TNFR2 is predominantly expressed by a subset of human and mouse CD4+CD25+FoxP3+ T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2+ Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30–40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2 Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4+CD25TNFR2+ cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2+ Tregs was considerably more potent than that of reportedly highly suppressive CD103+ Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2+ Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors.

Tumor necrosis factor is a pleiotropic cytokine that is a major participant in the initiation and orchestration of inflammation and immunity (1). TNF is also implicated in inflammation-associated cancers produced either by tumor cells and/or by infiltrating leukocytes (2). TNF mediates its biological functions through its receptors, TNFR1 and TNFR2. TNFR1, through its death domain, mediates the cytotoxic effect whereas TNFR2 is largely confined to cells of the immune system and its biological role is still not fully understood (1). TNF has been shown to have proinflammatory effects; nevertheless, increasing evidence reveals that TNF also has unexpected immunosuppressive effects and that these may be based on the capacity of TNF in concert with IL-2 to activate and expand mouse CD4+CD25+ T regulatory cells (Tregs)3 (3) presumably by interacting with TNFR2, which is preferentially expressed by both human and mouse Tregs (3, 4). Thus, up-regulation of Treg activity by TNF may mediate the delayed immunosuppressive effect of TNF on inflammatory responses and the expansion of Tregs in the tumor microenvironment. Consequently, we hypothesized that TNFR2 expression may identify the more functionally active Tregs in tumor-infiltrating lymphocytes (TILs).

Female wild-type C57BL/6 mice, 8- to 12-wk old, were provided by the Animal Production Area of the National Cancer Institute (NCI) (Frederick, MD). NCI-Frederick is accredited by American Association for the Accreditation of Laboratory Animal Care International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” published by the National Research Council (5). The C57BL/6-derived Lewis lung carcinoma (LLC) cell line was obtained from American Type Culture Collection. Abs purchased from BD Biosciences consisted of anti-CD3 (clone 145-2C11), CD4 (clone GK1.5), anti-CD25 (clone PC61), CD45RB (clone 16A), CD62L (clone MEL-14), CD44 (clone IM4), CD69 (clone H1.2F3), CD103 (clone M290), TNFR2 (clone TR75-89), CTLA4 (clone UC10-4F10–11) and CD16/CD32 (clone 2.4G2). Anti CD3e (clone 145-2C11), Foxp3 staining set (clone FJK-16s), and glucocorticoid-induced TNFR family-related protein (GITR) (clone DTA-1) Abs were purchased from eBioscience.

C57BL/6 mice were inoculated s.c. in the right rear flank with 100,000 LLC cells. After 2 wk, tumors were excised, minced, and digested in RPMI 1640 supplemented with 1 mg/ml collagenase IV and 0.1 mg/ml DNase I.

CD4 subsets were purified from spleen and lymph nodes (LNs; inguinal, axillary, and mesenteric regions) or TILs with a Cytomation MoFlo cytometer, yielding a purity of ∼98% for both subsets. For in vitro assays of the inhibition of proliferation by Treg, CFSE-labeled (2 μM; 8 min at room temperature) responder cells (5 × 104 cells/well) were seeded in a U-bottom 96-well plate with 2 × 105 cells/well APCs (T cell-depleted, irradiated spleen cells) plus 0.5 μg/ml functional grade anti-CD3e Ab. Subsets of CD4 cells were added to the wells at the desired ratio. After 48 h, CFSE dilution was determined with FACS.

After blocking the Fc receptor, cells were incubated with appropriately diluted Abs. Data were acquired on a FACSort device (BD Bioscience) and data analysis was conducted using CellQuest software (BD Biosciences).

As shown for normal C57BL/6 mice in Fig. 1,A, the expression of TNFR2 was largely restricted to splenic CD4+ cells. Similar results were observed by LN cells (data not shown). CD4+CD25+ T cells were the primary cell type expressing TNFR2, while fewer CD4+CD25 T cells expressed this receptor (Fig. 1,B). The proportions of CD25+TNFR2+ cells, CD25+TNFR2 cells, and CD25TNFR2+ splenic CD4 cells were 3.6, 6.1, and 2.8%, respectively (Fig. 1 C).

FIGURE 1.

Phenotypic characterization and distribution of normal C57BL/6 mouse CD4+CD25+TNFR2+ Tregs. A, Splenocytes were stained with anti-CD4 and -TNFR2 Abs and analyzed by FACS. B, Splenocytes were stained with anti-CD3, -CD4, -CD25 and -TNFR2 Abs. The expression of TNFR2 by CD4+CD25+ cells (gray histogram) and CD4+CD25 cells (solid line histogram) was analyzed by FACS, gating on CD3+ cells. Dashed line represents isotype control. C, Splenocytes were stained with anti-CD3, -CD4, -CD25, and -TNFR2. Expression of CD25 and TNFR2 was analyzed with FACS by gating on CD3+CD4+ cells. D, Splenocytes were stained with anti-CD4, -CD25, -TNFR2, and -CD45RB Abs or anti-CD62L, -CD44, -CD69, -CD103, -GITR, -CTLA-4, or -FoxP3 Abs. CTLA-4 and FoxP3 were stained intracellularly. Expression of phenotypic markers was analyzed with FACS by gating on indicated CD4 subsets. E, Cells from the indicated lymphoid tissues and peripheral blood were stained with anti-CD3 (or -CD8 for thymocytes), -CD4, -CD25 and -TNFR2 Abs. The percentage of TNFR2+ cells was analyzed with FACS by gating on a CD3+CD4+CD25+ population (except for thymocytes which were gated on CD8CD4+CD25+ cells). Error bars indicate SE derived from three mice (n = 3). Numbers in the quadrants represent the percentages of positive staining cells (%). The numbers in the histograms show the percentages of positive cells (%) and mean fluorescence intensity. Data shown are representatives of at least three separate experiments with similar results.

FIGURE 1.

Phenotypic characterization and distribution of normal C57BL/6 mouse CD4+CD25+TNFR2+ Tregs. A, Splenocytes were stained with anti-CD4 and -TNFR2 Abs and analyzed by FACS. B, Splenocytes were stained with anti-CD3, -CD4, -CD25 and -TNFR2 Abs. The expression of TNFR2 by CD4+CD25+ cells (gray histogram) and CD4+CD25 cells (solid line histogram) was analyzed by FACS, gating on CD3+ cells. Dashed line represents isotype control. C, Splenocytes were stained with anti-CD3, -CD4, -CD25, and -TNFR2. Expression of CD25 and TNFR2 was analyzed with FACS by gating on CD3+CD4+ cells. D, Splenocytes were stained with anti-CD4, -CD25, -TNFR2, and -CD45RB Abs or anti-CD62L, -CD44, -CD69, -CD103, -GITR, -CTLA-4, or -FoxP3 Abs. CTLA-4 and FoxP3 were stained intracellularly. Expression of phenotypic markers was analyzed with FACS by gating on indicated CD4 subsets. E, Cells from the indicated lymphoid tissues and peripheral blood were stained with anti-CD3 (or -CD8 for thymocytes), -CD4, -CD25 and -TNFR2 Abs. The percentage of TNFR2+ cells was analyzed with FACS by gating on a CD3+CD4+CD25+ population (except for thymocytes which were gated on CD8CD4+CD25+ cells). Error bars indicate SE derived from three mice (n = 3). Numbers in the quadrants represent the percentages of positive staining cells (%). The numbers in the histograms show the percentages of positive cells (%) and mean fluorescence intensity. Data shown are representatives of at least three separate experiments with similar results.

Close modal

Tregs are heterogeneous and consist of activated/memory as well as naive cells. As shown in Fig. 1,D, TNFR2+ Treg cells were CD45RBlow, CD62Llow, and CD44high and expressed relatively high levels of CD69, CD103, GITR and CTLA-4, indicative of the memory and activated phenotype. In contrast, naive Tregs were CD45RBhigh, CD62Lhigh, and CD44low and expressed relative low levels of CD69, CD103, GITR, and CTLA-4 and were TNFR2. CD4+CD25TNFR2+ cells contained fewer CD45RBhigh and CD62Lhigh and more CD44high and CD69+ cells compared with CD4+CD25+TNFR2 cells. Furthermore, TNFR2 showed a closer relationship than CD25 to the subset of memory and activated mouse peripheral FoxP3+ Treg cells. Both TNFR2+ and TNFR2 subsets of CD4+CD25+ cells expressed comparably high levels of FoxP3. However, the intensity of FoxP3 expression was consistently higher in CD4+CD25+TNFR2+ cells than in CD4+CD25+TNFR2 cells. Although only ∼1% of CD4+CD25TNFR2 cells were FoxP3+ cells, 25∼40% of CD4+CD25TNFR2+ cells expressed FoxP3 (Fig. 1 D).

In normal C57BL/6 mice, ∼80% of CD8CD4+CD25+ thymocytes were TNFR2+, which resembles the reported high TNFR2 expression by human thymic Tregs (4). In peripheral LNs, spleen, and Peyer’s patches, 30∼40% of CD4+CD25+ cells expressed TNFR2. In contrast, in mouse peripheral blood <10% of CD4+CD25+ cells were TNFR2+ (Fig. 1 E).

Upon stimulation with APC and anti-CD3, only CD4+CD25TNFR2 T cells were readily activated to proliferate and produce IFN-γ, whereas CD4+CD25+TNFR2+ and CD4+CD25+TNFR2 cells were unreactive. Interestingly, TNFR2-expressing CD4+CD25 T cells were also nonresponsive to TCR stimulation (data not shown).

Next, we examined the suppressive potential of these CD4 subsets. Flow-sorted CD4 subsets were cocultured as shown in Fig. 2,A. As we expected, CD4+CD25+TNFR2+ T cells were potent suppressor cells and inhibited the replication of responder CD4+CD25TNFR2 T cells. Surprisingly, CD4+CD25+TNFR2 T cells, comprising the majority of classical Tregs (60–70% of CD4+CD25+FoxP3+ T cells), had only minimal suppressive activity. Although not usually considered to be Tregs, TNFR2-expressing CD4+CD25 T cells had moderate and consistently more potent suppressive capacity than CD4+CD25+TNFR2 cells. Due to the technical difficulty of sorting FoxP3+ cells from normal C57BL/6 mice, we have not examined whether FoxP3+ cells present in CD4+CD25TNFR2+ subset are solely responsible for the suppressive activity. We very carefully repeated this experiment and obtained highly consistent results (Fig. 2 B). Furthermore, BALB/c mouse TNFR2+ Tregs also had a memory/activated phenotype and were up to10-fold more suppressive than TNFR2-Tregs (data not shown).

FIGURE 2.

CD4+CD25+TNFR2+ T cells were highly potent suppressor cells. Flow-sorted CD4+CD25TNFR2 or CD4+CD25 T cells (5 × 104 cells/well) were labeled with CFSE and cultured alone or cocultured with the indicated numbers or ratios of flow-sorted CD4 subsets from spleen and LNs of normal C57BL/6 mice. The percentages of CFSE-diluted cells are shown in the histograms. A, C, and D show a representative data of at least three separate experiments with similar results and B shows a summary of percentage inhibition of replication (%) from six separate experiments.

FIGURE 2.

CD4+CD25+TNFR2+ T cells were highly potent suppressor cells. Flow-sorted CD4+CD25TNFR2 or CD4+CD25 T cells (5 × 104 cells/well) were labeled with CFSE and cultured alone or cocultured with the indicated numbers or ratios of flow-sorted CD4 subsets from spleen and LNs of normal C57BL/6 mice. The percentages of CFSE-diluted cells are shown in the histograms. A, C, and D show a representative data of at least three separate experiments with similar results and B shows a summary of percentage inhibition of replication (%) from six separate experiments.

Close modal

It has been reported that CD103 expression can define the most potent suppressive subset of CD4+CD25+ Tregs (6). Because CD4+CD25+TNFR2+ T cells expressed the highest level of CD103, it is possible that the expression of CD103 may more accurately predict the suppressive potential of CD4 subsets. We confirmed the previous report (6) that CD4+CD25+CD103+ T cells were more potent suppressors than CD4+CD25+CD103 T cells (data not shown). However, the inhibition of proliferation by CD103+ Tregs was significantly less potent than that by TNFR2+ Tregs, as shown by both CFSE dilution assay (Fig. 2,C) and [3H]thymidine incorporation assay and also in the inhibition of cytokine production (p < 0.01∼0.05; data not shown). Furthermore, CD4+TNFR2+ T cells exhibited more suppressive effects than CD4+CD103+ T cells, and both of these subsets were more suppressive than the more heterogeneous CD4+CD25+ T cells (Fig. 2 D). Consequently, TNFR2 is superior to both CD103 and CD25 in defining functional CD4 T suppressor cells.

Tregs accumulate intratumorally and promote tumor growth by inhibiting antitumor immune response (7). Based on reports that TNFR2 is expressed by TILs isolated from several human solid tumors (8), we hypothesized that TNFR2+ Tregs with enhanced suppressive potential may be more prevalent in the tumor microenvironment. For this purpose, we examined the C57BL/6 mouse LLC tumor model. As predicted, 75∼100% of CD4+CD25+ TILs expressed TNFR2. The proportion of TNFR2+ cells present in the subset of CD4+CD25 TILs was also increased over the low levels present in splenic and LN cells (Fig. 3 A). Similar results were also observed in the BALB/c mouse 4T1 breast tumor model (data not shown).

FIGURE 3.

Highly suppressive TNFR2+ Tregs accumulated in the TILs of the LLC mouse model. A, LN cells, spleen cells (SPL), and TILs from LLC tumor-bearing mice were stained with CD4, CD25 and TNFR2. Expression of TNFR2 was analyzed with FACS by gating on CD4+CD25+ or CD4+CD25 cells from LLC bearing mouse TIL (LLC TIL), spleen (LLC SPL), or LNs (LLC LN) or from tumor-free C57BL/6 mouse spleen (Ctrl SPL) or LNs (Ctrl LN). B and C, CD4+CD25 T cells from normal control mouse LNs (5 × 104 cells/well) were labeled with CFSE and cultured alone or cocultured with the indicated source of Tregs at the desired ration. The percentage of CFSE-diluted cells was shown in the histograms. Data shown are representatives of three separate experiments with similar results.

FIGURE 3.

Highly suppressive TNFR2+ Tregs accumulated in the TILs of the LLC mouse model. A, LN cells, spleen cells (SPL), and TILs from LLC tumor-bearing mice were stained with CD4, CD25 and TNFR2. Expression of TNFR2 was analyzed with FACS by gating on CD4+CD25+ or CD4+CD25 cells from LLC bearing mouse TIL (LLC TIL), spleen (LLC SPL), or LNs (LLC LN) or from tumor-free C57BL/6 mouse spleen (Ctrl SPL) or LNs (Ctrl LN). B and C, CD4+CD25 T cells from normal control mouse LNs (5 × 104 cells/well) were labeled with CFSE and cultured alone or cocultured with the indicated source of Tregs at the desired ration. The percentage of CFSE-diluted cells was shown in the histograms. Data shown are representatives of three separate experiments with similar results.

Close modal

We next tested the prediction that the inhibitory activity of LLC CD4+CD25+ TILs should be greater than that of peripheral LN CD4+CD25+ cells. As shown in Fig. 3,B, there was no difference in the suppressive potency of Tregs over various ratios of T effector (Teff) to Treg cells from peripheral LNs of tumor-bearing and control mice. In contrast, TIL CD4+CD25+ cells retrieved from LLC tumors exhibited far more potent suppressive activity than LN CD4+CD25+ cells from tumor-free mice (Fig. 3,C). Thus, the suppressive effects of peripheral LN Tregs and TIL Tregs correlated well with their levels of TNFR2 expression. As shown in Fig. 3, B and C, the potent inhibitory effect exerted by CD4+CD25+ TILs was not Ag specific because both targeted responder cells and APCs were from tumor-free mice. The phenotype of TIL CD4+CD25+ cells, which were 75∼100% TNFR2+, resembled that of normal mouse LN TNFR2+ Tregs and were indicative of an activated/memory subset (data not shown).

Although other factors in the tumor microenvironment may also contribute to the activation of TIL Tregs, the presence of TNF in the inflammatory tumor site is most likely to contribute to up-regulating the number of functional Tregs by activating TNFR2 (3). It has been reported that TGFβ, a cytokine crucial for de novo generation of Tregs, is also able to induce TNF and TNFR2 expression on CD4 cells (9) and may therefore be responsible for the increase of TNFR2+ Tregs in the tumor. TNFR2 has also been reported to be a costimulator for Ag-driven T cell responses (1); thus, this receptor can also serve as a costimulator for the response of Treg to tumor Ag. Indeed, it has been shown that anti-TNF as well as anti-TNFR2 Abs can inhibit proliferation of TNFR2-expressing TILs isolated from human solid tumors (8). Despite reports that anti-TNF therapy increases Treg activity in rheumatoid arthritis (10), our preliminary observations also indicate that anti-TNF therapy actually decreased the proportion of FoxP3+ Tregs in TILs of various mouse tumor models (our unpublished data). Thus blockade of TNF-TNFR2 interaction may result in the down-regulation of Treg activity and this may provide the mechanistic basis for the reported therapeutic efficacy of anti-TNF therapy in cancer (2).

Several lines of evidence suggest that immunosuppressive action of TNF is mediated by TNFR2. For example, in the experimental autoimmune encephalomyelitis (EAE) mouse model TNFR1-deficient mice were completely resistant to the induction of disease, whereas TNFR2-deficient mice exhibited more severe EAE (11). The immunosuppressive action of TNFR2-dependent effects of TNF is most compatible with our observations that TNF is a potent and selective activator of Tregs (3).

It was reported that CD103 expression defines a potent suppressive subset of Tregs (6) and that CCR6 expression defines an effector/memory-like subset of Tregs (12). Expression of CD103 partially correlated with TNFR2 expression by Tregs (Fig. 1 D); however, the reported low thymic level of CD103+ Tregs (6) as well as the high level in peripheral blood of CCR6+ Tregs (12) is distinct from TNFR2+ Tregs in distribution. Furthermore, CD103 (integrin αEβ7) serves as a homing receptor for mucosa-seeking populations and has no clear relationship to the suppressive function of Tregs (6). In addition, whereas CD103 is only expressed by mouse Tregs, TNFR2 is also expressed by human Tregs (4) and correlates with their greater suppressive effects (our unpublished data). CCR6 is expressed by both memory type Tregs as well as Teff cells and serves to direct trafficking of CCR6+ cells to inflammatory sites. Although CCR6+ Tregs were described as effector/memory-like cells, CCR6+ Tregs and CCR6 Tregs do not show any differences in suppressive activity (12). Unlike CD103 and CCR6, TNFR2 is a functional receptor for TNF and therefore is likely to respond to TNF by activating and expanding Tregs at inflammatory sites (3). Nevertheless, TNFR2 knockout mice have normal functional CD4+CD25+ Tregs. Thus TNFR2 is not necessary to maintain Treg activity but may play a crucial role in the activation and expansion of Tregs at inflammatory or tumor sites. Presumably, this function of TNFR2 reflects down-regulatory effect of TNF, but not IL-1 and IL-6, on the inflammatory response (3).

Foxp3 is reported as both a specific marker and a commitment factor for the Treg cell lineage (7). Both TNFR2+ and TNFR2 CD4+CD25+ T cells from normal C57BL/6 mice expressed comparable high levels of FoxP3. Nevertheless, the suppressive function of FoxP3+TNFR2 cells is not appropriately “turned on” or intrinsically deficient. Furthermore, CD4+CD25TNFR2+ cells were more suppressive than CD4+CD25+TNFR2 cells, even though the former contained much fewer FoxP3+ cells than the latter. Thus, FoxP3 expression by resting mouse CD4 cells may not be sufficient to confer suppressive capacity. Our data suggest that the expression of TNFR2 is more relevant to a suppressive phenotype than the expression of CD25 and even FoxP3.

Apparently, TGFβ and IL-10 did not contribute to the in vitro suppressive activity of TNFR2+ Tregs, because neutralizing Abs against TGFβ and IL-10 failed to attenuate the activity of TNFR2+ Tregs (data not shown). Soluble TNFR2 potentially shed by TNFR2+ Tregs is also unlikely to influence Treg activity, because the level of soluble TNFR2 in the supernatant of cocultures containing TNFR2+ Tregs was not increased in our in vitro Treg function assay (48 h; data not shown). CTLA4 expression is only detected in the Treg compartment of CD4 cells in normal mice and its expression is implicated in the suppressive function of Tregs (7). This suppressive molecule was expressed by >80% of TNFR2+ Tregs, which is in agreement with the potent suppressive capacity of this subset of Tregs. Intriguingly, the superior suppressive activity of CD4+CD25TNFR2+ cells over CD4+CD25+TNFR2 cells correlates directly with the intensity of CTLA4 expression by these two subsets (Fig. 1 D). TNFR2, just like CTLA4, is selectively expressed by functional Tregs rather than Teffs, even in the absence of CD25 and FoxP3.

Taken together, our data demonstrate that TNFR2 expression defines a unique subset of mouse Tregs with an activated/memory phenotype and highly potent suppressive activity. In mouse tumor models, the proportion of tumor-infiltrating suppressive TNFR2+ Tregs was dramatically increased, suggesting therapeutic value by targeting TNFR2+ Tregs in tumor immunotherapy.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health (NIH), under contract N01-CO-12400. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Additional funding was provided by a Deutsche Forschungsgemeinschaft Grant Ma760/16-1. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government.

3

Abbreviations used in this paper: Treg, CD4+CD25+ T regulatory cell; GITR, glucocorticoid-induced TNFR family-related protein; LLC, Lewis lung carcinoma; LN, lymph node; Teff, CD4+CD25 T effector cell; TIL, tumor-infiltrating lymphocyte.

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