Induction of Inflammatory Mediators Regulatory T Cells Inhibits the +

Cutting Edge: TNFR-Shedding by CD4+ CD25+ Regulatory T Cells Inhibits the Induction of Inflammatory Mediators

This information is current as Geertje J. D. van Mierlo, Hans U. Scherer, Marjolijn of September 24, 2021. Hameetman, Mary E. Morgan, Roelof Flierman, Tom W. J. Huizinga and René E. M. Toes J Immunol 2008; 180:2747-2751; ; doi: 10.4049/jimmunol.180.5.2747

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE

JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: TNFR-Shedding by CD4؉CD25؉ Regulatory T Cells Inhibits the Induction of Inflammatory Mediators1 Geertje J. D. van Mierlo,2* Hans U. Scherer,2* Marjolijn Hameetman,2* Mary E. Morgan,* Roelof Flierman,*† Tom W. J. Huizinga,* and Rene´E. M. Toes3* ؉ ؉ CD4 CD25 regulatory T (Treg) cells play an essential and a mechanism that depends on CTLA-4 and membrane- role in maintaining tolerance to self and nonself. In sev- bound TGF-␤ (5, 6). eral models of T cell-mediated (auto) immunity, Treg cells We previously showed that adoptive transfer of Treg cells de- creases levels of acute phase proteins such as the serum amyloid exert protective effects by the inhibition of pathogenic Downloaded from T cell responses. In addition, Treg cells can modulate T P component in mice that had been injected with CFA (our cell-independent inflammation. We now show that unpublished observations) or had underwent total body irradi- ؉ ؉ CD4 CD25 Treg cells are able to shed large amounts of ation (7). For that reason, we hypothesized that Treg cells shed a soluble mediator that can inhibit the induction of acute phase TNFRII. This is paralleled by their ability to inhibit the ␣ action of TNF-␣ both in vitro and in vivo. In vivo, Treg responses. TNF- is one of the most prominent initiators of the cells suppressed IL-6 production in response to LPS injec- acute phase reaction that can, via the action of IL-6, promote http://www.jimmunol.org/ tion in mice. In contrast, Treg cells from TNFRII-defi- the release of several acute phase proteins from the liver (8–10). In this study, we describe a novel mechanism by which Treg cient mice were unable to do so despite their unhampered cells can counteract the action mediated by TNF-␣. capacity to suppress T cell proliferation in a conventional in vitro suppression assay. Thus, shedding of TNFRII represents a novel mechanism by which Treg cells can Materials and Methods inhibit the action of TNF, a pivotal cytokine driving Mice inflammation. The Journal of Immunology, 2008, 180: C57BL/6 mice and TNFRII knockout (KO) mice on a C57BL/6 background 2747–2751. (B6.129S2-Tnfrsf1btm1Mwm/J) were maintained at Leiden University Medical by guest on September 24, 2021 Center animal facility in accordance with national legislation under the super- vision of the University’s animal experimental committee. urrent understanding of the basic processes that control immune tolerance has been fueled by identifica- Isolation of murine Treg cells and culture ϩ ϩ 4 tion of CD4 CD25 regulatory T (Treg) cells as an Murine CD4ϩCD25ϩ and CD4ϩCD25Ϫ T cells were isolated from spleen C ϩ ϩ ϩ important component of self-tolerance. CD4 CD25 T cells and lymph nodes of 6- to 14-wk-old mice by positive selection of CD4 T cells have been shown to regulate peripheral self-tolerance, protect (MACS), fluorescent labeling (anti-CD4 anti-CD25), and subsequent FACS sorting (FACSAria cell sorter; BD Biosciences) on the basis of CD25 expres- against autoimmunity, and suppress immune responses to au- sion. Purified T cell subsets were activated in the presence of Dynabeads mouse toantigens, alloantigens, tumor Ags, and infectious agents CD3/CD28 (Dynal Biotech) and 50 IU/ml IL-2. (1–4). Despite accumulating evidence for the immunoregulatory Isolation of human Treg cells and culture ϩ ϩ properties of CD4 CD25 Treg cells, the mechanism by ϩ high ϩ Ϫ ϩ ϩ Isolation of human CD4 CD25 or CD4 CD25 T cells from buffy coats which CD4 CD25 Treg cells inhibit T cell-independent in- of healthy human donors was performed as previously described (11). FACS- ϩ ϩ ϩ ϩ Ϫ flammation is not well defined. CD4 CD25 Treg cells are sorted CD4 CD25high and CD4 CD25 cells were cultured in the presence ␮ ␮ anergic to TCR stimulation in vitro and capable of inhibiting of 1 g/ml anti-CD28 (CLB-CD28/1; Sanquin), 5 g/ml plate-bound anti- CD3 (OKT-3, BD Biosciences), and 100 U/ml IL-2 for up to 5 days. The proliferation and cytokine production of other T cells by secre- metalloproteinase inhibitor marimastat was added to cultures where indicated tion of anti-inflammatory cytokines (e.g., IL-10 and TGF-␤) at a final concentration of 10 ␮g/ml.

*Department of Rheumatology and †Department of Nephrology, Leiden University Med- 2 G.J.D.v.M., H.U.S., and M.H. contributed equally to this work. ical Center, Leiden, The Netherlands 3 Address correspondence and reprint requests to Dr. Rene´E. M. Toes, Department of Received for publication November 20, 2007. Accepted for publication December Rheumatology, Postal Zone C1-R, Leiden University Medical Center, P.O. Box 9600, 28, 2007. 2300 RC, Leiden, The Netherlands. E-mail address: [email protected] ϩ ϩ The costs of publication of this article were defrayed in part by the payment of page charges. 4 Abbreviations used in this paper: Treg cell, CD4 CD25 regulatory T (cell); KO, This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. knockout; MFI, mean fluorescence intensity; sTNFR, soluble TNF-receptor; Teff, effector Section 1734 solely to indicate this fact. T (cell); WT, wild type.

1 This study was supported by Dutch Arthritis Foundation Grant 02-I-402 and by research Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 funding from the European Community FP6 funding project 018661 Autocure. The work of R.E.M.T. was supported by a Vidi Grant from the Netherlands Organization for Sci- entific Research. H.U.S. was supported by a Pfizer Articulum Fellowship.

www.jimmunol.org 2748 CUTTING EDGE: TNFRII SHEDDING BY REGULATORY T CELLS

Although sTNFR were also detected in the cultures of acti- ϩ Ϫ vated CD4 CD25 cells, this T cell subset produced far fewer ϩ Ϫ sTNFR. CD4 CD25 T cells proliferate more vigorously than ϩ ϩ CD4 CD25 Treg cell populations, resulting in 8–10 times higher cell numbers after 6 days of culture. When corrected for ϩ ϩ cell numbers, CD4 CD25 Treg cells produced ϳ50 times ϩ Ϫ more sTNFR than their CD4 CD25 counterparts on a per cell basis (data not shown). FIGURE 1. CD4ϩCD25ϩ T cells shed TNFRII. Levels of soluble TNFRII ϩ ϩ in cell culture supernatants are shown. CD4ϩCD25ϩ and CD4ϩCD25Ϫ cells CD4 CD25 Treg cell-derived sTNFRII inhibits the action of were cultured with anti-CD3/anti-CD28 coated beads and IL-2. Supernatant TNF-␣ in vitro p ϭ ,ءء ;p Ͻ 0.05 ,ء) was analyzed for the presence of TNFRII by ELISA 0.0001). Depicted is one representative experiment of three. We next wished to examine the biologic activity of Treg cell- derived sTNFRII. For this purpose we performed a bioassay using TNF-␣-sensitive WEHI cells (12). Survival of WEHI cells Suppression assay was measured after incubation with ranging amounts of ϩ Ϫ ϩ ϩ rTNF-␣ in the presence or absence of culture supernatants de- After 3 to 4 days of in vitro activation, CD4 CD25 and CD4 CD25 T ϩ ϩ ϩ Ϫ cells were cultured with equal numbers of freshly isolated splenocytes in the rived from activated CD4 CD25 and CD4 CD25 T cells. 3 ϩ Ϫ presence of 1 ␮g/ml PHA. [ H]Thymidine incorporation of triplicates was Supernatant from CD4 CD25 T cells induced ϳ50% Downloaded from measured 3–4 days later. Suppression assays were performed for each sorted ϩ ϩ WEHI cell death without the addition of rTNF-␣, reflecting population of CD4 CD25 cells to ensure the suppressive capacity of isolated Ϫ increased shedding of TNF-␣ by activated CD25 effector T Treg cells. ϩ ϩ (Teff) cells as compared with CD4 CD25 Treg cells. TNF-␣ Flow cytometry -induced death of WEHI cells was largely prevented, however, Murine cells were stained using mAb against CD4, CD25, CD120b (TR75- when, next to titrated amounts of recombinant TNF-␣, culture ϩ ϩ 89), FoxP3 (FJK-16S, eBioscience), or an isotype control. Human cells were supernatant of CD4 CD25 Treg cells was added to the wells. http://www.jimmunol.org/ stained using mAb against CD4 (RPA-T4), CD25 (2A3), CD120b (MR2-1, (Fig. 2A). To confirm that the inhibition of cell death was in- AbD Serotec and 22235, R&D Systems), CCR7 (3D12), HLA-DR (L243), ϩ ϩ CD45RO (UCHL1), CD45RA (HI100), and CD62L (Dreg 56). Intracellular deed mediated by sTNFRII, we next isolated CD4 CD25 FoxP3-staining was performed using eBioscience FoxP3-staining kit (PCH101 Treg cells from TNFRII KO mice. No inhibition of cell death or appropriate isotype control). Abs were purchased from BD Biosciences un- was observed after the addition of culture supernatants from ac- less otherwise stated. ϩ ϩ tivated CD4 CD25 cells derived from TNFRII KO mice TNFRI and TNFRII secretion (Fig. 2B). Nonetheless, these cells were at least as potent as ϩ ϩ Souble TNFR (sTNFR) I and sTNFRII in culture supernatants were measured CD4 CD25 cells from control WT animals in a conven-

using standard ELISA kits (Hycult Biotechnology for murine sTNFR and tional in vitro T cell suppression assay, indicating that by guest on September 24, 2021 ϩ ϩ R&D Systems DuoSet for human sTNFR). CD4 CD25 cells from TNFRII KO animals were bona fide Bioactivity of sTNFR Treg cells with the ability to suppress Teff cells (Fig. 2C). Ad- dition of etanercept, a soluble TNFRII-Ig fusion protein used In vitro activity of sTNFR was measured using TNF-␣-sensitive WEHI 164 clone 13 cells as previously described (12). In vivo activity was determined by clinically to treat rheumatoid arthritis, had comparable effects injecting mice i.v. with 1 ϫ 106 Treg or control cells (CD4ϩCD25Ϫ T cells) of on the survival of WEHI cells (Fig. 2B). These data indicate either wild-type (WT) animals or TNFRII KO animals after 4 days of in vitro that the ability of Treg cells to inhibit T cell proliferation is un- ␮ activation. As a control, mice were injected i.v. with 250 g of etanercept, a affected in TNFRII KO mice and further show that Treg cell- TNFRII-Ig fusion protein. One hour later mice were injected i.p. with 150 ␮g of LPS (Salmonella typhosa, Sigma-Aldrich). Four and 6 h after LPS injection, derived sTNFRII is functional because it is able to prevent the blood samples were collected to determine serum levels of IL-6 using BD Bio- action of TNF-␣ in vitro. sciences mouse IL-6 ELISA set. CD4ϩCD25ϩ Treg cell-derived sTNFRII modulates LPS-induced Results and Discussion IL-6 production in vivo ϩ ϩ Shedding of sTNFRII by murine CD4 CD25 Treg cells but not by ␣ ϩ Ϫ TNF- modulates the kinetics of IL-6 expression following CD4 CD25 cells LPS injection in mice (13, 14). IL-6, in turn, induces the ex- ϩ ϩ Adoptive transfer of CD4 CD25 Treg cells can inhibit the pression of acute phase proteins. Thus, we reasoned that the induction of acute phase responses in mice (7). Because TNF-␣ reduction of the acute phase response observed previously (7) stimulates acute phase responses (8–10), we hypothesized that could be due to Treg cell-derived sTNFRII. To analyze this ϩ ϩ CD4 CD25 Treg cells could directly inhibit TNF-␣. FACS possibility, we injected mice i.p. with LPS 1 h after injection of ϩ ϩ ϩ ϩ analysis revealed that CD4 CD25 Treg cells, as opposed to CD4 CD25 Treg cells from either WT or TNFRII KO ϩ Ϫ CD4 CD25 T cells, strongly express TNFRII (data not mice. Serum IL-6 levels were analyzed at different time points shown), leading us to predict that Treg cells may be able to shed following LPS injection. ϩ ϩ sTNFRII. Therefore, we activated purified CD4 CD25 and At 4 and 6 h after LPS-injection, treatment with etanercept ϩ Ϫ CD4 CD25 T cell populations in vitro and analyzed super- significantly reduced the amount of IL-6 produced in response natants of these cultures for the presence of sTNFR. Although to LPS, confirming that, indeed, TNF-␣ is involved in the in- no sTNFRI could be observed (data not shown), sTNFRII was duction of IL-6 following LPS injection (Fig. 3). Likewise, ϩ ϩ ϩ detectable in culture supernatants of CD25 cells from day 1 adoptive transfer of CD4 CD25 Treg cells isolated from WT onward (Fig. 1). No TNFR shedding was noted in the presence animals significantly decreased IL-6 production. In contrast, ϩ ϩ of IL-2 only (data not shown), indicating that TCR triggering is CD4 CD25 Treg cells isolated from TNFRII KO animals required for TNFR shedding. lacked this ability, indicating that Treg cell-derived TNFRII is The Journal of Immunology 2749

FIGURE 4. Phenotype of TNFRII-expressing human CD4ϩ T cells. A, Cell surface expression of TNFRII on freshly isolated human PBMC from healthy donors. TNFRII expression is highest on CD4ϩCD25high cells that coexpress FoxP3. B, Phenotypical analysis of CD4ϩTNFRIIϩ (open histo- ϩ Ϫ

gram) vs CD4 TNFRII (filled histogram) T cells. Gating was based on an Downloaded from appropriate isotype control. Data are representative of four independent experiments.

TNFR-shedding by human CD4ϩCD25high T cells Given the potential implications of our findings with murine FIGURE 2. Biological activity of Treg cell-derived sTNFRII. A, sTNFRII Treg for the treatment of TNF-mediated inflammatory disor- http://www.jimmunol.org/ in the supernatant of CD4ϩCD25ϩ T cell cultures can prevent TNF-␣ in- ϩ ϩ ϩ Ϫ ders, we next investigated whether TNFR shedding would also duced cell death. CD4 CD25 and CD4 CD25 cells were activated in vitro be a feature of human Treg. for 4 days. Supernatant (sup) was added to cultures of WEHI cells in the pres- We first analyzed TNFRII-expression on freshly isolated hu- ence of varying amounts of rTNF-␣. Depicted is the percentage of WEHI cells ϩ surviving the culture for 20 h. B, TNFRII shed by Treg cells is required to man CD4 T cells. TNFRII expression was found to be highest ϩ high prevent TNF-␣-induced cell death. CD4ϩCD25ϩ cells from naive WT or on CD4 CD25 T cells but could also be detected on ϩ TNFRII KO mice were isolated and the supernatant of cell cultures was used in CD4 CD25int (where “int” is intermediate) and on a minor ϩ Ϫ ,p Ͻ 0.001; Et., fraction of CD4 CD25 T cells (Fig. 4A). In line with this ,ء) ␣-the WEHI cell bioassay in the presence of 0.5 pg/ml rTNF etanercept). Representative data from 11 independent experiments are shown TNFRII expression was highest on, but not exclusively con- by guest on September 24, 2021 ϩ ϩ ϩ ϩ in A and B. C, CD4 CD25 cells from TNFRII KO and WT mice are equally fined to, CD4 FoxP3 T cells. Phenotypic analysis using capable of suppressing Teff. A conventional T cell suppression assay was performed ϩ ϩ ϩ markers relevant for T cell function revealed that CD4 as control for Treg cell activity using CD4 CD25 T cells from WT or TNFRII ϩ ϩ Ϫ TNFRII T cells were largely CD45RO CD45RA (Fig. KO mice as suppressor cells. Percentages depict the percentages of inhibition of ϩ Ϫ proliferation. Representative data from nine experiments are shown. 4B). Whereas CD4 TNFRII T cells uniformly expressed ϩ CCR7 but no HLA-DR and were mostly CD62Lhigh, CD4 ϩ TNFRII T cells exhibited a more heterogeneous expression of involved in the inhibition of the IL-6 response following injec- these markers. Interestingly, FoxP3 expression was restricted to ϩ ϩ ϩ ϩ high ϩ tion of LPS. Together, these data show that CD4 CD25 TNFRII T cells. Thus, CD4 CD25 FoxP3 T cells that Treg cells can inhibit the action of TNF-␣ and dampen inflam- display both effector and central memory markers (15) consti- mation by releasing sTNFRII. tutively express TNFRII and have in part lost expression of the homing receptors CD62L and CCR7. ϩ Similar to the experiments performed in mice, CD4 ϩ Ϫ CD25high and CD4 CD25 T cells were subsequently purified by FACS sorting and activated in vitro for up to 5 days. TCR stimulation induced strong up-regulation of TNFRII sur- ϩ face expression on CD4 CD25high and, to a lesser extent, on ϩ Ϫ CD4 CD25 T cells during 5 days in culture (Fig. 5A). Cul- tures were performed in the presence or absence of marimastat, an inhibitor of metalloproteinases such as TNF-␣ converting enzyme (TACE, ADAM17), the enzyme responsible for the cleavage of TNFR from the cell surface. Inhibition of TNFR shedding by marimastat led to a strong accumulation of ϩ TNFRII on the cell surface of CD4 CD25high T cells as determined by an increase in mean fluorescence intensity (MFI) of ϩ Ϫ FIGURE 3. Treg cells inhibit LPS-induced IL-6 production through the TNFRII staining. Activation of CD4 CD25 T cells un- TNFR-shedding. CD4ϩCD25ϩ cells were isolated from either WT animals or TNFRII KO animals and activated in vitro for 4 days as described. Cells (1 ϫ der the same conditions, however, led to an only weak increase 106) were injected into mice receiving 150 ␮g of LPS i.p. 1 h later. Serum IL-6 in TNFRII MFI, indicating lower shedding activity. In line levels were determined 4 and 6 h after LPS injection. One representative exper- with this observation, large amounts of sTNFRII were detect- ϩ iment of four is shown (conc., concentration; Et., etanercept). able in culture supernatants of CD4 CD25high T cells, with 2750 CUTTING EDGE: TNFRII SHEDDING BY REGULATORY T CELLS

ϩ tion, as FACS sorting and subsequent activation of CD4 CD25int T cells led to substantially lower amounts of sTNFRII ϩ in culture supernatants than activation of CD4 CD25high T cells (data not shown). Interestingly, surface staining with ϩ HLA-DR of CD4 CD25high T cells from two donors activated ϩ in the presence or absence of marimastat revealed that CD4 ϩ CD25highHLA-DR T cells had substantially higher shedding ϩ Ϫ capacity than CD4 CD25highHLA-DR T cells (Fig. 5D). Within the Treg cell compartment, HLA-DR has previously been described to define a population with enhanced suppressive ability. Our data indicate that the TNFR shedding capacity differs between these two subsets. This might be relevant for the in vivo function of Treg cells and further emphasizes that human Treg cell populations are composed of functionally distinct subsets (16). In summary, we describe in this article a new mechanism of ϩ ϩ action of CD4 CD25 Treg cells. Our results are in line with

recent observations describing that TNF-␣ can transiently si- Downloaded from lence the suppressive activity of Treg cells through signaling via TNFRII (17, 18). Interestingly, suppressive function was found to be restored after several days (18). Our findings showing that TNFRII is shed several days after the activation of Treg cells fit well with these observations, as the shedding of TNFRII would allow Treg cells to counteract the action of TNF-␣, thereby cir- http://www.jimmunol.org/ cumventing its inhibitory effect on Treg cell function. This way, Treg cells could regain their suppressive ability to regulate the function of Teff cells and, at the same time, suppress the effects of TNF-␣, a crucial mediator of acute and chronic inflammation. ϩ ϩ We have previously shown that CD4 CD25 T cells can be used effectively in the treatment of collagen-induced arthritis

FIGURE 5. Characteristics of TNFRII shedding by human (CIA), a model for systemic arthritis in mice (7, 19). Collagen- by guest on September 24, 2021 ϩ CD4 CD25high T cells. A, MFI of TNFRII staining after culture in the pres- induced arthritis is primarily an Ab driven disease (20, 21) and ence or absence of marimastat (M). The inset depicts the increase in MFI on the role of T cells is, most likely, restricted to the provision of marimastat-treated CD25high or CD25Ϫ (CD25neg) cells as the area between help to B cells that produce collagen type II-specific Abs. As (betw.) the respective curves. B, Levels of sTNFRII in supernatants of cells after ϩ ϩ p Ͻ 0.01). C, Levels of sTNFRII produced from day CD4 CD25 Treg cells are able to reduce arthritis severity in ,ءء ;p Ͻ 0.05 ,ء) activation to day (d) adjusted for cell count. Cells were manually counted at each time the effector phase of the disease without affecting circulating point in duplicate. The amount of sTNFRII produced from one day to the next anti-CII Abs, it is likely that the shedding of sTNFR by adop- was divided by the average cell number present in the wells at these time points tively transferred Treg cells is involved in the inhibition of Ϫ ϫ using the formula: (conc. sTNFRII dayx ϩ 1 conc. sTNFRII dayx)/(1/2 arthritis. ϫ 5 ϩ ϫ 5 (cell count ( 10 ) dayx cell count ( 10 ) dayx ϩ 1)) (where “conc.” is con- Finally, our data obtained with human Treg cells indicate p Ͻ 0.05). Results presented in A–C are representative of five ,ء ;centration independent experiments. D, Increase in MFI of TNFRII staining on that the mechanism we show in mice is essentially similar in the CD4ϩCD25highHLA-DRϩ (DRϩ) and CD4ϩCD25highHLA-DRϪ (DRϪ)T human setting. Although constitutive TNFRII expression is ϩ high cells from two separate donors after 5 days of activation in the presence and not confined to Treg cells, human CD4 CD25 T cells ex- absence of marimastat. Increase in MFI of HLA-DRϩ cells was normalized to hibit a much stronger and more sustained shedding activity ϩ Ϫ 100 to account for overall differences in HLA-DR expression levels between upon activation when compared with CD4 CD25 T cells. donors. This is of potential interest in the context of TNF-mediated autoimmune diseases such as rheumatoid arthritis, in which TNFRII-Ig fusion proteins are used effectively in therapeutic ϩ Ϫ much lower levels being produced by CD4 CD25 T cells settings. Together, these data provide a rationale for the thera- (Fig. 5B). sTNFRI was almost undetectable in cultures of both peutic use of Treg cells in systemic autoimmune diseases. cell types (data not shown). The difference in TNFRII levels was even more prominent when adjusting TNFRII levels in supernatants for cell counts, taking into account the stronger ϩ Ϫ Acknowledgments proliferation of CD4 CD25 T cells. In addition, calcula- We thank G. de Roo, R. van der Linden, and M. van der Hoorn for FACS sort tion of the amount of TNFRII shed from day to day revealed purification of T cell subsets. Roeland Hanemaaijer (TNO, Leiden, The Neth- ϩ Ϫ that the shedding activity of CD4 CD25 T cells reached a erlands) provided the metalloproteinase inhibitor marimastat. peak between day 3 and 4, whereas TNFRII-shedding of ϩ CD4 CD25high T cells still increased (Fig. 5C). It is unlikely that the sTNFRII levels determined originate from activated ϩ Disclosures Teff cells contaminating the CD4 CD25high T cell popula- The authors have no financial conflict of interest. The Journal of Immunology 2751

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