Cutting Edge: CD95 Maintains Effector T Cell Homeostasis in Chronic Immune Activation

This information is current as Ramon Arens, Paul A. Baars, Margot Jak, Kiki Tesselaar, of September 24, 2021. Martin van der Valk, Marinus H. J. van Oers and René A. W. van Lier J Immunol 2005; 174:5915-5920; ; doi: 10.4049/jimmunol.174.10.5915

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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 © 2005 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: CD95 Maintains Effector T Cell Homeostasis in Chronic Immune Activation Ramon Arens,1*†‡ Paul A. Baars,* Margot Jak,* Kiki Tesselaar,* Martin van der Valk,§ Marinus H. J. van Oers,† and Rene´A. W. van Lier1*

The elimination of activated T cells is important to main- reports suggested that the death of primed T cells in vivo is tain homeostasis and avoid immunopathology. CD95 independent from the CD95 system (10–13). (Fas/APO-1) has been identified as a death mediator for TNFR-associated factor (TRAF)2 binding TNFR family activated T cells in vitro but the function of CD95 in members, such as OX40, 4–1BB, and CD27, influence T cell death of mature T cells in vivo is still controversial. Here division, survival, and effector function (14). These receptors we show that triggering of the costimulatory TNF can antagonize mitochondrion-dependent cell death by up-reg- Downloaded from family member CD27 sensitized T cells for CD95-in- ulating anti-apoptotic Bcl-2 family molecules such as Bcl-xL duced . CD95-deficient (lpr/lpr) T cells mas- and Bfl (Refs. 15 and 16 and unpublished data). sively expanded and differentiated into IFN-␥-secreting In the present study, we questioned whether CD95-induced effector cells in transgenic mice that constitutively express cell death has a role in the regulation of expansion of T cells stimulated via CD27. Whereas previous reports showed that

the CD27 , CD70. Concomitantly, CD95-defi- http://www.jimmunol.org/ CD27 stimulation enhanced the magnitude of T cell responses cient CD70 transgenic mice became moribund by 4 wk of (17, 18), we here found that CD27-CD70 interaction sensitizes age with severe liver pathology and bone marrow failure. T cells for CD95-mediated apoptosis, thereby providing evi- These findings establish that CD95 is a critical regulator dence for an in-built feedback mechanism. Thus, CD27-CD70 of effector T cell homeostasis in chronic immune interactions can operate as a double-edged sword by either in- activation. The Journal of Immunology, 2005, 174: ducing T cell survival while increasing sensitivity to death by 5915–5920. CD95.

fter initial clonal expansion in response to Ag most of Materials and Methods by guest on September 24, 2021 the responding T cells die by apoptosis and only few Mice Ag-specific T cells are preserved as memory T cells. Ϫ Ϫ A The mice strains used were C57BL/6, CD70 Tg (21), CD27 / (17), F5 TCR The death of the activated T cells is important to maintain ho- Tg (30) (kindly provided by Dr. D. Kioussis, National Institute for Medical meostasis and avoid immunopathology generated by toxic side Research, London, U.K.), IFN-␥Ϫ/Ϫ, and lpr/lpr mice (purchased from The effects of effector T cell molecules (1). Jackson Laboratory). All mice strains are on a C57BL/6 background. CD70 Tg mice were crossed with CD27Ϫ/Ϫ, F5 TCR Tg, lpr/lpr, and IFN-␥Ϫ/Ϫ mice. All Signaling through members of the death domain contain- mice were bred in the animal facility of The Netherlands Cancer Institute (Am- ing TNF receptor (TNFR) family such as CD95, TNFR1, sterdam) under specific pathogen-free conditions and were handled in accor- and DR4/5 induces apoptosis by the recruitment of a signal dance with institutional and national guidelines. CD27Ϫ/Ϫ, IFN-␥Ϫ/Ϫ, and transduction complex that activates enzymes (2). Ac- lpr/lpr mutant mice were genotyped by PCR analysis of tail DNA. CD70 Tg and F5 TCR Tg mice were genotyped by FACS analysis using CD70 mAb and tive induce cleavage of structural and regulatory V␤11 mAb, respectively. within cells and leads ultimately to apoptosis. Hu- mans and mice with mutations in the encoding for ei- Cell preparation and flow cytometry ther CD95 or its ligand progressively accumulate lympho- Single cell suspensions were prepared from freshly isolated spleens, lymph cytes in the periphery and are susceptible to develop nodes (axillary, brachial, inguinal, and mesenteric), and thymus by mincing and autoimmune diseases (2). In vitro CD95 was identified as an gently pressing the tissues through cell strainers. Bone marrow (femurs and tib- essential mediator of apoptosis for activated T cells (3–5). ias) single cell suspensions were prepared by flushing the bones with a needle. Subsequent studies using mice deficient in CD95 (lpr mice) Erythrocytes were lysed with ammonium chloride buffer. Surface staining and intracellular staining for cell surface molecules and cytokines was performed as or CD95 ligand (gld mice) showed that CD95-CD95L in- described (21) using Abs to CD4, CD8, CD62L, CD44, CD45R/B220, CD43 teractions regulate T cell homeostasis (6–9). However, other (clone 1B11), CD95, IL-2, TNF, IFN-␥, and IL-10 (BD Pharmingen). Flow

Departments of *Experimental Immunology and †Hematology, Academic Medical Cen- 1 Address correspondence and reprint requests to Dr. Ramon Arens or Dr. Rene´A.W.van ter, University of Amsterdam, Amsterdam, The Netherlands; ‡Department of Immunol- Lier, Department of Experimental Immunology, Academic Medical Center, University of ogy and §Laboratory of Experimental Animal Pathology, The Netherlands Cancer Insti- Amsterdam, P.O. Box 22700, 1100 DD Amsterdam, The Netherlands. E-mail addresses: tute, Amsterdam, The Netherlands [email protected] and [email protected], respectively. Received for publication February 9, 2005. Accepted for publication March 7, 2005. 2 Abbreviations used in this paper: TRAF, TNFR-associated factor; Tg, transgenic; LN, lymph nodes. 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.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 5916 CUTTING EDGE: REQUIREMENT OF CD95 IN CHRONIC T CELL ACTIVATION cytometric analysis was performed on a FACSCalibur with Cell Quest software benzidine substrate for visualization. After counterstaining with hematoxylin, (BD Biosciences). slides were mounted. For negative control, the primary mAb was omitted. Lymphocyte purification Results and Discussion To purify wild-type T cells, lymph node cell suspensions from 6- to 8-wk-old CD27 stimulation sensitizes T cells for CD95-mediated apoptosis wild-type mice were incubated with rat-anti-mouse MHC class II and rat-anti- mouse B220 Abs for 30 min at 4°C, washed and incubated with goat-anti-rat We first tested whether CD27 stimulation by its ligand CD70 IgG microbeads (Miltenyi Biotec) for 20 min at 4°C. After washing, cells were influenced CD95 cell surface expression on T cells and/or mod- magnetically separated with the MACS (negative selection T cells). To purify B cells, splenic cell suspensions from CD70 Tg and CD27Ϫ/Ϫ mice were incu- ulated susceptibility to CD95-induced apoptosis. CD3-stimu- bated with mouse CD19 microbeads (Miltenyi Biotec) for 20 min at 4°C. After lated wild-type T cells were cultured in the presence or absence washing, cells were magnetically separated with the MACS (positive selection B of CD27 stimulation by the addition of CD70 transgenic (Tg) cells). The T and B cell fractions were consistently found to be Ͼ95% CD3ϩ ϩ or control B cells, respectively. CD95 expression on T cells in- and Ͼ95% B220 , respectively, as evidenced by flow cytometry. creased with CD27 stimulation (Fig. 1A). Moreover, an in- T cell activation, CD95 expression, and CD95-mediated apoptosis assay creased proportion of the CD3/CD27 stimulated T cells un- T cells (1 ϫ 106 cells/ml) were stimulated with 1 ␮g/ml coated CD3 mAb derwent CD95-induced apoptosis compared with CD3 (purified from hybridoma supernatants, clone CRL 1975) and cultured 1:1 treatment alone (Fig. 1B), indicating that CD70 sensitizes T Ϫ Ϫ Ϫ Ϫ with either purified CD27 / ϫ CD70 Tg B cells or purified CD27 / B cells cells for CD95-mediated apoptosis in vitro. To test CD95 sus- in IMDM medium containing 10% FCS. CD95 expression on T cells was an- alyzed after 24 h by flow cytometry with PE-conjugated anti-CD95 mAb (clone ceptibility in vivo, we adoptively transferred influenza-specific Jo2; BD Pharmingen) and allophycocyanin-conjugated antiCD3 mAb (BD TCR Tg T cells and CD95-deficient (lpr/lpr) influenza-specific Downloaded from Ϫ Ϫ Ϫ Ϫ Pharmingen). For CD95-mediated apoptosis-purified hamster anti-mouse TCR Tg T cells into either CD27 / or CD27 / ϫ CD70 Tg ␮ 6 CD95 mAb (5 g/10 cells, clone Jo2; BD Pharmingen) was added, and after mice. The number of influenza-specific T cells was assessed 6 48 h, cells were subjected to flow cytometric analysis. Propidium iodide was used to identify apoptotic cells and allophycocyanin-conjugated anti-CD3 and 11 days after infection of the recipient mice with influenza mAb to identify T cells. virus. CD95 deficiency did not affect the size of the influenza- specific population in the spleen when T cells were transferred Ϫ Ϫ

Adoptive transfers http://www.jimmunol.org/ into CD27 / animals but strongly enhanced T cell expansion Ϫ Ϫ For the adoptive transfers, 8 ϫ 106 enriched T cells from spleen and LN cells in CD70 Tg ϫ CD27 / mice (Fig. 1C). Thus, in analogy to (Ͼ75% V␤11ϩCD8ϩ T cell) of TCR Tg and lpr/lprϫ TCR Tg were injected i.v. into either CD27ϩ/Ϫ or CD27Ϫ/Ϫϫ CD70 Tg recipients. Two days after what has been shown for CD40 signaling in the B cell system cellular transfer, recipient mice were infected with 25 hemagglutination unit (19, 20), CD27 signaling appeared to protect Ag-expanded T influenza virus. At day 6 and day 11 after viral challenge, the expansion of the cells from passive cell death but concomitantly primed these donor population was quantitated in spleen by flow cytometry using Abs to cells for CD95-mediated deletion. CD8 and V␤11. The donor population was distinguished from the endogenous V␤11 expressing CD8ϩ T cells by difference in fluorescence intensity. Neonatal death of CD95-deficient CD70 Tg mice

Histological analysis and immunohistochemistry To assess the importance of the CD95 system in an in vivo by guest on September 24, 2021 Tissues were fixed in 4% phosphate-buffered formalin, embedded in paraffin, model for chronic T cell activation, CD95-deficient (lpr/lpr) sectioned at 4 ␮m, and stained with H&E according to standard procedures. mice were bred with CD70 Tg mice. In CD70 Tg mice, con- For immunohistochemistry, tissues were deparaffinized in xylene and rehy- stitutive CD27 triggering by CD70 on B cells causes a progres- drated. Endogenous peroxidase activity was blocked by using 3% (v/v) H2O2 in methanol for 10 min. Before staining, paraffin sections were pretreated by heat- sive conversion of naive T cells into effector-memory cells, induced epitope retrieval. Slides were incubated with 5% normal goat serum/ which ultimately results in exhaustion of the immune system PBS for 30 min, and subsequently sections were incubated overnight with a and lethal immunodeficiency as seen in persisting, active viral 1:400 dilution of CD3 or B220 at 4°C. mAb immunoreactivity was detected with the streptavidin-biotin immunoperoxidase (sABC) method by using bio- infections (21, 22). CD95-deficient CD70 Tg animals were tinylated goat anti-rat IgG (Dako, 1:100) as secondary antibody, and diamino- born with an expected Mendelian frequency and looked healthy

FIGURE 1. Increased susceptibility of CD95-mediated apoptosis due to persistent CD27 stimulation. A, Elevated CD95 expression on CD27 stimulated T cells. Lymph node T cells were stimulated with anti-CD3 mAb in the presence of CD70ϩ B cells (i.e., CD27Ϫ/Ϫ ϫ CD70 Tg B cells, solid line histogram) or absence (i.e., CD27Ϫ/Ϫ B cells, shaded histogram). CD95 expression on T cells was analyzed at day 2 after stimulation by flow cytometry. Line and dotted line histogram represents isotype-control stainings of T cells in presence or absence of CD70ϩ B cells, respectively. B, CD27 stimulation by CD70 facilitates CD95-mediated apoptosis in vitro. Lymph node T cells were cultured with anti-CD3 mAb alone or with anti-CD3 mAb and anti-CD95 mAb in the presence or absence of CD70ϩ B cells. After 2 days, apoptotic cells were analyzed by flow cytometry. Mean Ϯ SDs of three experiments are shown. C, CD95-deficient (lpr/lpr) TCR Tg or TCR Tg T cells were transferred into either non-CD70 Tg (CD27Ϫ/Ϫ) or CD70 Tg (CD27Ϫ/Ϫϫ CD70 Tg) recipients. At days 6 and 11 after influenza virus infection, numbers of the donor TCR Tg CD8ϩ T cells in spleens of recipient mice were determined. Results are displayed as mean Ϯ SEM (n ϭ 4). The Journal of Immunology 5917 at birth. However, after 3 wk the CD95-deficient CD70 Tg synthesize IFN-␥ and IL-10 and a somewhat reduced capacity mice became sick and died at around 4 wk of age (Fig. 2A). In to produce TNF and IL-2 (Fig. 3C and data not shown). Fi- contrast, CD70 Tg mice with normal CD95 expression died at nally, the progressive B cell depletion observed in CD70 Tg around 25 wk of age. The spleen and lymph nodes (LN) of mice (21) was greatly accelerated in CD95-deficient CD70 Tg CD95-deficient CD70 Tg mice had a normal size but histology mice (Fig. 2B and Fig. 3D). revealed that normal splenic and LN architecture was disrupted Expression of TCR-V␤ elements did not differ between (Fig. 2B and data not shown). Furthermore, a severe atrophy of CD95-deficient CD70 Tg mice and controls (data not shown), the thymus was observed, and diffuse T lymphocytic infiltrates which suggests that, as in CD70 Tg mice (22), innocuous en- were seen in the liver but not in heart, kidney, lungs, or gut. A vironmental or autoantigens may drive effector T cell forma- strong decreased cellularity of the hematopoietic lineages was tion. In support of this, crossing CD95-deficient CD70 Tg seen in bone marrow (Fig. 2B). mice with Tg mice expressing an MHC class I-restricted influ- ϩ enza-specific F5 TCR, restrained CD8 effector T cell forma- ϩ CD95 constrains effector T cell formation in CD70 Tg mice tion whereas in these animals the CD4 compartment largely Concerning the composition of the peripheral lymphoid com- consisted of effector-type T cells (Fig. 3E). As an apparent con- partments, the spleens of 2-wk-old CD95-deficient CD70 Tg sequence of the reduced effector T cell formation in comparison mice contained normal T cell numbers whereas in LN a weakly to CD95-deficient CD70 Tg, premature death was postponed reduced T cell pool was observed (Fig. 3, A and B). Strikingly, to around 12 wk of age in CD95-deficient CD70 Tg ϫ TCR the percentage of activated effector T cells (defined by a Tg mice (Fig. 2A). Thus, CD95 deficiency in mice with a Downloaded from CD62lowCD44brightCD43high phenotype) was increased com- chronically activated immune system results in excessive Ag-in- pared with the various control animals (wild type, CD70 Tg, duced effector T cell formation and leads to premature death. and lpr/lpr). Comparison of 2-wk-old with 3-wk-old mice showed that the accumulation of effector T cells was progressive Effects of IFN-␥ in CD70-driven effector T cell formation and ϩ and was most prominent in the CD8 T cell subset (Fig. 3, A immunopathology and B). In agreement with the enhanced effector T cell forma- Ϫ/Ϫ http://www.jimmunol.org/ ϩ ϩ Suppressor of cytokine signaling (SOCS-1) mice have an tion, splenic and LN CD4 and CD8 T cells from CD95- exaggerated response to IFN-␥ and develop a complex neonatal deficient CD70 TG mice had a strong increase in the ability to disease (23) that has, with respect to bone marrow and liver pa- thology, conspicuous similarities to the pathology observed in CD95-deficient CD70 Tg mice. Previously, we have shown that IFN-␥ is responsible for progressive B cell depletion in CD70 Tg mice (21). Since the formation of IFN-␥-producing effector T cells is strongly enhanced in CD95-deficient CD70

Tg mice, we crossed CD95-deficient CD70 Tg mice on an by guest on September 24, 2021 IFN-␥ background to test whether IFN-␥ is responsible for the pathology in these mice. The survival of CD95-deficient CD70 Ϫ Ϫ Tg ϫ IFN-␥ / mice was slightly improved compared with CD95-deficient CD70 Tg mice (3.9 Ϯ 0.5 wk CD95-deficient CD70 Tg vs 6 Ϯ 1.1 wk CD95-deficient CD70 Tg ϫ IFN- Ϫ Ϫ ␥ / ) (Fig. 4A). Remarkably, histological analysis revealed that the pathology was completely distinct. The size of spleens and Ϫ Ϫ LN of CD95-deficient CD70 Tg ϫ IFN-␥ / mice were sub- stantially larger than of CD95-deficient CD70 Tg and CD70 Ϫ Ϫ Tg ϫ IFN-␥ / mice. Moreover, focal perivascular infiltrates of lymphocytic blasts were found in liver, lung and kidney (Fig. 4B and data not shown). B cell numbers in CD95-deficient Ϫ Ϫ CD70 Tg ϫ IFN-␥ / mice were only slightly higher than in CD95-deficient CD70 Tg mice (2.2 ϫ 106Ϯ 0.6 ϫ 106 CD95-deficient CD70 Tg vs 3.1 ϫ 106Ϯ 1.5 ϫ 106 CD95- Ϫ Ϫ deficient CD70 Tg ϫ IFN-␥ / per spleen; 0.27 ϫ 106Ϯ 0.21 ϫ 106 CD95-deficient CD70 Tg vs 0.6 ϫ 106Ϯ 0.12 ϫ Ϫ Ϫ 106 CD95-deficient CD70 Tg ϫ IFN-␥ / for LN), indicat- ing an IFN-␥ independent B cell reduction in these mice. T cell ϩ numbers, notably CD8 T cells, in spleen and LN were FIGURE 2. Premature death accompanied with disrupted splenic architec- strongly increased with an overrepresentation of effector-type T ture, T cell infiltrates in the liver and decreased bone marrow cellularity in cells (Fig. 4C). These T cells produced, as in CD95-deficient CD95-deficient CD70 Tg mice. A, Accelerated morbidity of CD95-deficient CD70 Tg mice, levels of TNF and IL-2 that were somewhat (lpr/lpr) CD70 Tg mice compared with CD70 Tg mice. B, The histological reduced compared with control mice. However, IL-10 secre- appearance of spleen, liver, and bone marrow in wild-type, lpr/lpr (CD95-de- tion was not detectable in these T cells (data not shown). The ficient), and CD70 Tg mice is contrasted with the disruption splenic architec- massive expansion of T cells would be in line with the suggested ture, diffuse T cell infiltrates in the liver and hypocellular bone marrow of ␥ CD95-deficient CD70 Tg mice. Autopsies were performed and included both function of IFN- in promoting the contraction of effector T gross and microscopic evaluations. Representative sections are shown from cell populations (24, 25), which may at least partly be explained spleen, liver, and bone marrow of animals 3 wk of age. by facilitating CD95-mediated apoptosis (26). The difference 5918 CUTTING EDGE: REQUIREMENT OF CD95 IN CHRONIC T CELL ACTIVATION

FIGURE 3. Massive effector T cell formation in CD95-deficient CD70 Tg

mice. T cell analysis of wild-type, CD70 Downloaded from Tg, lpr/lpr (CD95-deficient), and CD95-deficient CD70 Tg (CD62Lhigh CD43neg/low) mice. Absolute numbers of naive and effector (CD62LlowCD43high) CD4ϩ and CD8ϩ T cells were deter- mined by flow cytometry in spleen (A) and lymph nodes (B) of 2- and 3-wk-old http://www.jimmunol.org/ mice. C, Functional analysis of splenic CD4ϩ and CD8ϩ T cells. Splenocytes were stimulated for 5 h with PMA/iono- mycin in the presence of brefeldin A and subsequently analyzed by intracellular cy- tokine staining for TNF, IL-2, IFN-␥, and IL-10. D, Severe B cell depletion in lpr/lprϫ CD70 Tg mice. Splenic and lymph node B cells were measured by flow cytometry (B220high). E, Representative by guest on September 24, 2021 FACS profiles showing CD44 vs CD62L staining of splenic CD4ϩ and CD8ϩ T cells of 8-wk-old lpr/lpr, lpr/lprϫ CD70 Tg, TCR Tg, lpr/lprϫ TCR Tg andlpr/lprϫ CD70 Tg ϫTCR Tg. Data shown represent mean values plus SDs of 4–8 mice.

in pathology between IFN-␥-competent and IFN-␥-deficient The mechanism of priming for CD95-induced cell death by mice infer that although excessive IFN-␥ has detrimental effects CD27 signaling is unknown. A direct effect on inhibition of on liver and bone marrow functions also other pathological passive cell death is unlikely since Bcl-2 Tg T cells are not pro- changes related to disturbed homeostasis of effector T cells are tected from apoptosis by CD95 (27). IL-2 has been described as associated with mortality. a key regulator of effector T cell homeostasis by blocking passive The Journal of Immunology 5919

chronically activated via costimulatory TNFR family members that signal via TRAF molecules, such as CD27. Repeated trig- gering of these TNFRs may occur in situations of persisting pathogens since many of the TNFR ligands are activation mol- ecules that are up-regulated by Ag-specific activation (14). Thus homeostasis of effector T cells is dependent on the proper bal- ance between signals provided by TRAF-binding and death do- main-containing TNFR family members.

Acknowledgments We thank the staff of the Animal Facility of The Netherlands cancer Institute for excellent animal care.

Disclosures The authors have no financial conflict of interest.

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