HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fritzsching, B. Articles by Suri-Payer, E. Search for Related Content PubMed PubMed Citation Articles by Fritzsching, B. Articles by Suri-Payer, E.

Cutting Edge: In Contrast to Effector T Cells, CD4⁺CD25⁺FoxP3⁺ Regulatory T Cells Are Highly Susceptible to CD95 Ligand- but Not to TCR-Mediated Cell Death¹

Benedikt Fritzsching^2,*,, Nina Oberle^*, Nadine Eberhardt^*, Sabine Quick^*, Jürgen Haas, Brigitte Wildemann, Peter H. Krammer^2,* and Elisabeth Suri-Payer^*

^* Tumor Immunology Program German Cancer Research Center, and Division of Molecular Neuroimmunology, Department of Neurology, University of Heidelberg, Heidelberg, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD4⁺CD25⁺FoxP3⁺ regulatory T cells (T_reg) suppress T cell function and protect rodents from autoimmune disease. Regulation of T_reg during an immune response is of major importance. Enhanced survival of T_reg is beneficial in autoimmune disease, whereas increased depletion by apoptosis is advantageous in cancer. We show here that freshly isolated FACS-sorted T_reg are highly sensitive toward CD95-mediated apoptosis, whereas other T cell populations are resistant to CD95-induced apoptosis shortly after isolation. In contrast, TCR restimulation of T_reg in vitro revealed a reduced sensitivity toward activation-induced cell death compared with CD4⁺CD25^– T cells. Thus, the apoptosis phenotype of T_reg is unique in comparison to other T cells, and this might be further explored for novel therapeutic modulations of T_reg.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The function and manipulation of CD4⁺CD25⁺FoxP3⁺ regulatory T cells (T_reg)³ during an immune response have gained a lot of attention (1, 2). Depletion of T_reg leads to autoimmunity in mice (1), and dysfunction of T_reg has been linked to human autoimmune diseases (3, 4, 5). Whereas therapeutical expansion of T_reg may be advantageous in autoimmunity, accumulation of immunosuppressive T_reg in tumors could be detrimental (6). Depletion of T_reg should be beneficial in cancer, but specific therapeutic tools such as T_reg-depleting Abs are limited. Thus, new strategies to modulate survival or apoptosis of T_reg are warranted.

The investigation of apoptotic properties of T_reg is important, because they may be used to modulate the ratio of T_reg to effector T cells (T_eff). Taams et al. (7) reported a high susceptibility of human T_reg to spontaneous cell death or cytokine-deprivation death, and murine T_reg can be depleted due to their susceptibility to cyclophosphamide toxicity or gamma irradiation (8, 9). Conversely, other groups reported apoptosis resistance of murine T_reg when cells were treated with dexamethasone or anti-CD95 Ab (10, 11). While information on the apoptosis sensitivity of murine T_reg is inconsistent, CD95-mediated apoptosis of T_reg has not been studied in humans.

Apoptosis mediated by the interaction of CD95 (Apo-1/Fas) with CD95 ligand (CD95L) is well characterized in T cells (12). CD95 is widely expressed (13), whereas expression of CD95L is tightly regulated (14). Although 20–60% of naive CD4⁺ T cells express CD95, >90% of them are resistant to CD95-mediated apoptosis (15). However, several days after activation, they become sensitive toward CD95-mediated apoptosis and up-regulate CD95L after TCR restimulation (12). Subsequently, CD95L triggers apoptosis of CD95⁺ activated T cells, a phenomenon called activation-induced cell death (AICD). AICD is a major mechanism to eliminate the expanded pool of effector lymphocytes during the contraction phase of the immune response and to maintain lymphocyte homeostasis (12).

To further clarify the physiology of T_reg, we studied their apoptosis phenotype ex vivo. We show for the first time that freshly isolated T_reg are highly sensitive toward CD95L-mediated apoptosis unlike their resistant T_eff counterparts. In contrast, we find that T_reg are substantially less sensitive to AICD than T_eff.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Abs and reagents

The mAbs against CD4, CD62L, and CD95L (Nok-1) were obtained from BD Pharmingen, and anti-CD25 Ab from Miltenyi Biotec. CD95L was produced as a leucine zipper-tagged ligand of CD95 (15, 16). The anti-CD3 Ab OKT3 and the agonistic monoclonal anti-CD95 Ab (anti-Apo-1) were purified from hybdridoma supernatants by protein A affinity purification (15). The monoclonal anti-FoxP3 Ab was a kind gift from A. Banham (Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, U.K.). The pan-caspase inhibitor zVAD-fmk was obtained from Bachem, annexin V Alexa Fluor 488 from Molecular Probes, and propidium iodide (PI) and protein A were obtained from Sigma-Aldrich.

Lymphocyte separation

PBL were obtained from healthy individuals. CD4⁺CD25⁺ cells were first enriched using MACS beads (Miltenyi Biotec), and subsequently CD4⁺CD25^high cells were sorted with a FACS-Diva cell sorter (BD Biosciences).

Cell culture and cytotoxicity assays

Freshly isolated T cells were cultured in IL-2 (100 IU) containing ex Vivo-15 medium (Cambrex) supplemented with 1% Glutamax (Invitrogen Life Technologies). For apoptosis induction, T cells were stimulated with 1 µg/ml anti-CD95 Ab and 10 ng/ml protein A or 1/10 dilution of CD95L (15). Unstimulated cells were incubated with an isotype control Ab or CD95L-free control medium yielding spontaneous apoptosis of 15–30% in 20 h. To enhance viability of T_reg and T_eff, some experiments were performed in wells coated with anti-CD3 Ab (30 µg/ml). To measure AICD, T cells were expanded for 6 days (day 6 T cells) with 0.1 µg/ml anti-CD3 Ab and 1 µg/ml anti-CD28 Ab in combination with irradiated JY feeder cells (kind gift from C. Falk, Institute for Molecular Immunology, GSF National Research Center for Environment and Health, Munich, Germany) and 300 IU of IL-2 (3, 17). For induction of AICD, day 6 T cells were transferred into wells containing 30 µg/ml plate-bound anti-CD3 Ab (pb-anti-CD3) and were cultured for 24 h. Cell death was assessed by annexin V/PI costaining and forward- to side-scatter profile. Specific cell death was calculated as described previously (15): (% experimental cell death – % spontaneous cell death)/(100% – % spontaneous cell death) x 100.

Cell surface staining

Cells were stained with PE-labeled anti-CD25 Ab or anti-CD62L Ab, PE-Cy5-labeled anti-CD4 Ab, and FITC-labeled anti-CD95 Ab, or their respective isotype control Abs, and analyzed with a FACScan cytometer with at least 10,000 T_reg or T_eff counted.

RNA preparation and quantitative RT-PCR

Total RNA was isolated using the Absolutely RNA Microprep kit (Stratagene) and cDNA was prepared using random oligo(dT) primers (Invitrogen Life Technologies). FoxP3 message expression was quantified by detection of incorporated SYBR Green using the ABI Prism 5700 sequence detector system (Applied Biosystems). The relative expression level was determined by normalization to GAPDH with results presented as fold expresssion of T_eff mRNA levels. FoxP3 primer sequences were as follows: 5'-AGC TGG AGT TCC GCA AGA AAC (forward) and 5'-TGT TCG TCC ATC CTC CTT TCC (reverse).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD4⁺CD25⁺FoxP3⁺ T_reg are CD95 positive

Previous reports using magnetic bead-isolated CD4⁺CD25⁺ T cells showed CD95 expression on human and murine CD25⁺ cells (7, 16). We confirmed these data on CD4⁺ T_reg FACS-sorted for very high CD25 expression. These cells induced strong suppression of both T cell proliferation and cytokine production (data not shown) and expressed 100-fold higher FoxP3 levels than T_eff cells (see Fig. 2B). Almost all T_reg expressed scurfin protein in the nucleus as determined by immunocytochemistry (data not shown). Naive T_eff were sorted by gating on CD4⁺CD25^– T cells, which were negative for scurfin expression and showed extremely low FoxP3 levels (see Fig. 2B). T_reg expressed higher levels of CD95 molecules on the cell surface as compared with naive T_eff (Fig. 1A). T_reg remained CD95 positive during short-term (day 6) and long-term (day 20) in vitro expansion (Fig. 1A).

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Sensitivity to CD95L-induced apoptosis without TCR prestimulation is a unique feature of CD4+CD25+FoxP3+Treg. A, Using four-way sorting, four different subpopulations with regard to their CD25 expression were simultaneously obtained from human PBL: CD25– Teff, intermediate T cells with low CD25+ expression (Tint CD25+), intermediate T cells with high CD25+ expression (Tint CD25++), and Treg with the highest CD25 expression. B, Sorted subpopulations were analyzed by quantitative PCR for their FoxP3 mRNA content. C, Specific cell death was measured after CD95 cross-linking (1 µg/ml) for 20 h. One representative result of three is shown. Error bars are SD of triplicate or duplicate samples.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. CD4+CD25+FoxP3+ Treg are sensitive to CD95L-induced apoptosis without prestimulation in vitro. A, Freshly isolated (day 0) Treg and in vitro-expanded Treg (day 6 and 20) were stained with anti-CD95-FITC Ab and analyzed by FACS. Dashed line, Isotype control; filled profile, Teff (CD4+CD25–); open profile, Treg (CD4+CD25high). B, Treg () and Teff () were stimulated with soluble CD95L or anti-CD95 Ab cross-linked with protein A for 20 h, and specific cell death was determined as described in Materials and Methods. C, Dose-response curve of CD95L-induced cell death of Treg (•) and Teff (). D, For inhibition of CD95L-mediated apoptosis, Treg were incubated with indicated amounts of anti-CD95L Ab or caspase inhibitor zVAD-fmk before CD95L stimulation. E and F, Treg and Teff were stimulated for the indicated time points with anti-CD95 Ab (1 µg/ml) cross-linked with protein A. E, T cells were stained with annexin V/PI, analyzed by FACS, and gated for viable and early apoptotic cells (PI–), whereas PI+ necrotic or late apoptotic cells were excluded. Apoptosis was measured at different time points on gated PI– cells. F, Annexin V+PI– Treg (•) and annexin V+PI– Teff () were plotted against time. Error bars are SD of triplicate or duplicate samples.

To test whether freshly isolated T_reg are sensitive to CD95-mediated apoptosis without previous TCR stimulation, we incubated FACS-sorted T_reg and T_eff with cross-linked anti-CD95 Ab (anti-Apo-1) for 20 h. As reported previously (12), freshly isolated T_eff did not die upon addition of anti-CD95 Ab or soluble CD95L. However, T_reg treated under the same conditions showed a very high induction of apoptosis to both stimuli (Fig. 1B). We have carefully titrated both CD95L (Fig. 1C) and anti-CD95 Ab and performed experiments side by side with both reagents. Although both reagents yielded similar results, soluble CD95L might be the more physiological mimic to trigger CD95-mediated apoptosis than anti-CD95 Ab. After 4–6 days of TCR stimulation in vitro CD4⁺ T cells are usually sensitive toward the extrinsic pathway of apoptosis, which is initiated by the binding of CD95L to CD95 and is then executed by a cascade of caspases (12). To test whether CD95L-induced apoptosis of freshly isolated T_reg involves these events, we used a neutralizing Ab against CD95L as well as zVAD-fmk as an inhibitor of caspase activity. Both treatments blocked CD95-mediated apoptosis in a concentration-dependent manner (Fig. 1D). Once apoptosis has been triggered, annexin V staining and uptake of PI allows distinguishing between early apoptotic and late apoptotic or necrotic cells. Apoptotic cell death started within the first hour after CD95 triggering and led to 30% early apoptotic cells (annexin⁺PI^–) by 10 h (Fig. 1, E and F). After 20 h, most of the apoptotic cells had lost membrane integrity (annexin V⁺PI⁺), resulting in a total of 40–60% specific cell death, which did not increase further until 48 h (data not shown). Similar observations were also made with freshly isolated CD4⁺CD25⁺ T cells from murine spleen and lymph nodes demonstrating a consistent phenotype between murine and human T_reg (data not shown).

Sensitivity to CD95L-induced apoptosis without TCR prestimulation is a unique feature of CD4⁺CD25⁺FoxP3⁺ T_reg

Using high-speed four-way FACS, we simultaneously sorted four different subpopulations from peripheral blood: CD25^– T_eff, intermediate T cells with low CD25⁺ expression (T_int CD25⁺), intermediate T cells with high CD25⁺ expression (T_int CD25⁺⁺), and T_reg with the highest CD25 expression (Fig. 2A). Quantitative PCR analysis revealed very high FoxP3 mRNA expression in T_reg. Low amounts of FoxP3 mRNA were also detectable in the adjacent T_int CD25⁺⁺ subpopulation, whereas T_int CD25⁺ and CD25^– T_eff cells were essentially FoxP3^– (Fig. 2B). Similarly, only the T_reg subpopulation showed significant suppressive capacity and anergy as determined by proliferation assays (data not shown). Among the four freshly isolated T cell subpopulations, CD95-induced apoptosis was limited to cells with the highest expression of T_reg markers (CD25, FoxP3) (Fig. 2C). We suggest that only the CD25⁺⁺ cells are T_reg and that cells within the T_int CD25⁺⁺ sort gate comprise a mixture of T_reg and activated T_eff. This "contamination" of T_reg in the T_int CD25⁺⁺ subpopulation could explain the small increase in apoptosis upon CD95 cross-linking.

AICD is diminished in CD4⁺CD25⁺FoxP3⁺ T_reg

When previously activated lymphocytes encounter a second TCR signal, they express CD95L and kill each other by AICD. To test whether T_reg undergo AICD, we prestimulated CD25⁺⁺ cells with anti-CD3/anti-CD28 Abs in combination with FcR-bearing, cross-linking feeder cells and IL-2 (3, 17). This activation protocol allowed proliferation of T_reg and resulted in expansion of T_reg within 6 days of stimulation. At day 6, T_reg were restimulated by pb-anti-CD3 Ab for 24 h. Surprisingly, T_reg were significantly less sensitive to AICD than T_eff, although both cell types showed similar apoptosis sensitivity toward CD95L treatment (Fig. 3B). Because freshly isolated T_reg died in response to CD95 stimulation, we tested whether they would also be killed by pb-anti-CD3 Ab. However, stimulation of day 0 T_reg with pb-anti-CD3 Ab for 24 h yielded even less cell death compared with T_reg cultured in IL-2 medium alone (Fig. 3A). Next, we tested T_reg expanded for 20 days (100- to 1000-fold expansion) before restimulation with pb-anti-CD3 Ab. We repeatedly observed CD62L⁺ and CD62L^– cells in anti-CD3/anti-CD28-expanded T_reg cultures, which were both suppressive. Restimulation of both subpopulations with pb-anti-CD3 Ab did not induce AICD, whereas CD95 killing was preserved (Fig. 3C). In summary, we found that freshly isolated T_reg are highly sensitive to CD95 cross-linking in contrast to their resistant CD4⁺CD25^– T_eff counterparts. However T_reg are less sensitive to AICD compared with T_eff.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. AICD is low in CD4+CD25+FoxP3+ Treg. A, Freshly isolated Treg and Teff were stimulated with pb-anti-CD3 Ab (30 µg/ml) for 24 h or with CD95L for 20 h. B, In an in vitro model of AICD, human Treg and Teff were incubated with 0.1 µg/ml anti-CD3 Ab and 1 µg/ml anti-CD28 Ab together with irradiated JY feeder cells and IL-2 for 6 days and then restimulated as described in A. C, Human Treg were expanded for 20 days similar to B, and CD62L+ cells as well as CD62L– cells were FACS-sorted from expanded Treg. Next, cells were restimulated as described in A. Error bars are SD of triplicate or duplicate samples.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Papiernik and colleagues (11) observed a resistance of prestimulated CD4⁺CD25⁺ T cells derived from C57BL/6 mice toward apoptosis triggered by anti-CD95 Ab. However, this study included neither freshly isolated FACS-sorted T cells nor human T cells. Given the relatively high percentage of T_reg dying spontaneously even under conditions of T_reg proliferation and expansion (data not shown), some of the CD95-sensitive CD4⁺CD25⁺ cells might have died during their prestimulation phase. This might lead to an underestimation of CD95-triggered cell death. In addition, we used CD95L, which might bind and multimerize CD95 more efficiently than the anti-murine CD95 Ab used by Papiernik and colleagues (11). We consistently observed sensitivity of T_reg toward CD95-triggered apoptosis in both freshly isolated human T_reg (day 0) and short-term-stimulated T_reg (days 2–6) as well as in long-term-expanded T_reg (day 20).

In vivo, the apparent selective sensitivity of T_reg to CD95L might be an important mechanism to eliminate T_reg during the acute effector phase of an immune response at a time when T_eff are resistant to CD95-mediated apoptosis. In particular, in the presence of a fulminant acute infection, T_reg could be detrimental to the organism and a rapid elimination of T_reg in danger situations seems appropriate. In this case, T_reg might be killed either by soluble CD95L or adjacent membrane-bound CD95L expressed, e.g., on dying infected cells. Reduced elimination of T_reg during acute infection might hamper T_eff cells to clear the infection as recently shown in a model of leishmaniasis (19, 20).

Our second finding demonstrates a relative resistance of human T_reg to AICD. Neither freshly isolated T_reg nor T_reg activated for 6 days or expanded for 3 wk showed significant AICD upon TCR stimulation or restimulation. In contrast, activated T_eff are AICD sensitive (12) and thus die due to the weekly TCR restimulation (3, 21), whereas T_reg expand (data not shown). This observation further supports a reduced AICD sensitivity of T_reg. Interestingly, T_reg also express CD95L mRNA upon TCR restimulation (data not shown). Further studies including quantitative analysis of CD95L mRNA expression and CD95L protein expression might clarify the reason for reduced AICD sensitivity of T_reg. Other mechanisms including proteolytical cleavage of CD95L and CD95 into antagonistic products could further explain the relative AICD resistance of T_reg (22, 23).

Because T cell responses to pathogens could not only prime T cells for pathogen elimination, but also induce autoaggressive T_eff, prolonged survival of AICD-resistant T_reg in the critical down-phase of an immune response is conceivable. In support of this idea, T_reg have been described to escape from clonal deletion induced by Ag-specific (re)stimulation in vivo (24, 25, 26).

Even in the absence of AICD, T_reg numbers might be contracted during the down-phase of the immune response. First, the rapid decline of T_eff cells leads to much lower IL-2 levels, which may become limiting, and thus eliminates surplus T_reg via death by cytokine deprivation (27). Second, our data offer the possibility that CD95L-expressing T_eff might kill neighboring T_reg. Although still hypothetical, T_eff might keep T_reg in check by modulating their survival, whereas T_reg might mainly suppress effector function of T_eff, resulting in a tightly balanced ratio of both T cell populations. Obviously, further work is warranted to explore the use of the CD95/CD95L system for specifically modulating T_reg cell number and function in vivo.

	Acknowledgments

 
Drs. A. Banham, G. Roncador, G. Moldenhauer, and H. Weyd are gratefully acknowledged for their generous gift of Abs, and C. Falk for JY cells. We thank B. Franz, J. Mohr, and Dr. J. Sykora for helping to establish the immunocytochemistry protocol for scurfin; S. Prinz, M. Rutz, I. Hefft, K. Hexel, and M. Scheuermann for technical assistance; Drs. R. Arnold, K. Gu"low, and A. Golks, E. Kleinmann, and E. Pauly for critical reading of the manuscript; C. Frey and C. Fritsch for computational assistance; and Dr. M. Korporal for helpful discussion.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
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 work was supported by grants from the Gemeinnützige Hertie-Stiftung (1.319.110/01/11 and 1.01.1/04/003), Biogen GmbH, Landesstiftung Baden-Wuerttemberg, and Serono GmbH, and by the Young Investigator Award of the Faculty of Medicine, University of Heidelberg (to B.F.).

² Address correspondence and reprint requests to Dr. Benedikt Fritzsching or Dr. Peter H. Krammer, Tumor Immunology Program D030, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail addresses: Benedikt.Fritzsching{at}dkfz.de and P.Krammer{at}dkfz.de

³ Abbreviations used in this paper: T_reg, regulatory T cell; T_eff, effector T cell; CD95L, CD95 ligand; AICD, activation-induced cell death; PI, propidium iodide; pb-anti-CD3, plate-bound anti-CD3 Ab.

Received for publication January 24, 2005. Accepted for publication April 27, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Shevach, E. M.. 2000. Regulatory T cells in autoimmunity. Annu. Rev. Immunol. 18: 423-449.[Medline]
2. Bach, J. F.. 1995. Organ-specific autoimmunity. Immunol. Today 16: 353-355.[Medline]
3. Viglietta, V., C. Baecher-Allan, H. L. Weiner, D. A. Hafler. 2004. Loss of functional suppression by CD4⁺CD25⁺ regulatory T cells in patients with multiple sclerosis. J. Exp. Med. 199: 971-979.[Abstract/Free Full Text]
4. Ehrenstein, M. R., J. G. Evans, A. Singh, S. Moore, G. Warnes, D. A. Isenberg, C. Mauri. 2004. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNF therapy. J. Exp. Med. 200: 277-285.[Abstract/Free Full Text]
5. Kriegel, M. A., T. Lohmann, C. Gabler, N. Blank, J. R. Kalden, H. M. Lorenz. 2004. Defective suppressor function of human CD4⁺CD25⁺ regulatory T cells in autoimmune polyglandular syndrome type II. J. Exp. Med. 199: 1285-1291.[Abstract/Free Full Text]
6. Curiel, T. J., G. Coukos, L. Zou, X. Alvarez, P. Cheng, P. Mottram, M. Evdemon-Hogan, J. R. Conejo-Garcia, L. Zhang, M. Burow, et al 2004. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10: 942-949.[Medline]
7. Taams, L. S., J. Smith, M. H. Rustin, M. Salmon, L. W. Poulter, A. N. Akbar. 2001. Human anergic/suppressive CD4⁺CD25⁺ T cells: a highly differentiated and apoptosis-prone population. Eur. J. Immunol. 31: 1122-1131.[Medline]
8. Ghiringhelli, F., N. Larmonier, E. Schmitt, A. Parcellier, D. Cathelin, C. Garrido, B. Chauffert, E. Solary, B. Bonnotte, F. Martin. 2004. CD4⁺CD25⁺ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur. J. Immunol. 34: 336-344.[Medline]
9. Kipnis, J., H. Avidan, Y. Markovich, T. Mizrahi, E. Hauben, T. B. Prigozhina, S. Slavin, M. Schwartz. 2004. Low-dose gamma-irradiation promotes survival of injured neurons in the central nervous system via homeostasis-driven proliferation of T cells. Eur. J. Neurosci. 19: 1191-1198.[Medline]
10. Chen, X., T. Murakami, J. J. Oppenheim, O. M. Howard. 2004. Differential response of murine CD4⁺CD25⁺ and CD4⁺ to dexamethasone-induced cell death. Eur. J. Immunol. 34: 859-869.[Medline]
11. Banz, A., C. Pontoux, M. Papiernik. 2002. Modulation of Fas-dependent apoptosis: a dynamic process controlling both the persistence and death of CD4 regulatory T cells and effector T cells. J. Immunol. 169: 750-757.[Abstract/Free Full Text]
12. Krammer, P. H.. 2000. CD95’s deadly mission in the immune system. Nature 407: 789-795.[Medline]
13. Watanabe-Fukunaga, R., C. I. Brannan, N. Itoh, S. Yonehara, N. G. Copeland, N. A. Jenkins, S. Nagata. 1992. The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J. Immunol. 148: 1274-1279.[Abstract]
14. Suda, T., T. Takahashi, P. Golstein, S. Nagata. 1993. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75: 1169-1178.[Medline]
15. Schmitz, I., H. Weyd, A. Krueger, S. Baumann, S. C. Fas, P. H. Krammer, S. Kirchhoff. 2004. Resistance of short term activated T cells to CD95-mediated apoptosis correlates with de novo protein synthesis of c-FLIPshort. J. Immunol. 172: 2194-2200.[Abstract/Free Full Text]
16. Igney, F. H., C. K. Behrens, P. H. Krammer. 2003. The influence of CD95L expression on tumor rejection in mice. Eur. J. Immunol. 33: 2811-2821.[Medline]
17. Hoffmann, P., R. Eder, L. A. Kunz-Schughart, R. Andreesen, M. Edinger. 2004. Large-scale in vitro expansion of polyclonal human CD4⁺CD25^high regulatory T cells. Blood 104: 895-903.[Abstract/Free Full Text]
18. Rathmell, J. C., C. B. Thompson. 2002. Pathways of apoptosis in lymphocyte development, homeostasis, and disease. Cell 109:(Suppl.): S97-S107.
19. Mendez, S., S. K. Reckling, C. A. Piccirillo, D. Sacks, Y. Belkaid. 2004. Role for CD4⁺CD25⁺ regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity. J. Exp. Med. 200: 201-210.[Abstract/Free Full Text]
20. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, D. L. Sacks. 2002. CD4⁺CD25⁺ regulatory T cells control Leishmania major persistence and immunity. Nature 420: 502-507.[Medline]
21. Edinger, M., P. Hoffmann, J. Ermann, K. Drago, C. G. Fathman, S. Strober, R. S. Negrin. 2003. CD4⁺CD25⁺ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat. Med. 9: 1144-1150.[Medline]
22. Green, D. R., N. Droin, M. Pinkoski. 2003. Activation-induced cell death in T cells. Immunol. Rev. 193: 70-81.[Medline]
23. Janssen, O., J. Qian, A. Linkermann, D. Kabelitz. 2003. CD95 ligand—death factor and costimulatory molecule?. Cell Death Differ. 10: 1215-1225.[Medline]
24. Papiernik, M., M. do Carmo Leite-de-Moraes, C. Pontoux, A. M. Joret, B. Rocha, C. Penit, M. Dy. 1997. T cell deletion induced by chronic infection with mouse mammary tumor virus spares a CD25-positive, IL-10-producing T cell population with infectious capacity. J. Immunol. 158: 4642-4653.[Abstract]
25. Papiernik, M., M. L. de Moraes, C. Pontoux, F. Vasseur, C. Penit. 1998. Regulatory CD4 T cells: expression of IL-2R chain, resistance to clonal deletion and IL-2 dependency. Int. Immunol. 10: 371-378.[Abstract/Free Full Text]
26. Grundstrom, S., L. Cederbom, A. Sundstedt, P. Scheipers, F. Ivars. 2003. Superantigen-induced regulatory T cells display different suppressive functions in the presence or absence of natural CD4⁺CD25⁺ regulatory T cells in vivo. J. Immunol. 170: 5008-5017.[Abstract/Free Full Text]
27. Hildeman, D. A., Y. Zhu, T. C. Mitchell, J. Kappler, P. Marrack. 2002. Molecular mechanisms of activated T cell death in vivo. Curr. Opin. Immunol. 14: 354-359.[Medline]

This article has been cited by other articles:

				O. Bohana-Kashtan, S. Morisot, R. Hildreth, C. Brayton, H. I. Levitsky, and C. I. Civin Selective Reduction of Graft-versus-Host Disease-Mediating Human T Cells by Ex Vivo Treatment with Soluble Fas Ligand J. Immunol., July 1, 2009; 183(1): 696 - 705. [Abstract] [Full Text] [PDF]

				D. Mougiakakos, C. C. Johansson, and R. Kiessling Naturally occurring regulatory T cells show reduced sensitivity toward oxidative stress-induced cell death Blood, April 9, 2009; 113(15): 3542 - 3545. [Abstract] [Full Text] [PDF]

				L. Strauss, C. Bergmann, and T. L. Whiteside Human Circulating CD4+CD25highFoxp3+ Regulatory T Cells Kill Autologous CD8+ but Not CD4+ Responder Cells by Fas-Mediated Apoptosis J. Immunol., February 1, 2009; 182(3): 1469 - 1480. [Abstract] [Full Text] [PDF]

				M. Korporal, J. Haas, B. Balint, B. Fritzsching, A. Schwarz, S. Moeller, B. Fritz, E. Suri-Payer, and B. Wildemann Interferon Beta-Induced Restoration of Regulatory T-Cell Function in Multiple Sclerosis Is Prompted by an Increase in Newly Generated Naive Regulatory T Cells Arch Neurol, November 1, 2008; 65(11): 1434 - 1439. [Abstract] [Full Text] [PDF]

				E. S. Yolcu, X. Gu, C. Lacelle, H. Zhao, L. Bandura-Morgan, N. Askenasy, and H. Shirwan Induction of Tolerance to Cardiac Allografts Using Donor Splenocytes Engineered to Display on Their Surface an Exogenous Fas Ligand Protein J. Immunol., July 15, 2008; 181(2): 931 - 939. [Abstract] [Full Text] [PDF]

				C. Reardon, A. Wang, and D. M. McKay Transient Local Depletion of Foxp3+ Regulatory T Cells during Recovery from Colitis via Fas/Fas Ligand-Induced Death J. Immunol., June 15, 2008; 180(12): 8316 - 8326. [Abstract] [Full Text] [PDF]

				K. Venken, N. Hellings, T. Broekmans, K. Hensen, J.-L. Rummens, and P. Stinissen Natural Naive CD4+CD25+CD127low Regulatory T Cell (Treg) Development and Function Are Disturbed in Multiple Sclerosis Patients: Recovery of Memory Treg Homeostasis during Disease Progression J. Immunol., May 1, 2008; 180(9): 6411 - 6420. [Abstract] [Full Text] [PDF]

				B. Santner-Nanan, N. Seddiki, E. Zhu, V. Quent, A. Kelleher, B. F. de St Groth, and R. Nanan Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype Int. Immunol., March 1, 2008; 20(3): 375 - 383. [Abstract] [Full Text] [PDF]

				N. Oberle, N. Eberhardt, C. S. Falk, P. H. Krammer, and E. Suri-Payer Rapid Suppression of Cytokine Transcription in Human CD4+CD25 T Cells by CD4+Foxp3+ Regulatory T Cells: Independence of IL-2 Consumption, TGF-beta, and Various Inhibitors of TCR Signaling J. Immunol., September 15, 2007; 179(6): 3578 - 3587. [Abstract] [Full Text] [PDF]

				S. R. J. Taylor, D. R. Alexander, J. C. Cooper, C. F. Higgins, and J. I. Elliott Regulatory T Cells Are Resistant to Apoptosis via TCR but Not P2X7 J. Immunol., March 15, 2007; 178(6): 3474 - 3482. [Abstract] [Full Text] [PDF]

				J. Gonzalez, E. Tamayo, I. Santiuste, R. Marquina, L. Buelta, M. A. Gonzalez-Gay, S. Izui, M. Lopez-Hoyos, J. Merino, and R. Merino CD4+CD25+ T Cell-Dependent Inhibition of Autoimmunity in Transgenic Mice Overexpressing Human Bcl-2 in T Lymphocytes J. Immunol., March 1, 2007; 178(5): 2778 - 2786. [Abstract] [Full Text] [PDF]

				S. Liu, D. R. Breiter, G. Zheng, and A. Chen Enhanced Antitumor Responses Elicited by Combinatorial Protein Transfer of Chemotactic and Costimulatory Molecules J. Immunol., March 1, 2007; 178(5): 3301 - 3306. [Abstract] [Full Text] [PDF]

				A. Chen, S. Liu, D. Park, Y. Kang, and G. Zheng Depleting Intratumoral CD4+CD25+ Regulatory T Cells via FasL Protein Transfer Enhances the Therapeutic Efficacy of Adoptive T Cell Transfer Cancer Res., February 1, 2007; 67(3): 1291 - 1298. [Abstract] [Full Text] [PDF]

				L. Strauss, T. L. Whiteside, A. Knights, C. Bergmann, A. Knuth, and A. Zippelius Selective Survival of Naturally Occurring Human CD4+CD25+Foxp3+ Regulatory T Cells Cultured with Rapamycin J. Immunol., January 1, 2007; 178(1): 320 - 329. [Abstract] [Full Text] [PDF]

				M. O. Kilinc, K. S. Aulakh, R. E. Nair, S. A. Jones, P. Alard, M. M. Kosiewicz, and N. K. Egilmez Reversing Tumor Immune Suppression with Intratumoral IL-12: Activation of Tumor-Associated T Effector/Memory Cells, Induction of T Suppressor Apoptosis, and Infiltration of CD8+ T Effectors J. Immunol., November 15, 2006; 177(10): 6962 - 6973. [Abstract] [Full Text] [PDF]

				B. Li, A. S. Lalani, T. C. Harding, B. Luan, K. Koprivnikar, G. Huan Tu, R. Prell, M. J. VanRoey, A. D. Simmons, and K. Jooss Vascular Endothelial Growth Factor Blockade Reduces Intratumoral Regulatory T Cells and Enhances the Efficacy of a GM-CSF-Secreting Cancer Immunotherapy. Clin. Cancer Res., November 15, 2006; 12(22): 6808 - 6816. [Abstract] [Full Text] [PDF]

				B. Fritzsching, N. Oberle, E. Pauly, R. Geffers, J. Buer, J. Poschl, P. Krammer, O. Linderkamp, and E. Suri-Payer Naive regulatory T cells: a novel subpopulation defined by resistance toward CD95L-mediated cell death Blood, November 15, 2006; 108(10): 3371 - 3378. [Abstract] [Full Text] [PDF]

				F. Venet, A. Pachot, A.-L. Debard, J. Bohe, J. Bienvenu, A. Lepape, W. S. Powell, and G. Monneret Human CD4+CD25+ Regulatory T Lymphocytes Inhibit Lipopolysaccharide-Induced Monocyte Survival through a Fas/Fas Ligand-Dependent Mechanism J. Immunol., November 1, 2006; 177(9): 6540 - 6547. [Abstract] [Full Text] [PDF]

				A. S. Mohamood, C. J. Trujillo, D. Zheng, C. Jie, F. M. Murillo, J. P. Schneck, and A. R. A. Hamad Gld mutation of Fas ligand increases the frequency and up-regulates cell survival genes in CD25+CD4+ TR cells Int. Immunol., August 1, 2006; 18(8): 1265 - 1277. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fritzsching, B. Articles by Suri-Payer, E. Search for Related Content PubMed PubMed Citation Articles by Fritzsching, B. Articles by Suri-Payer, E.