CD8+CD28− Regulatory T Lymphocytes Prevent Experimental Inflammatory Bowel Disease in Mice

Background & Aims: Immune responses to innocuous intestinal antigens appear tightly controlled by regulatory T lymphocytes. While CD4+ T lymphocytes have recently attracted the most attention, CD8+ regulatory T-cell populations are also believed to play an important role in control of mucosal immunity. However, CD8+ regulatory T-cell function has mainly been studied in vitro and no direct in vivo evidence exists that they can control mucosal immune responses. We investigated the capacity of CD8+CD28− T cells to prevent experimental inflammatory bowel disease (IBD) in mice. Methods: CD8+CD28− regulatory T cells were isolated from unmanipulated mice and tested for their capacity to inhibit T-cell activation in allogeneic mixed lymphocyte cultures in vitro and to prevent IBD induced by injection of CD4+CD45RBhigh cells into syngeneic immunodeficient RAG-2 mutant mice. Results: CD8+CD28− T lymphocytes inhibited proliferation and interferon gamma production by CD4+ responder T cells in vitro. CD8+CD28− regulatory T cells freshly isolated from spleen or gut efficiently prevented IBD induced by transfer of colitogenic T cells into immunodeficient hosts. Regulatory CD8+CD28− T cells incapable of producing interleukin-10 did not prevent colitis. Moreover, IBD induced with colitogenic T cells incapable of responding to transforming growth factor β could not be prevented with CD8+CD28− regulatory T cells. CD8+CD28+ T cells did not inhibit in vitro or in vivo immune responses. Conclusions: Our findings show that naturally occurring CD8+CD28− regulatory T lymphocytes can prevent experimental IBD in mice and suggest that these cells may play an important role in control of mucosal immunity. Background & Aims: Immune responses to innocuous intestinal antigens appear tightly controlled by regulatory T lymphocytes. While CD4+ T lymphocytes have recently attracted the most attention, CD8+ regulatory T-cell populations are also believed to play an important role in control of mucosal immunity. However, CD8+ regulatory T-cell function has mainly been studied in vitro and no direct in vivo evidence exists that they can control mucosal immune responses. We investigated the capacity of CD8+CD28− T cells to prevent experimental inflammatory bowel disease (IBD) in mice. Methods: CD8+CD28− regulatory T cells were isolated from unmanipulated mice and tested for their capacity to inhibit T-cell activation in allogeneic mixed lymphocyte cultures in vitro and to prevent IBD induced by injection of CD4+CD45RBhigh cells into syngeneic immunodeficient RAG-2 mutant mice. Results: CD8+CD28− T lymphocytes inhibited proliferation and interferon gamma production by CD4+ responder T cells in vitro. CD8+CD28− regulatory T cells freshly isolated from spleen or gut efficiently prevented IBD induced by transfer of colitogenic T cells into immunodeficient hosts. Regulatory CD8+CD28− T cells incapable of producing interleukin-10 did not prevent colitis. Moreover, IBD induced with colitogenic T cells incapable of responding to transforming growth factor β could not be prevented with CD8+CD28− regulatory T cells. CD8+CD28+ T cells did not inhibit in vitro or in vivo immune responses. Conclusions: Our findings show that naturally occurring CD8+CD28− regulatory T lymphocytes can prevent experimental IBD in mice and suggest that these cells may play an important role in control of mucosal immunity. During development of T and B lymphocytes in primary lymphoid organs, the genes encoding their antigen receptors undergo random somatic rearrangements. The resulting, still immature repertoire is therefore very large and contains many cells specific for self-antigens. Probably the majority of these potentially self-reactive cells are negatively selected by induction of anergy or apoptosis.1Hogquist K.A. Baldwin T.A. Jameson S.C. Central tolerance: learning self-control in the thymus.Nat Rev Immunol. 2005; 5: 772-782Google Scholar, 2Hardy R.R. Hayakawa K.K. B cell development pathways.Annu Rev Immunol. 2001; 19: 595-621Google Scholar However, a significant number of potentially self-reactive lymphocytes leave the primary lymphoid organs and are kept under control by “peripheral tolerance mechanisms.”3Stockinger B. T lymphocyte tolerance: from thymic deletion to peripheral control mechanisms.Adv Immunol. 1999; 71: 229-265Google Scholar Probably the most important of these mechanisms is assured by regulatory T lymphocytes capable of suppressing adaptive and also innate immune responses.4Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses.Annu Rev Immunol. 2004; 22: 531-562Google Scholar, 5Piccirillo C.A. Shevach E.M. Naturally-occurring CD4+CD25+ immunoregulatory T cells: central players in the arena of peripheral tolerance.Semin Immunol. 2004; 16: 81-88Google Scholar Regulatory T cells are known to control immune responses to self-antigens (eg, those leading to autoimmune disease or eliminating transformed cells4Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses.Annu Rev Immunol. 2004; 22: 531-562Google Scholar, 5Piccirillo C.A. Shevach E.M. Naturally-occurring CD4+CD25+ immunoregulatory T cells: central players in the arena of peripheral tolerance.Semin Immunol. 2004; 16: 81-88Google Scholar, 6Terabe M. Berzofsky J.A. Immunoregulatory T cells in tumor immunity.Curr Opin Immunol. 2004; 16: 157-162Google Scholar) but also to nonself-antigens (eg, during pregnancy or upon infection7Mills K.H. Regulatory T cells: friend or foe in immunity to infection?.Nat Rev Immunol. 2004; 4: 841-855Google Scholar, 8Aluvihare V.R. Kallikourdis M. Betz A.G. Regulatory T cells mediate maternal tolerance to the fetus.Nat Immunol. 2004; 5: 266-271Google Scholar). These cells are also known to control immune responses to innocuous (probably nonself) antigens in intestinal mucosa and, in experimental animal models, their absence can lead to inflammatory bowel disease (IBD).9Coombes J.L. Robinson N.J. Maloy K.J. Uhlig H.H. Powrie F. Regulatory T cells and intestinal homeostasis.Immunol Rev. 2005; 204: 184-194Google Scholar Moreover, patients with IBD appear to have defects in lamina propria regulatory T-cell function.10Brimnes J. Allez M. Dotan I. Shao L. Nakazawa A. Mayer L. Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease.J Immunol. 2005; 174: 5814-5822Google Scholar A large number of murine models for IBD have been developed, allowing for a dissection of cellular and molecular mechanisms involved in this disease.11Elson C.O. Cong Y. McCracken V.J. Dimmitt R.A. Lorenz R.G. Weaver C.T. Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota.Immunol Rev. 2005; 206: 260-276Google Scholar In the most extensively used experimental model, IBD is induced by injection of naive (CD4+CD45RBhigh) T cells into syngeneic immunodeficient (eg, SCID or RAG deficient) mice.9Coombes J.L. Robinson N.J. Maloy K.J. Uhlig H.H. Powrie F. Regulatory T cells and intestinal homeostasis.Immunol Rev. 2005; 204: 184-194Google Scholar Three weeks posttransfer, characteristic signs of IBD start to appear: weight loss, diarrhea, and prostrated posture of the mice. Histologic analysis of the colon usually shows significant polymorphonuclear and mononuclear cell infiltration and hyperplasia of mucosa, severe elongation of crypts, and disappearance of goblet cells. Interferon (IFN)-γ production by colitogenic T cells has been shown to play a crucial role in this animal model for IBD.12Powrie F. Correa-Oliveira R. Mauze S. Coffman R.L. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell- mediated immunity.J Exp Med. 1994; 179: 589-600Google Scholar IBD induced by injection of CD4+CD45RBhigh cells into immunodeficient mice can be prevented by injection of naturally occurring CD4+CD25+ regulatory T lymphocytes.9Coombes J.L. Robinson N.J. Maloy K.J. Uhlig H.H. Powrie F. Regulatory T cells and intestinal homeostasis.Immunol Rev. 2005; 204: 184-194Google Scholar CD4+CD25+ T cells from interleukin (IL)-10 deficient mice do not prevent colitis, demonstrating the nonredundant role of this anti-inflammatory cytokine in prevention of IBD.13Asseman C. Mauze S. Leach M.W. Coffman R.L. Powrie F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation.J Exp Med. 1999; 190: 995-1004Google Scholar Moreover, colitis induced with T cells expressing a transgenic dominant negative form of the transforming growth factor (TGF)-β receptor II (dnTβRII), and therefore incapable of responding to TGF-β, cannot be prevented with CD4+CD25+ regulatory T cells, indicating a crucial role for TGF-β.14Fahlen L. Read S. Gorelik L. Hurst S.D. Coffman R.L. Flavell R.A. Powrie F. T cells that cannot respond to TGF-β escape control by CD4(+)CD25(+) regulatory T cells.J Exp Med. 2005; 201: 737-746Google Scholar Another CD4+ regulatory T-cell population capable of preventing IBD in mice has also been described.15Groux H. A O.G. Bigler M. Rouleau M. Antonenko S. de Vries J.E. Roncarolo M.G. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis.Nature. 1997; 389: 737-742Google Scholar CD4+CD25+ regulatory T cells have also been found in human intestines.16Maul J. Loddenkemper C. Mundt P. Berg E. Giese T. Stallmach A. Zeitz M. Duchmann R. Peripheral and intestinal regulatory CD4+ CD25(high) T cells in inflammatory bowel disease.Gastroenterology. 2005; 128: 1868-1878Abstract Full Text Full Text PDF Scopus (494) Google Scholar Combined, these data suggest that CD4+ regulatory T cells may play an important role in prevention of IBD. Whereas the best characterized regulatory T cells are of CD4+CD25+ phenotype, T lymphocytes with immunosuppressive potential have also been identified in the CD8+ population. Repeated in vitro stimulation of human peripheral blood lymphocytes with allogeneic antigen-presenting cells (APCs) gradually leads to a loss of proliferative capacity. This phenomenon is caused by CD8+CD28− regulatory T lymphocytes.17Vlad G. Cortesini R. Suciu-Foca N. License to heal: bidirectional interaction of antigen-specific regulatory T cells and tolerogenic APC.J Immunol. 2005; 174: 5907-5914Google Scholar In the mouse, CD8+CD28− cells have been shown to reduce severity of experimental autoimmune encephalomyelitis.18Najafian N. Chitnis T. Salama A.D. Zhu B. Benou C. Yuan X. Clarkson M.R. Sayegh M.H. Khoury S.J. Regulatory functions of CD8+CD28– T cells in an autoimmune disease model.J Clin Invest. 2003; 112: 1037-1048Google Scholar CD8+ T cells with immunosuppressive capacity also appear to play a role in oral tolerance.19Faria A.M. Weiner H.L. Oral tolerance.Immunol Rev. 2005; 206: 232-259Google Scholar Another CD8+ regulatory T-cell population in the mouse is characterized by high-level expression of CD122, the IL-2 receptor β-chain.20Rifa’i M. Kawamoto Y. Nakashima I. Suzuki H. Essential roles of CD8+CD122+ regulatory T cells in the maintenance of T cell homeostasis.J Exp Med. 2004; 200: 1123-1134Google Scholar, 21Endharti A.T. Rifa I.Ms. Shi Z. Fukuoka Y. Nakahara Y. Kawamoto Y. Takeda K. Isobe K. Suzuki H. Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells.J Immunol. 2005; 175: 7093-7097Google Scholar Other naturally occurring and experimentally induced murine and human CD8+ regulatory T-cell populations have also been described.22Hu D. Ikizawa K. Lu L. Sanchirico M.E. Shinohara M.L. Cantor H. Analysis of regulatory CD8 T cells in Qa-1-deficient mice.Nat Immunol. 2004; 5: 516-523Google Scholar, 23Li J. Goldstein I. Glickman-Nir E. Jiang H. Chess L. Induction of TCR Vbeta-specific CD8+ CTLs by TCR Vbeta-derived peptides bound to HLA-E.J Immunol. 2001; 167: 3800-3808Google Scholar, 24Dhodapkar M.V. Steinman R.M. Antigen-bearing immature dendritic cells induce peptide-specific CD8+ regulatory T cells in vivo in humans.Blood. 2002; 100: 174-177Google Scholar, 25Dhodapkar M.V. Steinman R.M. Krasovsky J. Munz C. Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells.J Exp Med. 2001; 193: 233-238Google Scholar, 26Gilliet M. Liu Y.J. Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells.J Exp Med. 2002; 195: 695-704Google Scholar, 27Xystrakis E. Dejean A.S. Bernard I. Druet P. Liblau R. Gonzalez-Dunia D. Saoudi A. Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation.Blood. 2004; 104: 3294-3301Google Scholar Therefore, several naturally occurring as well as induced immunoregulatory CD8+ T-cell populations have been identified. However, only limited data are available on the capacity of CD8+ regulatory T cells to inhibit immune responses in vivo. CD8+ regulatory T-cell populations are also believed to be involved in control of mucosal immune responses. Human CD8+ T cells with in vitro regulatory capacity have been shown to proliferate in cultures with intestinal epithelial cells.28Allez M. Brimnes J. Dotan I. Mayer L. Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells.Gastroenterology. 2002; 123: 1516-1526Google Scholar Importantly, lamina propria–derived CD8+ T cells from normal individuals, but not from patients affected with IBD, have in vitro suppressive activity.10Brimnes J. Allez M. Dotan I. Shao L. Nakazawa A. Mayer L. Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease.J Immunol. 2005; 174: 5814-5822Google Scholar Whereas these data strongly suggest a crucial role for regulatory CD8+ T cells in mucosal tolerance, direct evidence that these cells can control IBD (eg, in animal models) has not yet been reported. We have analyzed the capacity of naive CD8+CD28− and CD8+CD28+ T lymphocytes, freshly isolated from unmanipulated mice, to inhibit proliferation and IFN-γ production by CD4+ responder T cells in allogeneic mixed lymphocyte cultures. We also evaluated if naive CD8+CD28− and CD8+CD28+ T lymphocytes can prevent experimental IBD in mice and assessed regulatory effector mechanisms used. All mice (females) were used at 6–10 weeks of age except where indicated differently. C57BL/6 and DBA/2 mice were purchased from Janvier (Le Genest St Isle, France). RAG-2–deficient and major histocompatibility complex (MHC)-deficient (IAβ°β2m°) C57BL/6 mice were bred in our specific pathogen-free animal facility and were originally obtained from the Centre de Développement des Technologies Advancées–Centre National de la Recherche Scientifique (Orléans, France). IL-10–deficient C57BL/6 mice were purchased from Charles-River (L’Arbresle, France). dnTβRII-transgenic C57BL/6 mice29Gorelik L. Flavell R.A. Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease.Immunity. 2000; 12: 171-181Google Scholar were obtained from Dr Fiona Powrie (Oxford, England) and maintained in the animal facility of the Institut de Pharmacologie et de Biologie Structurale (Toulouse, France). For in vivo studies with cells derived from these mice, 4-week-old animals were used. The health status of mice in the animal facility was periodically monitored according to Federation of European Laboratory Animal Science Associations guidelines30Nicklas W. Baneux P. Boot R. Decelle T. Deeny A.A. Fumanelli M. Illgen-Wilcke B. Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units.Lab Anim. 2002; 36: 20-42Google Scholar and generally found free of monitored pathogens. Occasionally, Trichomonas spp or (unidentified) Helicobacter spp (but never Helicobacter hepaticus) were found. Isolation of intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) was performed as described previously.31Poussier P. Edouard P. Lee C. Binnie M. Julius M. Thymus-independent development and negative selection of T cells expressing T cell receptor alpha/beta in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes.J Exp Med. 1992; 176: 187-199Google Scholar In brief, colon specimens were washed extensively in Hank’s balanced salt solution without Ca2+ and Mg2+ (Invitrogen, Cergy-Pointoise, France), opened longitudinally, and cut in pieces of 5 mm. Fragments were incubated for 15 minutes at 37°C with stirring in Hank’s balanced salt solution without Ca2+ and Mg2+ supplemented with 1 mmol/L dithiothreitol (Sigma-Aldrich, Lyon, France). The tissue was then washed twice for 45 minutes in Hank’s balanced salt solution without Ca2+ and Mg2+ containing 0.75 mmol/L EDTA (Invitrogen) at 37°C with stirring. The supernatant (released IELs) was collected and washed in medium. For the isolation of LPLs, fragments were washed for 20 minutes in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum, 10 mmol/L HEPES, 2 mmol/L l -glutamine, penicillin, streptomycin, 50 μmol/L 2-mercaptoethanol, 1 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate and incubated twice for 2 hours in complete RPMI 1640 supplemented with 0.05 mg/mL collagenase (Sigma). The supernatant (released LPLs) was collected and washed in medium. The following reagents were purchased from eBiosciences (San Diego, CA): fluorescein isothiocyanate (FITC)-conjugated antibody specific for CD44 (IM7), CD8 (53.6.7), IFN-γ (XMG1.2), and CD45RB (IM7); phycoerythrin (PE)-conjugated anti-CD28 (37.51); allophycocyanine-conjugated anti-CD4 (GK1.5), anti-CD8 (53.6.7), anti-CD25 (PC61), and anti–IL-10 (JES5-16E3); biotin-conjugated anti-CD28 (375.1), anti-CD122 (5H4), anti-CD62L (MEL-14), and anti-Thy1.1 (HIS51); and streptavidin-PE and streptavidin-PE-Cy5.5. The following reagents were purchased from BD PharMingen (Heidelberg, Germany): APC-Cy7–conjugated antibody specific for CD8 (53.6.7) and Pacific Blue–conjugated anti-CD4 (RM4-5). Anti-human latency-associated peptide (LAP) (27232) was purchased from R&D Sciences (Minneapolis, MN) and biotin-labeled anti-mouse immunoglobulin G1 from Southern Biotech (Birmingham, AL). For fluorescence-activated cell sorter analysis, cells were incubated with antibodies in staining buffer (phosphate-buffered saline and 2.5% fetal calf serum) for 20 minutes and then washed. Intracellular IFN-γ and IL-10 staining was performed as described in the following text. Labeled cells were analyzed on a FACSCalibur using CellQuest software (BD Biosciences, San Diego, CA) or on an LSR II (BD Biosciences) using Diva (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR). CD28− and CD28+ CD8+ cells were isolated as follows. Erythrocyte-depleted splenocytes were incubated with a cocktail of the following rat monoclonal antibodies: anti-FcγRII/III (2.4G2), anti-CD4 (GK1.5), and anti-MHC class II (M5). Thus, labeled cells were eliminated using Dynabeads coated with sheep anti-rat immunoglobulin G antibody (Dynal Biotech, Oslo, Norway). The resulting population was incubated with FITC-labeled anti-CD8 and biotinylated anti-CD28, followed by streptavidin-PE, and CD8+CD28+ and CD8+CD28− cells were electronically sorted using a Coulter Epics Altra (Beckman Coulter, Fullerton, CA). Alternatively, the resulting population was incubated with biotinylated anti-CD28 and FITC-labeled anti-CD8 (53.6.7), washed, incubated with streptavidin-PE, and washed, and thus PE-labeled CD28+ cells were magnetically depleted using anti-PE labeled microbeads (Miltenyi, Bergisch-Gladbach, Germany). Resulting CD28− cells were enriched in CD8+ cells by incubation with anti–FITC-labeled microbeads and subsequent magnetic positive selection (Miltenyi). Thus, a purity of >93% was routinely obtained. CD4+ T cells used in in vitro assays were enriched from erythrocyte-depleted splenocytes by Dynabead-mediated depletion of FcγRIII+, MHC class II+, and CD8+ cells, as described previously. CD4+CD45RBhigh T cells used to induce colitis were obtained as follows: ACK-treated splenocytes were depleted of CD8+, MHC class II+, and FcγRIII+ cells as described previously, CD4+ cells enriched by incubation with anti–CD4-PE followed by magnetic sorting using anti–PE-labeled microbeads (Myltenyi), cells incubated with anti–CD45RB-FITC, and CD4+CD45RBhigh T cells electronically sorted using a Coulter Epics Altra (Beckman Coulter, Paris, France). CD4+ responder (105) and CD8+CD28− regulatory (or CD8+CD28+ control) cells (105) were cultured in the presence of APCs (5 × 105) in triplicate in 96-well round-bottom plates for 96 hours and 1 μCi of 3H thymidine was added to the cultures for the last 16 hours. Thymidine incorporation was assessed using a Direct Beta Counter Matrix 9600 (Packard, Downers Grove, IL). Alternatively, T-cell division in vitro was assessed by flow cytofluorography of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled cells. Isolated wild-type or dnTβRII transgenic CD4+ effector cells were stained in vitro with the cytoplasmic dye CFSE (Sigma-Aldrich) by incubating them for 10 minutes at 37°C with 5 μmol/L CFSE. The reaction was quenched by washing in ice-cold RPMI supplemented with 10% fetal calf serum. CFSE-labeled responders (105) were cultured with isolated CD8+CD28− regulatory cells (105) in the presence of MHC-deficient APCs (5 × 105) and 0.5 μg/mL anti-CD3ϵ antibody 2C11. After 3 days of culture, proliferation of CD4+ responder cells was assessed by fluorescence-activated cell sorter gating on CD4-APC+ responders. Cells from indicated cultures were restimulated with phorbol myristate acetate (50 ng/mL) and ionomycin (1 μg/mL; both from Sigma) for 4 hours at 37°C, and brefeldin A was added during the last 2 hours (10 μg/mL; Sigma). Cells were subsequently stained for indicated surface markers, fixed with 2% paraformaldehyde for 15 minutes at 4°C, permeabilized with 0.5% saponin and 1% bovine serum albumin in phosphate-buffered saline for 30 minutes at room temperature, and finally incubated for 30 minutes at room temperature with FITC-conjugated anti–IFN-γ or APC-conjugated anti–IL-10 in permeabilization buffer. C57BL/6 RAG-2−/− mice were injected intravenously with 4 × 105 syngeneic wild-type or dnTβRII-transgenic CD4+CD45RBhigh T cells either alone or with 2 × 105 syngeneic wild-type or IL-10–deficient CD8+CD28− or CD8+CD28+ cells, isolated as described previously. T cell–reconstituted RAG-2–deficient mice were weighed weekly and killed after 6 weeks. A 1-cm piece of the distal colon was removed and fixed in 10% buffered formol. Paraffin-embedded sections (5 μm) were cut and stained with H&E and used for microscopic assessment of colitis. Colons were graded semiquantitatively as no, minor, moderate, or severe colitis in a blinded fashion. Minor colitis was defined as minimal scattered mucosal inflammatory cell infiltrates with or without minimal epithelial hyperplasia. Moderate colitis was defined as mild to moderate scattered to diffuse inflammatory cell infiltrates, sometimes extending into the submucosa and associated with erosions, with mild epithelial hyperplasia and mild mucin depletion from goblet cells. Severe colitis was defined as marked inflammatory cell infiltrates that were often transmural and associated with severe ulceration, marked epithelial hyperplasia and mucin depletion, and loss of intestinal glands. To assess the relation of CD8+CD28− T cells to other previously reported CD8+ regulatory T lymphocytes, we analyzed the phenotype of these cells by flow cytometry (Figure 1). C57BL/6 splenocytes were stained with antibodies specific for CD4, CD8, and CD28 or an isotype-matched control antibody (Figure 1A). CD8+ T cells generally expressed slightly lower levels of CD28 than CD4+ cells. However, no clear CD8+CD28− population could be distinguished. CD8+CD28− cells were therefore defined as those expressing CD28 at background levels. The thus-defined CD28− population represented 26% ± 3% of CD8+ splenocytes. In 2 previous publications, CD122+ CD8+ T cells were shown to have suppressive activity.20Rifa’i M. Kawamoto Y. Nakashima I. Suzuki H. Essential roles of CD8+CD122+ regulatory T cells in the maintenance of T cell homeostasis.J Exp Med. 2004; 200: 1123-1134Google Scholar, 21Endharti A.T. Rifa I.Ms. Shi Z. Fukuoka Y. Nakahara Y. Kawamoto Y. Takeda K. Isobe K. Suzuki H. Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells.J Immunol. 2005; 175: 7093-7097Google Scholar We therefore analyzed expression of CD122, the IL-2 receptor β chain, on CD8+ T cells (Figure 1B). All CD8+ T cells expressed CD122, albeit most at low levels. Whereas all CD8+CD28− cells were CD122low, a fraction of CD8+CD28+ cells expressed high levels of CD122. Inversely, CD122high cells all expressed very high levels of CD28 (not shown). We also analyzed expression of markers that allow for distinction of naive, activated, and memory T cells (Figure 1B). Among CD28+ cells, a population of CD44high activated/memory CD8+ T cells was found. CD44high cells expressed high levels of CD122 (not shown). In contrast, CD28− cells were all CD44low. No difference in CD45RB expression between CD28+ versus CD28− CD8+ T cells was observed, and these cells were mostly CD45RBhigh. Moreover, CD8+CD28− T cells were mostly CD25low and CD62Lhigh. Therefore, CD8+CD28− regulatory T cells had a naive quiescent phenotype and were clearly distinct from regulatory CD8+CD122+ T cells. CD8+CD28− T lymphocytes were isolated from wild-type mice and tested for their capacity to inhibit proliferation and IFN-γ production by CD4+ responder cells in allogeneic mixed lymphocyte cultures (Figure 2). Splenocytes were depleted of CD4+, FcγRII/III+, and MHC class II+ cells, and remaining cells were sorted by flow cytometry based on expression of CD8 and CD28 (Figure 2A). Freshly isolated C57BL/6 (B6, H-2b) CD4+ T cells were stimulated with DBA/2 (H-2d) APCs in vitro in the presence of CD8+CD28+ or CD8+CD28− T cells, and proliferation and IFN-γ production was measured. As shown in Figure 2B, CD8+CD28− (but not CD8+CD28+) cells inhibited proliferation in these cultures (as measured by 3H-thymidine incorporation). CD8+CD28− cells acted in a dose-dependent manner, and close to maximum suppression of proliferation was already observed at a CD8+CD28− to CD4 cell ratio of 1 to 8 (Figure 2C). Next, we evaluated the capacity of CD8+CD28− cells to inhibit production of IFN-γ (which is crucial for induction of experimental IBD in immunodeficient mice12Powrie F. Correa-Oliveira R. Mauze S. Coffman R.L. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell- mediated immunity.J Exp Med. 1994; 179: 589-600Google Scholar) by CD4+ cells. Addition of CD8+CD28− regulatory T cells to allogeneic mixed lymphocyte cultures resulted in a reduction to background levels of the frequency of IFN-γ–producing cells among CD4+ T cells (Figure 2D). In contrast, CD8+CD28+ T cells did not inhibit differentiation of IFN-γ–producing alloreactive CD4+ effector T cells (Figure 2D). These data show that freshly isolated CD8+CD28− regulatory T cells efficiently inhibit proliferation and IFN-γ production by CD4+ responder T cells. Immunomodulation by several regulatory T-cell populations involves IL-10 and TGF-β. We therefore investigated if CD8+CD28− cells can produce these cytokines. Regulatory T cells were isolated from spleen and activated in the presence of MHC-deficient APCs and anti-CD3ϵ antibody ex vivo. After 1 week of culture, T cells were restimulated with phorbol myristate acetate/ionomycin in presence of the Golgi blocker brefeldin A and subsequently stained intracellularly with an antibody specific for IL-10. We observed that a substantial proportion (15% and 20% in 2 independent experiments) of activated CD8+CD28− cells produced IL-10 (Figure 3A). We also evaluated production of TGF-β by ex vivo activated CD8+CD28− cells. LAP is a proteolytic product of the pro–TGF-β1 protein, and its surface expression is therefore limited to TGF-β1–expressing cells.32Li M.O. Wan Y.Y. Sanjabi S. Robertson A.K. Flavell R.A. Transforming growth factor-beta regulation of immune responses.Annu Rev Immunol. 2006; 24: 401-448Google Scholar As shown in Figure 3A, a substantial proportion (20% and 25% in 2 independent experiments) of activated CD8+CD28− cells expressed LAP. Combined, these data show that ex vivo activated CD8+CD28− regulatory T cells expressed IL-10 and TGF-β1. We next assessed if IL-10 and/or TGF-β are involved in suppression of T-cell activation by CD8+CD28− T cells in vitro. Phenotypic analysis of splenocytes (using the same markers as those used in Figure 1B) revealed no difference between CD8+CD28− cells from wild-type and IL-10–deficient mice (data not shown). CD4+ T cells were stimulated in vitro with MHC-deficient APCs plus anti-CD3ϵ antibody, in the absence or presence of wild-type or IL-10–deficient CD8+CD28− regulatory T cells (at a 1:1 ratio), and proliferation in these cultures was analyzed 3 days later by assessment of 3H-thymidine incorporation (Figure 3B). Wild-type but also IL-10–deficient regulatory T cells very substantially inhibited proliferation. T cells from mice transgenic for dnTβRII do not respond to TGF-β.29Gorelik L. Flavell R.A. Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease.Immunity. 2000; 12: 171-181Google Scholar Proliferation of dnTβRII-transgenic CD4 responder T cells was substantially inhibited by wild-type regulatory T cells. Howe