CD11c+ Dendritic Cells Accelerate the Rejection of Older Cardiac Transplants via Interleukin-17ACLINICAL PERSPECTIVE
Background—Organ transplantation has seen an increased use of organs from older donors over the past decades in an attempt to meet the globally growing shortage of donor organs. However, inferior transplantation outcomes when older donor organs are used represent a growing challenge.
Methods and Results—Here, we characterize the impact of donor age on solid-organ transplantation using a murine cardiac transplantation model. We found a compromised graft survival when older hearts were used. Shorter graft survival of older hearts was independent of organ age per se, because chimeric young or old organs repopulated with young passenger leukocytes showed comparable survival times. Transplantation of older organs triggered more potent alloimmune responses via intragraft CD11c+ dendritic cells augmenting CD4+ and CD8+ T-cell proliferation and proinflammatory cytokine production, particularly that of interleukin-17A. Of note, depletion of donor CD11c+ dendritic cells before engraftment, neutralization of interleukin-17A, or transplantation of older hearts into IL-17A−/− mice delayed rejection and reduced alloimmune responses to levels observed when young hearts were transplanted.
Conclusions—These results demonstrate a critical role of old donor CD11c+ dendritic cells in mounting age-dependent alloimmune responses with an augmented interleukin-17A response in recipient animals. Targeting interleukin-17A may serve as a novel therapeutic approach when older organs are transplanted.
With the success of organ transplantation, age limits have essentially been lifted. Older organs have increasingly been used to compensate for the increasing organ shortage.1 Organ age, on the other hand, has been linked to intrinsic chronic interstitial inflammation in general, referred to as inflamm-aging, with reduced clearance of self-antigens and exogenous antigens as hallmark findings.2 Moreover, older donor organs not only have shown greater fragility and increased susceptibility to injury but also have elicited more potent systemic recipient immune responses through augmented immunogenicity. At the same time, mechanistic links between organ age and alloimmune responses remain unclear.
Clinical Perspective on p 131
Consistent with previous reports, we have previously shown in a large-scale clinical analysis that rates of acute rejection increase in parallel to donor age regardless of recipient age.3 It is well established that aging is associated with significant and broad changes in the immune system.4,5 The impact of these changes on transplantation outcome, however, remains poorly understood.
Increasing evidence indicates that there is an age-associated dysregulation in T-helper (Th) 1 and Th2 cytokine synthesis.6 Although the interleukin (IL)-2 signaling machinery has been shown to be critical for Th1 differentiation and proliferation, IL-2 has been established as an inhibitor of Th17 development.7 Of significance for studies of immunosenescence and alloimmune responses, CD4+ T cells isolated from splenocytes of old mice produced higher amounts of IL-17.8
Using a fully allogenic heart transplantation mouse model, we demonstrate that old cardiac allografts were rejected more rapidly, consistent with our clinical observations. Our data indicate that older CD11c+ dendritic cells (DCs) were the primary instigators of augmented alloimmune responses via IL-17A. Collectively, our data demonstrate that older organs skew the recipient immune response toward CD4+IL-17A+ T cells via resident cardiac allograft CD11c+ cells in an age-dependent manner. At the same time, blocking IL-17A prolonged survival of older organs, thus revealing novel therapeutic opportunities.
Animal use and care were in accordance with National Institutes of Health and Institutional Animal Care and Use Committee guidelines.
Eight- to 12-week-old wild-type (WT) male C57BL/6 or DBA/2J WT male mice were purchased from Charles River Laboratories (Wilmington, MA). Eighteen-month-old WT male C57BL/6 mice were purchased from the National Institute of Aging (NIA, Bethesda, MD). Eight- to 12-week-old BALB/c IL-17−/− mice were bred at the Harvard School of Public Health Animal Facility. Breeder pairs were kindly provided by Yoichiro Iwakura, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Heterotopic Heart Transplantation
Using a modified cuff technique, we heterotopically transplanted fully vascularized cardiac grafts from either old or young B6 donor mice into young DBA/2J, young BALB/c WT, or BALB/c IL-17−/− recipients. Hearts were anastomosed to the recipient’s common carotid artery and internal jugular vein. Transplantation into the recipient’s cervical region facilitated reliable functional assessment through palpation. Ischemic times were kept consistently at 40 minutes with an anastomosis time of 12 minutes. Graft function was measured daily by palpation, and allograft rejection was defined as the complete cessation of palpable contractility. Graft survival is shown as the median survival time in days.
In Vivo Treatment Protocols
For the generation of chimeric donors, young and old prospective cardiac donor C57BL/6 mice were lethally irradiated (11 Gy), eliminating all passenger leukocytes within the heart. Bone marrow was procured from young syngeneic C57BL/6 mice and transfused intravenously 24 hours after irradiation (10×106 cells per animal). Mice were then used as cardiac allograft donors after 6 weeks, when cardiac tissue had been repopulated with leukocytes derived from the transplanted bone marrow. Successful repopulation was validated by immunohistochemistry.
For the depletion of DCs, young and old cardiac donor C57BL/6 mice were treated intravenously with 0.5 mg liposomal clodronate (Encapsula NanoSciences, Nashville, TN) at days 8, 5, and 1 before graft procurement. This regimen ensured depletion of intragraft CD11c+ DCs as documented by flow cytometry analysis.
For blockage of IL-17A, 100 μg anti–IL-17A (R&D Systems, Minneapolis, MN) was administered intravenously to the recipient animals every other day after cardiac transplantation starting at day 0 until the day of complete rejection. The control group for this experiment was injected with isotype control antibodies using the same protocol.
ELISpot Assay and ELISA
ELISpot assays were performed with mouse interferon-γ (IFNγ) or mouse IL-6 ELISpot Kits (BD Biosciences, San Diego, CA). Briefly, 0.5×106 unselected splenocytes from DBA/2J recipients were used as responder cells and restimulated with 0.5×106 irradiated splenocytes from naïve donor-type B6 animals within 96-well ELISpot plates for 24 hours under standard cell culture conditions (with RPMI 1640 medium supplemented with 10% FCS, 1% penicillin/streptomycin, and 1% l-glutamine; 37°C and 5% CO2). Alternatively, 1×106 splenocytes from naïve DBA/2J mice used as responder cells to 1×104 flow-sorted splenic CD11c+ DCs (untreated or stimulated for 24 hours with 100 ng/mL lipopolysaccharide) isolated from either young or old naïve B6 mice. The ELISpot assay was adapted to measure the frequency of alloreactive T cells producing IFNγ or IL-6. The resulting spots were counted on a computer-assisted enzyme-linked immunospot image analyzer (Cellular Technology), and frequencies were expressed as numbers of cytokine-producing spots per 0.5×106 or 1×106 responder cells.
For detection of IL-17A in vitro, ELISA was performed. Mouse IL-17A was measured with commercial kits (eBioscience). Briefly, ELISA plates were coated with 100 μL anti-cytokine capture antibody at 4°C overnight. Plates were then washed 5 times with 0.05% PBS-Tween and coated for 1 hour with the blocking buffer provided by the manufacturer. Samples or standards were added in duplicates (100 μL per well) and incubated at 4°C overnight. Wells were washed 5 times with PBS-Tween and incubated with 100 μL anti-cytokine detection antibody at 4°C overnight. Wells were then washed 5 times with PBS-Tween and incubated with 100 μL avidin-horseradish peroxidase at room temperature for 30 minutes. Thereafter, wells were washed 7 times with PBS-Tween and incubated with 100 μL per well of a substrate. The reaction was stopped after 15 minutes with 1 mol/L H2SO4, and absorbance was measured with a multiplate microplate fluorescence reader (Synergy HT, Biotek, Winooski, VT) at 405 nm.
Single-cell leukocyte suspensions were obtained from spleens of young (age, 8–12 weeks) DBA/2J WT mice by negative selection with anti-mouse antibodies against CD8, CD11b, CD11c, CD19, CD24, CD25, CD44, CD45R, CD49b, TCRγ/δ, and TER119 using the EasySep Mouse Naïve CD4+ T Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer’s protocol. For isolation of CD11c+ DCs, single-cell suspensions were obtained from hearts of young (age, 8–12 weeks) and old (age, 18 months) C57BL/6 WT mice. Briefly, hearts were procured and washed 3 times with Ca2+- and Mg2+-free PBS. Tissue was then cut into 5-mm pieces, placed in tissue extraction buffer (5 mmol/L EDTA, 2 mmol/L 2-ME in PBS), and incubated with continuous, brisk stirring at 37°C for 30 minutes. The suspension was then filtrated through a 70-μm filter. CD11c+ DCs were then isolated with the EasySep Mouse CD11c Positive Selection Kit according to the manufacturer’s protocol.
Kaplan-Meier survival graphs were constructed, and log-rank comparisons of groups were used to calculate P values. The Student t test was used for comparison of means between 2 groups. No multiple-testing correction was performed. A value of P<0.05 was considered statistically significant. Data were expressed as mean±SEM. GraphPad Prism version 6 (GraphPad Software, La Jolla, CA) was used for statistical analysis.
Additional methods are detailed in the online-only Data Supplement.
Cardiac Allografts From Old Donors Are Rejected More Rapidly
We have previously shown in a large-scale clinical study that episodes of acute rejection were more frequent when older organs were used for transplantation.3 To shed light on this clinical observation and to understand underlying mechanisms, we transplanted fully major histocompatibility complex–mismatched cardiac allografts from either young (10–12 weeks) or old (18 months) C57BL/6 donor mice into young DBA/2J recipients. In this heterotopic and fully vascularized cardiac transplantation model, old hearts showed a significantly shorter graft survival (Figure 1A). Furthermore, old cardiac allografts displayed more advanced International Society for Heart and Lung Transplantation rejection scores compared with allografts procured from young donors (Figure 1B). Moreover, intragraft immunohistological analysis demonstrated increased CD4+ and CD8+ T cell infiltrates (Figure 1C). Collectively, these findings documented age-dependent differences in cardiac allograft survival correlating with increased lymphocellular infiltration and advanced structural changes.
Accelerated Rejection of Cardiac Allografts From Old Donors Is Associated With an Enhanced Systemic Alloimmune Response
To determine donor age–related effects on systemic alloimmune responses within recipients, frequencies of splenic CD4+/CD8+ effector T cells and CD4+ regulatory T cells were assessed by day 7 after transplantation. Although frequencies of CD4+CD25+FoxP3+ regulatory T cells were age independent, recipients of cardiac allografts from old donors showed significantly increased frequencies of CD8+CD44highCD62low and higher rates of CD4+CD44highCD62low effector T cells (Figure Ia in the online-only Data Supplement). Importantly, transplantation of old cardiac allografts also led to elevated frequencies of CD8+IFNγ+ T cells among recipient splenocytes as assessed by intracellular cytokine staining (Figure Ib in the online-only Data Supplement).
Properties of recipient splenocytes were further characterized in vitro by restimulation with donor-type antigen and subsequent measurements of cytokine production and proliferative responses. ELISpot data indicated increased frequencies of responder T cells that produced IFNγ and IL-6 on donor-type antigen stimulation (Figure Ic in the online-only Data Supplement). Moreover, an increased proliferation of alloreactive splenocytes responding to donor-type antigen was noted (Figure Id in the online-only Data Supplement). Taken together, these results indicated that older cardiac allografts enhanced systemic alloimmune responses of recipient mice.
Passenger Leucocytes Mediate Donor Age–Dependent Transplant Survival and Alloimmune Responses
Passenger leukocytes have been shown to play an important role in allograft rejection.9–11 The age of passenger leukocytes has previously been shown to affect allograft rejection.12–14 However, age-related changes in parenchymal tissue may also be of relevance in allograft rejection.15–17 Thus, to delineate whether the observed effects of donor age on allograft survival and systemic alloimmune responses were linked to age-dependent changes in parenchymal tissue or mediated by age-dependent modifications of passenger leukocytes residing within the graft, we generated chimeric C57BL/6 donor animals through lethal total-body irradiation and subsequent hematopoietic reconstitution with bone marrow procured from young, naïve, syngeneic C57BL/6 mice. By 6 weeks, young or old hearts from chimeric animals were transplanted into young WT DBA/2J animals. To validate this chimeric model, CD11c+ staining was performed 6 weeks after repopulation (Figure 2A).
When chimeric old or young hearts repopulated with young passenger leukocytes had been transplanted into young DBA/2J mice, graft survival was no longer donor age dependent (Figure 2B). Similarly, histomorphological changes, as assessed by International Society for Heart and Lung Transplantation rejection scores at day 7, were no longer age dependent (Figure 2C). Moreover, systemic immune responses after the engraftment of chimeric grafts demonstrated comparable frequencies of CD4+ and CD8+ effector T cells (Figure IIa in the online-only Data Supplement). No significant difference was observed in frequencies of CD4+CD25+FoxP3+ cells (Figure IIa in the online-only Data Supplement). In addition, after restimulation of recipient responder splenocytes with donor-type antigen, frequencies of IFNγ-producing alloreactive splenocytes did not differ in recipients of either old or young chimeric hearts (Figure IIb in the online-only Data Supplement). Of note, there was a significant decrease in frequencies of IL-6–producing alloreactive splenocytes in recipients of old chimeric hearts (Figure IIb in the online-only Data Supplement). Moreover, proliferation rates of recipient-derived splenocytes after transplantation of chimeric hearts were independent of organ age (Figure IIc in the online-only Data Supplement).
Collectively, these results suggest that the impact of donor age on alloimmune responses is not related to the age of cardiac parenchyma but rather to the age of passenger leukocytes.
CD11c+ DCs Mediate Donor Age–Related Effects of Augmented Alloimmune Responses
As a result of their ability to migrate to secondary lymphoid tissues and to present alloantigens, donor DCs are considered instigators of adaptive alloimmune responses.10,11,18 Thus, to determine whether CD11c+ DCs play a role in mediating the observed augmented alloimmune response when older organs are transplanted, donor DCs were depleted by liposomal clodronate before transplantation. Consistent with previous studies,19,20 liposomal clodronate pretreatment resulted in a dramatic depletion (>98%) of CD11c+ DCs in donor hearts (Figure 3A). Of note, pretreatment with liposomal clodronate had only minor effects on macrophage populations residing within cardiac tissue (Figure 3A). Next, CD11c+ DC-depleted donor cardiac allografts were transplanted into young recipients, and survival was assessed. Depletion of CD11c+ DCs prolonged survival of old cardiac allografts to levels comparable to that observed when young allografts were transplanted (Figure 3B). Moreover, International Society for Heart and Lung Transplantation rejection scores were comparable in both young and old cardiac allografts after clodronate treatment (Figure 3C). In addition, systemic alloimmune responses were age independent after donor treatment with clodronate as assessed by splenic T-cell populations (Figure IIIa in the online-only Data Supplement), intracellular cytokine staining (Figure IIIb in the online-only Data Supplement), frequencies of IFNγ– and IL-6–producing alloreactive splenocytes (Figure IIIc in the online-only Data Supplement), and proliferation of alloreactive splenocytes (Figure IIId in the online-only Data Supplement). Taken together, these findings suggest that intragraft CD11c+ DCs are the primary mediators of age-dependent immune responses and accelerated allograft rejection observed when old cardiac allografts are transplanted.
Aged CD11c+ DCs Demonstrate Increased Allostimulatory Capacities In Vitro
To investigate mechanisms of donor DC-mediated effects on alloimmunity in detail and to delineate the impact of immunosenescence on DCs, flow-sorted splenic naïve CD11c+ DCs were characterized. Both naïve and lipopolysaccharide-stimulated CD11c+ DCs isolated from old C57BL/6 mice used as allogeneic stimulators to splenocytes from young naïve DBA/2J mice evoked significantly stronger alloimmune responses with elevated frequencies of IFNγ-producing responder cells (Figure 4A), in addition to increased proliferation rates (Figure 4B). Preactivation with lipopolysaccharide increased the overall allostimulatory level of both young and old DCs in a dose-dependent manner. Moreover, flow cytometry revealed a significant increase in surface expression of activation and maturation markers, including I-Ab (major histocompatibility complex class II), CD40, CD80, and CD86, on old DCs (Figure 4C), supporting the concept of intrinsic functional modifications in older DCs.
IL-17A Is Critical in Enhancing the Rejection of Older Allografts
Elevated frequencies of Th17 cells have been observed in old mice and humans.21,22 More important, Th17 cells have been linked to critical pathways in allograft rejection.23 To dissect the role of Th17 cells in this process in more detail, we explored whether elevated levels of intragraft IL-17A were immanent to organ age per se or linked to age-dependent alloimmune responses elicited by old passenger leukocytes residing within the graft.
Cardiac allografts were collected before rejection (day 7), and IL-17A mRNA levels were quantified by real-time polymerase chain reaction. Results indicated a dramatic increase in IL-17A mRNA levels in old cardiac allografts (>30-fold increase; Figure 5A). Of note, old and young cardiac allografts showed equally low levels of IL-17A mRNA after the engraftment of organs that were pretreated with liposomal clodronate (Figure 5A). Next, isolated DCs of hearts from old (18 months) or young (8–12 weeks) C57BL/6 mice were cocultured with isolated naïve CD4+ cells (ratio, 1:5) for 7 days in media alone, under Th17-polarizing conditions alone, in lipopolysaccharide alone, or under Th17-polarizing conditions in the presence of lipopolysaccharide. Lipopolysaccharide-activated DCs from older cardiac allografts in Th17-polarizing conditions increased frequencies of CD4+IL-17A+ T cells dramatically (49.3% versus 24.2% in DCs from younger hearts; P<0.0001; Figure 5B). Of note, neither old or young DCs in media alone, in Th17-polarizing conditions alone, or in the presence of lipopolysaccharide alone demonstrated significant differences in the proliferation of CD4+IL-17A+ T cells, suggesting that both activation of donor DCs and Th17-polarizing conditions are required during the rejection of older allografts. These results were confirmed by ELISA (Figure 5C). Taken together, these findings suggest a prominent role for IL-17A in mounting an augmented immune response linked to the engraftment of older donor organs.
Blocking IL-17 in Recipient Animals Prolongs the Survival of Old but Not Young Cardiac Allografts
Our results thus far suggested a critical role of IL-17A in alloimmune responses after the transplantation of older grafts. To evaluate the role of IL-17A in vivo, we first performed a functional blockade of IL-17A by treating recipients of young or old C57BL/6 allografts with anti–IL-17A starting on the day of transplantation. Survival of older allografts was significantly prolonged after the application of anti–IL-17A. Of note, anti–IL-17A treatment did not significantly prolong the survival of young allografts (Figure 6A).
To explore the critical role of IL-17A in vivo further, hearts of old and young donors were transplanted into IL-17−/− BALB/c mice. Indeed, the absence of IL-17 prolonged graft survival of old hearts (median survival time, 12 versus 9 days in IL-17−/− mice compared to WT recipients; P<0.05; Figure 6B), whereas survival of young cardiac allografts remained unaltered (WT versus IL-17−/− mice recipients; median survival time, 11.5 versus 11 days; P=NS; Figure 6C).
These in vivo results emphasize the prominent role for IL-17A in donor age–dependent alloimmune responses.
The growing discrepancy between demand and supply in organ transplantation has resulted in the increased use of organs from old donors.24,25 Over the last decade, more than half of all transplanted kidneys were procured from donors >50 years of age.26 At the same time, transplantation outcomes of extended criteria donor organs were found to be inferior to those observed in organs from younger donors.12 In cardiac transplantation, donations have dropped dramatically since 2000. Although significantly more older recipients are receiving cardiac transplants, the age of heart donors is in general lower compared with the age of other solid-organ transplant donors.27 Nevertheless, understanding age-specific aspects of immunogenicity and repair may provide desperately needed novel resources for cardiac transplantation. In a large, retrospective analysis, we have recently shown that the transplantation of older organs is linked to more frequent rates of acute rejection.3 Despite vast clinical implications, experimental studies dissecting the effects of donor age on allograft immunogenicity and recipient immune responses remain scarce.28 Thus, dissecting mechanistic aspects of donor age-related immune responses appears of major clinical relevance.
Our experimental data reflect clinical observations showing that older organs are rejected earlier. Albeit small, the difference in graft survival of 2 days had been highly significant. We wish to point out that we used a cardiac transplantation model with a fully major histocompatibility complex–mismatched donor/recipient combination leading to graft dysfunction after acute rejection in <2 weeks. Moreover, aspects of age-dependent injury/repair, functional reserve, or mismatching in donor/recipient size are likely to be superseded by the vigorous alloimmune response in this model.
Furthermore, using a chimeric mouse model, we demonstrated that age-dependent alloimmune responses were not linked to the age of cardiac parenchyma. This model of complete depletion of intragraft leukocytes through lethal total-body irradiation and subsequent reconstitution with leukocytes derived from transplanted bone marrow has been established and used extensively by other investigators in the analysis of repopulation and residual antigen-presenting cells (APCs) using flow cytometry and real-time polymerase chain reaction.29,30 For this work, repopulation of prospective allografts was confirmed by immunohistochemical staining (Figure 2A).
Impaired graft function correlated with organ age is most likely related to both limited functional reserve and augmented immunogenicity. Physiological changes related to aging have been put forward as nonimmunological factors influencing graft survival. In theory, increased susceptibility to tissue damage during the transplantation procedure after ischemia, in addition to impaired repair mechanisms, may be linked to a proinflammatory milieu, including increased DAMP (damage-associated molecular pattern) signaling, eliciting and perpetuating secondary allospecific immune responses. Although physiological aspects of aging may, at least in theory, affect organ function after transplantation, our results suggest that the age of cardiac parenchyma per se did not mediate donor age–dependent differences of alloimmunity because old donor hearts containing young passenger leukocytes did not augment alloimmune responses.
Previous publications have illustrated the central role of IFNγ and CD4+ and CD8+ cells in allograft rejection.23,31,32 In these studies, a feedback loop leading to the production of IL-2 by Th1 cells after allogenic stimulation has been characterized. Those events promote alloreactive cytotoxic CD8+ cells, which in turn produce IFNγ, enhancing Th1 responses even further. Our data confirm these findings by showing increased frequencies of CD4+IFNγ+ and CD8+IFNγ+ effector cells after the engraftment of both young and old cardiac allografts. Of note, we observed higher frequencies of CD8+IFNγ+ T cells when old allografts were transplanted.
Recent clinical and experimental studies have shown the relevance of a novel CD4+ T-helper population called Th17 as instigators of allograft rejections. Th17 cells have been characterized by their signature cytokine IL-17A, a potent proinflammatory cytokine.33–35 Of note, studies outside of transplantation have shown that aging is associated with a general increase in levels of Th17 cells.36–38 However, the role of Th17 responses with respect to organ age remains unclear. Our results show a significant increase in intragraft IL-17A mRNA levels in old transplanted allografts. These results suggest a strong relationship between IFNγ-producing CD4+ and CD8+, as well as IL-17A-producing CD4+ cells, in accelerating the rejection of older organs. To characterize the functional relevance and the origin of the highly elevated IL-17A levels in older allografts in more detail, we cocultured intragraft DCs of old and young C57BL/6 mice with splenic naïve CD4+ cells. Those experiments allowed us to distinguish whether elevated IL-17 levels were intrinsically related to aging or linked to the encounter with old passenger DCs. Our data demonstrated a significant increase in CD4+IL-17A+ cells after DC activation in Th17-polarizing conditions, thus confirming that old passenger DCs trigger a more potent IL-17A response. The role of IL-17A in allograft rejection was subsequently tested in recipient animals deficient in IL-17A or those that were pretreated with anti–IL-17A. Both recipients showed not only an improved allograft survival compared with untreated controls but also a prolonged survival of older compared with younger allografts, thus demonstrating the critical role of IL-17A in the rejection of older grafts. Taken together, these results underscore a novel and clinical relevant role of IL-17A in the rejection of older allografts.
It is recognized that donor-derived APCs residing in the interstitium of allografts, in particular macrophages and DCs, are instigators of primary allospecific immune responses via direct alloantigen presentation to recipient responder cells within secondary lymphatic tissue.11,14,39 However, because of their potent ability to instigate an adaptive immune response by activating naïve T cells, DCs are generally regarded as primary initiators of allograft rejection.39,40 Both clinical and experimental studies have shown that old APCs elicit enhanced alloimmune responses. Moreover, APCs collected from healthy elderly individuals cocultured with purified T cells have elicited enhanced T-cell proliferation in both syngeneic and allogeneic settings.41 However, these studies failed to characterize phenotypic and functional aspects driving this process. In our studies, we focused on the role of DCs and characterized phenotypic and functional aspects. Our functional assays demonstrated that old DCs increased the proliferation and production of IFNγ in allogeneic responder cells (CD4+ and CD8+ cells). Of note, augmented IFNγ production was also observed after stimulation with lipopolysaccharide, suggesting age-dependent allostimulatory capacities of old DCs even after unspecific preactivation. Moreover, depletion of old CD11c+ DCs by liposomal clodronate treatment blocked the Th17 response of recipients and extended allograft survival, suggesting that DCs play a central role in this process. Furthermore, old CD11c+ DCs presented advanced activation and maturity, as shown by increased expression of major histocompatibility complex class II, CD80, CD86, and CD40. These data are in accordance with results by others showing an increased immunogenicity of old DCs in experimental graft-versus-host disease models.42
Administered in a liposomal formulation, clodronate is selectively taken up by phagocytic leukocytes, particularly DCs and macrophages.43 After intracellular disruption of the liposomes through lysosomal phospholipases, accumulating clodronate leads to selective apoptosis of target cells.44 The observed depletion of DCs in our model (Figure 3A) may be related to decreased permeability of cardiac capillaries to liposomal clodronate.45,46 Thus, effects in our model targeted predominantly circulating phagocytic leukocytes. With the turnover of resident macrophage being much slower than that of DCs,47 we do not assume a sizable effect of clodronate on cardiac macrophage populations after 1 week of treatment. Although our detailed in vitro and in vivo analysis supported the critical role of old DCs as instigators of organ age–dependent alloimmune responses, we cannot entirely rule out that other APCs, including macrophages, play a role in organ age–dependent immune responses.
Although data originating from experimental models have inherent shortcomings and might not always imply biological or even clinical significance, this work explains mechanistic findings of a previous clinical observation. Our experimental data show that donor age affects not only organ quality but also recipient alloimmune responses through passenger leukocytes independently of unspecific or procedure-related injuries. This observation is intriguing because interventions may therapeutically influence graft survival and overall transplantation outcome. Furthermore, we were able to identify the critical role of the age of DCs rather than organ age per se as a driving force of organ age–dependent alloimmune responses. These findings not only may advance our overall understanding of allograft rejection but also can potentially lead to new specific translational strategies in recipients of older cardiac transplants.
Sources of Funding
This work was supported by grants from the National Institutes of Health (RO-1AG039449) and the Fundacion Carlos Slim de la Salud (both to Dr Tullius). Dr Abdi was supported by a grant from the National Institutes of Health (5R01AI091930-04).
Guest Editor for this article was Charles Steenbergen, MD, PhD.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.114.014917/-/DC1.
- Received December 16, 2014.
- Accepted April 29, 2015.
- © 2015 American Heart Association, Inc.
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The growing global shortage of organs for transplantation has resulted in an increased use of older donor organs. Although inferior outcomes have been observed when older organs are transplanted, it remains unclear whether they are caused by an augmented immunogenicity in addition to compromised organ quality and a compromised capacity for repair. Previous clinical observations have demonstrated more frequent acute rejections when older organs are used. Here, we provide experimental evidence that older organs trigger more potent alloimmune responses via intragraft CD11c+ dendritic cells while augmenting CD4+ and CD8+ T-cell proliferation and proinflammatory cytokine production, particularly that of interleukin-17A. Depletion of donor CD11c+ dendritic cells before engraftment, neutralization of interleukin-17A, or transplantation of older hearts into IL-17A−/− mice delayed rejection and reduced previously observed organ age–dependent alloimmune responses. Our experimental data reveal donor CD11c+ dendritic cells as the primary instigators of organ age–dependent alloimmune responses and interleukin-17A as a potential novel therapeutic target when older organs are transplanted.