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IMMUNOBIOLOGY
From the Laboratory of Tumor Immunology and
Immunotherapy and the Laboratory of Cellular Physiology and Immunology,
Rockefeller University; New York, NY; and the Hematology Division,
Memorial Sloan-Kettering Cancer Center, New York, NY.
Regulatory T cells (TRs) can suppress the function of
other effector T cells in the setting of autoimmunity, transplantation, and resistance to tumors. The mechanism for the induction of
TRs has not been defined. We previously reported that an
injection of immature dendritic cells (DCs) pulsed with influenza
matrix peptide (MP) led 7 days later to antigen-specific silencing of effector T-cell function in the blood of 2 healthy human subjects. Here, we found that interferon- Regulatory T cells (TRs) have now been
clearly identified in mice and humans.1-3 These cells can
inhibit strong responses mediated by CD4+ and
CD8+ effector T cells, thus preventing allograft rejection,
graft-versus-host disease (GVHD), chronic inflammatory disease, and
autoimmunity (for a review, see Roncarolo and Levings,1
Waldmann and Cobbold,2 and Sakaguchi3). Recent
studies have identified TRs in human blood, where they have
2 main functional properties4-6: they proliferate poorly in
response to mitogenic stimuli and they can dampen the responses
of effector T cells.7 Although most studies have
characterized CD4+ TRs,8,9
CD8+ T cells with regulatory properties have also been
described.10-16
Certain populations of TRs, particularly those
expressing CD4 and the CD25 interleukin 2 (IL-2)-receptor chain, are
generated in the thymus, where the cortical epithelium has been
identified as a critical antigen-presenting cell (APC).17
TRs, often identified by their capacity to produce IL-10,
can also be induced peripherally in the settings of transplantation and
GVHD,1,2 but the APC requirements have not been
identified. It is important to identify pathways that control the
formation of TRs, since these would provide novel
strategies for antigen-specific immune suppression or immune tolerance.
Dendritic cells (DCs) are powerful APCs for the induction of effector T
cells.18 To initiate immunity, DCs must carry out 2 sets
of linked events.19 One is the capture of antigens and successful formation of major histocompatibility complex (MHC)-peptide complexes; the second is to undergo a process termed "maturation" to acquire many additional properties to stimulate
immunity.19 Immature DCs appear to be inactive as inducers
of immunity in vivo.20 However, in a standard
tissue-culture assay involving initiation of the mixed leukocyte
reaction, immature DCs were not inactive but instead induced the
formation of TRs, with both the anergic and regulatory
properties mentioned above.21
In parallel, we tested the effects of immunizing volunteers with
immature DCs. We reported findings in 2 healthy volunteers who received
a single subcutaneous injection of 2 × 106 immature DCs
pulsed with an HLA-A*0201-restricted influenza matrix peptide
(MP).22 In contrast to previous findings using mature DCs,23 injection of immature DCs was associated with
antigen-specific inhibition of effector T-cell function. The
peptide-specific, interferon- In the study described here, we showed that the loss of effector
function and induction of IL-10 producers is self-limited, since there
was a return to preimmunization status by 6 months after immunization.
Importantly, we found that 1 week after immunization, the blood did
contain CD8+ T cells with TR function, able to
block the function of CD8+ effectors that secrete IFN- Study design and injection of DCs
Follow-up and immune monitoring
Assays for TRs Peripheral blood mononuclear cells (PBMCs) obtained 7 days after immunization (TR sample), before immunization, or at recovery (eg, day 180) were thawed and cultured (2-3 × 105 cells/well) either separately or together in the presence of peptide-pulsed, autologous monocyte-derived mature DCs at a PBMC-to-DC ratio of 60:1. Antigen-specific INF- -producing
cells were quantified by using a standard ELISPOT assay as described
previously.23 In addition to the immunizing peptide
(influenza MP; GILGFVFTL), additional control HLA-A*0201-restricted
peptides were from Epstein-Barr virus latent membrane protein 2 (EBV
LMP-2; CLGGLLTMV) and human immunodeficiency virus 1 (HIV-1) gag (SLYNTVATL).
For some experiments, TRs containing PBMCs (from day-7 samples) were depleted of CD8+ T cells with use of immunomagnetic beads (Miltenyi Biotec, Auburn, CA) before they were added to the cocultures. For some experiments, the TR samples were separated from the recovery specimens by a transwell to check for soluble suppressor factors. In these cultures, APCs were added on either side of the transwell. In some experiments, the cocultures of TRs and recovery cells were performed in the presence of neutralizing anti-IL-10 antibody (10 µg/mL; R&D Systems, Minneapolis, MN) or 100 U/mL recombinant IL-2 (rIL-2; Chiron, Emeryville, CA). Statistical analysis The Student t test was used to compare results in different groups. The significance level was set at P < .05.
Both healthy volunteers had been primed to influenza at baseline
because influenza MP-specific effector T cells were detectable on
ELISPOT assays and peptide-specific CTLs could be expanded by a week of
culture with mature DCs. However, 1 week after the injection of
MP-pulsed immature DCs, these effector functions in the blood were
silenced. This loss of function was reversible, with values
returning to preinjection levels by 3 to 4 months after injection in
both subjects (Figure 1). In a reciprocal
fashion, silencing and recovery of effector T-cell function were
associated with the appearance and then decline in peptide-specific
IL-10 producers, which were no longer detectable after 90 to 100 days postimmunization (Figure 1). The DC injections were not associated with
any clinical toxicity or clinical or serologic evidence of autoimmunity
in either subject. Thus, the inhibition of effector T-cell function
after a single injection of immature DCs was found to be
self-limited.
Because we had previously shown that the loss of circulating
MP-specific effector T-cell function was not associated with a decline
in circulating MHC tetramer-binding cells,22 we tested whether the effector silencing after injection of immature MP-pulsed DCs was mediated by the induction of TRs. To assess this
directly, we mixed T cells from samples obtained 1 week after
immunization (when the effector silencing was maximal) with samples
obtained before immunization. The PBMCs obtained on day 7 inhibited
MP-specific producers of INF-
Further characterization of the suppression was carried out only in samples from Im2, from whom we had additional cells available. The suppression of T-cell function was not due simply to competition for APCs or consumption of IL-2, since it was specific for immunizing peptide and observed only when suppressor samples (day 7) but not nonsuppressor (preimmunization) samples were added to the recovery samples (day 180; Figure 2B). T-cell suppression was dose dependent and observed even when the ratio of day-7 to day-180 cells was 1:10. Suppression was lost if day-7 cells were depleted of CD8+ T cells or if cell contact between day-7 and recovery T cells was prevented in transwell cultures (Figure 2C). Although the day-7 specimens had been shown to contain MP-specific IL-10 producers, addition of neutralizing anti-IL-10 antibody led to only a slight recovery of MP-specific effectors. However, the suppression was fully reversed by addition of 100 U/mL rIL-2. Thus, we found that peptide-specific CD8+ TRs induced in vivo by immature DCs inhibit CD8+ T cells in a cell-contact-dependent manner, that is, a manner largely independent of IL-10.
These data provide direct evidence for the existence of
antigen-specific CD8+ T-cell-mediated immune regulation
and of the induction of such T cells in vivo in humans by immature
DCs. Once induced, these cells have a limited life span in the
circulation. Thus, naturally occurring TRs may require
continued antigen presentation by trafficking immature DCs. Because
peptide-specific, IL-10-producing cells are also induced by immature
DCs, we refer to these suppressor cells as TRs, in keeping
with previously established nomenclature. The regulation we observed
required cell-cell contact and was largely independent of IL-10. These
features are similar to those of CD4+ TRs
induced by immature DCs in vitro.21 A subset of
CD8+CD28 The site where immature DCs generate TRs in vivo is not known. One possibility is that the DCs might traffic to lymph nodes to meet T cells recirculating by means of high endothelial venules. An alternative, which we favor because TRs have an activated phenotype, is that the DCs activate TRs that circulate from blood to extravascular spaces (here, the skin) and then return to the lymph node by means of the lymphatics. Although our studies help to confirm the presence of TRs and IL-10-producing cells in the CD8 compartment, compared with previously described CD4+ TRs, further work is needed to clarify their relation to CD25+ suppressor cells and their mechanism of action. Our data suggest DC maturation as a key therapeutic target for the regulation of immunity.19 The inhibition of maturation in antigen-capturing DCs may promote the induction of TRs in vivo. Impairment of CD8+ T-cell suppressor function has been observed in patients with human autoimmune diseases such as lupus and multiple sclerosis.15,16 A role for TRs in acceptance of human allografts has also been suggested.24 In a reciprocal fashion, reduction of TRs may improve resistance to cancer and chronic infections, as was observed in a study of experimental tumors in mice.25
We thank Joseph Krasovsky for excellent technical assistance.
Submitted October 31, 2001; accepted February 22, 2002.
Supported in part by an investigator award from the Cancer Research Institute and grants from the National Institutes of Health (CA81138 to M.V.D., CA84512 to R.M.S., and MO-1-RR00102 to the Rockefeller General Clinical Research Center).
R.M.S. has a financial interest in Merix Biosciences, whose product was studied in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Madhav Dhodapkar, Laboratory of Tumor Immunology and Immunotherapy, Rockefeller University, 1230 York Ave, New York, NY, 10021; e-mail: dhodapm{at}mail.rockefeller.edu.
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4.
Taylor PA, Noelle RJ, Blazar BR.
CD4+CD25+ immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade.
J Exp Med.
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5.
Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G.
Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood.
J Exp Med.
2001;193:1303-1310
6.
Levings MK, Sangregorio R, Roncarolo MG.
Human CD25+CD4+ T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function.
J Exp Med.
2001;193:1295-1302
7.
Shevach EM.
Certified professionals: CD4+CD25+ suppressor T cells.
J Exp Med.
2001;193:F41-F46 8. Groux H, O'Garra A, Bigler M, et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. 1997;389:737-742[CrossRef][Medline] [Order article via Infotrieve].
9.
Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M.
Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor
10.
Gaur A, Ruberti G, Haspel R, Mayer JP, Fathman CG.
Requirement for CD8+ cells in T cell receptor peptide-induced clonal unresponsiveness.
Science.
1993;259:91-94
11.
Koh D-R, Fung-Leung W-P, Ho A, Gray D, Acha-Orbea H, Mak T-W.
Less mortality but more relapses in experimental allergic encephalomyelitis in CD8
12.
Borthwick NJ, Lowdell M, Salmon M, Akbar AN.
Loss of CD28 expression on CD8+ T cells is induced by IL-2 receptor 13. Koide J, Engleman EG. Differences in surface phenotype and mechanism of action between alloantigen-specific CD8+ cytotoxic and suppressor T cell clones. J Immunol. 1990;144:32-40[Abstract].
14.
Colovai AI, Ciubotariu R, Liu Z, Cortesini R, Suciu-Foca N.
CD8+CD28
15.
Filaci G, Bacilieri S, Fravega M, et al.
Impairment of CD8+ T suppressor cell function in patients with active systemic lupus erythematosus.
J Immunol.
2001;166:6452-6457
16.
Balashov KE, Khoury SJ, Hafler DA, Weiner HL.
Inhibition of T cell responses by activated human CD8+ T cells is mediated by interferon-
17.
Bensinger SJ, Bandeira A, Jordan MS, Caton AJ, Laufer TM.
Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4+25+ immunoregulatory T cells.
J Exp Med.
2001;194:427-438 18. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245-252[CrossRef][Medline] [Order article via Infotrieve]. 19. Mellman I, Steinman RM. Dendritic cells: specialized and regulated antigen processing machines. Cell. 2001;106:255-258[CrossRef][Medline] [Order article via Infotrieve].
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The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli.
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21.
Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH.
Induction of interleukin-10 producing, non-proliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature dendritic cells.
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22.
Dhodapkar MV, Steinman RM, 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;193:233-238 23. Dhodapkar MV, Steinman RM, Sapp M, et al. Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. J Clin Invest. 1999;104:173-180[Medline] [Order article via Infotrieve]. 24. VanBuskirk AM, Burlingham WJ, Jankowska-Gan E, et al. Human allograft acceptance is associated with immune regulation. J Clin Invest. 2000;106:145-155[Medline] [Order article via Infotrieve].
25.
Sutmuller RP, van Duivenvoorde LM, van Elsas A, et al.
Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25+ regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses.
J Exp Med.
2001;194:823-832
© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
,
IL-6, tumor necrosis factor 
, and prostaglandin E2.
suppressor T cells that mediate
suppression in a cell-contact-dependent fashion has also been
described.
/