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Rapamycin-Conditioned Dendritic Cells Are Poor Stimulators of Allogeneic CD4⁺ T Cells, but Enrich for Antigen-Specific Foxp3⁺ T Regulatory Cells and Promote Organ Transplant Tolerance¹

Hth R. Turnquist^2,*,, Giorgio Raimondi^2,*,, Alan F. Zahorchak^*,, Ryan T. Fischer^*,, Zhiliang Wang^*, and Angus W. Thomson^3,*,,

^* Thomas E. Starzl Transplantation Institute, Department of Surgery, and Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The ability of dendritic cells (DC) to regulate Ag-specific immune responses via their influence on T regulatory cells (Treg) may be key to their potential as therapeutic tools or targets for the promotion/restoration of tolerance. In this report, we describe the ability of maturation-resistant, rapamycin (RAPA)-conditioned DC, which are markedly impaired in Foxp3^– T cell allostimulatory capacity, to favor the stimulation of murine alloantigen-specific CD4⁺CD25⁺Foxp3⁺ Treg. This was distinct from control DC, especially following CD40 ligation, which potently expanded non-Treg. RAPA-DC-stimulated Treg were superior alloantigen-specific suppressors of T effector responses compared with those stimulated by control DC. Supporting the ability of RAPA to target effector T and B cells, but permit the proliferation and suppressive function of Treg, an infusion of recipient-derived alloantigen-pulsed RAPA-DC followed by a short postoperative course of low-dose RAPA promoted indefinite (>100 day) heart graft survival. This was associated with graft infiltration by CD4⁺Foxp3⁺ Treg and the absence of transplant vasculopathy. The adoptive transfer of CD4⁺ T cells from animals with long-surviving grafts conferred resistance to rejection. These novel findings demonstrate that, whereas maturation resistance does not impair the capacity of RAPA-DC to modulate Treg, it profoundly impairs their ability to expand T effector cells. A demonstration of this mechanism endorses their potential as tolerance-promoting cellular vaccines.

	Introduction

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Dendritic cells (DC)⁴ are rare, uniquely well-equipped APC that induce and regulate immune reactivity (1, 2). Their capacity to regulate T cell responses reflects an ability to provide critical instructive signals mediated by B7 (3, 4) and TNF family members (5, 6, 7, 8, 9) and specific cytokines (10). The expression of these signals is regulated by environmental factors sensed through DC surface receptors, including TLRs (11), TNF receptor family members, especially CD40 (7), and specific cytokine receptors (12). Adequate triggering of these receptors results in DC "maturation" and enhances their ability to activate effector CD4⁺ and CD8⁺ T cells. Alternatively, immature and "semi-immature" DC populations, which lack adequate T cell stimulatory ability, can suppress peripheral Ag-specific immune responses through the induction of T cell anergy and deletion (2, 13, 14). In transplantation, the capacity of immature, donor-derived (15, 16, 17, 18, 19) and, more recently, recipient DC (20, 21, 22, 23) to down-regulate alloantigen-specific immune responses and prolong graft survival has been demonstrated in animal models.

Interestingly, both "immature" and "mature" DC can initiate the expansion of CD4⁺CD25⁺ Forkhead/winged helix protein-3 (Foxp3)⁺ T regulatory cells (Treg) (24, 25, 26, 27). Treg constitute a naturally arising leukocyte population that regulates immune response to self and other Ags, including alloantigen (reviewed in Ref. 28, 29, 30). Thus, T cell responses can be viewed as the culmination of a DC-orchestrated expansion and induction of effector functions of both Foxp3^– and Foxp3⁺ T cells. To date, studies on the capacity of DC to expand/induce Treg have implemented transgenic T cell lines or highly purified populations of freshly isolated CD4⁺CD25⁺ T cells (26, 27, 31). Thus, the manner in which DC maturation affects Treg expansion and function in the context of more physiological/heterogenous (i.e., CD25^– and CD25⁺ CD4⁺) T cell populations is not well defined.

The immunophilin ligand rapamycin (RAPA), a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus, exhibits potent immunosuppressive properties and is used therapeutically in renal transplantation (32, 33, 34). Specific inhibition by RAPA of the serine/threonine protein kinase mammalian target of RAPA (mTOR) in T cells blocks costimulation and cytokine-induced signaling but allows TCR-mediated signal transduction (32). Consequently, RAPA promotes T cell anergy and/or deletion (32, 35, 36). Unlike other commonly used immunosuppressants, RAPA does not appear to interfere with tolerance induction (36, 37) and permits the in vitro expansion and suppressive function of Treg (38, 39, 40).

Recently, we have demonstrated that RAPA inhibits DC maturation and T cell stimulatory capacity in vitro and in vivo at clinically relevant levels (41, 42). In addition, the exposure of DC to RAPA confers resistance to maturation following exposure to LPS (41). Similar changes have been reported for RAPA-treated human monocyte-derived DC (43). In mice, recipient-derived, alloantigen-pulsed RAPA-DC prolong organ allograft survival, an effect that is enhanced by multiple DC infusions (42).

We show herein that DC generated in the presence of RAPA are poor allostimulators, resistant to maturation following CD40 ligation. Although their T cell allostimulatory capacity is markedly impaired, RAPA-DC skew the balance of Foxp3⁺ Treg relative to T effectors (in heterogenous T cell populations) by maintaining the ability to stimulate Treg similar to control (CTR) DC. Based on the ability of RAPA to inhibit endogenous DC and T and B cells but allow the proliferation and suppressive function of Treg, we hypothesized that postoperative low-dose RAPA might facilitate long-term graft survival in combination with RAPA-DC infusion. We show that a single infusion of recipient-derived, alloantigen-pulsed RAPA-DC, combined with a short course of minimally effective RAPA, promotes indefinite organ graft survival. This is associated with graft infiltration by Treg and the absence of transplant vasculopathy. Thus, the ability of RAPA-DC to permit Treg activation while minimizing effector cell generation may underlie this tolerogenic effect.

	Materials and Methods

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Animals

Male 8- to 12-wk-old C57BL/10 (B10; H2K^b), C3H/HeJ (C3H; H2K^k), and BALB/c (H2K^d) mice were purchased from The Jackson Laboratory and maintained in the specific pathogen-free Central Animal Facility of the University of Pittsburgh School of Medicine (Pittsburgh, PA). Experiments were conducted under an institutional animal care and use committee-approved protocol and in accordance with National Institutes of Health-approved guidelines.

Generation and purification of bone marrow (BM)-derived DC and macrophages

Myeloid DC were propagated from C3H BM cells as described (42, 44). On day 2 of culture 10 ng/ml RAPA (Sigma-Aldrich) or control medium was added. Every 2 days, 80% of the culture supernatant was replaced with fresh cytokine-containing medium (with or without RAPA). On day 4, nonadherent cells were removed. On day 7, the cells were washed thoroughly and CD11c⁺ DC were purified to >90% using anti-CD11c immunomagnetic beads (Miltenyi Biotec). The CD11c⁺ cells were incubated overnight and, where indicated, stimulated with anti-mouse CD40 mAb (no azide, low endotoxin format, clone HM40-3; 5 µg/ml; BD Pharmingen). Alloantigen-pulsed DC were generated by incubation for 18–24 h at 37°C with cell-free B10 splenocyte lysates prepared as described (42). BM-derived macrophages (M) were also generated from C3H BM cells by culture in M-CSF (20 ng/ml) for 7 days followed by CD11c⁺ depletion and CD11b⁺ selection using immunomagnetic beads (Miltenyi Biotec). RAPA-M were propagated by the addition of 10 ng/ml RAPA on days 2–7 of culture. M-CSF and RAPA were replaced on days 4 and 6.

Phenotypic analysis of DC and M

Cell surface Ag expression was analyzed on days 7 and 8 as described (45). FITC-, PE-, PE-cyanine (Cy)5-conjugated, or biotinylated mAbs were used to detect the expression of CD11b (clone M170), CD11c (clone HL3 or N418), CD80 (clone16-10A1), CD86 (clone GL1), I-A^k (clone 11-5.2), CCR7 (clone 4B12; BioLegend), and F4/80 (clone BM8; Invitrogen Life Technologies). Appropriately conjugated, isotype-matched IgGs were used as negative controls. All mAbs, as well as isotype control IgGs and streptavidin-PE-Cy5, were from BD Pharmingen, unless otherwise specified. Data were acquired with a BD FACScan or a LSR II flow cytometer (BD Biosciences Immunocytometry Systems) and analyzed using FCSPress 1.4 (R. Hicks, Cambridge, U.K.) or FlowJo 8.1.1 (Tree Star) software packages.

RNase protection assay

The procedure adopted for RNase protection assay (RPA) has been described in detail (46). Briefly, RNA was isolated from snap-frozen, purified DCs using a total RNA isolation kit (BD Pharmingen). RPA was performed using the RiboQuant Multiprobe RPA system (BD Pharmingen) and cDNAs encoding mouse CCR1, CCR1b, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, GM-CSF, IFN-, IL-1, IL-1R, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p35, IL-12p40, IL-13, IL-15, TGF-1, TGF-2, TGF-3, TNF-, and the housekeeping genes L32 and GAPDH were used as templates. Quantification of bands was performed by densitometric assessment of scanned autoradiographs using Scion Image version 1.63 software (National Institutes of Health, Bethesda, MD). The signals from specific mRNA were normalized to the signals from housekeeping genes run on each lane to adjust for loading differences.

ELISA

IL-12p70 concentrations were quantified in 18- to 24-h supernatants from DC cultures (containing 1 x 10⁶ cells/ml) using the ELISA Ready-SET-Go! kit (eBioscience) according to the manufacturer’s protocol.

T cell purification

CD4⁺ T cells were purified from spleen and lymph node cell suspensions by negative selection. Non-CD4⁺ cells were labeled with anti-CD11b, anti-TER-119, anti-Gr-1, anti-I-A/I-E, anti-CD8, anti-B220, and anti-Gr-1 mAbs (all from BD Pharmingen). Following incubation with mouse depletion Dynabeads (Dynal Biotech), bead-bound cells were removed by magnetic isolation. In certain cases, the resultant CD4⁺ population was then incubated with biotin-conjugated anti-CD25 mAb, and CD4⁺CD25⁺ T cells were isolated by positive selection using anti-biotin microbeads and MS separation columns (Miltenyi Biotec). Purity was consistently 90–95%. Alternatively, nylon wool columns were used to obtain enriched "bulk" T cells from B10 spleens (purity 70–80% CD3⁺ cells). For adoptive transfer experiments, splenic and lymph node CD4⁺ T cells were purified (>90%) by positive selection using anti-CD4 immunomagnetic beads (Miltenyi Biotec).

Mixed leukocyte reaction

Graded numbers of -irradiated (20 gray) C3H DC were used as stimulators of purified allogeneic (B10) splenic T cells (2 x 10⁵/well) in a 72-h MLR using 96-well, round-bottom plates as described (41). For the final 16–18 h, individual wells were pulse-labeled with 1 µCi of [³H]thymidine. Radioisotope incorporation was determined using a beta scintillation counter. Results are expressed as mean cpm ± 1 SEM calculated from triplicate wells.

T cell proliferation assay

Purified CD4⁺ T cells were CFSE-labeled using the Vibrant CFDA SE Cell Tracer kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. B10 CD4⁺ T cells (2 x 10⁵) were cocultured with 2 x 10⁴ allogeneic (C3H) DC for 5 days in 96-well, round-bottom plates. Similarly, 2 x 10⁵ syngeneic CD4⁺ T cells were cocultured with 2 x 10⁴ B10 alloantigen-pulsed C3H DC for 5 days. At the end of the culture period, cells were collected and stained for surface markers and Foxp3 following the protocol recommended by eBioscience. For surface staining, FITC-, PE-, and CyChrome-conjugated mAbs (BD Pharmingen) recognizing CD4 (clone L3T4), CD25 (clone PC61), CD44 (clone IM7), or CD62L (clone MEL-14) were used. Both the anti-mouse Foxp3 mAb (clone FJK-16s) and the Foxp3 staining set were from eBioscience. Data on resultant samples were acquired and analyzed as described above.

Analysis of T cell apoptosis

CD4⁺ T cells stimulated with CTR-DC or RAPA-DC at a 10:1 ratio were harvested on day 4 of MLR and labeled with PE-conjugated anti-CD4 mAb or anti-CD25. The incidences of viable cells and early and late apoptotic cells were determined by using an Annexin V^FITC Apoptosis Detection kit (BD Pharmingen) and following the manufacturer’s recommended protocol. Following the staining of externalized phosphatidylserine with Annexin V^FITC and incubation in the vital dye 7-aminoactinomycin D (7-AAD), data were acquired and analyzed as described above.

Suppressor function assay

B10 CD4⁺ T cells were purified, stimulated with either C3H RAPA-DC or CTR-DC for 8 days as described above, and then rested for 3 days in recombinant mouse IL-2 (50U/ml; PeproTech). Following the rest period, CD25⁺ T cells were positively selected using anti-CD25 immunobeads and compared directly to freshly isolated CD4⁺CD25⁺ B10 T cells for their ability to suppress the proliferative responses of freshly isolated, CFSE-labeled CD4⁺ B10 T cells responding to either C3H (donor) or BALB/c (third party) T cell-depleted splenocytes. Suppressive capacity was assessed after 3 days of coculture as described (19, 47), following staining for surface CD4 and intracellular Foxp3, by assessment of CSFE dilution.

Vascularized heart transplantation

Heterotopic (intra-abdominal) heart transplantation was performed from B10 to C3H mice, as described (15). Seven days before transplantation (day –7), animals were injected i.v. via the lateral tail vein with 2 x 10⁶ immunobead-sorted donor (B10) or third party (BALB/c) alloantigen-pulsed RAPA-DC. Groups of mice also received a subtherapeutic regimen of 1 mg/kg/day i.p. RAPA (Sigma-Aldrich) in a vehicle containing 0.02% Tween 80 and 0.26% polyethylene glycol (both from Sigma-Aldrich) for 10 consecutive days (days 0 to 9). Additional groups received either unpulsed or alloantigen-pulsed CTR-DC, RAPA alone, or vehicle alone. Transplant survival was assessed by daily transabdominal palpation. Rejection was defined by the complete cessation of cardiac contraction and confirmed histologically. CD4⁺ T cells from mice with long-surviving grafts (>100 days), normal mice, or animals rejecting their transplants were purified and infused i.v. into normal syngeneic recipients that received donor or third party strain heart grafts 1 day later.

Immunofluorescence staining for Foxp3⁺ cells in tissue sections

Cardiac allografts and native heart tissue were snap frozen in OCT medium (Sakura Finetek). Cryostat sections (8 µ m) were fixed in 96% ethanol and blocked with 10% (v/v) normal goat serum and the avidin/biotin blocking kit (Vector Laboratories). Following incubation with anti-CD4 mAb (RM4-5; BD Pharmingen), sections were incubated with Cy2 F(ab')₂ anti-rat IgG (Jackson ImmunoResearch Laboratories) in addition to biotin-conjugated anti-Foxp3 mAb (clone MF333F; Alexis). Staining for Foxp3 was visualized with Cy3 streptavidin and cell nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes). Following fixation in 2% (v/v) paraformaldehyde, the slides were mounted in glycerol and PBS, and examined with a Zeiss Axiovert 135 microscope equipped with appropriate filters and a cooled charge-coupled device camera (Photometrics CH250). Signals from different fluorochromes were acquired independently, and montages were edited using the Adobe Photoshop software program (Adobe Systems).

Statistical analyses

Results are expressed as means ± 1 SEM. The significances of differences between means were determined using Student’s t test and the JMP IN 4.04 Statistical Package (SAS Institute). p < 0.05 was considered significant. GraphPad Prism 2.0C Software package (GraphPad Software) was used to generate survival curves and the significance of differences in graft survival between groups was determined by Kaplan-Meier analysis and the log-rank test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
RAPA inhibits MHC class II and costimulatory molecule expression by murine BM-derived DC and diminishes their T cell allostimulatory function

Myeloid DC were generated from mouse BM cells (lineage depleted) in 7 day cultures with GM-CSF plus IL-4. In cultures treated with 10 ng/ml RAPA, the cells appeared to be uniformly smaller than those in CTR cultures (Fig. 1A; side scatter vs forward scatter). Similar observations have been made of T cells cultured in RAPA (38). CTR-DC and RAPA-DC exhibited similar levels of surface CD11c (Fig. 1A) and could be purified consistently to >90% from 7 day cultures. In addition, CD11c⁺ CTR-DC and RAPA-DC expressed similar surface levels of CD11b (Fig. 1A), suggesting that RAPA did not affect the differentiation of DC precursors to myeloid DC. A significant reduction in CD11c⁺ cells was observed in RAPA-treated cultures. From 10 x 10⁶ plated BM cells, 7.80 ± 0.76 x 10⁶ CD11c⁺ cells were obtained after purification under control conditions, whereas 4.53 ± 0.99 x 10⁶ CD11c⁺ cells were isolated from RAPA-treated cultures (n = 8; p < 0.05).

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Phenotype and function of RAPA-DC. BM-derived myeloid DC were generated from C3H mice (H2k) with GM-CSF plus IL-4 (1000 U/ml), in the absence (CTR-DC) or presence of 10 ng/ml RAPA (RAPA-DC), as described in the Materials and Methods. A, Both CTR-DC and RAPA-DC (CD11c+) were purified by immunobead selection to >90% from 7 day cultures. CD11c+ CTR- and RAPA-DC expressed similar surface levels of CD11b; however, RAPA-DC were smaller (based on side scatter (SSC)/forward scatter (FSC)) and exhibited much lower MHC class II (I-Ak) and CD86 compared with CTR-DC. B, Compared with CTR-DC, RAPA-DC showed very weak T cell (C57BL/10; H-2b) allostimulatory activity in a 72-h MLR. Results shown are means ± SEM (, p < 0.05, RAPA-DC vs T cells only; *, p < 0.05, RAPA-DC vs CTR-DC; Student’s t test). Data in A and B are from individual experiments representative of at least three performed.

Inhibitory effects of RAPA on DC maturation and T cell allostimulatory capacity are resistant to CD40 ligation

The foregoing phenotypic and functional observations are consistent with the ability of RAPA to inhibit DC maturation. However, the stability of RAPA-DC under inflammatory conditions is likely to be crucial for their successful application as tolerogenic agents. Inflammatory signals that result from surgical trauma constitute strong DC maturation stimuli. In addition, there is evidence that TLRs, in addition to being critical for the recognition of microbial pathogens, are stimulated following transplantation by endogenous inflammatory ligands (48). We have previously shown that RAPA-DC are resistant to maturation following exposure to IL-4 (41) or LPS (41). CD40 ligation strongly induces DC maturation and can ablate the tolerogenic properties of DC (49). To investigate the response of RAPA-DC to CD40 ligation, RAPA-DC and CTR-DC were incubated for 18–24 h with agonistic anti-CD40 mAb. Whereas CTR-DC up-regulated surface expression of CD80, CD86, and MHC class II, RAPA-DC retained low expression of MHC class II, CD80, and especially CD86 following CD40 ligation (Fig. 2A). Normalization of independent experiments (n = 5) by the conversion of flow data to the percentage of the mean fluorescence intensity (MFI) for the indicated group relative to the MFI for CTR-DC in the same experiment confirmed that the decreases in CD80 and CD86 on RAPA-DC, both before and after CD40 ligation, were statistically significant (Fig. 2B). An equivalent robust resistance to CD40 ligation was observed for B10-derived RAPA-DC (data not shown), confirming that the effect was not strain specific. Likewise, the failure of RAPA-DC, unlike CTR-DC, to up-regulate the secretion of the Th1-driving cytokine IL-12p70 after CD40 ligation supports resistance to CD40 ligation-induced maturation (Fig. 2C). Although quantitative analysis of corresponding IL-12p40 mRNA expression revealed lower levels in RAPA-DC compared with CTR-DC, TGF- 1 mRNA transcripts were increased in RAPA-DC (data not shown). The message for other cytokines examined did not differ significantly between CTR-DC and RAPA-DC with the exception of IL-1, which was increased in the latter cells (data not shown). As expected, the allostimulatory activity of CD11c⁺ CTR-DC was markedly increased following CD40 ligation, whereas RAPA-DC displayed greatly reduced allostimulatory capacity after incubation with anti-CD40 mAb (Fig. 2D).

View larger version (32K): [in this window] [in a new window]   FIGURE 2. RAPA-DC are resistant to maturation induction. CTR-DC or RAPA-DC were purified from culture (day 7), washed extensively, and then incubated (in the absence of RAPA) for 18–24 h with anti-CD40 mAb or control isotype IgG. A, RAPA-DC exhibited much lower surface expression of MHC II, CD80, and especially CD86 both before and after CD40 ligation. Data shown are gated on CD11c+ cells and numbers indicate MFI for each condition. Dashed line indicates isotype control. B, Flow cytometric data were normalized by calculating the percentage of the MFI obtained for CD11c+ gated cells of the indicated group relative to the MFI obtained for CD11c+ CTR-DC in the same experiment. The mean of five independent experiments is presented and the significances of differences were determined by Student’s t test (*, p < 0.05 vs CTR-DC; #, p < 0.05 vs CTR-DC plus anti-CD40). C, IL-12p70 protein in culture supernatants from triplicate wells assessed by ELISA. D, RAPA-DC displayed much inferior T cell allostimulatory activity in MLR using 2 x 105 B10 T cells as responders even following their exposure to anti-CD40 (p < 0.05 by Student’s t test: x, CTR-DC vs RAPA-DC; , RAPA-DC vs T cells only; *, CTR-DC vs CTR-DC plus anti-CD40; , RAPA-DC vs RAPA-DC plus anti-CD40). Data are from one experiment representative of two performed. E, Comparative RPA analysis of CCR7 mRNA expression in CTR-DC and RAPA-DC revealed similar levels. Densitometric analysis of scanned autoradiographs generated values expressed as arbitrary units relative to corresponding housekeeping gene transcripts. Data are from a single experiment representative of two separate experiments. F, MFI for surface CCR7 on CD11c+ cells correlated with mRNA levels detected by RPA analysis. Dashed line, Isotype control; dark line, unstimulated; and gray line, CD40-ligated DC.

RAPA-DC enhance the proportion of Treg relative to non-Treg, even after CD40 ligation

Because there is an incomplete understanding of how DC maturation influences the outcome of their interactions with heterogenous T cell populations (i.e., those containing both Treg and non-Treg), we performed flow cytometric analysis of total CD4⁺ B10 T cells cocultured for 5 days with unstimulated or CD40-ligated C3H RAPA-DC or CTR-DC. At the end of the culture period, there were greatly reduced CD25^high cells in RAPA-DC-stimulated cultures compared with unstimulated, and especially CD40-ligated CTR-DC-stimulated MLR (Fig. 3A). These observations are consistent with the reduced allostimulatory capacity of RAPA-DC, even following exposure to a powerful maturation stimulus (Fig. 2D). Interestingly, within the CD25⁺ T cell population RAPA-DC-stimulated cultures maintained a higher incidence of Treg (CD4⁺CD25⁺Foxp3⁺ T cells). This difference was especially pronounced between cultures stimulated with CTR-DC and RAPA-DC exposed to anti-CD40 mAb (Fig. 3A). The culture of purified CD4⁺ T cells with CTR-M or RAPA-M, or the addition of 10 ng/ml RAPA to purified CD4⁺ B10 T cells in the absence of APC, did not enhance the incidence of CD4⁺CD25⁺Foxp3⁺ T cells during the 5-day culture period (data not shown).

View larger version (59K): [in this window] [in a new window]   FIGURE 3. RAPA-DC maintain higher proportions of Treg relative to non-Treg even after CD40 ligation. CD4+ B10 T cells were stimulated for 5 days with unstimulated or anti-CD40-ligated C3H RAPA-DC or CTR-DC. A, Flow cytometric analysis shows reduced CD25high cells in RAPA-DC-stimulated cultures but a higher incidence of Treg (CD4+CD25+Foxp3+) in the CD25high population. B, When compared with CD4+ T cells stimulated for 5 days with CTR-DC, non-Treg (Foxp3–), but not Treg (Foxp3+), exposed to RAPA-DC exhibited reduced surface expression of the activation marker CD44. Numbers indicate the percentage of cells in M2. The majority of Foxp3– T cells exposed to RAPA-DC retained a naive phenotype based on CD62L expression (numbers indicate percentage of cells in M1). Alternatively, Foxp3+ T cells from RAPA-DC-stimulated cultures have reduced CD62L expression in comparison to CTR-DC-stimulated Treg (numbers indicate percentage of cells in M2 plus M3). Data are from one experiment representative of at least two completed.

RAPA-DC support Treg but fail to increase non-Treg

An important consideration is how the overall balance between Treg and non-Treg is maintained in the CD25⁺ T cell population given the relationship between the number of Treg to effector cells and the ability of Treg to suppress effector responses (50). When freshly isolated B10 CD4⁺ T cell populations from spleens and lymph nodes were examined, 11.4 ± 2.0% of CD4⁺ cells were Foxp3⁺ (data not shown). Based on these percentages, the absolute numbers of CD4⁺ Treg and non-Treg added to each well were determined for the starting populations in MLR (day 0; Fig. 4A). In a similar fashion, the number of viable cells counted at day 5 and the percentages of CD4⁺CD25⁺Foxp3^– and CD4⁺CD25⁺Foxp3⁺ cells were used to obtain the absolute number of non-Treg and Treg cells, respectively. Over the 5-day culture period, CTR-DC-stimulated cultures demonstrated a slight increase (1.2-fold) in non-Treg, but these numbers were not significantly different from the starting numbers (Fig. 4A, left panel). Cultures stimulated with CTR-DC exposed to CD40 ligation displayed a significantly increased number of CD4⁺CD25⁺Foxp3^– over time (2.4-fold; Fig. 4A, right panel) compared with cultures stimulated with CTR-DC, RAPA-DC, or CD40-ligated RAPA-DC (Fig. 4A). In contrast, RAPA-DC-stimulated cultures, even those stimulated with CD40-ligated RAPA-DC, displayed a significant reduction of non-Treg during the culture period. Interestingly, no significant net changes in Treg numbers over time or under different culture conditions were observed (Fig. 4A).

View larger version (39K): [in this window] [in a new window]   FIGURE 4. RAPA-DC induce increased apoptosis and decreased non-Treg numbers, especially compared with anti-CD40-stimulated CTR-DC. Purified 2 x 105 CD4+ B10 T cells were stimulated at a 10:1 ratio with either unstimulated or anti-CD40-ligated C3H RAPA-DC or CTR-DC. A, Based on the percentage obtained under each culture condition and the number of viable cells in each well, the absolute number of CD25highFoxp3+ (Treg) and CD25highFoxp3– (non-Treg) was calculated. Unlike those stimulated with CTR-DC, and especially CD40-ligated CTR-DC, RAPA-DC-stimulated cultures did not exhibit increases in non-Treg but displayed significantly reduced non-Treg numbers. Treg numbers were not significantly different between RAPA-DC- or CTR-DC-stimulated cultures after 5 days under any condition. Data are means ± SEM from at least two independent experiments. Significant differences established using Student’s t test (*, p < 0.05; #, p < 0.05 vs unstimulated CTR-DC). B, When Annexin V and 7-AAD staining was used in flow cytometric analysis to determine the level of T cell apoptosis on day 4 of culture, an increased incidence of late apoptotic CD4+ cells (Annexin V+7-AAD+) and a decreased incidence of viable cells (Annexin V–7-AAD–) were detected in cultures stimulated with RAPA-DC, especially when CD40-ligated RAPA-DC were compared with CD40-ligated CTR-DC. The data are representative of three separate experiments.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. RAPA-DC-stimulated CD25+ T cells exhibit alloantigen-specific regulatory function compared with those expanded by CTR-DC. CD4+ T cells were purified from B10 mice and stimulated with either C3H RAPA- or CTR-DC for 8 days. T cells from these cultures were harvested, counted, and rested for 3 days in IL-2 (50 U/ml). Following anti-CD25 purification the rested T cells were analyzed by flow for Foxp3+ expression (A) or compared with freshly isolated CD4+CD25+ B10 T cells (B) for their ability to suppress proliferative responses of freshly isolated, CFSE-labeled CD4+ B10 T cells responding to either C3H (donor) or BALB/c (third party) T cell-depleted splenocytes. A, Flow cytometric analysis demonstrates increased incidences of Treg relative to non-Treg in RAPA-DC stimulated cultures. Gated for CD4+ cells, 10,000 events. B, Assessment of the proliferation of naive B10 T to C3H (donor) or BALB/c (third party) splenocytes by FACS analysis of CFSE dilution demonstrated that CD4+CD25+ T cells expanded by RAPA-DC displayed alloantigen-specific suppressive capacity. The percentage of cells in the CD4high/proliferating gate (R1) or CD4low/nonproliferating gate (R2) is indicated. In contrast, neither CTR-DC-expanded CD4+CD25+ nor freshly isolated Treg modulated the responses of naive T cells to donor or third party splenocytes at a 1:20 ratio. Data are representative of two experiments performed.

There is recent evidence that "mature" CD86^high allogeneic DC can induce the proliferation of purified CD4⁺CD25⁺ Treg, especially in the presence of exogenous IL-2 (27). Despite the lack of a net change in Treg numbers over the 5-day culture period when CFSE-labeled CD4⁺ B10 T cells (containing both CD25⁺ and CD25^– cells) were stimulated with C3H CTR-ligated or CD40 ligated-CTR-DC, Treg proliferation was observed (R1; Fig. 5A). However, the great majority of proliferating CTR-DC-stimulated CD4⁺ T cells (CD25⁺ gated) were Foxp3^– cells (R3; Fig. 5A). Interestingly, flow analysis of CD25⁺ cells from RAPA-DC-stimulated cultures revealed a consistent increase in the incidence of proliferating Treg over that observed with CTR-DC. Significantly, this property of RAPA-DC was retained after CD40 ligation (Fig. 5B). Based on the percentages determined by flow cytometry for each condition and the number of cells counted in each well, the absolute numbers of proliferating Treg (R1) and non-Treg (R3) were calculated and used to generate a ratio (proliferating CD4⁺Foxp3⁺/proliferating CD4⁺Foxp3^–) that supported the ability of RAPA-DC, even following CD40 ligation, to favor Treg stimulation relevant to non-Treg. When compared directly, proliferating Treg (R1) in RAPA-DC- stimulated cultures expressed increased levels of Foxp3 compared with those from CTR-DC-stimulated cultures (Fig. 5C).

View larger version (39K): [in this window] [in a new window]   FIGURE 5. Compared with CTR-DC, RAPA-DC favor proliferation of Treg relative to non-Treg. CFSE-labeled CD4+ B10 T cells (2 x 105) were stimulated for 5 days with 2 x 104 C3H RAPA-DC or CTR-DC generated under the conditions indicated. A, Flow cytometric analysis (of CD25+ gated cells) revealed that, compared with CTR-DC, RAPA-DC shifted the balance of T cell proliferation in favor of Treg. RAPA-DC retained this capacity after CD40-ligation. B, Based on percentages determined by flow cytometry and the number of cells counted in each well, the absolute numbers of proliferating Treg and non-Treg (Foxp3–) were calculated and used to generate a ratio that confirmed that RAPA-DC-stimulated T cell proliferation was skewed toward Treg, even following CD40 ligation. Data are means ± SEM from two representative experiments. C, When compared directly, proliferating Treg (upper left quadrant; region (R1)) in RAPA-DC- stimulated cultures (A) had increased levels of Foxp3 vs those in CTR-DC-stimulated cultures. The far left open histogram depicts isotype control. Treg from CTR-DC- and RAPA-DC-stimulated cultures are depicted by the middle and the filled histograms, respectively. Numbers denote MFI.

To ascertain whether RAPA-DC could induce Foxp3 expression and convert naive CD4⁺CD25^– T cells into Treg, freshly isolated CD4⁺CD25^– B10 T cells (<1% CD25⁺; <6% Foxp3⁺) were stimulated with C3H CTR-DC or RAPA-DC for 5 days. Analysis of the incidence of Foxp3⁺ cells at the end of the culture period revealed no increase in Foxp3⁺ cells above background levels in either CTR-DC- or RAPA-DC-stimulated cultures (Fig. 6A). Moreover, as shown in Fig. 6B, compared with CTR-DC, RAPA-DC failed to induce a significant proliferation of purified CD4⁺CD25^– T cells but caused significant proliferation of purified CD4⁺CD25⁺ cells. Thus, RAPA-DC do not appear to convert CD4⁺CD25^– T cells into Treg through the induction of Foxp3.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. RAPA-DC induce little Foxp3 expression and fail to stimulate significant proliferation in CD4+CD25– cells. Total CD4+, CD4+CD25–, and CD4+CD25+ T cells were purified from B10 mice and stimulated with C3H CTR-DC or RAPA-DC for 5 days. A, From total CD4+ cells CD4+CD25– cells were purified to >99% purity. These populations had greatly reduced but still detectable Foxp3 expression on day 0. Following 5 days of culture, an increased incidence of Foxp3+CD4+ cells was observed in the total CD4+ populations stimulated with RAPA-DC. However, RAPA-DC did not increase the incidence of Foxp3+ cells in the CD4+CD25– population over that in the starting population. B, In a 48-h MLR (10:1 T cells/DC), RAPA-DC, even following anti-CD40 ligation, failed to stimulate CD4+CD25– cell proliferation significantly above that of unstimulated cells (middle panel). In a 72-h MLR (10:1 T cells/DC), whereas RAPA-DC had significantly reduced ability to stimulate proliferation in total CD4+ cells (top panel), these cells did not differ significantly from CTR-DC in their capacity to induce proliferation of CD4+CD25+ cells (bottom panel). Results shown are means ± SEM by Student’s t test (*, p < 0.05 vs T cells only; #, p < 0.05 vs CTR-DC; and , p < 0.05 vs anti-CD40-ligated CTR-DC). Data are from one representative experiment of two performed.

In total, the above findings support a rapid, DC-induced dilution of Treg by non-Treg under "inflammatory" conditions when both Treg and non-Treg are present. Moreover, these data reveal that although maturation-resistant RAPA-DC stimulate minimal proliferation of non-Treg and fail to support the survival of these cells even in the face of CD40 ligation, they retain the ability to stimulate Treg similar to that of CTR-DC.

RAPA-DC-stimulated CD25⁺ T cells suppress T cell proliferative responses in an alloantigen-specific manner

To ascertain whether Treg stimulated by either CTR-DC or RAPA-DC were capable of Ag-specific regulation of T cell proliferation, CD4⁺ B10 T cells were stimulated with either C3H RAPA-DC or CTR-DC for 5 days, counted, and then rested for 3 days in low-dose IL-2. Following anti-CD25 purification, the rested T cells were analyzed by flow for Foxp3⁺ expression. Compared with CTR-DC-stimulated cultures, RAPA-DC-stimulated cultures again exhibited markedly increased incidences of Treg relative to non-Treg (Fig. 7A). In addition, we also compared the ability of DC-stimulated, CD25-purified cells to suppress the proliferative responses of naive CFSE-labeled CD4⁺ B10 T cells with that of freshly isolated CD4⁺CD25⁺ B10 T cells. CD25⁺ T cells purified from these cultures were mixed at a 1:10 (data not shown) or 1:20 ratio with 1.5 x 10⁵ CFSE-labeled CD4⁺ B10 T cells that were then stimulated with 1.5 x 10⁵ C3H (donor) or BALB/c (third party) splenocytes. Four days later, CFSE dilution in the CD4⁺ Foxp3^– population was analyzed and the percentage of cells in the CD4^high/proliferating gate (R1) or CD4^low/nonproliferating gate (R2) was determined. An assessment of the proliferative response of non-Foxp3⁺ naive B10 T cells to C3H or BALB/c splenocyte stimulation by analysis of CFSE dilution showed that CD4⁺CD25⁺ T cells expanded by RAPA-DC displayed alloantigen-specific suppressive capacity (Fig. 7B). In contrast, neither CTR-DC-stimulated CD4⁺CD25⁺ cells nor, as described (27, 51), purified, freshly isolated Treg modulated the proliferative responses of naive T cells to donor or third party splenocytes at a 1:20 ratio (Fig. 7B). Weak, nonalloantigen-specific inhibition of responses was observed with CTR-DC-stimulated cells at a 1:10 ratio (data not shown). Freshly isolated Treg exhibited a potent ability to suppress proliferative responses to both C3H or BALB/c splenic APC at a 1:1 ratio and a weak capacity to inhibit responses to C3H APC at a 1:10 ratio (data not shown). These findings show that RAPA-DC preferentially allow the proliferation/activation of alloantigen-specific CD25⁺ Treg, with little concomitant stimulation of non-Treg.

Alloantigen-pulsed RAPA-DC promote the proliferation of autologous Treg expressing increased Foxp3

To determine whether a similar preferential expansion of Treg is induced in T cells by autologous RAPA-DC presenting alloantigen, CD4⁺ C3H T cells were stimulated for 5 days with either CTR-DC or RAPA-DC pulsed overnight with B10 alloantigen (42). Again, RAPA-DC-stimulated cultures had greatly decreased incidences of CD25⁺ T cells but increased percentages of Treg in the CD25⁺ population (Fig. 8A). Similar to our findings for allogeneic T cells, alloantigen-pulsed RAPA-DC shifted the balance of syngeneic T cell proliferation in favor of Treg (Fig. 8B). An analysis of proliferation via an assessment of CFSE dilution revealed that RAPA-DC induced a slightly greater percentage of proliferating Treg and a greatly reduced percentage and division of non-Treg CD25⁺ cells (Fig. 8B). When compared directly, proliferating Treg (R1) in RAPA-DC-stimulated cultures displayed increased levels of Foxp3 vs those from CTR-DC-stimulated cultures (Fig. 8C).

View larger version (32K): [in this window] [in a new window]   FIGURE 8. Recipient-derived, alloantigen-pulsed RAPA-DC promote the proliferation of Treg expressing increased Foxp3. CD4+ C3H T cells were stimulated for 5 days with either CTR-DC or RAPA-DC pulsed with B10 alloantigen as described in the Materials and Methods. A, RAPA-DC-stimulated cultures had decreased incidences of CD25+ T cells but increased percentages of Treg in the CD25+ population. B, Analysis of proliferation via assessment of CFSE dilution revealed that RAPA-DC induced similar percentages of proliferating Treg but greatly reduced percentages and divisions of non-Treg CD25+ cells. B and C, When compared directly, proliferating Treg (R1) in RAPA-DC stimulated cultures had increased levels of Foxp3 vs those from CTR-DC-stimulated cultures. Data are representative of two experiments performed.

In recent studies we have demonstrated the therapeutic potential of recipient-derived, alloantigen-pulsed RAPA-DC by showing that their infusion could prolong the survival of MHC- and non-MHC-mismatched (B10 to C3H) heart transplants in an Ag-specific fashion (42). We have documented that RAPA inhibits the immunostimulatory capacity of DC in vivo (41). Additional recent studies suggest that RAPA, while targeting the activity of most T and B cells, allows the expansion and suppressive function of Treg (38, 39, 40). Together, these reports and the current data showing the "Treg-sparing" stimulatory capacity of RAPA-DC suggested that postoperative RAPA might augment the tolerogenic potential of alloantigen-pulsed, recipient-derived RAPA-DC. In support of this hypothesis, the infusion of recipient-derived RAPA-DC pulsed with complex donor alloantigen (on day –7) followed by a short, minimally effective postoperative course of low-dose RAPA (1 mg/kg/day i.p.; 10 days), which alone did not prolong transplant survival significantly, induced long-term graft survival (median graft survival time (MST) >100 days) (Fig. 9A). Minimal histological evidence of transplant vascular pathology was observed in these animals (Fig. 9B). The infusion of either unpulsed or donor alloantigen-pulsed, recipient-derived CTR-DC on day –7 did not prolong graft survival significantly (CTR-DC, MST = 12 days; donor alloantigen-pulsed CTR-DC, MST = 10 days; data not shown). When examined for the presence of CD4⁺Foxp3⁺ T cells, long-surviving (>100 days) heart grafts from mice treated with RAPA-DC plus low-dose RAPA had striking infiltration of CD4⁺ Foxp3⁺ cells. Foxp3⁺ cells were not evident in rejected hearts or in normal native control hearts (Fig. 9B). Moreover, adoptive transfer of 10 x 10⁶ CD4⁺ lymphocytes from long-term survivors delayed significantly the rejection of B10 cardiac allografts in naive C3H mice (Fig. 9C), supporting the involvement CD4⁺ regulatory cells in the prevention of graft rejection.

View larger version (58K): [in this window] [in a new window]   FIGURE 9. Administration of RAPA-DC with a short postoperative course of minimally effective RAPA promotes indefinite allograft survival associated with CD4+ regulatory cells. A, C3H RAPA-DC pulsed with B10 alloantigen were injected i.v. into syngeneic mice 7 days before B10 heart graft transplantation. RAPA (1 mg/kg/day) was administered i.p. for 10 days (day 0 to day 9) either alone (n = 4) or in combination with RAPA-DC (n = 9). Vehicle-only treated mice were included as controls (n = 4). B, Long-surviving heart grafts (>100 days) from mice treated with alloantigen-pulsed RAPA-DC plus minimal RAPA displayed minimal parenchymal damage, infiltrates of mononuclear cells in the myocardial interstitium (top right), and little evidence of vasculopathy. The infiltrating cells were composed mainly of CD4+Foxp3+ T cells (yellow; bottom right). Data are representative of four animals in each group. Original magnification x400; x1000 for insets. C, Transfer of purified CD4+ T cells from the spleens and lymph nodes of mice (in A) bearing long-term (>100 days) surviving grafts to naive C3H mice conferred resistance to subsequent B10 heart graft rejection. As controls, CD4+ cells from mice rejecting their grafts or from untreated mice were used.

	Discussion

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When generated ex vivo in clinically relevant concentrations of RAPA, highly purified RAPA-DC show an impaired ability to undergo phenotypic and functional maturation following strongly agonistic CD40 ligation that mimics stimulatory interaction with activated T cells (52, 53, 54). This resistance to maturation was observed following the withdrawal of RAPA, indicating that it is a characteristic of the generated DC. We have demonstrated previously the robust resistance of murine myeloid RAPA-DC to maturation following exposure to LPS or IL-4 (41, 42). We now extend these findings to include the profound resistance of RAPA-DC to maturation induced by CD40 ligation, a stimulus likely to be encountered by DC introduced into allogeneic recipients, possibly under inflammatory conditions. Thus, it is well accepted that the ligation of CD40 on unmodified DC by CD40L (CD154) expressed on activated CD4⁺ T cells induces DC maturation and negates their tolerogenic properties (49). Also, we report that RAPA-DC, although expressing low levels of MHC and costimulatory molecules compared with CTR-DC, exhibit similar expression of the chemokine receptor CCR7 that binds the chemokines CCL19 and CCL21, promoting homing to secondary lymphoid tissue (55, 56). These data support our previous observation that RAPA exposure does not hinder the ability of myeloid DC to migrate to secondary lymphoid tissue (42). Recently, others (57) have reported that RAPA may even facilitate CCR7 expression and enhance the migration of DC to regional lymphoid tissue. The demonstrated ability of RAPA-DC to resist maturation following TLR and CD40 ligation but display CCR7 and lymphoid tissue homing ability is of critical importance to their potential as tolerogenic vectors.

We also report here for the first time a shift in balance of the proliferation of alloantigen-specific, CD4⁺CD25⁺Foxp3⁺ Treg relative to non-Treg by RAPA-DC. Both allogeneic and alloantigen-pulsed syngeneic RAPA-DC induced the proliferation of Treg and, in both cases, showed markedly reduced ability to expand non-Treg cells. This was distinct from CTR-DC, which, although also able to induce Treg proliferation, expanded CD25⁺Foxp3^– cells much more profoundly, a property enhanced by CD40 ligation-induced maturation. Also compared with CTR-DC, RAPA-DC induced higher levels of T cell apoptosis in MLR. An analysis of activation marker expression by T cells cultured with RAPA-DC or CTR-DC supported the capacity of RAPA-DC to stimulate Treg, without parallel stimulation of non-Treg, a differential effect that enriches for alloantigen-specific Foxp3⁺ Treg by favoring the survival/expansion of suppressive, alloantigen-specific Foxp3⁺ Treg. These data imply that the ability of DC to favor Treg may reside in more "immature" DC and that it decreases with increased DC maturation. This is highly relevant to the potential use of DC to regulate immune responses under inflammatory conditions and in relation to heterogeneous populations of responsive T cells. Thus, DC that are resistant to full maturation offer a distinct advantage over conventional "immature" DC. Predisposition of the latter to maturation under inflammatory conditions or in response to interaction with activated T cells limits their effectiveness in targeting Treg relative to effector cells and, as such, their potential as tolerogenic vectors.

CD4⁺Foxp3⁺ Treg are considered to be resistant to TCR-induced proliferation and naturally anergic in vitro (58, 59). However, this anergic state can be overcome by exogenous IL-2 (60). Relatedly, Brinster et al. (61) have shown that LPS- or CpG-matured BM-derived DC can induce the proliferation of purified Treg but that such proliferation is dependent on IL-2 produced by contaminating CD25⁺ effector cells. Herein we have observed the preferential expansion of Treg by RAPA-DC when non-Treg are present in physiological proportions. These cells, when minimally stimulated by RAPA-DC, represent a source of IL-2 necessary to overcome the anergic state of Treg. In addition, Kubo et al. (62) have reported that the exposure of Treg to IL-1 and IL-6 facilitates the reversal of Treg anergy. We observed that IL-1 was significantly up-regulated in RAPA-DC regardless of stimulation. Thus, although RAPA-DC express minimal CD86, which is characteristic of DC that facilitate Treg expansion (26, 27, 61), the expression of IL-1 by RAPA-DC and IL-2 production by non-Treg may support the observed expansion of alloantigen-specific Treg.

The present findings are highly relevant to recent reports that, both in vitro (38, 39, 40) and in vivo (63, 64), RAPA targets effector T cells but preserves Treg suppressive function (39, 40) and allows the proliferation of Treg. These include murine and human CD4⁺CD25⁺Foxp3⁺ cells (38, 64, 65) and type-1 Treg (Tr1) (63). A recent clinical report suggests the Treg-sparing capacity of RAPA regimens compared with those based on calcineurin inhibitors (66). However, based on our previous (41) and current observations, reports that concern the in vivo administration of RAPA require interpretation in the context of the influence of RAPA on both DC and Treg. Taken together, all of these findings support a critical role for the mTOR pathway in determining the balance between tolerance and immunity. The highly specific blocking of mTOR by RAPA in both T lymphocytes and DC during cell differentiation and stimulation results in conditions that favor the tolerogenic function of DC and the proliferation/function of Treg. Accordingly, analysis of the gene expression and proteomic profile of RAPA-DC may provide important insights into the central involvement of mTOR in immunity and tolerance and, in addition, identify signaling pathways and cell products that may be critical to the differential stimulation of Treg by DC.

RAPA-DC retain the ability (compared with CTR-DC) to drive Treg proliferation while preserving a comparatively large population of nonproliferating Treg compared with CTR-DC. This may reflect an ability of RAPA-DC to drive the expansion of a small number of alloreactive Treg clones. Alternatively, it could indicate that RAPA-DC can induce Foxp3 expression in previously Foxp3^– cells. However, although it has been demonstrated that weakly immunogenic DC and suboptimal stimulation of T cells can induce Foxp3 expression in peripheral CD25^– cells (67, 68), we found no evidence that RAPA-DC stimulation of CD4⁺CD25^– could increase the incidence of Foxp3⁺ cells in this population.

Recently, Yamazaki et al. (27) demonstrated that alloantigen-specific Treg could be expanded from highly purified CD25⁺ T cell populations by using CD86⁺ (mature) DC in MLR. When transferred into sublethally irradiated hosts, these Treg suppressed graft-vs-host disease. These observations and our current finding that CD40-ligated CTR-DC induce the proliferation of Treg support the idea that "mature" DC may be suitable for the ex vivo expansion of Treg for therapeutic application. However, our findings show that RAPA-DC also stimulate potent alloantigen-specific Treg and suggest that these stably immature DC may, in comparison with mature DC, lessen the risk of the unwanted proliferation of contaminating non-Treg populations.

Importantly, in the present studies we demonstrate the capacity of recipient-derived, alloantigen-pulsed RAPA-DC, when combined with a short postoperative subtherapeutic course of RAPA, to induce long-term allograft survival with minimal evidence of transplant vasculopathy. Unlike RAPA, the use of calcineurin inhibitors (such as cyclosporine or tacrolimus) may antagonize the efficacy of tolerogenic strategies in transplantation (36). A single infusion of alloantigen-pulsed RAPA-DC in combination with postoperative low-dose RAPA gave superior results to those achieved previously for RAPA-DC in combination with tacrolimus, where indefinite graft survival was not observed (42). The capacity of RAPA-DC together with RAPA to modulate endogenous Treg was evident by the infiltration of CD4⁺Foxp3⁺ cells in long-term surviving cardiac grafts. Moreover, purified CD4⁺ T cells from RAPA-DC/RAPA-treated animals with long-surviving grafts transferred resistance to rejection in naive recipients.

In total, the stable resistance of RAPA-DC to maturation-inducing stimuli, TLR ligands (42), cytokines (41), and now CD40 ligation and their ability to activate alloantigen-specific Treg while showing markedly impaired ability to stimulate/expand T effector cells may represent a key regulatory mechanism underlying their tolerogenicity. These cells offer advantages over "conventional" immature DC as potential tolerogenic vectors.

	Acknowledgments

 
We acknowledge the support of the Starzl Transplantation Institute Flow Cytometry Core Facility (Dr. Hongmei Shen) and the Central Animal Facility of the University of Pittsburgh School of Medicine. In addition, we thank Dr. Adrian E. Morelli and William Shufesky for advice and assistance with immunofluorescence staining of tissue sections.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants R01DK49745, R01AI41011, and R01AI60994 (to A.W.T.). H.R.T. was supported by National Institutes of Health Grant T32CA082084 and a nonconcurrent American Society of Transplantation Basic Science Fellowship, G.R. by a research training fellowship from The Transplantation Society, and R.T.F. by National Institutes of Health Grant T32DK71492.

² H.R.T. and G.R. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Angus W. Thomson, Departments of Surgery and Immunology, University of Pittsburgh School of Medicine, Thomas E. Starzl Transplantation Institute, 200 Lothrop Street, Biomedical Science Tower, Room W1540, Pittsburgh, PA 15213. E-mail address: thomsonaw{at}upmc.edu

⁴ Abbreviations used in this paper: DC, dendritic cell; 7-AAD, 7-aminoactinomycin D; BM, bone marrow; B10, C57BL/10; C3H, C3H/HeJ; CTR, control; Cy, cyanine; DAPI, 4',6-diamidino-2-phenylindole, dihydrochloride; Foxp3, Forkhead/winged helix protein-3; M, macrophage; MFI, mean fluorescence intensity; MST, median graft survival time; mTOR, mammalian target of rapamycin; RAPA, rapamycin; RPA, RNase protection assay; Treg, T regulatory cell.

Received for publication November 13, 2006. Accepted for publication March 27, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
2. Morelli, A. E., A. W. Thomson. 2003. Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction. Immunol. Rev. 196: 125-146. [Medline]
3. Chen, L.. 2004. Coinhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat. Rev. Immunol. 4: 336-347. [Medline]
4. Greenwald, R. J., G. J. Freeman, A. H. Sharpe. 2005. The B7 family revisited. Annu. Rev. Immunol. 23: 515-548. [Medline]
5. Ohshima, Y., Y. Tanaka, H. Tozawa, Y. Takahashi, C. Maliszewski, G. Delespesse. 1997. Expression and function of OX40 ligand on human dendritic cells. J. Immunol. 159: 3838-3848. [Abstract]
6. Tesselaar, K., Y. Xiao, R. Arens, G. M. van Schijndel, D. H. Schuurhuis, R. E. Mebius, J. Borst, R. A. van Lier. 2003. Expression of the murine CD27 ligand CD70 in vitro and in vivo. J. Immunol. 170: 33-40. [Abstract/Free Full Text]
7. Quezada, S. A., L. Z. Jarvinen, E. F. Lind, R. J. Noelle. 2004. CD40/CD154 interactions at the interface of tolerance and immunity. Annu. Rev. Immunol. 22: 307-328. [Medline]
8. Borst, J., J. Hendriks, Y. Xiao. 2005. CD27 and CD70 in T cell and B cell activation. Curr. Opin. Immunol. 17: 275-281. [Medline]
9. Hendriks, J., Y. Xiao, J. W. Rossen, K. F. van der Sluijs, K. Sugamura, N. Ishii, J. Borst. 2005. During viral infection of the respiratory tract, CD27, 4-1BB, and OX40 collectively determine formation of CD8⁺ memory T cells and their capacity for secondary expansion. J. Immunol. 175: 1665-1676. [Abstract/Free Full Text]
10. Moser, M., K. M. Murphy. 2000. Dendritic cell regulation of TH1-TH2 development. Nat. Immunol. 1: 199-205. [Medline]
11. Takeda, K., T. Kaisho, S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21: 335-376. [Medline]
12. Kapsenberg, M. L.. 2003. Dendritic-cell control of pathogen-driven T-cell polarization. Nat. Rev. Immunol. 3: 984-993. [Medline]
13. Lutz, M. B., G. Schuler. 2002. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?. Trends Immunol. 23: 445-449. [Medline]
14. Steinman, R. M., D. Hawiger, M. C. Nussenzweig. 2003. Tolerogenic dendritic cells. Annu. Rev. Immunol. 21: 685-711. [Medline]
15. Fu, F., Y. Li, S. Qian, L. Lu, F. Chambers, T. E. Starzl, J. J. Fung, A. W. Thomson. 1996. Costimulatory molecule-deficient dendritic cell progenitors (MHC class II⁺, CD80^dim, CD86^–) prolong cardiac allograft survival in nonimmunosuppressed recipients. Transplantation 62: 659-665. [Medline]
16. Lutz, M. B., R. M. Suri, M. Niimi, A. L. Ogilvie, N. A. Kukutsch, S. Rossner, G. Schuler, J. M. Austyn. 2000. Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo. Eur. J. Immunol. 30: 1813-1822. [Medline]
17. O’Connell, P. J., W. Li, Z. Wang, S. M. Specht, A. J. Logar, A. W. Thomson. 2002. Immature and mature CD8⁺ dendritic cells prolong the survival of vascularized heart allografts. J. Immunol. 168: 143-154. [Abstract/Free Full Text]
18. Mirenda, V., I. Berton, J. Read, T. Cook, J. Smith, A. Dorling, R. I. Lechler. 2004. Modified dendritic cells coexpressing self and allogeneic major histocompatability complex molecules: an efficient way to induce indirect pathway regulation. J. Am. Soc. Nephrol. 15: 987-997. [Abstract/Free Full Text]
19. Lan, Y. Y., Z. Wang, G. Raimondi, W. Wu, B. L. Colvin, A. De Creus, A. W. Thomson. 2006. "Alternatively activated" dendritic cells preferentially secrete IL-10, expand Foxp3⁺CD4⁺ T cells, and induce long-term organ allograft survival in combination with CTLA4-Ig. J. Immunol. 177: 5868-5877. [Abstract/Free Full Text]
20. Garrovillo, M., A. Ali, H. A. Depaz, R. Gopinathan, O. O. Oluwole, M. A. Hardy, S. F. Oluwole. 2001. Induction of transplant tolerance with immunodominant allopeptide-pulsed host lymphoid and myeloid dendritic cells. Am. J. Transplant. 1: 129-137. [Medline]
21. Peche, H., B. Trinite, B. Martinet, M. C. Cuturi. 2005. Prolongation of heart allograft survival by immature dendritic cells generated from recipient type bone marrow progenitors. Am. J. Transplant. 5: 255-267. [Medline]
22. Beriou, G., H. Peche, C. Guillonneau, E. Merieau, M. C. Cuturi. 2005. Donor-specific allograft tolerance by administration of recipient-derived immature dendritic cells and suboptimal immunosuppression. Transplantation 79: 969-972. [Medline]
23. McCurry, K. R., B. L. Colvin, A. F. Zahorchak, A. W. Thomson. 2006. Regulatory dendritic cell therapy in organ transplantation. Transpl. Int. 19: 525-538. [Medline]
24. Mahnke, K., Y. Qian, J. Knop, A. H. Enk. 2003. Induction of CD4⁺/CD25⁺ regulatory T cells by targeting of antigens to immature dendritic cells. Blood 101: 4862-4869. [Abstract/Free Full Text]
25. Wang, Z., A. T. Larregina, W. J. Shufesky, M. J. Perone, A. Montecalvo, A. F. Zahorchak, A. W. Thomson, A. E. Morelli. 2006. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells. Am. J. Transplant. 6: 1297-1311. [Medline]
26. Yamazaki, S., T. Iyoda, K. Tarbell, K. Olson, K. Velinzon, K. Inaba, R. M. Steinman. 2003. Direct expansion of functional CD25⁺ CD4⁺ regulatory T cells by antigen-processing dendritic cells. J. Exp. Med. 198: 235-247. [Abstract/Free Full Text]
27. Yamazaki, S., M. Patel, A. Harper, A. Bonito, H. Fukuyama, M. Pack, K. V. Tarbell, M. Talmor, J. V. Ravetch, K. Inaba, R. M. Steinman. 2006. Effective expansion of alloantigen-specific Foxp3⁺ CD25⁺ CD4⁺ regulatory T cells by dendritic cells during the mixed leukocyte reaction. Proc. Natl. Acad. Sci. USA 103: 2758-2763. [Abstract/Free Full Text]
28. Wood, K. J., S. Sakaguchi. 2003. Regulatory T cells in transplantation tolerance. Nat. Rev. Immunol. 3: 199-210. [Medline]
29. Walsh, P. T., D. K. Taylor, L. A. Turka. 2004. Tregs and transplantation tolerance. J. Clin. Invest. 114: 1398-1403. [Medline]
30. Wing, K., Z. Fehervari, S. Sakaguchi. 2006. Emerging possibilities in the development and function of regulatory T cells. Int. Immunol. 18: 991-1000. [Abstract/Free Full Text]
31. Fehervari, Z., S. Sakaguchi. 2004. Control of Foxp3⁺ CD25⁺CD4⁺ regulatory cell activation and function by dendritic cells. Int. Immunol. 16: 1769-1780. [Abstract/Free Full Text]
32. Sehgal, S. N.. 1998. Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin. Biochem. 31: 335-340. [Medline]
33. Chueh, S. C., B. D. Kahan. 2005. Clinical application of sirolimus in renal transplantation: an update. Transpl. Int. 18: 261-277. [Medline]
34. Kahan, B. D., J. S. Camardo. 2001. Rapamycin: clinical results and future opportunities. Transplantation 72: 1181-1193. [Medline]
35. Wells, A. D., X. C. Li, Y. Li, M. C. Walsh, X. X. Zheng, Z. Wu, G. Nunez, A. Tang, M. Sayegh, W. W. Hancock, et al 1999. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat. Med. 5: 1303-1307. [Medline]
36. Li, Y., X. C. Li, X. X. Zheng, A. D. Wells, L. A. Turka, T. B. Strom. 1999. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat. Med. 5: 1298-1302. [Medline]
37. Blaha, P., S. Bigenzahn, Z. Koporc, M. Schmid, F. Langer, E. Selzer, H. Bergmeister, F. Wrba, J. Kurtz, C. Kiss, et al 2003. The influence of immunosuppressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade. Blood 101: 2886-2893. [Abstract/Free Full Text]