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Cutting Edge: Contact-Mediated Suppression by CD4⁺CD25⁺ Regulatory Cells Involves a Granzyme B-Dependent, Perforin-Independent Mechanism¹

David C. Gondek^2,*, Li-Fan Lu^2,*, Sergio A. Quezada^*, Shimon Sakaguchi and Randolph J. Noelle^3,*

^* Department of Microbiology and Immunology, Dartmouth Medical School and Norris Cotton Cancer Center, Lebanon, NH 03756; and Department of Experimental Pathology, Institute for Frontier Medical Sciences, and Department of Transplantation and Immunology, Faculty of Medicine, Kyoto University, Kyoto, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
CD4⁺CD25⁺ regulatory T cells (T_reg) are potent immunosuppressive cells that are pivotal in the regulation of peripheral tolerance. In this report, we identify granzyme B (GZ-B) as one of the key components of T_reg-mediated suppression. Induction of regulatory activity is correlated with the up-regulation of GZ-B expression. Proof of a functional involvement of GZ-B in contact-mediated suppression by T_reg is shown by the reduced ability of T_reg from GZ-B^–/– mice to suppress as efficiently as T_reg from WT mice. GZ-B-mediated suppression is perforin independent, because suppression by T_reg from perforin^–/– and WT is indistinguishable. Additionally, suppression mediated by T_reg appears to be mediated, in part, by the induction of apoptosis in the CD4⁺CD25^– effector cell. In summary, GZ-B is one of the key mechanisms through which CD4⁺CD25⁺ T_reg induce cell contact-mediated suppression.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Recent studies have underscored the importance of regulatory T cells (T_reg)⁴ in preventing the emergence of autoimmune disease, dampening the intensity of immune responses to pathogens and mediating peripheral transplantation tolerance. Multiple subsets of T_reg have been implicated in these processes and include the thymically derived CD4⁺CD25⁺ T_reg (1), as well an inducible regulatory T cell (Tr1) subset (2). A major focus of study has been to molecularly characterize the mechanisms that mediate T_reg suppression of immunity. It has been shown that Tr1 suppress predominantly by a cytokine-dependent mechanism characterized by IL-10 and TGF- secretion (3). Similarly, a TGF--dependent mechanism has also been implicated in suppression by CD4⁺CD25⁺ T_reg (4, 5, 6). In addition to suppression via soluble factors, the CD4⁺CD25⁺ T_reg have been shown to mediate suppression via a contact-dependent mechanism (7). The molecular basis for contact-dependent suppression by CD4⁺CD25⁺ T_reg is not known.

Glucocorticoid-induced TNF-like receptor (GITR or TNFSF18) is a member of the TNFR family that is constitutively expressed on T_reg and inducibly expressed on CD4⁺CD25^– effector T cells (T_eff) (8, 9). Triggering of GITR has been shown to extinguish their contact-dependent suppressive activity (8, 10). Based on this overt change in biological function, transcriptional profiling of resting, activated T_reg, and anti-GITR-treated activated T_reg has led to the identification of a number of candidate molecules that may be involved in contact-dependent suppression. One such molecule that was identified as up-regulated in activated T_reg and whose expression was reduced via GITR-triggering is granzyme B (GZ-B).

GZ-B is a serine protease, secreted mainly by NK cells and CTLs (11), and is largely responsible for the induction of apoptosis in the target cell. However, recent reports have shown that human CD4⁺ T cells are also able to synthesize GZ-B and perforin (12, 13). Furthermore, studies by Ley and coworkers as well as others (14, 15) have shown that GZ-B is highly up-regulated in activated human T cells bearing a Tr1 phenotype. Moreover, Ley and coworkers (16) have shown CD4⁺CD25⁺ T_reg in the human system mediate suppression with requirement for granzyme A (GZ-A). These results suggest a possible role for granzyme in mediating T cell suppression. The data presented in this study implicate that GZ-B plays a pivotal role in the suppressive capacity of murine CD4⁺CD25⁺ T_reg.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice and materials

Congenic strains CD45.1 or CD45.2 C57BL/6 and perforin^–/– mice, 8–10 wk old, were purchased from The Jackson Laboratory. C57BL/6 GZ-B^–/– mice (15) were bred and maintained in our facility at Dartmouth Medical School.

Cell isolation, gene array, and real time

Single-cell suspensions were prepared from 8- to 10-wk-old mice and applied to CD4 enrichment. CD4⁺CD25^– and CD4⁺CD25⁺ T cells were further purified by magnetic separation with MACS (Miltenyi Biotec) according to the manufacturer’s instructions. Enriched cell populations and purified cells were phenotypically analyzed by FACS. The purities of CD4⁺CD25^– and CD4⁺CD25⁺ T cells were 90–95%, respectively. Freshly isolated cells have been inoculated (10⁶/ml; complete RPMI 1640/10% FBS supplemented with 100 U of IL2) into a 24-well plate precoated with 10 µg/ml anti-CD3 (clone 2C11) with or without 10 µg/ml anti-GITR (clone DTA-1; Ref.8) cultured at 37°C for 0, 12, and 48 h. Purified RNA were then analyzed by using Affymetrix mouse genome A430 oligonucleotide arrays or by real-time PCR analysis.

Cell culture and T cell suppression assay

GZ-B expression was assessed in freshly isolated CD4⁺CD25⁺ T cells or in cells cultured in vitro 24–72 h in the presence of plate-bound CD3 (1 µg/ml) with 100 U/ml IL-2.

Spleens and lymph nodes from wild-type, perforin^–/–, or GZ-B^–/– mice were magnetic bead sorted as stated above. Further purification of the T_eff subset was accomplished with a CD4⁺ T cell Isolation kit (Miltenyi Biotec). Effector cells were >95% pure at the end of this isolation. In a polyclonal T_reg suppressor assay, CD4⁺CD25^– T_eff cells (5 x 10⁴) were cocultured with irradiated T-depleted splenocytes (1 x 10⁵), 5 µg/ml anti-CD3, and indicated numbers of CD4⁺CD25⁺ cells for 3 days. In some experiments, 5 µg/ml anti-GITR was also added to the wells. Proliferation was assessed by incorporation of [³H]thymidine (1 µCi/well), which was added for the last 8 h of culture.

Cell surface, intracellular staining, and flow cytometry

Approximately 2 x 10⁵ cells from each of triplicate wells were collected and pooled. Cells were labeled with anti-CD45.1-allophycocyanin (clone A20; eBioscience). Samples were then resuspended in 1x annexin staining buffer and treated with Annexin V^FITC (BD Pharmingen) and propidium iodide (PI; Sigma-Aldrich). For GZ-B expression assay, following isolation for fresh T_reg or 24–72 h for cultured T_reg, cells were stained with anti-CD4-FITC (clone RM 4-5) and anti-CD25-PE (clone PC-61). Samples were then fixed and permeabilized (Cytofix/Cytoperm; BD Pharmingen) and stained with anti-human GZ-B-allophycocyanin (clone GB12; Caltag) diluted 1/200 in staining buffer. Throughout all steps, normal rat serum (5% v/v; Invitrogen Life Technologies) was used to block nonspecific binding. Samples were analyzed on FACScan (BD Biosciences). Anti-human GZ-B cross-reactivity with mouse GZ-B has been previously reported (15). For CFSE experiments, CD45.1⁺ cells were labeled with 5 µM CFSE and added to suppressor assay as described above.

Statistical analysis

Analysis of proliferation assays and real-time expression between the various treatment groups were analyzed by two-tailed, paired Student’s t test. Values of p < 0.05 were considered significant.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Anti-GITR causes down-regulation of GZ-B in in vitro-cultured T_reg

CD4⁺CD25⁺ T_reg are suppressive to naive CD4⁺ T_eff in vitro following polyclonal and Ag-specific activation. Furthermore, the in vitro suppressive capacity has been shown to be contact dependent and ablated following treatment with anti-GITR (8). Global gene analysis of activated T_reg treated or untreated with anti-GITR was used to identify candidate genes involved in suppression. We examined naive and activated T_eff (purified CD4⁺CD25^– T cells) and T_reg (purified CD4⁺CD25⁺ T cells) in the presence of anti-CD3 with or without anti-GITR for 12 or 48 h. Of the 22,700 genes examined, 259 were up-regulated >1.5-fold and 99 were down-regulated >1.5-fold in T_reg following treatment with anti-GITR and anti-CD3 relative to treatment with anti-CD3 alone. GZ-B, as has been shown previously, is up-regulated with T_reg activation via anti-CD3 alone (9, 17). Studies presented herein show that that GZ-B is down-regulated 2-fold with anti-CD3 in combination with anti-GITR (Fig. 1, A and B). The microarray data was confirmed by RT-PCR (Fig. 1C). At both the 12- and 48-h time point, the levels GZ-B expression are 2-fold greater with anti-CD3 alone treatment vs combining with anti-GITR stimulation. Moreover, protein expression of GZ-B recapitulates the results found via RT-PCR by increasing the abundance of GZ-B from 24 to 72 h (Fig. 1D). Additionally, after 12 h in culture, T_reg GZ-B mRNA expression is 20-fold greater than T_eff with CD3 stimulation alone (data not shown). We also examined expression levels of GZ-A and perforin at all time points. For both molecules, we see similar regulation to that of GZ-B with anti-GITR treatment; however, expression is at a much lower intensity at all time points (Fig. 1, A and B). These data were also confirmed by RT-PCR (data not shown). These data indicate that, immediately following activation, T_reg rapidly up-regulate GZ-B; however, GZ-A and perforin remain low in abundance relative to GZ-B expression.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Activation-induced up-regulation of GZ-B in Treg and its regulation by GITR. A, Cells were purified via MACS columns to >90% purity and cultured in vitro with anti-CD3 and IL-2 with or without anti-GITR for 12 h. RNA was prepared and hybridized to the Affymetrix A430 array. Relative expression indicates the mean log 2 ratio of changes in Treg expression between anti-CD3-alone treatment and anti-CD3 with anti-GITR. B, Gene chip signal intensity comparison of GZ-A, GZ-B, and perforin following treatments for 12 or 48 h. C, Real-time RT-PCR analysis of GZ-B expression in Treg treated as described above. Figure is representative of two independent experiments. D, GZ-B expression in freshly isolated Treg or in vitro cultured for 24 or 72 h with plate-bound anti-CD3 and 100 U of IL-2.

Coculture of T_reg with wild-type T_eff leads to suppression of proliferation in a dose-dependent manner. To functionally evaluate the role of GZ-B in the contact-mediated suppression by T_reg, the suppressive activity of T_reg from WT and GZ-B^–/– mice was compared (Fig. 2A). Data presented show that T_reg from WT mice at a 1:1 ratio suppress the proliferation of T_eff >90%, whereas T_reg from GZ-B^–/– mice suppress T_eff proliferation <50%. The reduced suppressive activity of T_reg from GZ-B^–/– mice is observed across a spectrum of T_reg:T_eff ratios, suggesting a functional role of GZ-B in contact-mediated suppression. A comparison of FoxP3 levels of GZ-B^–/– T_reg revealed no significant difference from those of WT T_reg (data not shown). Because loss of GZ-B does not completely extinguish T_reg suppression, additional contact-dependent mechanisms must be important in this system.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. GZ-B mediates Treg suppression via a perforin-independent mechanism. A, Treg isolated from wild-type or GZ-B–/– mice were cocultured with CD4+CD25– Teff and irradiated T-depleted APC with anti-CD3 for 72 h. Wells were pulsed with 1 uCi/well 3H for the last 8 h of culture, and analyzed as described in Materials and Methods. B, Treg isolated from wild-type or perforin–/– mice were cultured as above.

A recent report by Grossman et al. (16) indicates that human CD4⁺CD25⁺ T_reg mediate their suppressive effects via death induced by a GZ-A perforin-dependent mechanism. The differences between the use of GZ-A in humans and GZ-B in mice could be due to species differences, or subtle differences in the subsets and/or activation of T cells that were used. With regard to perforin dependency, the study by Ley (16) implicates perforin because of the fact that a calcium chelator relieves suppression. Although this is a reasonable assertion, they also show that CD18 is required, and it is known that this molecule requires calcium to form the tight synapse required for granzyme-mediated toxicity (23, 24, 25). In our studies using perforin knockout mice, suppression was indistinguishable from WT mice.

Induction of T_eff apoptosis is a component of contact-mediated suppression

Recent reports have re-examined apoptosis by T_reg of T_eff as a mechanism for suppression (15, 26). The molecule(s) that mediate the induction of T_eff apoptosis have not been resolved, and it is unlikely that FasL plays a central role (27). Based on the finding that GZ-B plays a functionally significant role in T_reg suppression, the ability of T_reg to induce T_eff apoptosis and cell death was re-examined. The induction of T_eff apoptosis by T_reg was determined following the in vitro coculture of activated T_eff and T_reg. Briefly, CD45.1⁺ (Ly5.2⁺) T_eff were cocultured with increasing numbers of CD45.2⁺ (Ly5.1⁺) T_reg, in the presence of anti-CD3. After 72 h of culture, apoptosis of the CD4⁺ T_eff was determined by multiparameter flow cytometry. The data show that there is a dose-dependent increase in cell death of the T_eff cells when cocultured with T_reg, such that 50% more T_eff are dead at a 1:1 ratio than at a 1:16 ratio of T_reg to T_eff (Fig. 3A). Moreover, addition of anti-GITR relieves the suppression and apoptosis as evidenced by enhanced proliferation and cell survival (data not shown). In parallel experiments, we examined thymidine incorporation in a standard suppressor assay with T_reg from wild-type mice treated with anti-CD3 to determine levels of suppressive activity concurrent with PI/annexin staining (Fig. 3B). To distinguish between the antiproliferative and antiapoptotic effect of T_reg, we examined suppression and death with CFSE-labeled T_eff counterstained with PI. In Fig. 3C, we demonstrate that the T_eff have a greater percentage of PI⁺ cells when cocultured with T_reg. Interestingly, in addition to the induction of cell death, the proliferation of PI^– T_eff was also inhibited, which indicates multiple mechanisms are involved in T_reg-mediated suppression.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Treg suppression is mediated by enhanced death in Teff. A, Ly5.2+ Teff and Ly5.1+ Treg and APC were cocultured for 72 h in a standard suppressor assay. Wells were harvested and stained with anti-Ly5.2 to identify the CD4+CD25– Teff. Cells were then stained with PI and Annexin VFITC. B, Proliferation of cells in wells run simultaneously with stained wells. Cells were pulsed with 1uCi/well 3H for the last 8 h of culture. C, CFSE-labeled cells were cultured for 72 h in standard suppressor assay, and then counterstained with PI.

	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 National Institutes of Health Grants CA91436-01 and AI48667.

² D.C.G. and L.-F.L. contributed equally to this manuscript.

³ Address correspondence and reprint requests to Dr. Randolph J. Noelle, Department of Microbiology and Immunology, Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756. E-mail address: rjn{at}dartmouth.edu

⁴ Abbreviations used in this paper: T_reg, regulatory T cell; T_eff, effector T cell; GITR, glucocorticoid-induced TNF-like receptor; GZ-B, granzyme B; GZ-A, granzyme A; PI, propidium iodide.

Received for publication August 18, 2004. Accepted for publication December 7, 2004.

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				E. Billerbeck, H. E. Blum, and R. Thimme Parallel Expansion of Human Virus-Specific FoxP3- Effector Memory and De Novo-Generated FoxP3+ Regulatory CD8+ T Cells upon Antigen Recognition In Vitro J. Immunol., July 15, 2007; 179(2): 1039 - 1048. [Abstract] [Full Text] [PDF]

				S. E.A. Street, N. Zerafa, M. Iezzi, J. A. Westwood, J. Stagg, P. Musiani, and M. J. Smyth Host Perforin Reduces Tumor Number but Does Not Increase Survival in Oncogene-Driven Mammary Adenocarcinoma Cancer Res., June 1, 2007; 67(11): 5454 - 5460. [Abstract] [Full Text] [PDF]

				K. G. Elpek, C. Lacelle, N. P. Singh, E. S. Yolcu, and H. Shirwan CD4+CD25+ T Regulatory Cells Dominate Multiple Immune Evasion Mechanisms in Early but Not Late Phases of Tumor Development in a B Cell Lymphoma Model J. Immunol., June 1, 2007; 178(11): 6840 - 6848. [Abstract] [Full Text] [PDF]

				D. Baatar, P. Olkhanud, K. Sumitomo, D. Taub, R. Gress, and A. Biragyn Human Peripheral Blood T Regulatory Cells (Tregs), Functionally Primed CCR4+ Tregs and Unprimed CCR4- Tregs, Regulate Effector T Cells Using FasL J. Immunol., April 15, 2007; 178(8): 4891 - 4900. [Abstract] [Full Text] [PDF]

				E. Marinova, S. Han, and B. Zheng Germinal Center Helper T Cells Are Dual Functional Regulatory Cells with Suppressive Activity to Conventional CD4+ T Cells J. Immunol., April 15, 2007; 178(8): 5010 - 5017. [Abstract] [Full Text] [PDF]

				G. Rudge, S. P. Barrett, B. Scott, and I. R. van Driel Infiltration of a Mesothelioma by IFN-{gamma}-Producing Cells and Tumor Rejection after Depletion of Regulatory T Cells J. Immunol., April 1, 2007; 178(7): 4089 - 4096. [Abstract] [Full Text] [PDF]

				D. Haribhai, W. Lin, L. M. Relland, N. Truong, C. B. Williams, and T. A. Chatila Regulatory T Cells Dynamically Control the Primary Immune Response to Foreign Antigen J. Immunol., March 1, 2007; 178(5): 2961 - 2972. [Abstract] [Full Text] [PDF]

				N. K. Crellin, R. V. Garcia, and M. K. Levings Altered activation of AKT is required for the suppressive function of human CD4+CD25+ T regulatory cells Blood, March 1, 2007; 109(5): 2014 - 2022. [Abstract] [Full Text] [PDF]

				R. Zeiser, V. H. Nguyen, J.-Z. Hou, A. Beilhack, E. Zambricki, M. Buess, C. H. Contag, and R. S. Negrin Early CD30 signaling is critical for adoptively transferred CD4+CD25+ regulatory T cells in prevention of acute graft-versus-host disease Blood, March 1, 2007; 109(5): 2225 - 2233. [Abstract] [Full Text] [PDF]

				F. Marangoni, S. Trifari, S. Scaramuzza, C. Panaroni, S. Martino, L. D. Notarangelo, Z. Baz, A. Metin, F. Cattaneo, A. Villa, et al. WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells J. Exp. Med., February 19, 2007; 204(2): 369 - 380. [Abstract] [Full Text] [PDF]

				Z.-X. Zhang, Y. Ma, H. Wang, J. Arp, J. Jiang, X. Huang, K. M. He, B. Garcia, J. Madrenas, and R. Zhong Double-Negative T Cells, Activated by Xenoantigen, Lyse Autologous B and T Cells Using a Perforin/Granzyme-Dependent, Fas-Fas Ligand-Independent Pathway J. Immunol., November 15, 2006; 177(10): 6920 - 6929. [Abstract] [Full Text] [PDF]

				T. L. Sukiennicki and D. J. Fowell Distinct Molecular Program Imposed on CD4+ T Cell Targets by CD4+CD25+ Regulatory T Cells J. Immunol., November 15, 2006; 177(10): 6952 - 6961. [Abstract] [Full Text] [PDF]

				P. Lewkowicz, N. Lewkowicz, A. Sasiak, and H. Tchorzewski Lipopolysaccharide-Activated CD4+CD25+ T Regulatory Cells Inhibit Neutrophil Function and Promote Their Apoptosis and Death J. Immunol., November 15, 2006; 177(10): 7155 - 7163. [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]

				S. Steffens, F. Burger, G. Pelli, Y. Dean, G. Elson, M. Kosco-Vilbois, L. Chatenoud, and F. Mach Short-Term Treatment With Anti-CD3 Antibody Reduces the Development and Progression of Atherosclerosis in Mice Circulation, October 31, 2006; 114(18): 1977 - 1984. [Abstract] [Full Text] [PDF]

				R. Reibke, N. Garbi, R. Ganss, G. J. Hammerling, B. Arnold, and T. Oelert CD8+ regulatory T cells generated by neonatal recognition of peripheral self-antigen PNAS, October 10, 2006; 103(41): 15142 - 15147. [Abstract] [Full Text] [PDF]

				C. M. Tschopp, N. Spiegl, S. Didichenko, W. Lutmann, P. Julius, J. C. Virchow, C. E. Hack, and C. A. Dahinden Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma Blood, October 1, 2006; 108(7): 2290 - 2299. [Abstract] [Full Text] [PDF]

				K. Loser and S. Beissert Therapeutic modulation of cutaneous autoimmunity by regulatory T cells Rheumatology, October 1, 2006; 45(suppl_3): iii20 - iii22. [Abstract] [Full Text] [PDF]

				A. Toda and C. A. Piccirillo Development and function of naturally occurring CD4+CD25+ regulatory T cells J. Leukoc. Biol., September 1, 2006; 80(3): 458 - 470. [Abstract] [Full Text] [PDF]

				E. Uss, A. T. Rowshani, B. Hooibrink, N. M. Lardy, R. A. W. van Lier, and I. J. M. ten Berge CD103 Is a Marker for Alloantigen-Induced Regulatory CD8+ T Cells. J. Immunol., September 1, 2006; 177(5): 2775 - 2783. [Abstract] [Full Text] [PDF]

				N. Sugimoto, T. Oida, K. Hirota, K. Nakamura, T. Nomura, T. Uchiyama, and S. Sakaguchi Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis Int. Immunol., August 1, 2006; 18(8): 1197 - 1209. [Abstract] [Full Text] [PDF]

				K. Wing, Z. Fehervari, and S. Sakaguchi Emerging possibilities in the development and function of regulatory T cells Int. Immunol., July 1, 2006; 18(7): 991 - 1000. [Abstract] [Full Text] [PDF]