KB, tif)
Supplemental Fig 3
NIHMS574897-supplement-Supplemental_Fig_3.tif (349.2KB, tif)
Supplemental Fig 4
NIHMS574897-supplement-Supplemental_Fig_4.tif (641.8KB, tif)
Supplemental Fig 5
NIHMS574897-supplement-Supplemental_Fig_5.tif (867.9KB, tif)
Supplemental Fig 6
NIHMS574897-supplement-Supplemental_Fig_6.tif (290.8KB, tif)
Supplemental Table 1
NIHMS574897-supplement-Supplemental_Table_1.docx (36.2KB, docx)

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