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The Journal of Immunology, 2006, 177: 5868-5877.
Copyright © 2006 by The American Association of Immunologists, Inc.

"Alternatively Activated" Dendritic Cells Preferentially Secrete IL-10, Expand Foxp3+CD4+ T Cells, and Induce Long-Term Organ Allograft Survival in Combination with CTLA4-Ig1

Yuk Yuen Lan*,{dagger}, Zhiliang Wang*, Giorgio Raimondi*, Wenhan Wu*, Bridget L. Colvin*, An De Creus* and Angus W. Thomson2,*,{dagger}

* Thomas E. Starzl Transplantation Institute and Department of Surgery and {dagger} Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In this study, we propagated myeloid dendritic cells (DC) from BALB/c (H2d) mouse bone marrow progenitors in IL-10 and TGF-beta, then stimulated the cells with LPS. These "alternatively activated" (AA) DC expressed lower TLR4 transcripts than LPS-stimulated control DC and were resistant to maturation. They expressed comparatively low levels of surface MHC class II, CD40, CD80, CD86, and programmed death-ligand 2 (B7-DC; CD273), whereas programmed death-ligand 1 (B7-H1; CD274) and inducible costimulatory ligand expression were unaffected. AADC secreted much higher levels of IL-10, but lower levels of IL-12p70 compared with activated control DC. Their poor allogeneic (C57BL/10; B10) T cell stimulatory activity and ability to induce alloantigen-specific, hyporesponsive T cell proliferation was not associated with enhanced T cell apoptosis. Increased IL-10 production was induced in the alloreactive T cell population, wherein CD4+Foxp3+ cells were expanded. The AADC-expanded allogeneic CD4+CD25+ T cells showed enhanced suppressive activity for T cell proliferative responses compared with freshly isolated T regulatory cells. In vivo migration of AADC to secondary lymphoid tissue was not impaired. A single infusion of BALB/c AADC to quiescent B10 recipients induced alloantigen-specific hyporesponsive T cell proliferation and prolonged subsequent heart graft survival. This effect was potentiated markedly by CTLA4-Ig, administered 1 day after the AADC. Transfer of CD4+ T cells from recipients of long-surviving grafts (>100 days) that were infiltrated with CD4+Foxp3+ cells, prolonged the survival of donor-strain hearts in naive recipients. These data enhance insight into the regulatory properties of AADC and demonstrate their therapeutic potential in vascularized organ transplantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)3 constitute a heterogeneous population of rare, uniquely well-equipped, bone marrow (BM)-derived APC, that play critical roles in central and peripheral tolerance in the normal steady state (1, 2). Due to their inherent plasticity, DC can instigate or regulate immune reactivity. In an effort to improve transplant outcomes and minimize dependency on antirejection drugs, various approaches have been adopted to generate tolerogenic DC that regulate alloimmune responses (3). Thus, immature myeloid DC, propagated from BM precursors in low concentrations of GM-CSF and expressing low levels of MHC and costimulatory molecules, induce alloantigen-specific T cell hyporesponsiveness (4) and prolong organ graft survival (5, 6). Pharmacologic agents, including aspirin (7), vitamin D3 (8, 9), and various immunosuppressive drugs, e.g., corticosteroids (10), cyclosporine A (11), rapamycin (12), and mycophenolate mofetil (13), inhibit DC maturation, promote their tolerogenicity (14), and enhance their ability to induce long-term allograft survival (15, 16).

DC exposed to immunosuppressive cytokines also exhibit potential for tolerance induction. Thus, IL-10, a multifunctional cytokine with diverse effects on many cell types (17), acts as an autocrine/paracrine inhibitor of DC maturation and function (18, 19, 20). Moreover, DC exposed to IL-10 (21, 22) or genetically modified to overexpress IL-10 (23), can induce Ag-specific T cell anergy, while the differentiation of regulatory T cells (Treg) by immature DC may require IL-10 (24). TGF-beta1 is essential for the maintenance of immune homeostasis (25). It blocks DC development from BM progenitors (26), while protecting these progenitors from apoptosis (27). Like IL-10, TGF-beta has been implicated in the generation and expansion of Treg (28, 29). Indeed, during tumor progression, murine myeloid DC induce CD4+CD25+ Treg in a TGF-beta-dependent manner (30). IL-10 and TGF-beta1 exert an additive, suppressive effect on Ag-specific T cell responses. Thus, IL-10- and TGF-beta1-treated CD4+ T cells, but not T cells treated with either cytokine alone, exhibit alloantigen-specific hyporesponsiveness and have markedly impaired ability to induce graft-vs-host disease (GVHD) (31). Significantly, however, neither IL-10- nor TGF-beta-transduced DC exert a marked inhibitory effect on allograft survival (32, 33).

Recently, evidence has emerged that regulatory or "alternatively activated" (AA) DC, generated in IL-10 and TGF-beta1, then exposed to LPS, protect mice from lethal GVHD (34, 35) or lethal endotoxemia (36). These recipient-matched DC display low levels of costimulatory molecules and impair allogeneic effector T cell functions. Importantly, their ability to regulate T cell functions is retained in vitro and in vivo, even under inflammatory conditions. Here, our goal was to extend insight into the functional biology of these AADC, and their capacity to modulate alloreactive T cell responses. In addition, we assessed, for the first time, their influence on organ allograft survival. Our data indicate that AADC not only induce alloantigen-specific hyporesponsive T cell proliferation, but also expand CD4+CD25+ forkhead winged helix protein-3+ (Foxp3+) functional Treg. Moreover, one infusion of AADC into prospective graft recipients could prolong MHC-mismatched heart transplant survival significantly, and, together with B7-CD28 pathway blockade, could prolong graft survival indefinitely. This regulatory effect was associated with graft infiltration by CD4+Foxp3+ cells and with adoptive transfer of resistance to graft rejection by host CD4+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Male BALB/c (H2d), C57BL/10J (B10; H2b), and C3H (H2k) 8- to 12-wk-old mice were purchased from The Jackson Laboratory and housed in the specific pathogen-free Central Animal Facility of the University of Pittsburgh. They received Purina rodent chow (Ralstan Purina) and tap water ad libitum. Experiments were conducted in accordance with the National Institutes of Health Guide for use and care of laboratory animals and under an Institutional Animal Care and Use Committee-approved protocol.

Reagents and mAbs

Mouse rGM-CSF was a gift from Schering-Plough, whereas recombinant murine IL-10 and recombinant human TGF-beta1 (referred to as TGF-beta in the text) were obtained from PeproTech. Complete medium (CM) comprised RPMI 1640 (BioWhittaker) supplemented with 10% v/v FCS (Nalgene), nonessential amino acids, L-glutamine, sodium pyruvate, penicillin-streptomycin and 2-ME (all Invitrogen Life Technologies). Anti-mouse mAbs were from BD Pharmingen, unless otherwise specified. Vybrant CFDA SE cell tracer kit was purchased from Molecular Probes. Magnetic microbeads and separation columns were from Miltenyi Biotec. TRIzol reagent was purchased from R&D Systems and Advantage RT-for-PCR kit from BD Clontech. Quantikine M IL-10, IL-12p70, and IFN-{gamma} ELISA kits were obtained from R&D Systems. Human CTLA4-Ig was a gift from Bristol-Myers Squibb Pharmaceutical Research Institute (Candiac).

Generation of DC

BM cells were flushed from femurs and tibias of normal BALB/c mice and subjected to red blood cell lysis. The cells were then plated at a density of 0.2 x 106/ml in petri dishes. As described by Sato et al. (34, 35), DC were generated in 10 ml of CM, supplemented with GM-CSF (1000 U/ml), IL-10 (20 ng/ml), and TGF-beta (20 ng/ml). Fresh medium and cytokines were added on day 4. On day 7, the nonadherent cells were collected, washed with PBS, and DC were positively selected with anti-CD11c magnetic microbeads (purity >90%). Because macrophages are adherent and express very low levels of CD11c, they were excluded from the DC preparations. The purified DC were replated at the same density in CM with Escherichia coli LPS (1 µg/ml; serotype 026:B6; Sigma-Aldrich) for 24 h. On day 8, the LPS-stimulated DC (AADC) were collected and washed extensively in PBS before use. Control DC (referred to hereafter as GMDC) were propagated under the same conditions in GM-CSF alone, then harvested and purified as described above before exposure to LPS for the final 24 h of culture. In some experiments, conventional bone marrow-derived DC (BMDC) propagated in GM-CSF plus IL-4 as described (37) were used to elicit T cell responses.

Detection of TLR4 expression

Total RNA was extracted from purified GM- or AADC using the TRIzol method, as described (37). cDNA was synthesized from the RNA samples with Advantage RT-for-PCR kit. PCR primers were: 5'-GCA TGG CTT ACA CCA CCT CT-3', sense, and 5'-GTG CTG AAA ATC CAG GTG CT-3', antisense. The PCR mix was run for 35 cycles (94°C, 30 s; 61°C, 30 s; 72°C, 30 s), with a final extension step of 7 min at 72°C. PCR samples were then analyzed on 1% w/v agarose gel stained with ethidium bromide.

Flow cytometric analysis

DC were double-stained with PE-anti-CD11c and either FITC-anti-CD40, -CD80, -CD86, -IAd, -programmed death ligand 1 (PD-L1) (B7-H1; CD274), -PD-L2 (B7-H2;CD273), or -ICOSL for phenotypic analysis. For surface adhesion molecule or chemokine receptor expression, the DC were stained with PE-anti-CD11c and either FITC-anti-CD11b, -CD31, -CD44, -CD54, or -CCR7. The incidence of positive cells and mean fluorescence intensity (MFI) were determined by flow cytometry using an EPICS Elite flow cytometer (Beckman Coulter).

Cytokine quantitation

Culture supernatants were stored at –80°C and levels of IL-10 and IL-12p70 were measured using ELISA kits, according to the manufacturer’s instructions. The sensitivity limits for IL-10 and IL-12p70 were 30 and 7.8 pg/ml, respectively.

Mixed leukocyte reaction

Nylon wool-purified, allogeneic T cells (B10; 2 x 105) were cocultured with gamma-irradiated (20 Gy), BALB/c GMDC or AADC at various T cell:DC ratios for 72 h in 96-well, round-bottom plates (Corning). [3H]Thymidine (Amersham Biosciences) was added to each well 18 h before cell harvesting and determination of T cell proliferation using a liquid scintillation counter. Data are expressed as mean cpm ±1 SD.

Analysis of T cell apoptosis

GM- or AADC were cocultured with allogeneic (B10) nylon wool-purified T cells at a ratio of 1:10 for 2–5 days. T cell apoptosis was monitored by staining of phosphatidylserine translocation with FITC-annexin V, in combination with propidium iodide (PI), as described (38) and according to the manufacturer’s instructions (BD Pharmingen). Cells were costained with PE-anti-CD4/CD8 mAb to allow analysis of T cell subsets by flow cytometry.

In vitro T cell restimulation

Three-day cocultures of nylon wool-purified splenic T cells (B10; 2 x 105) and irradiated GMDC or AADC (BALB/c) were set up in 96-well, round-bottom plates at a DC:T cell ratio of 1:10. DC were then depleted by negative CD11c immunobead selection and the T cells rested for 3 days in CM supplemented with low concentration IL-2 (5 U/ml; Genetics Institute). Primed T cells (2 x 105) were restimulated with irradiated donor (BALB/c) or third-party (C3H) bulk splenocytes, with or without exogenous IL-2 (100 U/ml) for 3 days. T cell proliferation was measured by [3H]thymidine incorporation, as with primary MLR. To quantify cytokine production by primed T cells upon restimulation in the absence of exogenous IL-2, supernatants were collected at 72 h and levels of IL-10 and IFN-{gamma} (sensitivity limit 4 pg/ml) determined by ELISA.

In vitro expansion and functional assessment of CD4+ Treg

Immunobead-purified, allogeneic CD4+ T cells (B10; 2 x 105) were cocultured with nonirradiated, GMDC or AADC (BALB/c; 2 x 104) for 3 days. The T cells were then surface-stained with Cyc-anti-CD4 and FITC-anti-CD25 mAbs and resuspended in Fix/Perm Buffer (eBioscience). Intracellular staining with PE-anti-Foxp3 mAb (eBioscience) and estimation of the incidence of CD4+CD25+Foxp3+ Treg was then determined by flow cytometry, as described (39). To quantify Treg proliferation, purified allogeneic CD4+ T cells were labeled with CFSE as described (39), before 5-day coculture with GM- or AADC in the absence or presence of either neutralizing anti-IL-10 mAb (10 µg/ml) or isotype control Ig, added at the start of the cultures. T cells collected at the end of the assay were stained for surface CD4, then intracellularly with PE-anti-Foxp3 mAb. Proliferation of CD4+Foxp3+/Foxp3– cells was evaluated from the CFSE dilution profile. To assess their regulatory function, CD4+CD25+ cells from 7-day AADC- or GMDC-stimulated cultures, or freshly isolated CD4+CD25+ T cells from normal B10 mice, were purified based on CD25 expression and tested for their ability to inhibit proliferation of normal, CFSE-labeled, syngeneic CD4+ T cells in response to stimulation with BALB/c BMDC.

DC trafficking

Three million CFSE-GM- or AADC, labeled as described (40), were injected s.c. into the rear footpads of normal allogeneic (B10) recipients. CFSE+ cells in the draining (popliteal) lymph nodes were determined 18 h later, by rare-event, flow cytometric analysis. Nondraining (inguinal) lymph node cells served as negative controls. Alternatively, 3 x 106 GMDC or AADC were injected i.v. via the lateral tail vein and 24 h later, the presence of donor MHC class II+ (IAd+) cells in spleen was examined by immunofluorescence staining, as described (41).

Ex vivo T cell restimulation

Two million GMDC or AADC were injected i.v. into normal allogeneic (B10) recipients. Seven days later, primed T cells (2 x 105) from recipient spleens were purified using nylon wool columns, then restimulated with irradiated donor (BALB/c) or third-party (C3H) bulk splenocytes, at various ratios. T cell proliferation was determined by [3H]thymidine incorporation during the final 18 h of culture.

Heart transplantation

Heterotopic (intra-abdominal) vascularized heart transplantation was performed from BALB/c to B10 mice as described (42), under methoxyflurane inhalation anesthesia (Medical Development). The heart was transplanted into the abdomen with end-to-side anastomosis of aorta to aorta and pulmonary artery to vena cava. Two million GMDC or AADC were injected i.v. via the lateral tail vein, 7 days before transplant (day 7). Human CTLA4-Ig (250 µg/mouse) was administered i.p. on day 6. Graft survival was assessed by daily transabdominal palpation. Rejection was defined as total cessation of graft contraction and confirmed by histological examination.

Immunohistochemistry

Heart grafts were fixed in 4% v/v paraformaldehyde and embedded in paraffin for sectioning. H&E stain was used for histologic analysis. To detect Foxp3+ cells in heart grafts, cryostat sections were fixed in 4% paraformaldehyde, treated with goat serum and the avidin/biotin blocking kit (Vector Laboratories) and incubated with anti-Foxp3 mAb (Alexis) overnight, followed by Cy 3F(ab')2 anti-rat IgG (Jackson ImmunoResearch Laboratories). Slides were then incubated with CD4 mAb followed by CyTM 2F (ab')2 anti-rat IgG (Jackson ImmunoResearch Laboratories). Nuclei were stained with DAPI (Molecular Probes).

Adoptive cell transfer

CD4+ T cells were isolated from lymph nodes and spleens of long-surviving (>100 days) graft recipients or control B10 mice using immunomagnetic beads (Miltenyi Biotec). Ten million cells were adoptively transferred by i.v. injection (lateral tail vein) to naive, syngeneic recipient mice (B10) that received BALB/c heart allografts 24 h later. Graft survival was assessed as described above.

Statistical analyses

Significance of differences between means was calculated using the paired Student t test. Differences between groups were considered significant at p < 0.05. Graft survival data were compiled using Kaplan-Meier analysis, and compared by log-rank test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Exposure to IL-10 and TGF-beta alters the surface phenotype, TLR4 expression, and cytokine secretion pattern of LPS-stimulated, BM-derived myeloid DC

We first examined the influence of IL-10 and TGF-beta on DC development from replicating BALB/c BM progenitors. IL-10 and TGF-beta did not affect DC viability, that ranged from 90 to 96% over the culture period. Before LPS activation, differences in surface phenotype were observed between the control and cytokine-treated populations, with lower expression of MHC class II (MHC II) (IAd), CD80, PD-L2, and ICOSL on the cytokine-treated DC (Fig. 1). Stimulation with LPS for 24 h increased levels of MHC II and classic costimulatory molecules (CD40 and CD80) on both populations (referred to hereafter as control GMDC and AADC), while the incidence of cells expressing MHC II, CD40, CD80, and CD86 and the MFI for each of these molecules was consistently greater on GMDC (Fig. 1). This indicated that AADC were comparatively resistant to maturation in response to TLR4 ligation. Levels of the B7 family cosignaling molecules PD-L1 and ICOSL were similar on LPS-stimulated GMDC and AADC, but AADC expressed less PD-L2 than GMDC. Both CD11c+ GMDC and AADC exhibited characteristic DC morphology, with prominent, eccentric, reniform, or multilobulated nuclei, though the latter DC appeared more rounded, with shorter dendrites (Fig. 2A). As it has been reported that TLR4 mRNA expression is increased in TGF-beta null mice (43) and down-regulated in TGF-beta-treated murine DC (44), we considered that exposure to both IL-10 and TGF-beta might also inhibit TLR4 expression. Indeed, when compared with GMDC, AADC showed significantly lower expression of TLR4 mRNA, as determined by RT-PCR (Fig. 2B).


Figure 1
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  		FIGURE 1. DC propagated in IL-10 and TGF-beta are less mature than control DC and more resistant to LPS-induced maturation. BALB/c DC propagated from BM in IL-10 and TGF-beta were costained with mAbs against CD11c and either MHC II (IAd) or TNFR (CD40) or B7 family members (CD80, CD86, PD-L1 (CD274), PD-L2 (CD273) and ICOSL) before and after LPS stimulation and analyzed by flow cytometry. Histograms shown were gated on the CD11c+ population and both percent-positive cells and MFI are indicated. The results are from one representative experiment of three performed.

 

Figure 2
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  		FIGURE 2. Morphology and TLR4 expression by AADC and control DC (GMDC). A, Cytospin preparations of both types of DC showed a high nucleus-cytoplasm ratio and reniform or multilobulated nuclei. Dendrites were abundant and well-developed on GMDC. AADC were more rounded, with less prominent dendrites. Giemsa, x200. B, TLR4 expression by GMDC and AADC was examined by RT-PCR. AADC expressed a lower level of TLR4 mRNA. Densitometric values for TLR4-specific bands are normalized to HPRT. The experiment was performed three times and the data shown are means ± 1 SD.

 
AADC produce more IL-10 and less IL-12p70 than GMDC and exhibit markedly impaired T cell allostimulatory ability

When we examined the cytokine production pattern of the LPS-stimulated DC over 24 h, AADC produced less IL-12p70 but substantially more IL-10 compared with GMDC (Fig. 3A). Based on their comparatively low surface expression of classic costimulatory molecules, high expression of PD-L1 (implicated in suppression of alloreactive T cell responses by DC (45)) and enhanced secretion of IL-10 compared with GMDC, we postulated that AADC would be less capable of naive allogeneic T cell stimulation. When tested in primary MLR, BALB/c AADC induced much weaker proliferative responses in B10 T cells compared with GMDC, at all DC:T cell ratios examined (Fig. 3B).


Figure 3
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  		FIGURE 3. AADC produce substantially more IL-10 and less IL-12p70 than GMDC and are comparatively poor stimulators of allogeneic T cell proliferation. A, Supernatants of LPS-stimulated GMDC or AADC cultures were harvested at 24 h, and levels of IL-10 and IL-12p70 were determined by ELISA. Data from three separate experiments are shown. B, Gamma-irradiated BALB/c GMDC or AADC were cocultured with nylon wool-purified allogeneic B10 T cells (2 x 105) at different ratios in 72 h MLR. T cell proliferation was measured by [3H]thymidine uptake during the last 18 h and expressed as cpm (cpm ± 1 SD). The results are from one experiment representative of four performed.

 
AADC do not enhance allogeneic T cell apoptosis, but promote Ag-specific, hyporesponsive T cell proliferation

To further address possible mechanisms underlying the much inferior capacity of AADC to induce T cell proliferation, T cell apoptosis was examined in 2–5-day cocultures of GMDC or AADC with allogeneic (B10) T cells. The incidence of annexin V+/PI– (apoptotic) CD4+ or CD8+ T cells at day 2 (Fig. 4A) and at other time points (data not shown) was similar in both GMDC- and AADC-stimulated cultures. Thus, pre-exposure to IL-10 and TGF-beta did not affect the influence of LPS-activated DC (AADC) on the apoptotic death of allogeneic T cells. We also examined whether T cell stimulation by AADC could lead to alloantigen-specific, hyporesponsive cell proliferation. As shown in Fig. 4B, AADC-primed (p) B10 T cells (pT) exhibited hyporesponsive proliferation to restimulation by donor (BALB/c) Ag. This hyporesponsive proliferation was donor-specific, as responses to third party (C3H) stimulators were unimpaired. The hyporesponsive proliferation to donor was not reversed by exogenous IL-2 (Fig. 4B). When specific cytokine levels were assayed, increased levels of IL-10 were consistently found in those cultures in which AADC-stimulated (compared with GMDC-stimulated) B10 pT were restimulated with donor alloantigen. (Fig. 4C). Levels of IFN-{gamma} production in cultures of AADC-stimulated pT were unaffected compared with GMDC-pT cultures.


Figure 4
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  		FIGURE 4. AADC do not enhance allogeneic T cell apoptosis but induce alloantigen-specific, hyporesponsive T cell proliferation. A, Gamma-irradiated BALB/c GMDC or AADC (2 x 104) were cocultured with nylon wool-purified, allogeneic B10 T cells (2 x 105) for 2 days. Apoptosis of CD4+ or CD8+ T cells was analyzed by staining with FITC-annexin V in combination with PI, as described in Materials and Methods. Percentages of apoptotic T cells (annexin V+/PI–) in GMDC, or AADC-stimulated cultures are shown. Data are from one experiment and are representative of two performed. B, Nylon wool-purified, allogeneic T cells (B10; 2 x 105) were cocultured with gamma-irradiated GMDC or AADC (BALB/c; 2 x 104) for 72 h. DC were then depleted by negative immunobead selection and T cells rested for 72 h in CM supplemented with low concentration IL-2 (5 U/ml). Primed T cells (pT) were then restimulated with irradiated, allogeneic (BALB/c) or third-party (C3H) bulk splenocytes, at a ratio of 1:1, with or without exogenous IL-2 (100 U/ml) in secondary 72 h coculture. T cell proliferation was measured by [3H]thymidine incorporation, as in primary MLR. AADC-stimulated pT demonstrated alloantigen-specific hyporesponsiveness. This hyporesponsiveness was not reversed by addition of IL-2. Data shown are from one representative experiment of two performed; *, p < 0.01. C, Supernatants were harvested from the 72 h T cell restimulation cultures (no added IL-2) and levels of IL-10 and IFN-{gamma} determined by ELISA. The results of four separate experiments are shown.

 
AADC promote expansion of CD4+CD25+Foxp3+ Treg in vitro

We next investigated whether the tolerogenic properties of AADC might be correlated with specific interactions with CD4+CD25+ Treg. GMDC or AADC were cultured with normal, allogeneic (B10) CD4+ T cells for 3 days, then the frequency of CD4+CD25+Foxp3+ Treg was analyzed by flow cytometry. A higher incidence of Foxp3+ cells (as a percentage of CD4+CD25+ cells) was detected consistently in AADC-stimulated cultures (Fig. 5A). Considering that the natural frequency of Foxp3+ cells is >90% of CD4+CD25+ cells in the mouse (46), our observation could indicate an ability of AADC to promote the survival and/or expansion of CD4+ Treg, relative to effector (Foxp3–) CD4+ T cells. To distinguish between these possibilities, we analyzed the level of proliferation of Foxp3+ and Foxp3–CD4+ T cells in MLR using CFSE-labeled allogeneic CD4+ T cells as responders. As indicated in the density plots (Fig. 5B), AADC retained the ability to induce proliferation of Foxp3+ Treg (comparable to GMDC), while Foxp3– T cell proliferation was significantly impaired. Consequently, stimulation of allogeneic T cells with AADC resulted in an increased ratio of Treg over effectors (Fig. 5C) that could contribute to their control of alloreactivity. As shown in Fig. 5D, addition of neutralizing anti-IL-10 mAb at the start of cultures did not significantly affect the expansion of Foxp3+ relative to Foxp3– cells by AADC. Moreover, compared with freshly isolated CD4+CD25+ T cells from normal B10 mice, AADC-expanded CD4+CD25+ T cells were more effective on a per cell basis (at ratios of 1:10 and 1:20) in suppressing syngeneic (B10) CD4+ T cell proliferation in response to allogeneic (BALB/c) BMDC.


Figure 5
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  		FIGURE 5. AADC preferentially expand Foxp3+ Treg in proliferating CD4+ T cell cultures. A, Nonirradiated BALB/c GMDC or AADC (2 x 104) were cocultured with bead-purified allogeneic B10 CD4+ T cells (2 x 105) for 3 days. Cells were surface-stained with Cyc anti-CD4 mAb and then stained intracellularly with PE-anti-Foxp3 mAb to determine the frequency of CD4+CD25+Foxp3+ Treg. A higher incidence of Foxp3+ cells (expressed as a percentage of CD4+CD25+ cells) was detected consistently in AADC- compared with GMDC-stimulated cultures. Histograms gated on CD4+CD25+ cells are from one representative experiment of two performed. B, To examine the level of proliferation of Foxp3+/Foxp3– CD4+ T cells, CFSE-labeled allogeneic CD4+ T cells were used as responders in 5-day cultures. AADC retained the ability to induce proliferation of Foxp3+ Treg (comparable to GMDC), but their ability to expand Foxp3– T cells was markedly impaired. Proliferating CFSE-labeled cells are shown in the density plots, representative of two experiments performed. This resulted (C) in an increased ratio of Foxp3+ Treg over Foxp3– CD4+ effectors in AADC-stimulated cultures compared with those stimulated with GMDC. The results are from two separate experiments performed. D, Addition of neutralizing anti-IL-10 mAb at the start of cultures did not inhibit the ability of AADC to enhance expansion of Foxp3+ cells relative to Foxp3– cells. E, AADC-primed (p)/expanded CD4+CD25+ T cells were more potent inhibitors of normal CD4+ T cell proliferation on a per cell basis than freshly isolated CD4+CD25+ T cells in response to stimulation with BALB/c BMDC. Data are representative of three experiments.

 
AADC express similar levels of surface adhesion molecules but lower CCR7 than GMDC and migrate to secondary lymphoid tissue of allogeneic hosts

AADC and GMDC uniformly expressed similar surface levels of the intercellular adhesion molecules CD11b, CD44 and CD54 that are important for DC transendothelial migration, whereas only minimal expression of CD31 was detected (Fig. 6A). Surface levels of CCR7, which promote homing to secondary lymphoid tissue in response to cognate chemokines (CCL19 and CCL21), were lower on AADC (Fig. 6A). To evaluate the migratory properties of the DC in vivo, we injected CFSE-labeled GMDC or AADC s.c. into the hind footpad of normal allogeneic (B10) recipients and compared the numbers of labeled DC that reached the draining (popliteal) lymph node within 18 h. Similar absolute numbers of labeled GMDC and AADC were found (Fig. 6B), which indicated that, despite lower surface CCR7, AADC could migrate as well as GMDC to draining secondary lymphoid tissue. Following i.v. injection of GMDC or AADC into allogeneic recipients, similar, albeit low numbers of migratory cells (IAd+) were detected in spleens at 24 h (Fig. 6C).


Figure 6
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  		FIGURE 6. AADC express similar levels of surface adhesion molecules but lower CCR7 than GMDC and migrate to secondary lymphoid tissue of allogeneic hosts. A, Surface adhesion molecule and CCR7 expression on GMDC and AADC were examined by two-color flow cytometry. Histograms shown are gated on CD11c+ populations, with the incidence of positive cells and MFI indicated. Results shown are from one experiment representative of two performed. B, Three million CFSE-labeled BALB/c GMDC or AADC were injected s.c. into the left and right footpad, respectively, of the same allogeneic B10 recipient. Labeled cells in the draining (popliteal) lymph nodes were determined 18 h later, by rare-event analysis. Percentages of labeled GMDC and AADC were similar, as shown in the dot plots. The mean number of CFSE-labeled cells per popliteal lymph node in GMDC- compared with AADC-injected mice (n = 3) did not differ significantly: GMDC: 319 ± 78; AADC: 258 ± 45. Labeled cells did not reach the nondraining (inguinal) lymph nodes that were analyzed for comparison. C, Three million BALB/c GMDC or AADC were infused i.v. into allogeneic (B10; IAb+) recipients and IAd+ (donor) cells were detected by immunofluorescence staining in spleens 24 h later; x200. Results shown are from one experiment representative of two performed; x200

 
AADC induce alloantigen-specific hyporesponsive T cell proliferation in vivo

To better understand the influence of AADC on T cell responses in mice receiving BALB/c GMDC or AADC infusions before heart transplant, we isolated primed T cells (pT) from the spleens of (B10) mice, 7 days after DC injection and quantified the ex vivo T cell proliferative response upon alloantigen restimulation. Primed T cells from AADC-treated mice exhibited less proliferation in response to restimulation by donor splenocytes than those from GMDC-treated animals (Fig. 7). By contrast, stimulation by third-party (C3H) alloantigen evoked a similar, much lower response by T cells from GMDC- and AADC-treated mice.


Figure 7
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  		FIGURE 7. AADC induce alloantigen-specific T cell hyporesponsiveness in vivo. Two million BALB/c GM/AADC were infused i.v. into groups of two normal allogeneic (B10) recipients. Primed T cells (pT) were then isolated from recipient spleens, 7 days later and restimulated ex vivo with gamma-irradiated donor (BALB/c) or third-party (C3H) bulk splenocytes (2 x 105) in 72 h MLR. T cell proliferation was determined by [3H]thymidine incorporation for the final 18 h. Primed T cells from AADC-treated recipients were less reactive to donor Ags. Results (means ± 1 SD) are from one representative experiment of two performed. Three million BALBc GMDC or AADS were infused i.v. into allogeneic (B10) recipients and IAd+ (donor) cells were detected by immunofluorescence staining 24 h later in recipient spleens; x200.

 
Infusion of AADC in combination with CTLA4-Ig can induce long-term (>100 day) heart graft survival; transfer of CD4+ T cells to naive recipients confers resistance to rejection

We then studied the therapeutic efficacy of AADC in the fully MHC-mismatched (BALB/c->B10), vascularized heart allograft model. Two million BALB/c GMDC or AADC were infused i.v. into quiescent B10 recipients, 7 days before transplantation (on day 0). Graft survival in AADC-treated recipients, but not GMDC-treated recipients, was prolonged significantly (Fig. 8, A and B). This effect was enhanced dramatically by a single injection of the B7-CD28 pathway blocking molecule CTLA4-Ig, 24 h after the AADC, resulting in a median graft survival time of >100 days. By contrast, administration of CTLA4-Ig 24 h after GMDC did not result in long-term transplant survival. Histological examination of long-surviving grafts in AADC + CTLA4-Ig-treated mice revealed minimal parenchymal injury, with absence of significant vasculopathy (Fig. 8C). Immunohistochemical staining for CD4 and Foxp3 revealed CD4+ Foxp3+ cell infiltrates within the tolerated grafts, but not within normal or rejecting hearts (Fig. 8D). Moreover, adoptive transfer of CD4+ T cells (10 x 106 i.v.) from lymphoid tissues of long-term (>100 day) graft survivors, but not from normal B10 mice, to naive B10 recipients, 1 day before transplant, significantly prolonged donor strain (BALB/c) but not third-party heart graft survival (Fig. 8E).


Figure 8
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  		FIGURE 8. Infusion of AADC in combination with CTLA4-Ig induces long-term (>100 day) organ allograft survival; CD4+ T cells transfer resistance to rejection. A and B, BALB/c GMDC or AADC (2 x 106) were infused i.v. into normal B10 recipients, 7 days before vascularized heart transplant. Graft survival was prolonged significantly in AADC-treated recipients. Additional treatment of AADC- but not GMDC-infused recipients with CTLA4-Ig, 1 day later, resulted in long-term graft survival (>100 days) in a high proportion of recipients. C, Histological examination of long-surviving heart grafts (>100 days) in mice treated with AADC and CTLA4-Ig revealed a low level of mononuclear cell infiltration, minimal parenchymal damage and the absence of vasculopathy. H&E, x200. D, Immunofluorescence staining (as described in Materials and Methods) for CD4 (green) and Foxp3 (red) revealed CD4+Foxp3+ cells (yellow; arrows) in the tolerated heart allografts, but not in normal or rejecting hearts; x200. E, Adoptive transfer of CD4+ T cells (10 x 106 i.v., day 1) from secondary lymphoid tissues of long-surviving graft (>100 days) recipients significantly prolonged donor-strain heart graft survival in naive B10 recipients; p < 0.005. MST, Median graft survival time. The p value is based on the log-rank test compared with control.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Exposure of replicating myeloid DC progenitors (35) or DC line cells (47) to anti-inflammatory agents, followed by their "activation" in response to LPS, is a comparatively novel approach to the generation of tolerogenic (or regulatory) DC for cell-based therapy of allograft rejection or GVHD. The present study was prompted by recent observations that such AADC, propagated from mouse BM in IL-10 and TGF-beta then stimulated with LPS, exhibit potent immunoregulatory properties. Thus, it has been reported that a single systemic injection of AADC can control the ability of transplanted T cells to induce lethal GVHD (34), or protect mice against lethal endotoxemia and peritonitis (36). Such potent regulatory DC appear to be more effective in controlling adverse inflammatory/immune responses in vivo than DC conditioned in IL-10 or TGF-beta alone, or modified to express transgenes encoding these cytokines and that exert a comparatively modest effect on allograft rejection without immunosuppression therapy (32, 33, 48). The influence of these reputedly potent AADC on organ allograft survival has not previously been examined. Our goal was to further assess the functional biology of these cells, particularly in relation to alloreactive T cell responses and to assess their impact on graft outcome in a vascularized heart transplant model.

In this study, we found that identical conditioning of murine myeloid DC propagated from the same strain (BALB/c) as reported previously (34) with IL-10 and TGF-beta, followed by LPS activation, resulted in comparatively low levels of TLR4 and cell surface MHC class II and classic costimulatory molecule (CD40, CD80, and CD86) expression. By contrast, no significant effects on surface expression of the newer B7 family cosignaling molecules, PD-L1 and ICOSL, that have been implicated in immune regulation (49), were detected compared with LPS-stimulated control GMDC. In addition, we found that AADC secreted much higher levels of IL-10, but lower levels of IL-12p70 than GMDC.

Decreased expression of TLR4, the receptor that interacts with bacterial cell wall LPS to initiate an NF-{kappa}B-dependent signaling pathway, was associated, in AADC, with LPS hyporesponsiveness. This resulted in reduced secretion of the Th1-inducing cytokine IL-12p70 and increased production of IL-10. Conversely, it has been shown that increased TLR4 mRNA in TGF-beta1 null mice is associated with LPS hyperresponsiveness (43). TGF-beta blocks NF{kappa}B activation and interferes with TLR ligand-induced responses (50). Moreover, Fujita et al. (36) have observed that in AADC, activation of LPS-induced signaling events involving MAPK and NF{kappa}B is impaired. They have suggested that potent expression of I{kappa}B proteins in AADC may suppress NF{kappa}B-mediated production of proinflammatory cytokines, such as IL-12 (as shown in the present study), IL-1beta, TNF-{alpha}, and IL-6. The preferential autocrine production of IL-10 by AADC that we observed, and that appears to involve cAMP-mediated activation of protein kinase A (36), may be involved in the defective production of these proinflammatory cytokines. This view is strengthened by the observation that AADC propagated from IL-10 knockout mice show reduced impairment of proinflammatory cytokine production (36).

Compared with control GMDC, the ability of AADC to induce normal allogeneic T cell activation/proliferation was markedly impaired, reflecting both comparatively low levels of classic TNFR (CD40) and B7 family (CD80, CD86) costimulatory molecules and the preferential secretion of IL-10/reduced IL-12p70 production by these cells. However, like control GMDC, AADC exhibited uniformly high levels of surface PD-L1, a novel B7 family molecule (51) that has been implicated in down-regulation of T cell responses. Indeed, our recent studies (45) show that comparatively high levels of PD-L1 relative to CD80/CD86 expression on murine (plasmacytoid) DC inhibits their ability to drive allogeneic T cell proliferation. Furthermore, there is evidence that PD-L1:PD-1 interactions may influence alloreactive T cell responses by inducing regulatory cells, and that blocking of PD-L1, but not PD-L2 signaling, prevents their induction (52). Interestingly, ICOSL expression, that was observed at comparatively low levels on both AADC and control GMDC in the present study, is not (unlike CD80 and CD86) dependent on NF-{kappa}B activation (53).

Our data indicate that AADC do not enhance apoptotic death of alloactivated T cells in vitro. Interestingly, it has been reported (36) that AADC treatment of mice with systemic inflammation reduces thymocyte apoptosis. Thus, modulation of T cell apoptosis does not appear to account for the poor allostimulatory activity of AADC. In our study, AADC induced alloantigen-specific hyporesponsive T cell proliferation that was not reversed by IL-2, enhanced IL-10 production by alloactivated T cells, and expanded CD4+CD25+Foxp3+ (Treg) cells in short-term culture. Moreover, compared with freshly isolated CD4+CD25+ T cells, AADC-expanded CD4+CD25+ T cells were more effective, on a per cell basis, in suppressing effector CD4+ T cell proliferation in response to stimulation with alloantigen. Such rapid expansion of Treg, within days of allostimulation, has recently been reported in vivo (54). These observations extend the functional characterization of AADC generated in IL-10 and TGF-beta and strongly suggest their potential to regulate alloimmune reactivity in vivo. IL-10 can directly affect DC to enhance their tolerogenic functions (22) and also promote the differentiation of adaptive T regulatory type-1 cells (24, 55). We observed a higher proportion of CD4+CD25+Foxp3+ Treg in AADC-stimulated allogeneic T cell cultures, consistent with poor effector (Foxp3–) T cell proliferation, compared with that observed in response to GMDC. The mechanism responsible for expansion of CD4+CD25+ Treg from CD4+ T cells primed with allogeneic AADC is unclear, but may involve conversion of naive CD4+ T cells to CD4+CD25+ Treg. Alternatively, allogeneic AADC may preferentially expand naturally existing CD4+CD25+ Treg. Our finding that neutralizing anti-IL-10 mAb did not block the expansion of Foxp3+ relative to Foxp3– cells indicates that Foxp3+ cell expansion in response to AADC is not dependent on this cytokine and is consistent with the presence of Treg in IL-10–/– mice (56).

Our data show that AADC expressed similar levels of surface intercellular adhesion molecules to GMDC, but reduced CCR7 expression-the latter consistent with their more immature phenotype and reduced responsiveness to CCL19 (57). However, AADC appeared to traffic normally in vivo to draining lymph nodes and spleens of quiescent, prospective organ allograft recipients. This is consistent with the observations of Sato et al. (35), who reported no change in the secondary lymphoid tissue homing ability of AADC following their infusion into allogeneic recipients. Homing of potentially tolerogenic DC to recipient lymph nodes, as observed in these studies, has been underscored as important for maximization of their therapeutic potential (58).

We found that a single infusion of allogeneic AADC to quiescent animals induced alloantigen-specific hyporesponsive T cell proliferation and prolonged subsequent organ allograft survival significantly in the absence of immunosuppressive therapy, but no grafts survived >43 days. This contrasts with the ability of similar numbers of recipient AADC, administered once only, 2 days after allogeneic BM transplantation, to prevent lethal GVHD and permit host survival for >60 days (35). However, the therapeutic effect of AADC in our model was markedly potentiated by a single injection of the recombinant fusion protein CTLA4-Ig, one day after the AADC. Thus, blockade of the B7-CD28 pathway led to >100 day graft survival in the majority of AADC-treated, but not in any GMDC-treated, heart graft recipients. Costimulation blockade with CTLA4-Ig or anti-CD154 mAb has been shown previously to potentiate the tolerogenicity of donor DC (15, 48, 59, 60, 61) and this strategy has been linked to induction of Treg (15). The present data are the first to show that long-term organ allograft survival can be achieved by host conditioning with AADC plus costimulation blockade. Sato et al. (35) found that IL-10-producing CD4+ T cells and CD4+CD25+CD152+ Treg were increased in AADC-treated recipients. Consistent with this finding, we observed that AADC could expand suppressive CD4+CD25+ Foxp3+ T cells. We further detected CD4+Foxp3+ cell infiltration in long-surviving (>100 days) heart allografts and demonstrated that host CD4+ T cells could transfer regulation of alloimmune reactivity in naive recipients, thus implicating CD4+ Treg in the long-term maintenance of allograft survival.

In conclusion, the present study extends insight into the functional biology of AADC and their interactions with allogeneic T cells. It also extends potential therapeutic applications of AADC to organ transplantation, where a single pretransplant infusion of AADC, plus one injection of CTLA4-Ig, can lead to long-term allograft survival, an effect that appears to be mediated at least in part, by CD4+ Treg.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 R01 DK49745, R01 AI41011, and R01 AI60994 (to A.W.T.) and by an American Heart Association Predoctoral Fellowship (515403U) (to Y.Y.L.). B.L.C. is the recipient of an American Society of Transplantation Basic Science Fellowship and G.R. is in receipt of a Research Training Fellowship from the Transplantation Society. Back

2 Address correspondence and reprint requests to Dr. Angus W. Thomson, University of Pittsburgh, W1544 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address: thomsonaw{at}msx.upmc.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; Treg, regulatory T cell; GVHD, graft-versus-host disease; AADC, alternatively activated DC; Foxp3, Forkhead winged helix protein-3; CM, complete medium; PD-L1, programmed death-ligand 1; MFI, mean fluorescence intensity; PI, propidium iodide; MHC II, MHC class II. Back

Received for publication March 28, 2006. Accepted for publication July 28, 2006.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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