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Ludwig Institute Clinical Trial Center, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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30 years ago as the active principle produced by a strain of Streptomyces hygroscopicus (2). In mammalian cells, rapamycin binds to FK506-binding protein-12, a highly conserved cytoplasmic receptor. The FK506-binding protein-12-rapamycin complex then binds to and inhibits the kinase activities of the mammalian target of rapamycin serine/threonine protein kinase, the activation of which is essential for protein synthesis and cell cycle progression. The presence of rapamycin during T cell activation blocks T cell cycle progression in the G1 phase, resulting in the induction of tolerance (3, 4). Because of its immunosuppressive properties, rapamycin is currently used to prevent graft rejection (5). In contrast, the cytostatic properties of rapamycin have also promoted its use, as well as that of other mammalian target of rapamycin inhibitors, for cancer therapy (6, 7, 8). As illustrated by these diverse clinical applications, the full spectrum of the effects of in vivo treatment with rapamycin and related compounds remains to date to be fully elucidated. Among unpredicted effects of rapamycin, Tian et al. (9) have reported a reduction of CD4+CD8+ double-positive thymocytes and increased proportions of CD4+CD25+ T cells in the periphery of rats treated with therapeutic doses of rapamycin. More recently, Battaglia et al. (10) have reported that stimulation of CD4+ T cells from spleens of OVA TCR transgenic mice in the presence of rapamycin results in the selective expansion of naturally occurring CD4+CD25+ regulatory T cells (Tregs)3 able to suppress allograft rejection in a model of allogeneic pancreatic islet transplantation. Based on these data, the authors have proposed that rapamycin may be used to selectively expand ex vivo naturally occurring Tregs for cellular therapy in T cell-mediated diseases. The relevance of these findings to the human system, however, has not been assessed to date. In addition, direct evidence of the ability of rapamycin to promote the selective expansion of naturally occurring human Tregs was not provided in previous studies.
CD4+ Tregs play a key role in inducing and maintaining immunologic tolerance and are also involved in the modulation of immunoresponses to cancer, pathogens, and alloantigens (11, 12, 13). In humans, different subsets of CD4+ Tregs have been described. They include Th3 cells secreting high levels of TGF-
1, which are induced by stimulation of CD4+ T cells in the presence of TGF-1 or by oral Ag administration and Tr-1 cells, secreting IFN-
and IL-10, that can be induced following stimulation in the presence of IL-10 (14, 15, 16). The Th3 and Tr-1 subsets are generated in the periphery and allow the development of peripheral tolerance to self Ags not or poorly expressed in the thymus. A distinct subset is composed by naturally occurring Tregs, generated as a distinct lineage in the thymus and that constitutively express CD25. Thymically derived Tregs are constitutively anergic (17). The key role of cells from this subset in the maintenance of tolerance is demonstrated by the development of autoimmune diseases following their depletion or functional alteration in normal animals (18). The phenotypic characterization of naturally occurring Tregs among human circulating lymphocytes (the most readily available source of cells for immunological assessments in humans) has been complicated by the fact that CD25 is not exclusively expressed by Tregs, but also by nonregulatory lymphocytes, following activation. Furthermore, whereas initial studies in humans have focused on the overall CD4+CD25+ population that represents, as in mice, 6–10% of total CD4+ T cells, later only CD4+ T cells expressing the highest levels of CD25 (CD4+CD25bright) and with an Ag-experienced phenotype have been considered to be genuine Tregs. In addition to the previously described Ag-experienced Tregs (19) that most likely contain some proportions of peripherally derived Tregs and also of activated non-Tregs, we have recently identified a subset of clearly distinguishable CD25+ cells contained in the naive CD4+CD45RA+CCR7+ T cell fraction, which we have named natural naive (Nn) Tregs (20). NnTregs represent the naive circulating compartment of human naturally occurring Tregs. NnTregs display, in the presence of IL-2, a higher proliferative potential than Ag-experienced Tregs, but, similar to those, they are anergic and suppressor ex vivo. The definition of the circulating NnTreg subset highly facilitates the analysis of naturally occurring Tregs in humans.
In this study, we have assessed the effect of rapamycin on the growth, phenotype, and function of human circulating CD4+ T cells, following TCR-mediated stimulation in the presence of IL-2. Consistent with the recent data obtained in mice (10), we have observed that stimulation of human CD4+ T cells in the presence of rapamycin results in a highly increased suppressor function as compared with that of CD4+ T cells stimulated in the absence of the drug. However, when we analyzed the origin of this phenomenon by assessing the effect of rapamycin on the growth and differentiation of non-Treg and Treg populations of defined differentiation stages, highly purified ex vivo from circulating CD4+ T cells, we found that the increased suppressive activity in rapamycin-treated cultures is not due to the selective expansion of naturally occurring Tregs, but to rapamycin-induced anergy and suppressor functions in conventional CD4+ T cell populations. This effect is dependent on the continuous presence of rapamycin in the cultures and is reversible upon withdrawal of the drug.
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Materials and Methods |
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Peripheral blood samples were obtained from the New York Blood Center. Mononuclear cells were isolated by density gradient sedimentation using a Ficoll-Hypaque gradient (Amersham Biosciences). CD4+ T cells were enriched by magnetic cell sorting using the MiniMACS Separator (Miltenyi Biotec). For the cell sorting experiments, CD4+ T cells were stained with anti-CD4 (BD Biosciences), anti-CD8, anti-CD45RA (Caltag Laboratories), and anti-CD25 (Beckman Coulter). After gating on the CD4+CD8– lymphocytes, cells were separated into four subsets on the basis of CD45RA and CD25 expression, as described previously (20), using a FACSAria (BD Biosciences). Data analysis was performed using the FACSDiva software (BD Biosciences). The purity of sorted populations was routinely >96%.
Assessment of cell growth, differentiation, and suppression
To assess the growth potential of CD4+ Treg and non-Treg populations in the absence or in the presence of rapamycin, total CD4+ T cells or sorted T cell subsets were labeled or not with CFSE (5 µM; Molecular Probes) and stimulated with plate-bound anti-CD3 (OKT3; 0.4 µg/ml) and anti-CD28 (CD28.2; 1 µg/ml) (eBiosciences) in the presence of IL-2 (100 IU/ml), which was maintained throughout the culture period, and irradiated allogeneic CD4–CD8– APCs. Where indicated, the culture was carried on in the presence of rapamycin (Sigma-Aldrich; 100 nM). Stimulation was repeated at weekly intervals. The phenotype of stimulated CD4+ T cells was assessed at day 7 after stimulation by staining with Abs to CD3 (Caltag Laboratories), CD4 (BD Biosciences), CD45RA (Caltag Laboratories), CCR7 (BD Biosciences), and CD25 (Beckman Coulter). Cell division was assessed at day 5 after stimulation by FACS analysis of CFSE dilution. The mean cycle number was calculated as the sum of nfn for n = 0 to nmax, where n is the cycle number and fn is the fraction of cells that have undergone n divisions. Cell growth was assessed at day 7 after stimulation by manual counting. The ability of Tregs to suppress the growth of responder total CD4+ cells was assessed by coculture of CFSE-labeled responders (2 x 104 cells/well) with suppressors at a suppressor to responder cell ratio of 1:1, in 96-well U-bottom plates in the presence of 2 x 104 cells/well irradiated allogeneic CD14+ cells as APCs and PHA (1 µg/ml; Sigma-Aldrich) or plate-bound anti-CD3/CD28 in the absence of exogenous IL-2. The growth (100 – percentage of undivided cells) in the wells with suppressor cells (experimental group) was compared with that in the wells without suppressors (control group). The percentage of growth was determined at day 5 after stimulation as follows: (growth of experimental group/growth of control) x 100.
Assessment of FOXP3 expression
RNA was prepared from in vitro-stimulated sorted CD4+ T cell subsets on day 7 after stimulation using RNeasy Mini Kit (Qiagen). cDNA synthesis was performed using Promega Reverse Transcription System A3500 (Promega), and cDNA integrity was tested by amplification of RPL32 in a 35-cycle PCR. Semiquantitative FOXP3 mRNA expression was assessed using the following primers: forward primer, 5'-TCA CCT ACG CCA CGG TCA T-3' and reverse primer, 5'-CAC AAA GCA CTT GTG CAG-3'. Quantitative real-time PCR was performed with a TaqMan assay on an ABI 7000 system (Applied Biosystems) using Assays-on-Demand Gene Expression probes for FOXP3 (Hs 00203958; Applied Biosystems). As a control of input cDNA, we used GAPDH, Taqman probe, and primers. The probe sequence was as follows: FAM-5'-AAG GTG AAG GTC GGA GTC AAC GGA TTT G-3'-TAMRA. Primer sequences for GAPDH were as follows: 5'-CCACATCGCTCAGACACCAT-3' and 5'-CCAGGCGCCCAATACG-3' (Applied Biosystems).
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Results |
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We initially assessed the effect of rapamycin on the growth and differentiation of total human circulating CD4+ T cells. To this purpose, CD4+ T cells were highly enriched from circulating lymphocytes of healthy donors using magnetic cell sorting and stimulated with plate-bound anti-CD3/CD28 Abs, APCs, and IL-2 in the absence or in the presence of rapamycin. To follow cell division, one part of the cells was labeled with CFSE before stimulation. Subsequently, the cultures underwent additional stimulation cycles, at 1-wk intervals. Cell division was measured at day 5 after stimulation, and total cell growth and phenotype were assessed at day 7 after stimulation. Cell division was clearly higher in the absence than in the presence of rapamycin, as reflected by both the percentage of undivided cells in the cultures and the mean cycle number (Fig. 1, A–C). At the end of each stimulation cycle, the total number of CD4+ T cells recovered in control cultures was in average 3- to 6-fold higher as compared with cultures in rapamycin (Fig. 1D). As assessed by staining with annexin V, 24 h after each stimulation, the presence of rapamycin had no detectable effect on activation-induced cell death (data not shown). At the end of the first stimulation cycle, CD4+ T cells stimulated in the absence of rapamycin contained lower proportions of naive (CD45RA+CCR7+) and central memory CD4+ T cells (CD45RA–CCR7+) as compared with those stimulated in the presence of rapamycin (Fig. 2, A and B). In contrast, the proportion of effector memory CD4+ T cells (CD45RA–CCR7–) was higher in the absence than in the presence of rapamycin. After additional stimulation, both in the absence and in the presence of rapamycin, however, the majority of the cells in the cultures acquired an effector memory phenotype (data not shown). Together these data point toward an effect of rapamycin in delaying T cell differentiation. CD4+ T cells stimulated and cultured in the presence of rapamycin contained higher proportions of CD25bright T cells as compared with control cultures. The increase of CD25bright T cells in cultures containing rapamycin was already evident after the first stimulation cycle (Fig. 2, C and D) and was maintained after further stimulation. The presence of rapamycin in the absence of TCR-mediated stimulation had no effect on the cell cultures (data not shown). The suppressor capacity of CD4+ T cells stimulated in vitro in the absence or in the presence of rapamycin was assessed 1 wk after the second cycle of stimulation. To this purpose, CD4+ T cells from rapamycin-treated and control cultures were cocultured with allogeneic CFSE-labeled responder CD4+ T cells and stimulated with either PHA or anti-CD3/CD28 Abs. As illustrated in Fig. 3, in both experimental settings, rapamycin-treated CD4+ T cells displayed a capacity to suppress the proliferation of responder T cells that was much increased as compared with that of control cultures.
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Battaglia et al. (10) have recently reported that the presence of CD4+ T cells with increased suppressive activity in rapamycin-treated murine cultures is due to a selective expansion of the low proportions of naturally occurring CD4+ Tregs present in the total CD4+ T cell populations at the beginning of the cultures. To assess the relevance of these findings in our experimental system, we stained highly enriched CD4+ T cells from healthy donors with Abs to CD45RA and CD25 and sorted them into four distinct subsets, as previously described (20): naive CD45RA+CD25–, NnTregs CD45RA+CD25+, and two Ag-experienced subsets, one CD45RA– expressing intermediate levels of CD25 (defined as CD45RA–CD25–), and the CD45RA–CD25bright population corresponding to Ag-experienced Tregs (Fig. 4A). We have shown previously that both ex vivo suppressor functions and expression of FOXP3, a transcriptional repressor considered as a Treg marker, are confined to the Treg and NnTreg subsets (20). The sorted populations were stimulated with anti-CD3/CD28 Abs, IL-2, and APCs in the presence or absence of rapamycin, as detailed above. As illustrated in Fig. 4B, after the first cycle of stimulation, all sorted populations, with the exception of CD45RA–CD25bright Tregs, which have poor proliferative capacity, had proliferated to a roughly similar extent in the absence of rapamycin. After additional stimulation in the absence of rapamycin, Ag-experienced CD4+CD25– T cell populations proliferated slightly more than naive CD4+CD25– T cells. Treg populations proliferated less than nonregulatory ones, although NnTregs proliferated more than Tregs (Fig. 4B). Proliferation of all populations was inhibited by the presence of rapamycin to an extent that was comparable or superior in regulatory as compared with nonregulatory populations (Fig. 4B). It is noteworthy that at the end of the first cycle of in vitro stimulation, both in the presence or in the absence of rapamycin, FOXP3 was expressed at high and comparable levels in all populations (Fig. 4C). These results are in line with recent data reported by Walker et al. (21), indicating that, at variance with the murine system, expression of FOXP3 in human conventional CD4+CD25– T cells is induced following in vitro stimulation. Following in vitro expansion in the absence of rapamycin, the Ag-experienced CD25– populations were not suppressive, but instead they slightly facilitated the growth of cocultured CD4+ T cells (Fig. 4D). We failed to detect significant suppressive activity with the populations derived from Ag-experienced Tregs. Interestingly, similar data (loss of suppressor function by Tregs following in vitro expansion) have been reported by Battaglia et al. (10), and are in line with the concept that Ag-experienced Treg populations contain variable proportions of non-Tregs that may overgrow after in vitro expansion. Surprisingly, in vitro-stimulated naive CD4+CD25– exhibited a rather significant suppressor activity. However, in vitro-stimulated NnTregs exhibited an even stronger suppressive activity. In addition and importantly, for all populations, suppressor functions were increased after stimulation in the presence of rapamycin. By taking into account that the CD25+ Treg populations represent, together, <5% of total CD4+ T cells, that they proliferate less than conventional CD4+ T cells, and that their growth is inhibited by rapamycin even more than that of conventional CD4+ T cells, our data clearly demonstrate that rapamycin-mediated enrichment of T cells with suppressor functions in stimulated CD4+ T cell cultures is not due to the selective expansion of naturally occurring CD4+CD25bright Tregs present in the total CD4+ T cell fraction, but, instead, to the ability of rapamycin to induce suppressor functions in the total CD4+ T cell population.
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A previous study from Powell et al. (22) has provided evidence that full activation of CD4+ T cells in the presence of rapamycin results in the induction of a hypoproliferative state. In contrast, it has been shown that T cells stimulated in vitro using immobilized anti-CD3 mAb can inhibit the proliferation of responder T cells in vitro, and that in vivo, adoptive transfer of these T cells into recipients of allogeneic skin grafts leads to prolonged skin graft survival (23). To further address the mechanisms of the induction of suppressive activity observed upon stimulation of CD4+ T cells in the presence of rapamycin, we assessed the ability of CD4+ T cell populations stimulated in the absence or in the presence of rapamycin to respond to stimulation with anti-CD3/CD28 Abs in the absence or in the presence of IL-2. As expected, CD4+ T cell populations stimulated in the absence of rapamycin efficiently responded to TCR-mediated stimulation both in the absence and in the presence of IL-2 (Fig. 5A). However, CD4+ T cell populations stimulated in the presence of rapamycin proliferated poorly following TCR-mediated stimulation in the absence of IL-2. Proliferation, however, was partially restored by the presence of IL-2 (Fig. 5A). These data demonstrate that stimulation in the presence of rapamycin induces a hypoproliferative state in conventional CD4+ T cells, similar to that constitutively exhibited by naturally occurring CD4+ Treg populations, consistent with what was previously reported by Powell et al. (22). Also consistent with the data from Powell et al. in contrast to the reduced proliferative capacity of rapamycin-treated T cells, their IL-2 production following TCR-mediated stimulation was similar to that of CD4+ T cells expanded in the absence of rapamycin (data not shown). To assess whether the hypoproliferative state induced by rapamycin was permanent or reversible, cells from rapamycin-treated populations were further stimulated in the absence of rapamycin for an additional week. Withdrawal of rapamycin from the cultures resulted indeed in a complete reversal of T cell hyporesponsiveness (Fig. 5A). Consistent with our previous data (Fig. 3), CD4+ T cells stimulated in the presence of rapamycin suppressed the proliferation of responder CD4+ T cells. The suppressor capacity of these cells, however, was abolished by an additional stimulation in the absence of rapamycin (Fig. 5B). Together, our data indicate that stimulation of conventional CD4+ T cells in the presence of rapamycin results in the induction of a hypoproliferative state together with suppressor functions. This condition, however, is temporary and reversible as it is dependent upon the continuous presence of rapamycin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Consistent with previous findings reported by Powell et al. (22), we found that the presence of rapamycin, concomitant with TCR-mediated stimulation, induces a hypoproliferative state in CD4+ T cells. The hypoproliferative state of rapamycin-treated cells is very likely in close relation with their suppressive functions. Indeed, it has been shown that T cells made unresponsive in vitro using immobilized anti-CD3 mAb vigorously suppress the proliferation of responder T cells in vitro and can in vivo, after adoptive transfer into recipients of allogeneic skin grafts, lead to prolonged survival of the graft (23). This is in line with the capacity of rapamycin-treated CD4+ T cells to prevent allograft rejection in vivo (10). Interestingly, the suppression observed by in vitro-anergized CD4+ T cells does not appear to be due to the release of inhibitory cytokines, but requires cell-cell contact as reported for naturally occurring Tregs (23). In addition, naturally occurring Tregs are constitutively anergic as they are selected as such in the thymus (24). The hypoproliferative state of CD4+ T cells in rapamycin-treated cultures is distinct from anergy. Indeed, rapamycin-induced hypoproliferation is also observed in the presence of costimulation, and does not inhibit costimulation-mediated IL-2 production (22). Thus, conventional CD4+ T cells that are converted into Tregs by TCR-mediated stimulation in the presence of rapamycin are not anergic, but share some functional characteristics with both T cells anergized in vitro and naturally anergic cells of the thymically derived CD4+CD25+ lineage, including suppressor functions. Although the molecular bases of suppression remain to be determined, it can reasonably be expected that at least a fraction of the involved molecules will be common among these different types of suppressor cells.
Finally, it is noteworthy that CD4+ T cells, which acquired suppressor functions through TCR-mediated stimulation in the presence of rapamycin, reverted into nonsuppressive cells after further stimulation and culture in the absence of the drug. The ability of rapamycin to induce profound, but completely reversible hypoproliferative state and suppressor functions in CD4+ T cells is an important characteristic of this drug and may explain its ability to induce graft tolerance in the absence of long-term immunosuppression (5). However, it is noteworthy that, in light of our results, the appropriateness of using rapamycin to prepare Tregs from stimulated CD4+ T cells for cellular therapy in T cell-mediated diseases, as suggested by Battaglia et al. (10), must be considered with caution. For this type of application, the use of purified autologous naturally occurring NnTregs (20) would seem more appropriate. Preclinical studies aimed at optimizing this approach are currently being conducted in our laboratory.
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Disclosures |
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Footnotes |
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1 D.V. and M.A. are supported by the Cancer Research Institute and the Ludwig Institute for Cancer Research. 
2 Address correspondence and reprint requests to Dr. Danila Valmori, Ludwig Institute Clinical Trial Center, Division of Medical Oncology, Department of Medicine, Columbia University College of Physicians and Surgeons, 650 West 168th Street, Black Building Room 20-09, New York, NY 10032; E-mail address: dv2117{at}columbia.edu or Dr. Maha Ayyoub, Ludwig Institute Clinical Trial Center, Division of Medical Oncology, Department of Medicine, Columbia University College of Physicians and Surgeons, 650 West 168th Street, Black Building Room 20-09, New York, NY 10032; E-mail address: msa2106{at}columbia.edu
3 Abbreviations used in this paper: Treg, regulatory T cell; Nn, natural naive.
Received for publication October 3, 2005. Accepted for publication April 21, 2006.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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) monoclonal antibody. Cancer Res. 59: 3128-3133. -secreting Th3 regulatory cells. Immunol. Rev. 182: 207-214. [Medline]-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]This article has been cited by other articles:
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  P. M. Porrett, X. Yuan, D. F. LaRosa, P. T. Walsh, J. Yang, W. Gao, P. Li, J. Zhang, J. M. Ansari, W. W. Hancock, et al. Mechanisms Underlying Blockade of Allograft Acceptance by TLR Ligands J. Immunol., August 1, 2008; 181(3): 1692 - 1699. [Abstract] [Full Text] [PDF]
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S. Basu, T. Golovina, T. Mikheeva, C. H. June, and J. L. Riley Cutting Edge: Foxp3-Mediated Induction of Pim 2 Allows Human T Regulatory Cells to Preferentially Expand in Rapamycin J. Immunol., May 1, 2008; 180(9): 5794 - 5798. [Abstract] [Full Text] [PDF]
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 A. Giorgini and A. Noble Blockade of chronic graft-versus-host disease by alloantigen-induced CD4+CD25+Foxp3+ regulatory T cells in nonlymphopenic hosts J. Leukoc. Biol., November 1, 2007; 82(5): 1053 - 1061. [Abstract] [Full Text] [PDF]
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 B. D. CARSON and S. F. ZIEGLER Impaired T Cell Receptor Signaling in Foxp3+ CD4 T Cells Ann. N.Y. Acad. Sci., April 1, 2007; 1103(1): 167 - 178. [Abstract] [Full Text] [PDF]
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 M. Noris, F. Casiraghi, M. Todeschini, P. Cravedi, D. Cugini, G. Monteferrante, S. Aiello, L. Cassis, E. Gotti, F. Gaspari, et al. Regulatory T Cells and T Cell Depletion: Role of Immunosuppressive Drugs J. Am. Soc. Nephrol., March 1, 2007; 18(3): 1007 - 1018. [Abstract] [Full Text] [PDF]
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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]
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M. Battaglia, A. Stabilini, B. Migliavacca, J. Horejs-Hoeck, T. Kaupper, and M.-G. Roncarolo Rapamycin Promotes Expansion of Functional CD4+CD25+FOXP3+ Regulatory T Cells of Both Healthy Subjects and Type 1 Diabetic Patients J. Immunol., December 15, 2006; 177(12): 8338 - 8347. [Abstract] [Full Text] [PDF]
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