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Immature and Mature CD8⁺ Dendritic Cells Prolong the Survival of Vascularized Heart Allografts¹

Peta J. O’Connell^2,*,, Wei Li^3,*,, Zhiliang Wang^*,, Susan M. Specht^*,, Alison J. Logar^*, and Angus W. Thomson^*,,

^* Thomas E. Starzl Transplantation Institute and Departments of Surgery and Molecular Genetics and Biochemistry, University of Pittsburgh Medical Center, Pittsburgh, PA 15213

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD8⁺ and CD8^- dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8^- (myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8⁺ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2^b) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2^k) recipients for Th1 responses. Paradoxically and in contrast to their CD8^- counterparts, mature CD8⁺ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8⁺ and CD8^- DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8⁺ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.

	Introduction

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Dendritic cells (DCs)⁴ are a heterogeneous population of rare leukocytes, uniquely specialized for the initiation of primary immune responses (1). They originate from circulating hemopoietic precursors and are distributed via the blood to virtually all tissues and organs. Therein, DCs reside as immature APCs with high endocytic and phagocytic capacity and the ability to efficiently process foreign and self Ag. After Ag uptake or organ transplantation, DCs migrate from the periphery to draining secondary lymphoid tissue, where they present Ag to naive or resting T lymphocytes. Considerable new information concerning the ontogeny of DCs, the factors that influence their maturation and tissue distribution, and the role of these important APCs in the induction and regulation of immune responses has been reviewed extensively (2, 3, 4).

Recent speculation that tolerance and immunity may be mediated by distinct DC subsets (5) is buoyed by descriptions of phenotypically and functionally distinct DC populations, in both rodents and humans. CD11c⁺ DCs with the phenotype CD8⁺CD11b^low or CD8^-CD11b^high have been identified in and isolated from both mouse lymphoid and nonlymphoid tissues, including bone marrow, thymus, blood, spleen, lymph node, Peyer’s patches, lung, and liver (6, 7, 8, 9, 10, 11). Their relative incidences vary with tissue distribution: CD8^- DCs are the predominant subset both in bone marrow and blood (7, 11), whereas CD8⁺ DCs are the principal thymic DCs (12). Adoptive transfer studies have demonstrated that CD8⁺ and CD8^- DCs may both develop from highly purified, committed lymphoid and myeloid precursors (13, 14). Moreover, CD8 expression may be induced on Langerhans cells (15, 16). Thus, whether differential expression of CD8 by DCs accurately reflects developmental commitment to functionally distinct DC subsets or the influence of microenvironmental factors remains to be determined.

CD8⁺ and CD8^- DCs reside in distinct microanatomic locations. CD8⁺ DCs localize in T cell areas of periarteriolar lymphocytic sheaths in the spleen and lymph nodes (17, 18, 19). CD8^- DCs are found in marginal zones, but they redistribute to the periarteriolar lymphocytic sheaths after exposure to proinflammatory stimuli, including LPS or parasite extracts (20, 21). In contrast to initial reports (22, 23), CD8⁺ DCs have been found to migrate from s.c. sites to draining lymph nodes (10, 24). They have also been shown to traffic to the spleen after i.v. administration (23, 24). CD8⁺ DCs are the major producer of IFN- (25) and are the only DC subset to cross-prime CD8⁺ T cells in vivo (26, 27).

The relationship between mouse DC subtype and their capacity to stimulate T cell proliferation and Th cytokine production is unclear. Although in vitro experiments have shown that both CD8⁺ and CD8^- DCs stimulate T cell responses efficiently, CD8⁺ DCs can also regulate T cell proliferation. Compared with their CD8^- counterparts, CD8⁺ DCs induce elevated levels of CD95 (Fas)-CD95 ligand (CD95L)-dependent apoptosis of CD4⁺ T cells (28) and restrict CD8⁺ T cell proliferation by limiting their ability to produce IL-2 (29). In contrast, adoptive transfer of allogeneic or Ag (keyhole limpet hemocyanin or OVA)-pulsed CD8⁺ and CD8^- DCs has demonstrated that both DC subsets prime T cells in vivo with equivalent efficiency (30, 31). CD8⁺ DCs were initially described as the major producer of IL-12p70 (18, 21, 30, 32) and were reported to induce predominantly Th1 responses, whereas CD8^- DCs drive Th2 or mixed Th1/Th2 responses (30, 31). However, recently, the capacity of all CD11c⁺ DCs to produce IL-12p70, and thus also their ability to induce Th1 responses, has been show to vary with Ag stimulus (33, 34).

In transplantation, donor-derived DCs have been regarded traditionally as the principal instigators of rejection (35, 36, 37). However, recent evidence has strengthened the view that either donor or host DCs, particularly those that are immature, can also regulate antidonor reactivity and prolong graft survival (38, 39, 40, 41, 42, 43). The majority of studies that have investigated the function and potential therapeutic utility of DCs in allo- or autoimmunity have used either bulk DCs isolated directly from tissues or myeloid DCs generated in vitro using GM-CSF (±IL-4). The present report describes the capacity of immature and mature mouse CD8⁺ and CD8^- DCs to stimulate allogeneic T cell responses, both in vitro and in vivo. It also examines, for the first time, the influence of these DCs administered before transplant on antidonor immune reactivity and organ allograft survival. The data reveal that both immature and mature CD8⁺ DCs, but only immature CD8^- DCs, can significantly prolong transplant survival in the absence of antirejection therapy. Conditioning with CD8⁺ DCs was not accompanied by evidence of either T cell deletion or immune deviation at the time of transplant. However, within 5 days of transplantation, donor-specific T cell responses were significantly impaired in mature CD8⁺ DC-treated mice. These novel observations provide evidence of an in vivo immunoregulatory activity of CD8⁺ DCs in the context of alloimmunity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

Male C57BL/10J (B10; H2^b), C3H/HeJ (C3H; H2^k), or BALB/cByJ (BALB/c; H2^d) mice, 8–12 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). They were housed in the specific pathogen-free facility of the University of Pittsburgh Medical Center (Pittsburgh, PA), and were provided with Purina rodent chow (Ralston Purina, St. Louis, MO) and tap water ad libitum.

Reagents

FITC, PE, or CyChrome (CyC)-conjugated mAbs to detect cell surface CD3 (145-2C11), CD4 (H129.19), CD8 (53-6.7), CD11b (M1/70), CD11c (HL3), CD40 (3/23), CD54 (3E2), CD80 (16-10A1), CD86 (GL1), H2K^b (A6-88.5), and IA^b -chain (AF6-120.1) expression by flow cytometry were purchased from BD PharMingen (San Diego, CA). PE-Texas Red (TR)-conjugated anti-CD8 (5H10) was purchased from Caltag Laboratories (Burlingame, CA). Biotinylated anti-CD205 (NLDC-145; Bachem, Bubendorf, Switzerland) and anti-CD95L (MFL3; BD PharMingen) were detected using streptavidin-PE (BD PharMingen). Recombinant mouse GM-CSF was provided by Dr. S. K. Narula (Schering-Plough, Kenilworth, NJ). Human rFlt3L, derived from Chinese hamster ovary cells, was provided by Immunex (Seattle, WA). RPMI 1640 (Life Technologies, Rockville, MD) was supplemented with 10% (v/v) FCS (Nalgene, Miami, FL), nonessential amino acids, L-glutamine, sodium pyruvate, penicillin-streptomycin, and 2-ME (all from Life Technologies) and is referred to subsequently as complete medium.

DC isolation and sorting

DCs were isolated from the spleens of animals given Flt3L (10 µg/mouse/day i.p. in HBSS) for 10 consecutive days (7). Spleens were disaggregated and digested for 15 min with 10 ml of type IV collagenase (200 µg/ml; Sigma-Aldrich, St. Louis, MO) in HBSS supplemented with 100 µg/ml DNase (Roche, Mannheim, Germany). After digestion, splenocytes were collected by centrifugation at 500 x g, and erythrocytes were lysed by hypotonic shock using 0.15 M NH₄Cl. DCs were isolated either immediately after splenocyte preparation or after overnight (18 h) incubation in complete medium containing GM-CSF (4 ng/ml). DCs were enriched from fresh or overnight-incubated splenocytes by metrizamide (16.5 or 14.5% (w/v), respectively) density centrifugation at 500 x g for 15 min at room temperature (20°C). For purification by sorting, the buffy layer was labeled with anti-CD11c FITC and anti-CD8 PE for 30 min at 4°C. Cells were washed, incubated for 5 min at 4°C with cation-free HBSS containing 1% (v/v) FCS and 10 mM EDTA to disaggregate cell clusters, and then resuspended in complete medium. CD8⁺CD11c⁺ or CD8^-CD11c⁺ DC populations, with high forward- and side-scatter profiles, were sorted using a Coulter EPICS Elite (Beckman Coulter, Hialeah, FL) to >95% purity.

Flow cytometric analyses

Leukocytes were first blocked with 10% (v/v) normal goat serum (Sigma-Aldrich) for 20 min at 4°C and then stained with mAb for 30 min at 4°C. Cells stained with appropriate isotype-matched Ig (BD PharMingen) were used as negative controls. After staining, the cells were fixed with 1% (w/v) paraformaldehyde and analyzed using a Coulter EPICS XL.MCL (Beckman Coulter) flow cytometer and EXPO 32 software (Applied Cytometry Systems, Sheffield, U.K.).

Adoptive transfer of DC subsets

Sorted CD8⁺ and CD8^- B10 DCs were washed extensively in HBSS and then injected (2 x 10⁶ in 400–500 µl of HBSS) into C3H mice via the lateral tail vein. After 7 days, mice received vascularized heterotopic B10 heart transplants, as described below. For ex vivo functional studies, spleens were removed either 7 or 12 days after adoptive transfer of DCs (corresponding to the time of heart transplant and 5 days posttransplant, respectively).

MLR

Bulk splenocytes or T cells from naive or DC-primed C3H mice were enriched by a single passage through nylon wool columns (45 min at 37°C) and used as responders. A total of 2 x 10⁵ cells were placed in each well of 96-well round-bottom plates, and varying numbers of gamma-irradiated (20 Gy), sorted CD8⁺ or CD8^- DCs or 2 x 10⁵ normal bulk splenocytes (C3H, B10, or BALB/c) were added as stimulators. In some experiments, human rIL-2 (50 U/ml; Genetics Institute, Cambridge, MA) was added at the start of culture to test for reversal of hyporesponsiveness. The cultures were incubated in complete medium for 72 h, unless otherwise specified, in a humidified atmosphere of 5% CO₂ in air. [³H]TdR (1 µCi in 10 µl) was added to each well for the final 18 h of culture. Cells were harvested using a multiple-well harvester, and [³H]TdR incorporation was determined in a liquid scintillation counter. Results are expressed as the mean counts per minute ± 1 SD from triplicate cultures.

Detection of intracellular cytokines

Cytokines were detected intracellulary in responder C3H T cells after 72-h MLR using normal bulk B10 splenocytes as stimulators (stimulator:responder ratio, 1:1). The T cells were restimulated with plate-bound hamster anti-mouse CD3 (clone 145-2C11, 10 µg/ml) and soluble hamster anti-mouse CD28 (clone 37.51, 10 µg/ml) for 5 h at 37°C, in the presence of brefeldin A (10 µg/ml; Sigma-Aldrich). Thereafter, cells were washed with 1% (v/v) FCS/PBS, fixed with 4% (w/v) paraformaldehyde (20 min, 4°C), and permeabilized with 0.15% (w/v) saponin/1% (v/v) FCS/PBS for 15 min at 4°C. The cells were then labeled by incubation for 30 min at 4°C with 1) anti-CD3 CyC, 2) anti-CD4 FITC, and 3) anti-CD8 PE-TR. Intracellular cytokines were detected by the addition of PE-conjugated anti-IFN- (XMG1.2), anti-IL-2 (Jes6-5H4), anti-IL-4 (BVD4-1D11), or anti-IL-10 (JES5-16E3) mAbs, all purchased from BD PharMingen. After staining, the cells were washed with 1% (v/v) FCS/PBS, fixed with 1% (w/v) paraformaldehyde, and analyzed immediately using a Coulter EPICS XL.MCL flow cytometer. Cells stained with appropriate isotype-matched Ig (BD PharMingen) were used as negative controls.

Heterotopic heart transplantation

Surgical procedures were performed under inhalation anesthesia using methoxyflurane (Pitman-Moore, Atlanta, GA). Vascularized heterotopic cardiac transplants to an abdominal site were performed as described (44). Contraction of the donor heart was monitored daily by abdominal palpation. Total cessation of cardiac contraction was defined as rejection.

Exposure of DC subsets to LPS or CD40L-transfected J558 cells

Bulk DCs were enriched from freshly isolated Flt3L-mobilized spleen cells by 16.5% metrizamide density centrifugation and then were incubated overnight with GM-CSF (4 ng/ml) alone or with either LPS (100 ng/ml, Escherichia coli serotype 026:B6; Sigma-Aldrich) or CD40L-transfected J558 cells (45) at a 1:1 ratio. DCs were then triple-immunolabeled and analyzed by flow cytometry as described above to determine their relative expression of cell surface MHC and costimulatory molecules.

In vivo trafficking of DC subsets

Freshly isolated splenocytes from Flt3L-treated mice were incubated overnight with GM-CSF (4 ng/ml). Bulk DCs were then enriched by 14.5% metrizamide density centrifugation (purity 95% CD11c⁺) and tracer-labeled using PKH26 (Sigma-Aldrich) following the manufacturer’s recommended protocol. DCs were washed extensively in PBS and then injected (5 x 10⁶ cells in 400–500 µl of HBSS) into naive C3H mice via the lateral tail vein. Thirty-six hours after DC administration, recipient spleens were removed and DCs enriched by 16.5% metrizamide density centrifugation. DCs were triple-immunolabeled as described above with anti-CD11c PE, anti-CD8 CyC, and biotinylated anti-CD86-streptavidin PE-TR (Immunotech, Marseille, France), and then analyzed by flow cytometry. Donor (B10) DCs were identified by PKH26 (green) fluorescence.

ELISA

Splenocytes, prepared from C3H mice 5 days after heart transplantation, were restimulated with bulk donor-type (B10) splenocytes as described for MLR. Supernatants were harvested after 72 h of coculture. To assess cytokine production over a discrete period (24 h) at the peak of T cell proliferation, cells were harvested after a 72-h coculture, washed, and resuspended in fresh complete medium for an additional 24-h stimulation with anti-CD3 and anti-CD28 mAbs. ELISA for mouse IFN-, IL-4, and IL-10 in culture supernatants were performed using reagents purchased from BD PharMingen and following the manufacturer’s recommended procedures. The detection limits were 190 pg/ml for IFN-, 3.9 pg/ml for IL-4, and 15 mg/ml for IL-10.

Assay for regulatory activity in the recipient spleen and transplanted heart

Heart grafts were perfused in situ under gaseous anesthesia (Metofane; Schering-Plough) via the native heart with 10 ml of HBSS and then 5 ml of collagenase (1 mg/ml). Transplanted hearts were then removed, minced into small pieces, and digested in collagenase containing DNase (100 µg/ml) at 37°C for 60 min. Cells were then filtered through a 70-µm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ). Graft-infiltrating cells (GICs) were then isolated by density centrifugation using Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada) for 30 min at 800 x g, and then they were washed twice with complete medium. Bulk splenocytes were prepared from the recipient spleen as described above. The presence of regulatory cells within GICs or bulk recipient splenocytes was assayed by the addition of 5 x 10⁴ gamma-irradiated (20 Gy) cells at the start of 72-h one-way MLR (B10C3H) as described earlier. Naive bulk B10 spleen cells were used as a source of stimulators (2 x 10⁶/ml), and nylon-wool enriched C3H splenocytes were used as responder T cells (2 x 10⁶/ml).

Statistical analyses

Statistical analysis was performed using two-tailed Student’s t test; p < 0.05 was considered significant. Graft survival data were compared by Kaplan-Meier analysis and the log-rank test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mobilization of CD8⁺ and CD8^- splenic DC subsets in Flt3L-treated mice

Mice treated with the naturally occurring hemopoietic growth factor Flt3L are a convenient source of DC subsets that exist only in very small numbers (1% total leukocytes) within normal murine lymphoid tissue (7, 12). These cells can readily be identified by their surface expression of CD11c, a marker that is typically restricted to DCs in the mouse (46, 47). As shown in Fig. 1, administration of 10 µg of Flt3L once daily for 10 days expanded two major CD11c⁺ MHC class II⁺ DC populations, CD8⁺CD11b^low and CD8^-CD11b^high (7), which comprised 8 and 10.5%, respectively, of total splenocytes. A minor population (typically 10–20%) of CD8^-CD11c⁺ DCs were also CD4⁺, consistent with a previously described subpopulation of splenic DCs (48, 49). Freshly isolated CD8⁺ DCs expressed higher levels of the multilectin receptor CD205 (50) compared with CD8^- DCs. As reported elsewhere, the death ligand CD95L, which has been implicated in the immunomodulatory function of murine DCs (28, 51), was detected at moderate levels on both CD8⁺ and CD8^- subsets.

View larger version (53K): [in this window] [in a new window]   FIGURE 1. Flow cytometric analysis of splenic DCs freshly isolated from B10 mice after 10 days of Flt3L administration. Splenocytes were triple-immunolabeled with 1) anti-CD11c FITC, 2) anti-CD8 CyC, and 3) anti-MHC class II (IAb), anti-CD4, anti-CD11b PE, biotinylated anti-CD95L, or anti-CD205 streptavidin-PE. The scatter plot identifies CD8+ (A) and CD8- (B) CD11c+ DC subsets in freshly isolated splenocytes. Histograms show the expression of specific markers (PE) on CD8+ (filled histograms) and CD8- (open histograms) CD11c+ DCs. A minor subpopulation of CD8- DCs (*, 16%) exhibited positive staining for CD4. Dotted profiles show isotype-matched controls. The results are representative of at least three separate experiments.

Phenotypic maturation of splenic CD8⁺ and CD8^- DC subsets after overnight incubation

Freshly isolated CD8⁺ and CD8^- DC subsets were phenotypically immature, as determined by their expression of moderate levels of MHC class II and CD54, together with low to moderate expression of the costimulatory molecules CD40, CD80, and CD86. This immature or "Ag-processing" phenotype is consistent with previous reports regarding DCs freshly isolated from both lymphoid and nonlymphoid tissues (10, 52, 53). As shown in Fig. 2, overnight incubation (18 h) of bulk DCs in the presence of GM-CSF resulted in elevated expression of surface MHC class II and CD54 and pronounced increases in costimulatory molecule (CD40, 80, and 86) expression by both CD8⁺ and CD8^- DC subsets. Consistent with our earlier observation regarding hepatic DC subsets, splenic CD8⁺ DCs consistently expressed slightly higher levels of MHC, accessory, and costimulatory molecules (10). The inclusion of GM-CSF during overnight culture previously has been shown to maintain the viability of both DC subsets isolated from either lymphoid or nonlymphoid tissues and to promote their maturation (10, 24, 50). Comparative functional studies on immature and mature CD8⁺ and CD8^- DC subsets were undertaken using sorted, freshly isolated, and overnight-incubated DC populations, respectively.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Flow cytometric analysis of MHC class I and II, costimulatory and accessory molecule expression by immature (freshly isolated) and mature (overnight incubated) splenic DC subsets. Splenocytes were triple-immunolabeled with 1) anti-CD11c FITC, 2) anti-CD8 CyC, and 3) anti-MHC class I (H2Kb), anti-MHC class II (IAb), anti-CD40, anti-CD54, anti-CD80, or anti-CD86 PE. Histograms show the expression of specific markers (PE) on CD8+ (A) and CD8- (B) DCs gated as shown in Fig. 1, either as freshly isolated cells (filled histograms) or overnight incubated (open histograms). The data demonstrate that culture of freshly isolated immature splenic DCs results in phenotypic maturation of both DC subsets. Dotted profiles show isotype-matched controls. The results are representative of at least three separate experiments.

Compared with immature CD8^- DCs, immature CD8⁺ DCs induce inferior proliferation of naive allogeneic T cells

Although they exhibited only moderate levels of surface MHC class II and were deficient in expression of costimulatory molecules, sorted, freshly isolated immature B10 DC subsets induced 10-fold (range, 3–15) higher proliferation of naive allogeneic (C3H) splenic T cells compared with bulk B10 spleen cells (Fig. 3, A and B). Interestingly, T cell proliferation in 72-h culture was significantly greater after stimulation with immature CD8^- DCs, despite their apparent lower levels of surface MHC and costimulatory molecule expression compared with CD8⁺ DCs (Fig. 2). This difference in T cell proliferation did not appear to be related to surface expression of CD95L, which was similar on both freshly isolated DC subsets (Fig. 1). Interestingly, when a 1/1 mixture of sorted CD8⁺ and CD8^- DCs was used as a stimulator, the resulting T cell proliferation curve fell midway between those curves observed when each subpopulation was used alone (data not shown). To determine whether the difference in proliferation induced by the freshly isolated DC subsets detected at 72 h might reflect different response kinetics, MLR cultures were harvested at 24, 48, and 72 h. At each time point, T cell proliferation was significantly less in response to stimulation with immature CD8⁺ DCs compared with immature CD8^- cells (Fig. 3B). Thus, consistent with previous reports (28), the differential proliferative response of naive allogeneic T cells to the immature splenic DC populations was evident throughout the course of the MLR and could not be attributed to distinct response kinetics.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Allostimulatory capacity for naive splenic T cells (C3H; 2 x 106 cells/ml) of gamma-irradiated CD8+ and CD8- DCs sorted from B10 spleen after 10 days of Flt3L administration. A, T cell proliferation was consistently higher after stimulation with immature (i; freshly isolated) CD8- DCs (*, p < 0.01) compared with the CD8+ subset. B, Coculture of immature CD8+ or CD8- DCs (5 x 105 cells/ml) over 24–72 h demonstrates that the difference in T cell proliferation was not due to distinct proliferation kinetics. C, By contrast, sorted mature (m; overnight-incubated) CD8+ and CD8- DCs were equivalent allostimulatory cells. The results were obtained from 72-h MLR unless otherwise indicated and are the mean ± 1 SD from triplicate cultures. The data are representative of at least three separate experiments.

Mature CD8⁺ and CD8^- DCs induce equivalent proliferation of naive allogeneic T cells

We next examined whether the differential proliferative response of naive allogeneic T cells to immature CD8⁺ and CD8^- DCs might be retained after phenotypic and functional maturation of the DCs. Consistent with the striking up-regulation of surface MHC class II Ags and costimulatory molecules observed after overnight incubation (Fig. 2), sorted CD8⁺ and CD8^- DCs exhibited equivalent and markedly increased allostimulatory activity (Fig. 3C). This was 20-fold greater than that of bulk allogeneic B10 spleen cells in 72-h MLR.

T cells from mice primed with CD8⁺ or CD8^- DCs exhibit similar ex vivo proliferative responses to donor alloantigens

The influence of immature and mature donor CD8⁺ or CD8^- DCs on in vivo T cell alloreactivity was examined. Normal C3H mice were injected i.v. with 2 x 10⁶ sorted immature or mature CD8⁺ or CD8^- B10 DCs. Seven days later, C3H splenic T cells were isolated and restimulated with bulk splenocytes from syngeneic (C3H), donor (B10), or third-party (BALB/c) mice. Day 7 was chosen for analysis, because i.v. infusion of myeloid DCs 7 days before transplantation previously has been shown to effectively inhibit antidonor reactivity in organ allograft recipients (40, 42, 54). As shown in Fig. 4, the proliferative response to donor Ags of T cells from DC-primed mice was significantly enhanced compared with naive T cells (p < 0.01 and p < 0.005, immature and mature DCs, respectively). Moreover, T cell priming was donor specific; the proliferative responses of naive T cells and of T cells from DC-injected mice to third-party stimulator cells were comparable. No significant difference was detected between the donor-specific, ex vivo T cell proliferative responses of mice primed with either CD8⁺ or CD8^- DC subsets. However, consistent with both their phenotype (Fig. 2) and in vitro allostimulatory activity (Fig. 3), mature DC subsets administered i.v. activated T cells more efficiently compared with immature DC subsets. The ex vivo proliferative responses of T cells from immature and mature DC-injected mice to B10 alloantigens were 2-fold and 7- to 8-fold greater, respectively, than those of naive T cells. These data indicate that donor-specific conditioning either with immature, moderately stimulatory DCs or with mature, strongly stimulatory DCs does not lead to differential loss of T cell alloreactivity in the ensuing 7-day period.

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Three-day MLR responses of splenic T cells from C3H recipients of immature (i; freshly isolated) (A), and mature (m; overnight-incubated) (B) sorted B10 CD8+ and CD8- DCs, 7 days after administration via the lateral tail vein. Splenic T cells (2 x 106 cells/ml) were enriched using nylon wool columns and cocultured with bulk splenocyte stimulator cells (2 x 106 cells/ml) syngeneic with the recipient (C3H), allogeneic (donor type; B10), or third party (BALB/c). The results are the mean ± 1 SD from triplicate cultures. *, p < 0.01; **, p < 0.005. The data are representative of at least three separate experiments.

T cells from mice primed with CD8⁺ or CD8^- DCs exhibit predominantly Th1/Tc1 cytokine responses after restimulation with donor alloantigens

It has been demonstrated previously that splenic CD8⁺ DCs are deficient in their ability to prime CD8⁺ T cells for IL-2 production in vitro (29). We examined the production of signal cytokines for Th1/Tc1 (IFN- and IL-2) and Th2/Tc2 (IL-4 and IL-10) responses within CD3⁺CD4⁺ and CD3⁺CD8⁺ splenic T cells isolated from C3H mice primed 7 days earlier with B10 CD8⁺ or CD8^- DCs and restimulated in vitro with B10 alloantigens. Cytokine production was assessed using intracellular immunostaining and flow cytometry after 72-h MLR. As described in Materials and Methods, cytokine production was assessed over a discrete period (5 h) at the peak of T cell proliferation. Cytokine production by restimulated T cells primed in vivo with immature DCs of either subset exhibited a similar low frequency (typically 1–5%) of mAb-positive cells (data not shown), which was consistent with the weak to moderate allostimulatory activity of the immature DC subsets, both in vitro (Fig. 3, A and B), and in vivo (Fig. 4A). In contrast, in vivo priming with mature B10 DC subsets led to substantially higher levels of T cell activation (Fig. 4B), with parallel increases in T cell cytokine production. As shown in Fig. 5, T cells primed by either mature CD8⁺ or CD8^- DC in vivo showed similar incidences of CD4⁺ and CD8⁺ subsets. These T cells exhibited predominantly Th1/Tc1-type cytokines (IFN- and IL-2) with a lower incidence of cells producing the Th2/Tc2 cytokine IL-10. The incidence of IL-4-positive T cells was not typically above background levels. In contrast, the incidence of cytokine-producing naive C3H T cells stimulated in vitro with bulk B10 splenocytes was very low (2% of total CD3⁺ T cells), which is consistent with the low levels of proliferation observed in Fig. 4. Thus, there was no evidence that i.v. infusion of allogeneic DCs of either subset impaired the capacity of either CD4⁺ or CD8⁺ T cells to produce IL-2 or other cytokines or that it induced immune deviation in response to alloantigen-specific restimulation 7 days later.

View larger version (68K): [in this window] [in a new window]   FIGURE 5. Detection of Th1/Tc1 (IFN- and IL-2) and Th2/Tc2 (IL-4 and IL-10) cytokines in splenic C3H T cells enriched from naive mice (A) or the recipients of sorted mature (m; overnight-incubated) B10 CD8+ DCs (B) and CD8- DCs (C) 7 days after DC administration via the lateral tail vein. C3H T cells (2 x 106 cells/ml) were stimulated in vitro with bulk splenocytes (2 x106 cells/ml) of the same allogeneic strain (B10) as the injected DCs. After 3-day MLR, the T cells were harvested and restimulated with anti-CD3 and anti-CD28 mAbs, as described in Materials and Methods, and then immunolabeled with 1) anti-CD3 CyC, 2) anti-CD4 FITC, 3) anti-CD8 PE-TR, and 4) anti-IFN-, anti-IL-2, anti-IL-4, or anti-IL-10-PE. Only anti-CD3 CyC+ T cells are represented in B and C; APCs and other minor contaminating populations in the MLR were gated out, according to their lack of CD3. Data are representative of three separate experiments.

Both immature CD8⁺ and CD8^- DCs markedly prolong heart allograft survival

It previously has been shown (40, 41, 42) that donor-type immature myeloid DCs, generated in vitro, can markedly prolong allograft survival if administered systemically 7 days before transplantation. To evaluate and compare the influence of in vivo-mobilized immature CD8⁺ and CD8^- DC subsets on allograft survival, C3H mice received 2 x 10⁶ sorted (>95% purity) B10 DCs i.v. 7 days before vascularized heterotopic B10 cardiac transplantation, in the absence of any immunosuppressive therapy. As shown in Fig. 6, immature DCs significantly prolonged the median graft survival time from 11 days in untreated controls to 29 and 20 days after administration of CD8⁺ or CD8^- DCs, respectively. Although adoptive transfer of immature CD8⁺ DCs induced more pronounced extension of graft survival compared with immature CD8^- DCs, this effect was not significantly different using log-rank analysis.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. A, Kaplan-Meier analysis of the survival of B10 cardiac allografts in untreated C3H recipients () compared with those administered i.v. with 2 x 106 sorted immature (i; freshly isolated) B10 CD8+ (•; p < 0.0001) or CD8- (; p < 0.005) DCs 7 days before transplantation. Comparison of the survival curves was performed using the log-rank test. There were seven to nine animals per group.

Mature CD8⁺ but not mature CD8^- DCs prolong heart allograft survival

In contrast to immature myeloid DCs, in vitro generated mature myeloid DCs of donor origin have been shown to accelerate organ allograft rejection (40, 41, 42). To date, there have been no reports regarding the influence of purified, in vivo mobilized DCs or CD8⁺ DCs on organ allograft survival. As shown in Fig. 7, i.v. administration of 2 x 10⁶ sorted mature CD8⁺ B10 DCs 7 days before heart transplantation prolonged B10 graft survival time (median, 26 days) to an extent similar to that observed after adoptive transfer of immature CD8⁺ DCs (Fig. 6; median, 29 days). By contrast, an equivalent number of sorted mature CD8^- DCs reduced median graft survival time (Fig. 7) compared with untreated controls (median, 7.5 and 11 days, respectively), although this difference was not statistically significant.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. A, Kaplan-Meier analysis of the survival of B10 cardiac allografts in untreated C3H recipients () compared with those administered i.v. with 2 x 106 sorted mature (m; overnight-incubated) B10 CD8+ (•; p < 0.0001) or CD8- (; p = 0.14) DCs 7 days before transplantation. Comparison of the survival curves was performed using the log-rank test. There were eight to nine animals per group.

Exposure to maturation-inducing stimuli (LPS or CD40 ligation) or adoptive transfer to allogeneic hosts does not affect surface expression of costimulatory molecules by mature DCs

We considered the hypothesis that mature (overnight-incubated) CD8⁺ DCs might not have achieved "terminal" maturation before adoptive transfer, which could have contributed to their "tolerogenic" effect. To ascertain the extent to which the overnight-incubated DC subsets represented fully mature APCs, the expression of MHC class II and costimulatory molecules by these cells was compared with the phenotype of bulk DCs cultured overnight in GM-CSF (4 ng/ml) with either LPS (100 ng/ml) or CD40L-transfected J558 cells. As shown in Fig. 8, A and B, neither stimulus further up-regulated surface expression of MHC class II, CD40, CD80, or CD86 on either CD8⁺ or CD8^- DCs. Furthermore, when overnight-incubated (GM-CSF alone), tracer (PKH26)-labeled B10 DCs were adoptively transferred (i.v.) to allogeneic (C3H) recipients, the relative expression of surface CD86 36 h later by CD8⁺ DCs or CD8^- DCs that trafficked to recipient spleens was similar (Fig. 8, C and D). This renders unlikely the possibility that prolongation of graft survival after adoptive transfer of overnight-incubated mature CD8⁺ DCs was due to an incomplete or lesser state of phenotypic maturation of these cells compared with CD8^- DCs.

View larger version (27K): [in this window] [in a new window]   FIGURE 8. Exposure to maturation-inducing stimuli (LPS or CD40 ligation) or adoptive transfer to allogeneic recipients does not affect surface expression of costimulatory molecules on mature (m) DCs. Splenocytes were enriched for DCs by 16.5% metrizamide density centrifugation, incubated overnight, and then triple-immunolabeled with 1) anti-CD11c FITC, 2) anti-CD8 CyC, and 3) anti-MHC class II (IAb), anti-CD40, anti-CD80, or anti-CD86 PE. Histograms show the expression of specific markers (PE) on CD8+ (A) and CD8- (B) gated CD11c+ DCs incubated overnight with GM-CSF (open histograms; 4 ng/ml) alone or together with LPS (filled histograms; 100 ng/ml) or the J558 CD40L-transfected plasmacytoma cells (dashed histograms). Isotype controls (dotted histograms) are shown at the far left. C and D, Tracer (PKH26)-labeled, overnight-incubated, mature B10 DCs enriched by 14.5% metrizamide density centrifugation were injected i.v. via the lateral tail vein into recipient (C3H) mice. Thirty-six hours later, C3H splenocytes were enriched for DCs by 16.5% metrizamide density centrifugation and triple-immunolabeled with 1) anti-CD11c PE, 2) anti-CD8 CyC, and 3) biotinylated anti-CD86 streptavidin PE-TR. Shaded histograms show that donor (PKH26+) CD8+, and CD8- DCs, which traffic to recipient spleen, express similar levels of CD86. The apparent difference in CD86 expression by B10 CD8+ and CD8- DCs (A and B vs C and D) results from the different fluorochromes used for detection. Dotted histograms show isotype-matched controls. The results are representative of two independent experiments (A and B) and one of four separate animals (C and D).

T cells from heart-allografted mice primed with mature CD8⁺ DCs exhibit impaired donor-specific proliferative responses that are not reversed by exogenous IL-2

To compare antidonor and third-party T cell responses of animals given mature donor-type CD8⁺ or CD8^- DCs 7 days before heart transplantation, host spleen cells were harvested 5 days posttransplant and cultured with syngeneic (C3H) donor (B10) or third-party (BALB/c) stimulators. Proliferative responses were quantified at 72 h, as shown in Fig. 9A. At the time of transplantation, T cells from DC-primed mice exhibited potent and equivalent donor-specific proliferative responses (Fig. 4B). By contrast, splenocytes obtained 5 days posttransplant from mature CD8⁺ DC-primed mice exhibited suppressed proliferative responses to donor Ags, equivalent to primary responses to third party Ags (Fig. 9A). Suppression of responsiveness was donor-specific and not reversible by exogenous IL-2 (50 U/ml) added at the start of culture (Fig. 9B). There was no significant difference between the proliferative responses of splenocytes from mice primed with either DC subset to third party Ags (Fig. 9A). In contrast, the posttransplant proliferative response of splenocytes from mature CD8^- DC-primed mice to donor Ags was significantly greater (p < 0.05) compared with third-party Ags. Mice primed with mature CD8^- DCs exhibited significantly higher (p < 0.05) proliferative responses to donor alloantigens compared with mature CD8⁺ DC-primed splenocytes. These data indicate that pretreatment of transplant recipients with mature CD8⁺ donor DCs, a procedure that did not in itself impair antidonor reactivity at the time of grafting, led to diminution of antidonor T cell proliferative responses by 5 days posttransplant that could not be ascribed to anergy.

View larger version (46K): [in this window] [in a new window]   FIGURE 9. T cells from heart-allografted mice primed with mature CD8+ DCs exhibit impaired donor-specific proliferative responses. Allograft recipients were primed 7 days before transplantation with sorted mature (m; overnight-incubated) B10 CD8+ or CD8- DCs. A, MLR responses of bulk C3H splenocytes (2 x 106 cells/ml) harvested 5 days after B10 heart transplantation and cocultured with bulk splenocyte stimulators (2 x 106 cells/ml) syngeneic with the recipient (C3H), allogeneic (donor type, B10), or third party (BALB/c). Data shown are from four individual animals in each DC treatment group. B, Addition of bioactive recombinant human IL-2 (50 U/ml) to ex vivo MLR (B10C3H) did not restore the proliferative response of C3H splenocytes to B10 alloantigens from mice pretreated with mature (m) B10 CD8+ DCs to B10 alloantigens. Data shown are representative of two independent experiments, with two animals in each group per experiment. All results were obtained from 72-h MLR and are the means ± 1 SD from triplicate cultures. *, p < 0.05; **, p < 0.01.

Prolongation of graft survival by mature CD8⁺ DCs is not associated with immune deviation, as determined by cytokine secretion in ex vivo MLR

Production of Th1 and Th2 cytokines in MLR cultures comprising responder (C3H) spleen cells harvested 5 days posttransplant from mice pretreated with mature CD8⁺ or CD8^- DCs was quantified by ELISA. Supernatants were collected 72 h after stimulation with allogeneic (B10) splenocytes or after a further 24-h stimulation with anti-CD3 and anti-CD28 mAbs. As shown in Fig. 10, compared with mice primed with CD8^- DCs, both Th1/Tc1 (IFN-) and Th2/Tc2 (IL-4 and IL-10) cytokine production in MLR cultures of CD8⁺ DC-primed animals were significantly lower. Although this is consistent with the observed inhibition of graft rejection by CD8⁺ DCs, the findings also suggest that immune deviation (skewing toward Th2/Tc2 responsiveness) does not accompany diminished antidonor responsiveness.

View larger version (27K): [in this window] [in a new window]   FIGURE 10. Levels of Th1/Tc1 (IFN-) (A) and Th2/Tc2 (IL-4 and IL-10) cytokines (B and C) in posttransplant MLR supernatants by ELISA. Bulk C3H splenocytes (2 x 106/ml) harvested 5 days after B10 heart transplantation were cocultured with bulk B10 splenocyte stimulators (2 x 106 cells/ml). Allograft recipients were primed 7 days before transplantation with sorted mature (m; overnight-incubated) B10 CD8+ DCs or CD8- DCs. Supernatants were harvested immediately after 72-h MLR or after a further 24-h restimulation with anti-CD3 or anti-CD28 mAbs. Cytokine production in MLR cultures of CD8+ DC-primed animals were consistently lower compared with CD8- DC-primed mice (*, p < 5 x 10-4; **, p < 5 x 10-6. The results are the means ± 1 SD from triplicate cultures. Data shown are representative of two independent experiments with two animals in each group. ND, Not detected.

Prolongation of graft survival by mature CD8⁺ DCs is not associated with generation of regulatory cells

In a further effort to address mechanisms that might underlie the capacity of mature CD8⁺ DCs to prolong graft survival, we looked for evidence of regulatory cells both in the graft infiltrate and in recipients’ spleens. The assay used has been employed extensively to identify regulatory cells in the context of transplantation outcome (55). Incorporation of GICs syngeneic with C3H responders in primary MLR (B10C3H) revealed inconsistent evidence of regulatory cell activity in mature CD8⁺ DC-treated animals (Fig. 11A) 5 days posttransplant. However, consistent reduction of the MLR was observed with GICs from mature CD8^- DC-treated mice, which we interpret as evidence of antidonor reactivity directed against B10 stimulator cells. No evidence of systemic regulatory cells was detected in recipient spleens (Fig. 11B). Taken together, these observations indicate that the impaired donor-specific T cell responsiveness observed in allografted recipients of mature CD8⁺ DCs is not accompanied by reversible T cell anergy, immune deviation, or the presence of systemic regulatory cells.

View larger version (48K): [in this window] [in a new window]   FIGURE 11. Prolongation of graft survival by mature CD8+ DCs is not associated with evidence of regulatory cells. GICs (5 x 104) (A) or spleen cells (5 x 104) (B) of C3H mice isolated 5 days after B10 heart transplantation were added at the start of B10 (2 x 106/ml) C3H (2 x 106/ml) primary MLR. Allograft recipients were primed 7 days before transplantation with sorted mature (m; overnight-incubated) B10 CD8+ DCs or CD8- DCs. Data shown are from two independent experiments with two animals in each group. All results were obtained from 72-h MLR and are the means ± 1 SD from triplicate cultures. *, p < 0.01; **, p < 0.005; compared with primary MLR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Freshly isolated splenic DC subsets expressed low to moderate levels of MHC class II and costimulatory molecules. Consistent with this immature phenotype, freshly isolated CD8⁺ and CD8^- DCs were low to moderate stimulators of allogeneic T cell proliferation, albeit superior to bulk spleen cells. The present finding that immature, Flt3L-induced donor CD8⁺ and CD8^- DCs can significantly extend vascularized heart graft survival time when administered 7 days before transplant is consistent with prior reports regarding the influence of in vitro-generated immature myeloid DCs on graft survival. Thus, in rodent models, in vitro generated immature myeloid DCs modulate antidonor T cell reactivity in vitro (38, 43, 60) and prolong heart (40, 41, 42, 60) and pancreatic islet (39) allograft survival. Typically, in the absence of immunosuppressive therapy, systemic administration of immature myeloid DCs of donor origin before transplantation does not achieve alloantigen-specific tolerance. The temporary or unstable nature of the hyporesponsiveness induced in nonimmunosuppressed recipients may reflect alternate immune stimulation via indirect alloantigen presentation by competent host APCs. Alternatively, the failure to achieve tolerance has been ascribed to the apparent in vivo maturation of the donor DCs into immunostimulatory APCs (40). The latter proposal is supported by a recent study that demonstrated permanent heart graft acceptance (>100 days) after injection of immature myeloid donor DCs that were refractory to typical maturation-inducing stimuli, 7 days before transplant (42). With respect to donor-derived myeloid DCs, the kinetic dependence of the therapeutic effect has been demonstrated in several independent studies (40, 42, 54). In this report, we have not ascertained the extent to which the therapeutic effect of CD8⁺ donor DCs is dependent on the temporal relationship between their administration and organ grafting. Current investigations in our laboratory are designed to examine this issue, concomitant with other variables likely to impact on efficacy, including dose, frequency, and route of DC administration and adjunctive immunosuppression.

As reported herein, overnight incubation of CD8⁺ or CD8^- DCs resulted in their maturation into potent and equivalent allostimulatory APCs, which expressed high levels of MHC and costimulatory molecules. Consistent with previous reports regarding in vitro-generated myeloid DCs (40, 42), the capacity of CD8^- DCs to prolong allograft survival was strictly associated with an immature, costimulatory, molecule-deficient phenotype. Mice pretreated with mature CD8^- DCs rejected their allografts with similar or accelerated kinetics compared with untreated controls (median, 7.5 and 11 days, respectively). In contrast, the present findings demonstrate for the first time that systemic administration of donor-type CD8⁺ DCs, irrespective of their maturation state, significantly prolong MHC-mismatched organ graft survival in the absence of exogenous immunosuppression. Interestingly, there was no significant difference between the capacity of either immature or mature CD8⁺ DCs to prolong graft survival (median, 29 and 26 days, respectively). These findings are consistent with the recent observation that a bone marrow resident leukocyte that shares phenotypic characteristics with CD8⁺ DCs and veto cells (CD3^-CD8⁺CD11c⁺TCR^-) facilitates the engraftment of purified allogeneic mouse hemopoietic stem cells (61). Furthermore, the "tolerizing" property of mature CD8⁺ DCs could not be ascribed to a lesser state of maturation of these cells compared with mature CD8^- DCs, as determined by phenotypic and functional analyses in the presence or absence of a variety of potent maturation-inducing stimuli. These findings are consistent with the recent observation that CD8⁺ and CD8^- DCs exhibit similar profiles of MHC, costimulatory, and accessory molecule expression after maturation both in vitro and after exposure to LPS in vivo (33). Despite this phenotypic similarity, the "tolerogenic" effect of pretransplant administration of mature CD8⁺ DCs was inhibited by the simultaneous infusion of relatively small numbers of the mature CD8^- subset.

Efforts to elucidate the mechanism(s) via which DCs may regulate alloreactivity and thereby prolong graft survival have focused on in vitro-generated immature myeloid DCs, either alone or combined with anti-inflammatory and/or immunosuppressive agents to suppress DC maturation (reviewed in Ref. 62). Collectively, these studies have indicated that immature or costimulatory molecule-deficient DCs have potential to prolong graft survival by the induction of Ag-specific T cell anergy (38, 43, 60), the promotion of alloreactive T cell apoptosis (63), or the induction of T regulatory cells (64). Using a panel of ex vivo functional assays, the present study has used the contrasting effects of mature CD8⁺ or CD8^- DCs on graft survival to investigate mechanisms via which CD8⁺ DCs impact donor-specific immune reactivity. Consistent with their in vitro function, ex vivo analyses of mice primed with either mature CD8⁺ or CD8^- DCs revealed vigorous donor Ag-restricted T cell proliferative responses of equivalent strength, which were predominantly Th1. However, after organ transplantation, recipients of mature CD8⁺ DCs exhibited significantly impaired proliferative and Th cytokine responses to donor alloantigens that could not be ascribed to anergy. Whether this reflects physical or functional T cell depletion is the subject of ongoing investigation. Furthermore, the capacity of mature CD8⁺ DCs to promote predominant Th1 responses in allogeneic recipients indicates that immune deviation is unlikely to provide a basis for prolonged graft survival.

Adoptive transfer of either CD8⁺ or CD8^- DCs to naive allogeneic recipients was associated with the induction of antidonor CTLs, as measured in ex vivo analyses against tumor cell targets of the appropriate MHC haplotype. However, there was no pattern of activity consistent with graft outcome (P. J. O’Connell, T. Takayama, K. Kaneko, and A. W. Thomson, unpublished observations). Prolongation of graft survival, concomitant with vigorous ex vivo antidonor CTL activity, has been observed previously in models of skin and organ transplantation (65, 66).

Although the relationship between the murine CD8⁺ and CD8^- subsets we have studied and subpopulations of human DCs is unclear, CD8^- DCs appear to correlate loosely with immunostimulatory CD11b⁺CD11c⁺IL-3R^low (CD123^low) circulating human DC1 or "monocytoid" (myeloid) DCs. Similar to mouse CD8^- DCs, DC1 are more effective at CD4⁺ T cell priming than human DC2 (67). In contrast, DC2s (CD11b^-CD11c^-IL-3R^high) selectively promote Th2 cell responses (67) and thus may have tolerogenic properties. Only the function of immature autologous human DC1 has been examined in vivo. Thus, Dhodapkar et al. (68) have reported that immature DC1 can promote specific T cell unresponsiveness to model Ags (influenza matrix protein) in healthy human volunteers, prompting suggestions that DC1 may have potential for therapy of allograft rejection or autoimmunity. Furthermore, recent in vitro evidence suggests that repeated stimulation of human CD4⁺ T cells with allogeneic immature DC1 leads to irreversible inhibition of their proliferation in vitro, associated with induction of IL-10-producing T regulatory 1 (Tr1) cells (64). Little information exists concerning the influence, if any, of mouse DCs on the generation of Tr1 cells, although a hepatic B220⁺ DC population generated in vitro has been recently reported to promote the induction of Tr1 cells (69). Moreover, there is evidence that CD8⁺ DCs can suppress the induction of T cell reactivity by tumor/self-peptide-loaded CD8^- DCs in vivo, although these findings may reflect the outcome of DC-DC interactions (70). Our efforts in this study to identify regulatory cells in transplanted animals administered with the CD8⁺ donor DCs that prolonged graft survival provided equivocal evidence of these cells within the graft and no evidence of systemic regulatory cells. These observations may reflect the transient nature of the impaired antidonor response after the single pretransplant administration of CD8⁺ donor DCs.

In addition to efficiently mobilizing CD8^- and CD8⁺ DC subsets in mice, Flt3L dramatically increases DC1 and DC2 precursors in humans (71, 72). As shown in the present study, CD8⁺ DCs are capable of prolonging allograft survival in the absence of immunosuppression and irrespective of their maturational status. Conceivably, differential mobilization and isolation (73) of such a regulatory DC subset from prospective allogeneic bone marrow or organ donors may allow their evaluation in tolerance-enhancing strategies in clinical transplantation.

	Acknowledgments

 
We thank Dr. Holger Hackstein (University of Pittsburgh) for assistance with statistical analyses and Dr. Jennifer E. Woodward (University of Pittsburgh) for critical suggestions and advice. We are grateful to the Immunex Corporation for providing Flt3L and for constructive discussion. J558 CD40L-transfected plasmacytoma cells were provided by Dr. Peter Lane (University of Birmingham, Birmingham, U.K.).

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (DK49745 and AI41011) and the Roche Organ Transplantation Research Foundation (ROTRF 13068349).

² Address correspondence and reprint requests to Dr. Peta J. O’Connell at the current address: John P. Robarts Research Institute, 100 Perth Drive, P.O. Box 5015, London, Ontario, Canada N6A 5K8. E-mail address: peta{at}rri.ca

³ Current address: Department of Surgery, University of Washington, Seattle, WA 98195.

⁴ Abbreviations used in this paper: DC, dendritic cell; CD95L, CD95 ligand; CyC, CyChrome; TR, Texas Red; GIC, graft-infiltrating cell; Tr1, T regulatory 1.

Received for publication May 22, 2001. Accepted for publication October 26, 2001.

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