CD11c+ Dendritic Cells Accelerate the Rejection of Older Cardiac Transplants via Interleukin-17A

Abstract

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.

Keywords: aging; immunology; rejection; transplantation.

Figure 1

Old cardiac allografts are rejected more rapidly. a, Hearts from old and young C57BL/6 mice were transplanted into young DBA/2J mice and mean survival times (MST) were analyzed. (n=7/group; **P<0.01; data are representative of two independent sets of experiments). b, 7 days after transplantation, mice were euthanized and transplanted hearts were procured; H&E staining was performed to asses lymphocytic infiltration and structural changes; samples were scored based on the International Society for Heart and Lung Transplantation (ISHLT) rejection score (n=8/group, magnification, 10x; *P<0.05). c, Paraffin sections were stained for CD4/CD8 to assess lymphocytic infiltration; data is shown as cells per high-power field (HPF) (n=8/group; *P<0.05; data are representative of two independent sets of experiments).

Figure 2

Old cardiac allografts from chimeric animals showed survival and rejection scores comparable to young cardiac allografts. Young and old C57BL/6 mice were lethally irradiated (11 Gy), thus eliminating passenger leukocytes. 24 hours after irradiation, bone marrow from young syngeneic C57BL/6 mice was injected intravenously to reconstitute passenger leukocytes within donor hearts. a, immunostaining for CD11c was performed 6 weeks after bone marrow transplantation to confirm successful repopulation of prospective donor hearts with DCs (representative slides; 10x). b-c, Hearts from young and old chimeric animals were transplanted into young WT DBA/2J mice; (b) mean survival time and (c) rejection scores by H&E staining were analyzed. (n=4/group; NS, non-significant; data are representative of two independent sets of experiments).

Figure 3

Clodronate depletion of DCs in older donors abolished previously observed accelerated rejection in younger recipients. Young and old C57BL/6 donors were treated intravenously with liposomal clodronate at -8, -5 and -1 days prior to graft procurement. a, This regimen insured depletion of intragraft CD11c⁺ DCs as documented by flow cytometry;, empty liposomes without clodronate were used as controls (n=3). b, DC-depleted hearts from young and old donors were transplanted into young DBA/2J mice and (b) mean survival time as well as (c) rejection scores as determined by H&E staining were analyzed. (n=5/group; NS, non significant; data are representative of two independent sets of experiments).

Figure 4

Old DCs induce enhanced alloimmune responses. a-b, flow-sorted splenic CD11c⁺ DCs from either young or old naïve C57BL/6 mice were used as allogeneic stimulators of young DBA/2 splenocytes in ELISpot and proliferation assays either without activation or lipopolysaccharide (LPS) activation at low (10ng/ml) and high (100ng/ml) concentrations; (a) frequencies of IFNγ-producing responder splenocytes (n=6/group; *P<0.05; ***P<0.001; experiments were performed in triplicates; data are representative of three independent sets of experiments) and (b) proliferation rates of responder splenocytes by ³H thymidine incorporation (n=6/group; *P<0.05; **P<0.001; experiments performed in triplicates; data are representative of three independent sets of experiments) were analyzed. c, Characterization of activation and maturation markers (I-A^b/MHC-II, CD40, CD80, and CD86) was performed using mean fluorescence intensity (MFI) (n=3/group; *P<0.05; ***P<0.001; grey tinted: young DCs, black line: old DCs; representative histogram plots of three independent sets of experiments are shown).

Figure 5

IL-17A has a critical role in age-dependent immune responses. a, 7 days after transplantation hearts were procured and intragraft levels of IL-17A were analyzed by real-time PCR (n=4/group; **, p<0.01; ***, p<0.001). b-c, DCs were isolated from old and young hearts (C57BL/6) and co-cultured with naïve CD4⁺ cells isolated from spleens (DBA/2J) in media alone, in Th17-polarizing conditions alone, in presence of lipopolysaccharide (LPS) alone or under Th17 polarizing conditions in presence of LPS. After 7 days, (b) frequencies of CD4⁺IL-17A⁺ cells were assessed by flow cytometry gating on CD4⁺ cells and (c) cytokine secretion was assessed by ELISA (n=3/group; **P<0.01, ***P<0.001 ****P<0.0001).

Figure 6

Targeting IL-17A in solid organ transplantation abolished donor age-related alloimmune responses. a, hearts from old and young C57BL/6 mice were transplanted into young DBA/2J recipient mice; recipient mice were treated with 100μg of anti-IL-17A or with isotype control antibodies (IgG) every other day starting at day 0 and mean survival times (MST) were analyzed (n=5/group; *P<0.05; NS=non-significant). b, hearts from old and young C57BL/6 mice were transplanted into young BALB/c IL-17A^-/-mice and MST was analyzed (n=4-5/group; *P<0.05).