CXCR4 blockade reduces the severity of murine heart allograft rejection by plasmacytoid dendritic cell-mediated immune regulation

Fu, Jian; Lehmann, Christian H. K.; Wang, Xinning; Wahlbuhl, Mandy; Allabauer, Ida; Wilde, Benjamin; Amon, Lukas; Dolff, Sebastian; Cesnjevar, Robert; Kribben, Andreas; Woelfle, Joachim; Rascher, Wolfgang; Hoyer, Peter F.; Dudziak, Diana; Witzke, Oliver; Hoerning, André

doi:10.1038/s41598-021-03115-z

Download PDF

Article
Open access
Published: 10 December 2021

CXCR4 blockade reduces the severity of murine heart allograft rejection by plasmacytoid dendritic cell-mediated immune regulation

Jian Fu^1,2,3^na1,
Christian H. K. Lehmann^4,9^na1,
Xinning Wang^1,10,
Mandy Wahlbuhl²,
Ida Allabauer²,
Benjamin Wilde¹,
Lukas Amon⁴,
Sebastian Dolff⁵,
Robert Cesnjevar^7,6,
Andreas Kribben¹,
Joachim Woelfle²,
Wolfgang Rascher²,
Peter F. Hoyer⁸,
Diana Dudziak^4,9,
Oliver Witzke⁵ &
…
André Hoerning^2,8

Scientific Reports volume 11, Article number: 23815 (2021) Cite this article

1875 Accesses
6 Citations
2 Altmetric
Metrics details

Subjects

Abstract

Allograft-specific regulatory T cells (T_reg cells) are crucial for long-term graft acceptance after transplantation. Although adoptive T_reg cell transfer has been proposed, major challenges include graft-specificity and stability. Thus, there is an unmet need for the direct induction of graft-specific T_reg cells. We hypothesized a synergism of the immunotolerogenic effects of rapamycin (mTOR inhibition) and plerixafor (CXCR4 antagonist) for T_reg cell induction. Thus, we performed fully-mismatched heart transplantations and found combination treatment to result in prolonged allograft survival. Moreover, fibrosis and myocyte lesions were reduced. Although less CD3⁺ T cell infiltrated, higher T_reg cell numbers were observed. Noteworthy, this was accompanied by a plerixafor-dependent plasmacytoid dendritic cells-(pDCs)-mobilization. Furthermore, in vivo pDC-depletion abrogated the plerixafor-mediated T_reg cell number increase and reduced allograft survival. Our pharmacological approach allowed to increase T_reg cell numbers due to pDC-mediated immune regulation. Therefore pDCs can be an attractive immunotherapeutic target in addition to plerixafor treatment.

Basiliximab impairs regulatory T cell (TREG) function and could affect the short-term graft acceptance in children with heart transplantation

Article Open access 12 January 2021

Jacobo López-Abente, Marta Martínez-Bonet, … Rafael Correa-Rocha

Donor myeloid derived suppressor cells (MDSCs) prolong allogeneic cardiac graft survival through programming of recipient myeloid cells in vivo

Article Open access 28 August 2020

Songjie Cai, John Y. Choi, … Jamil R. Azzi

Donor bone-marrow CXCR4+ Foxp3+ T-regulatory cells are essential for costimulation blockade-induced long-term survival of murine limb transplants

Article Open access 09 June 2020

Liqing Wang, Zhonglin Wang, … Wayne W. Hancock

Introduction

Induction and maintenance of donor-specific allo-immune tolerance is dependent on the balance of effector T cells and regulatory CD4⁺ T cells (T_reg cells)¹. The latter play an essential role in the development of long-term allograft acceptance² and tolerance³. Hence, establishing protocols for immunotherapies targeting T_reg cells in the setting of solid organ transplantation has gained an intense interest. Tipping this balance in favor of donor-specific regulation by an adoptive transfer of T_reg cells represents an attractive approach to achieve long-term allograft survival^4,5,6. However, critical issues, such as isolation and storage of ex vivo expanded human T_reg cells, represent a significant challenge for the clinical routine application^1,7,8. Another important problem is their potential functional instability, which might lead to an undesirable switch to a pro-inflammatory state after adoptive T_reg cell transfer⁷. Alternatively, the expansion of the patient's T_reg cells or the conversion of naïve CD4⁺ T cells into T_reg cells in vivo, for example by the administration of recombinant IL-2, have been proposed⁹. These in vivo expanded and/or induced T_reg cells are expected to be allo-educated by exposure to the allograft leading to donor allo-antigen-specific T_reg cells¹. Furthermore, immunosuppressive therapies routinely applied after transplantation can hamper the development of a durable immunomodulatory effect after adoptive transfer of T_reg cells^10,11,12.

Dendritic cells (DCs) play a pivotal role in orchestrating T cell immune responses. To ensure the balance between self-tolerance and pathogen eradication, DCs are not only able to expand effector T cells, they can also foster the differentiation of naturally occurring T_reg cells (nT_reg cells) in the thymus^13,14 or the differentiation of naïve CD4⁺ T cells into so-called induced T_reg cells (iT_reg cells) in the periphery¹⁵. This property has been mainly linked to immature conventional dendritic cells (cDCs)^14,15, which are characterized by a low-level presentation of antigenic peptides in the absence of co-stimulatory molecules. Therefore, the role of immature, and thus tolerogenic, cDCs has been tested in various murine models of autoimmune diseases¹³, such as Crohn's like ileitis¹⁶, autoimmune diabetes¹⁷ and collagen-induced arthritis (CIA)¹⁸, allergy¹⁹, but also graft versus host disease²⁰ as well as transplantation²¹. These studies as well as first Phase I–II clinical trials in patients with autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis or Crohn's disease^{22,23,24,25,26}, but also in the setting of organ transplantation²⁷, suggests that DCs represent a promising therapeutic alternative to conventional, unspecific immunosuppressive drugs when tolerogenic DCs are adoptively transferred. This is of special importance as the therapeutic setting with immunosuppressive drugs is often associated with severe side effects as well as limited efficacy.

Although the main function of plasmacytoid DCs (pDCs), which are a subpopulation of DCs, is the secretion of type I interferons in response to viral infections^{28,29,30,31,32}, pDCs have been proposed to play a role in immune tolerance^28,29,30,33. They are per se characterized by an altered costimulatory molecule expression profile and poor allo-stimulatory capacity when interacting with T cells. Thus, pDCs have been considered as a possible targets to induce and maintain allo-immune tolerance^{33,34,35,36,37,38}. Loschko et al. demonstrated that pDCs contribute to antigen-specific peripheral immune tolerance after antigen delivery to the surface molecule sialic acid binding Ig-like lectin H (Siglec-H)³⁹. This was mediated by inducing hyporesponsive CD4⁺ T cells exhibiting reduced expansion and Th1/Th17 cell plasticity³⁹. Besides, pDCs have been shown to inhibit immune responses in several murine disease models including asthma, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, and experimental autoimmune encephalomyelitis (EAE), a murine model for multiple sclerosis, graft-versus-host disease as well as allogeneic transplant rejection by reducing pathogenic effector T cell responses or by promoting T_reg cells^{29,40,41,42,43,44,45,46,47}.

Various immunosuppressive therapies are in clinical routine to counteract the allo-reactive inflammatory response, e.g. by fostering T_reg cell responses. One way is to apply calcineurin-inhibitors that may not only compromise the survival of allo-reactive effector T cells, but also negatively influence the function and survival of allo-reactive T_reg cells^10,11. In contrast, the mTOR inhibitor rapamycin and its derivatives have been shown to favor the development and compartmental expansion of T_reg cells, while inhibiting the activation of effector T cells and B cells, both, in mice and humans^11,48,49,50. Inhibitors of mTOR have not only been suggested to augment selective expansion of T_reg cells in vivo^51,52, they also prevent DC-maturation and hamper the upregulation of MHC class II and co-stimulatory molecules⁵³. Injection of such tolerogenic rapamycin-conditioned DCs into mice has been proposed to prolong graft survival in various transplantation models^54,55.

Due to constant self-renewal and expansion of hematopoietic progenitor cells, the bone marrow is a rich source of premature, rather tolerogenic cells, including T cells, B cells, pDCs and to a low extent also conventional DCs and macrophages. From about 1.5% of all bone marrow-residing CD4⁺ T cells⁵⁶, approximately one-third of them are CD4⁺FoxP3⁺ T_reg cells⁵⁷. Interestingly, pDCs are enriched in the bone marrow implicating a role in maintenance of the immuntolerogenic state²⁸.

Plerixafor (AMD3100) is a CXCR4 antagonist shown to disrupt the interaction of the C-X-C chemokine receptor CXCR4 with its ligand SDF1α, which is essentially produced by bone marrow stromal cells. After application of plerixafor it was noted that bone marrow-residing cells were released into the peripheral circulation^58,59. Thus, meanwhile plerixafor is clinically used to mobilize CXCR4-expressing hematopoietic stem and progenitor cells from the bone marrow into the peripheral blood for separation and subsequent transplantation in several hematologic disorders⁶⁰. CXCR4 is widely expressed in the hematopoietic cell compartment⁶¹. Hence, application of CXCR4 antagonists does not only lead to a mobilization of hematopoietic stem and progenitor cells but is accompanied by marked leukocytosis that affects all hematopoietic lineages including T_reg cells^60,62. Moreover, homing into and retention of both, T_reg cells⁵⁶ and DCs⁶³, in the bone marrow have been proposed to be dependent on CXCR4/SDF1α interaction as well as the constitutive expression of VCAM-1 and endothelial selectins²⁸. Since DCs and their progenitors express CXCR4, they will be translocated to the circulation after the application of plerixafor.

We hypothesized that the immunotolerogenic effects of the mTOR inhibitor rapamycin may synergize with the CXCR4 antagonist plerixafor to allow for a mobilization of T_reg cells out of the bone marrow to be enriched in a heart allograft. By applying a full-mismatch heart allograft murine model, we here provide evidence that plerixafor induced and maintained the expansion of FoxP3⁺ T_reg cells into the periphery and into the heart allograft. We observed that this expansion was accompanied by the recruitment of pDCs from the bone marrow into the peripheral blood, secondary lymphoid organs, and the allograft. Following this line, the in vivo depletion of pDCs led to an abrogation of the achieved prolongation of heart allograft survival by plerixafor rendering pDCs important targets for future immunotherapies in the context of transplantation as well as autoimmune diseases. Overall, we conclude that the combination of the CXCR4 antagonist plerixafor with the mTOR inhibitor rapamycin improves heart allograft survival.

Results

Treatment with plerixafor and rapamycin prolongs allograft survival by reducing the inflammatory allo-response

To investigate whether CXCR4 blockade mediates additional beneficial immune regulatory effects, we utilized an abdominal heterotopic murine heart transplantation model as previously described^64,95 and analyzed the survival of the heart transplants by palpation according to Martins et al.⁶⁵. The C57BL/6J recipient mice received either fully-mismatched BALB/c (allogeneic) or C57BL/6J (syngeneic) heart transplants and were treated two days before and every other day for 14 days post transplantation. As outlined in Fig. 1A, the mice either received only vehicles [no treatment, NT (allogeneic) or SC (syngeneic)], 0.4 mg/kg rapamycin + plerixafor vehicle [R (allogeneic) or SR (syngeneic)], 1 mg/kg plerixafor + rapamycin vehicle (P1), 5 mg/kg plerixafor + rapamycin vehicle (P5), 1 mg/kg plerixafor + 0.4 mg/kg rapamycin (P1R), or 5 mg/kg plerixafor + 0.4 mg/kg rapamycin [P5R (allogeneic) or SP5R (syngeneic)].

Our analysis revealed that hearts in allogeneic recipients with basic immunosuppression by rapamycin (R) demonstrated a better survival compared to vehicle-treated recipients (NT) (43.5 vs. 8 days; Fig. 1B). While the addition of low-dose plerixafor (P1R) yielded a moderate additional survival prolongation (49 vs. 43.5 days), the higher dose of plerixafor (P5R) led to a marked prolonged heart allograft survival (77.5 vs. 43.5 days, Fig. 1B). Furthermore, animals treated only with plerixafor harbored a slightly prolonged allograft survival for both, P1 (10 days) and P5 groups (10 days) compared vehicle-only controls (NT; 8 days). Thus, our data indicated a prolonged allograft survival by combining rapamycin with Plerixaflor.

To assess the transplants' general health status, we performed histological analyses. Therefore, allografts were removed 14 s post transplantation from vehicle controls (NT), plerixafor only (P5), rapamycin only (R), or rapamycin + plerixafor (P5R) treated animals and analyzed for fibrosis and myocyte lesions (Fig. 1C). We found less extensive signs of fibrosis (Fig. 1C, first panel) and a lower myocyte lesion score (Fig. 1C, second panel) in allografts of P5R animals. As expected, vehicle treated controls (NT) and plerixafor-only-treated animals (P5) showed an almost total tissue destruction (data not shown). Thus, our data revealed a better health status of the allografts upon treatment with rapamycin and plerixafor (P5R).

Furthermore, we evaluated the infiltration of CD3⁺ T cells (major drivers of allograft rejection) into the transplants by histology. Thereby, in line with less severe tissue destruction, we revealed a reduced CD3⁺ T lymphocyte infiltration after plerixafor + rapamycin (P5R) treatment compared to rapamycin-only (R) (Fig. 1C, third panel). Since mTOR inhibition might favor the expansion of CD4⁺ T_reg cells^11,48,66 and these cells are a critical prerequisite for long-term allograft survival, we investigated the effect of plerixafor on the number of allograft-residing FoxP3⁺ T_reg cells. Therefore, we visualized them in heart allograft sections by a FoxP3 antibody as such cells are also stained by CD3. This data is represented as percentage of FoxP3⁺ cells within all CD3 cells by division of the number of FoxP3⁺ cells by the total number of CD3⁺ T cells. Surprisingly, although the CD3⁺ T cell number was reduced in the P5R group (Fig. 1C, third panel), we found higher FoxP3⁺ T_reg cell numbers (Fig. 1B, forth panel) compared to rapamycin-only treated animals (R). This is clearly indicated by the FoxP3⁺/CD3⁺ ratio (Fig. 1C, fourth panel). Of note, also the absolute T_reg cell numbers were increased by about 2.5-fold (72.8 ± 8.0 (P5R) vs. 28.1 ± 3.3 (R) T_reg cells/mm²). Collectively, our data demonstrate synergistic effects of plerixafor and rapamycin fostering heart allograft survival and reducing inflammation induced tissue destruction.

Plerixafor treatment increased FoxP3⁺ T_reg cell numbers in circulation and allograft

Rapamycin can have beneficial effects on survival and proliferation of T_reg cells^11,50,51 and might expand this subset^{11,48,66,67,68,69}. To characterize this effect in our model, we analyzed T_reg cell numbers in blood, spleen, celiac lymph node, and the transplanted hearts 14 days post transplantation by flow cytometry. Therefore, we stained single cell suspensions using CD19, CD161b/c (NK1.1), CD3, CD4, CD25, and FoxP3 (Supplemental Fig. S1). Besides the fully mismatched heart transplantations (R, P5, and P5R), we included syngeneic control transplantations [vehicle-only (SC), rapamycin-only (SR), and rapamycin + plerixafor (SP5R)]. This comparison of allogeneic and syngeneic transplants allowed to shed light T_reg cells' specificity. While we found no increase of CD4⁺Foxp3⁺ Treg cell numbers in peripheral blood of transplanted versus non-transplanted C57BL/6 mice after treatment with rapamycin-only (R and SR) or vehicle-only (SC) (Fig. 2A, Supplemental Table 1), our data indicated an significant increase FoxP3⁺CD4⁺ T cells in blood by plerixafor (P5) and plerixafor + rapamycin (P5R and SP5R) (Fig. 2A, Supplemental Table 1). This increase was neither observed in spleen nor in celiac lymph nodes. Of note, syngeneic heart transplants (SC, SP5, and SP5R) did not contain T_reg cells (Fig. 2D, Supplemental Table 1). In contrast, infiltration of CD4⁺FoxP3⁺ T cells into the allograft was only augmented by P5R treatment in comparison plerixafor-only (P5) or rapamycin-only (R), when mice received fully mismatched heart allografts (Fig. 2D, Supplemental Table 1).

Thus, we conclude that plerixafor increased T_reg cell numbers in peripheral blood in our allogeneic and syngeneic heterotypic heart transplantation model. Rapamycin and plerixafor have a synergistic effect on the T_reg cell infiltration into fully mismatched heart transplants explaining the prolongation of heart allograft survival in the respective treatment groups.

Plerixafor mobilize PDCA-1⁺Siglec-H⁺ pDCs into blood and celiac lymph nodes, while the concomitant rapamycin application increases infiltration into the heart allograft

The application of rapamycin can lead to increased pDC numbers, which might be pro-tolerogenic by augmenting proliferation of T_regs cells^70,71,72. Therefore, we hypothesized that the heart allograft survival prolongation by rapamycin + plerixafor is pDC mediated. Thus, we investigated pDC numbers by flow cytometry in blood, spleen, celiac lymph nodes, bone marrow, and samples of the left transplanted heart ventricle 14 days after transplantation (Fig. 3, Supplemental Table 1). pDCs were analyzed using a staining with CD19, CD161b/c (NK1.1), CD317 (PDCA-1, Bst-2), and Siglec-H, and defined as PDCA-1⁺Siglec-H⁺ (Supplemental Fig. S1).

Our data revealed higher pDC numbers in peripheral blood of plerixafor-alone (P5), but also plerixafor + rapamycin (P5R) treated animals compared to rapamycin-only (R) (Fig. 3A). A similar increase was also observed after syngeneic heart transplantation (SP5R vs. SR) demonstrating independency from the allogeneic setting. Of note, in the allogeneic setting the blood pDCs increase was less pronounced in plerixafor + rapamycin (P5R) compared plerixafor-only (P5) treated animals (Fig. 3A, Supplemental Table 1). While pDC numbers were not elevated in celiac lymph nodes (Fig. 3C, Supplemental Table 1), but in the allograft (Fig. 3D, Supplemental Table 1) within the plerixafor + rapamycin group (P5R), these cells were likely mobilized from the BM (Fig. 3E, Supplemental Table 1) by the CXCR4 blockade. In spleen no changes of pDC numbers were observed (Fig. 3B, Supplemental Table 1). Of note, plerixafor increased pDC numbers in celiac lymph nodes (Fig. 3C, Supplemental Table 1) only in the allogeneic heart transplant setting. Finally, the corresponding bone marrow specimen (Fig. 3E, Supplemental Table 1) demonstrated decreased pDC numbers after plerixafor treatment (P5, P5R, and SP5R) compared to rapamycin-only treatment (R) only in allogeneic and syngeneic heart transplant settings. We also analyzed the accumulation of pDCs in heart allografts by flow cytometry. Thereby, we found higher pDC numbers after rapamycin + plerixafor treatment (P5R) (Fig. 3D, Supplemental Table 1). In contrast, the few immune cells in syngeneic heart transplants demonstrated unchanged pDC numbers. Additionally, we also investigated other immune cell subsets, such as B cells, CD4⁺ and CD8⁺ T cells, Ly6C^high and Ly6C^low monocytes, neutrophils, as well as cDC1 and cDC2 dendritic cells, in peripheral blood, transplanted heart, splenic single cell suspensions. No changes within the frequency of these subsets, which can potentially interact with Treg cells or dampen the immune response, could be associated with the prolongation of heart allograft survival induced by rapamycin + plerixafor treatment (Supplemental Fig. S2, Supplemental Table 1).

In summary, this data demonstrate a plerixafor-mediated pDC mobilization into peripheral blood, celiac lymph nodes and heart allografts. Furthermore, we could observe a plerixafor mediated enrichment of pDCs in celiac lymph nodes in the allogenic transplantation setting.

Depletion of PDCA-1⁺Siglec-H⁺ pDCs causes a decrease of FoxP3⁺ Treg numbers in the periphery and heart allograft abrogating the synergistic effect of plerixafor and rapamycin

To investigate the contribution of pDCs to the prolongation of allograft survival and the increase of FoxP3⁺ T_reg cell numbers, we performed pDCs depletion experiments in the allogeneic heart transplantation setting and investigated the allografts for myocyte lesions (Fig. 4B), allograft fibrosis (Fig. 4C), allograft infiltrating immune cells (Fig. 4D), and T_reg cell numbers (Fig. 4E) 14 days after transplantation. We depleted pDCs by an in vivo intraperitoneal injection with anti-PDCA-1 antibodies two days before and every second day till 14 after transplantation (Fig. 4A). We treated animals as before by added an anti-PDCA-1 depletion antibody (P5R-aPDCA, R-aPDCA, P5-aPDCA) and the corresponding isotype control antibody (P5R, R, P5) (Fig. 4A). Syngeneic heart transplantations were performed in vehicles treated animals (SC). The achieved pDC depletion with a single dose of anti-PDCA-1 mAb was very effective in peripheral blood, spleen, LN and thymus, whereas bone marrow Siglec-H⁺ pDCs were not completely depleted (Supplemental Fig. S3A). Of note, tissue macrophages of spleen, lymph node, thymus, and bone marrow tissues were not affected by the treatment with the anti-PDCA-1 depletion antibody (Supplemental Fig. S3A). The pDC depletion was maintained for the whole treatment period (data not shown). Tissue macrophages (MerTK⁺) did not express PDCA-1 in our experimental setting (data not shown), but their numbers were reduced by anti-PDCA-1 treatment in the different groups (Supplemental Fig. S3B). We suspect therefore that the reduction is an indirect effect due to the depletion of the pDCs. Only a few B cells were found in the transplanted heart tissues and were mainly negative for PDCA-1 expression. Only after plerixafor only (P5-Iso) treatment, PDCA-1 expression could be detected and those cells were also reduced in numbers by anti-PDCA-1 treatment (Supplemental Fig. S3C).

Our analysis revealed that the pDC depletion in rapamycin + Perixaflor (P5R) treated mice (P5R-aPDCA1) led to a stronger cardiac allograft lymphocyte infiltration and a higher myocyte lesion score (Fig. 4B). The fibrosis of the allograft was also increased by anti-PDCA-1 treatment in the R-aPDCA1 and the P5R-aPDCA-1 groups (Fig. 4C). Furthermore, P5R + anti-PDCA-1 treated mice (P5R-aPDCA1) displayed significantly less CD4⁺FoxP3⁺ T_reg cells in blood and allograft (Fig. 4D,E). These results demonstrated the association of CD4⁺FoxP3⁺ T_reg cell enrichment with the increase of PDCA-1⁺Siglec-H⁺ pDCs.

Finally, anti-PDCA-1 depletion (P5R-aPDCA) abrogated the prolongation of allograft survival compared to P5R treated animals receiving isotype injections (P5R; 63 vs. 20 days; Fig. 4F). Rapamycin only treated animals displayed no changes in heart allograft survival with (R-aPDCA1) or without (R) pDC depletion. Of note, a slight but significant reduction in median allograft survival time was observed in P5-aPDCA1 versus isotype antibody treated P5 animals (8 vs. 12 days). Therefore, our data suggest an essential role of pDCs for the synergistic prolongation of heart allograft survival by plerixafor treatment, but are dispensable in rapamycin only treatments. Of note, we could observe a tendency of an increased IL-10 expression by pDCs within the heart tissue after treatment with rapamycin + plerixafor (P5R) compared to rapamycin treatment (R) as displayed in Supplemental Fig. S6A. Of note, nearly all IL-10 expressing cells found in the transplanted hearts could be identified as pDCs. Furthermore, we also observed a tendency in a lower type I interferon expression in the transplanted heart tissues after rapamycin + plerixafor (P5R) treatment compared to rapamycin (R) alone.

In conclusion, we found that the in vivo depletion of pDCs abrogated both, T_reg cell expansion as well as the plerixafor-mediated prolongation of allograft survival. Therefore, our data indicate that plerixafor is capable to reduce the severity of allograft rejection only under at least a low dosed concomitant mTOR inhibition by recruiting bone marrow-residing PDCA-1⁺Siglec-H⁺ pDCs into both, secondary lymphoid organs and the allograft.

Discussion

The broad clinical application of adoptive T_reg cell immunotherapy is hindered by the risk of T_reg cell stability and the required consistency of purity and regulatory potency¹. Therefore, there is still an unmet need for the direct modulation of the host's immune system. We here investigated an alternative approach to reduce allo-immune responses after allogeneic heart transplantation in mice combining the CXCR4 antagonist plerixafor with low-dose rapamycin. We observed a recruiting of pDCs from the bone marrow into the allograft and identified these cells as key players.

Here, we demonstrated a synergistic effect of plerixafor for rapamycin treatment allowing for a longer allograft survival accompanied by an amelioration of myocardial damage, fibrosis and a significant higher allograft infiltration of Foxp3⁺ T_reg cells (Figs. 1 and 2). plerixafor mobilized pDCs from bone marrow and their depletion reverted all positive effects of plerixafor including the prolongation of allograft survival.

pDCs and T_reg cells allograft infiltration upon plerixafor treatment was dependent on the fully mismatched transplant setting (Figs. 2 and 3) and therefore, we concluded its dependency on alloantigen presence. The infiltration itself might be either triggered by the alloantigen presentation in secondary lymphoid organs or by the alloimmune response in the transplant inducing expression of inflammatory chemokine ligands, such as CXCL9-11 or MIP1α^73,74. Although CXCL12 fostered trafficking of CXCR4⁺ progenitor epithelial cells into tracheal transplants⁷⁵, CXCL12 expression was not altered in heart allografts (data not shown). Immune cell trafficking is dependent on chemokine-chemokine receptor networks⁷⁶. Thus, changes within the allografts may foster T_reg cell and pDC infiltration under mTOR inhibition as this was previously observed for T_reg cells in a murine islet allograft model⁷⁷.

In experimental ischemic cardiomyopathy, immune cell migration from bone marrow (including hematopoietic progenitor cells, HSCs) improved recovery after ischemia/reperfusion injury^78,79. In line with our data, a CXCR4 blockade fostered tissue repair after myocardial infarction due to T_reg cell mobilization⁸⁰, but also speeded up the healing process^78,81.

In line, we observed a longer allograft survival after plerixafor treatment and demonstrated further that pDC depletion resulted in reversal of these effects (Fig. 4). This suggests a certain effect of plerixafor alone synergizing with rapamycin immunosuppression. In rat liver transplantations a similar synergism of plerixafor with low-dose tacrolimus was reported and accompanied by a strong increase of allograft-residing T_reg cells⁸². In addition, in pancreatic islet transplantation into hyperglycemic C57BL/6 mice a CXCR4 antagonist prolonged graft survival⁵⁸. The authors also attributed this to hematopoietic stem cells (HSCs) due to the abrogation of these effects by αCD117 antibody mediated depletion of HSCs. However, the expression of CD117 not limited to these cells as it can also be found on many other precursor cell populations including the most import pDCs precursor population, suggesting that also pDCs will be affected. The authors excluded a role of T_reg cells by depletion with an anti-CD25 antibody, which did not fully abrogate the prolongation of allograft survival. Rapamycin as well as plerixafor can directly influence the functionality of T_reg cells. Whereas rapamycin has been reported to foster T_reg cell function^69,83,84,85, plerixafor was shown to partially reduce T_reg cell-mediated immunosuppression⁸⁶.

In this study, we also investigated the role of pDCs in our murine heart transplant model as other immune cell population could not directly by associated with prolonged allograft survival by rapamycin + plerixafor (Fig. 3 and Supplemental Fig. S2. pDCs have been reported to foster the suppressive function of T_reg cells^34,35. The experiments demonstrated that plerixafor not only resulted in the well-known peripheral shedding of hematopoietic stem cells^58,59, but also of PDCA-1⁺Siglec-H⁺ pDCs. We identified an immune regulatory role of mobilized pDCs, as their anti-PDCA-1-mediated depletion resulted in a reduction of T_reg cells in both, blood and allograft. pDCs have been described to suppress the peripheral immune response in several murine models of auto- and allo-inflammatory disorders¹³, such as autoimmune arthritis⁴⁴, autoimmune encephalomyelitis⁴⁰, and graft-versus-host disease^20,43. Whereas donor-derived cDCs have been traditionally regarded as principal instigators of allograft rejection, considerable evidence demonstrates a role for recipient’s pDCs in mediating the induction and/or maintenance of tolerance to allografts^3,34. Abe et al. first reported a prolongation of murine cardiac allograft survival by an adoptive transfer of BM-derived pDCs⁸⁷. In addition, Turnquist et al. observed an enrichment of antigen-specific FoxP3⁺ T_reg cells promoting heart allograft tolerance upon adoptive transfer of in vitro rapamycin preconditioned DCs⁸⁸. Furthermore, pDCs participate in lupus models in peripheral tolerance by inducing T_reg cells²⁹. Importantly, in operational tolerant pediatric liver recipients higher peripheral blood pDC numbers (compared to cDCs) have been observed⁸⁹, while elevated myeloid cell/pDC ratios were associated with early acute cellular rejection after pediatric small bowel transplantation⁹⁰ and late rejection after pediatric liver transplantation⁹¹, respectively.

There are different possibilities for the mechanism of pDC-driven induction of heart allograft-specific T_reg cells. They may result from a direct T_reg cell induction in the thymus by pDCs from the periphery, which have uptaken peripheral antigens, presenting those in the thymus promoting central tolerance⁴². Although, we observed an increased homing of pDCs into celiac lymph nodes and the cardiac allograft, we cannot specify where these interactions take place in our model. In line with these results, previous studies from Ochando et al. demonstrated homing of pDCs, which acquired donor-specific allo-antigens, to draining LNs where they induced de novo allo-antigen-specific T_reg cells under tolerizing conditions⁴⁷. Furthermore, additional studies from Liu et al. identified pDCs as crucial players for establishing cardiac allograft tolerance in mice lacking the lymph node homing receptor CCR7⁹². Furthermore, pDCs might be also involved in the recruitment, differentiation, and/or functional modulation of tissue macrophages involved in tissue repair as we observed a reduction of those cells after pDC depletion while excluding a direct depletion due to lacking PDCA-1 expression (Supplemental Fig. S3B).

In summary, we demonstrate for the first time an immunomodulatory potency of plerixafor under sub-therapeutical mTOR inhibition after allogeneic heart transplantation. Combining the immunosuppressive drug with the mobilization of bone-marrow-residing pDCs allowed to reduce allograft rejection leading to prolongation of allograft survival in vivo. Here, we do not exclude a role for hematopoietic stem cells on heart allograft survival, but rather classify pDCs as an essential population for the prolonged heart allograft survival by plerixafor. We proofed the essential involvement of pDCs in the immune regulatory mechanisms in check, because their depletion resulted in an impairment of T_reg cell expansion and prolongation of allograft survival. Furthermore, a strong and significant exacerbation of allograft histology scores after pDC depletion occurred.

These results may impact on future clinical immunosuppressive therapies since the direct adoptive transfer of T_reg cells implies several disadvantages^93,94. Therefore, it is of utmost importance to develop alternatives relying on the direct induction of donor-specific T_reg cells within the patient. Here, we have demonstrated that a combination of immunosuppressive drugs implementing plerixafor might be able to provide the possibility to achieve this in vivo.

Materials and methods

Animals

BALB/c (allogeneic) or C57BL/6J (syngeneic) mice served as heart donors and C57BL/6J as transplant recipients (all male and 8–12 weeks old). All animal experiments adhered to EU directive 2010/63/EU and were approved by the 'Regierung von Unterfranken' (approval #G1071/09) and were carried out in accordance to the ARRIVE guidelines. For anesthesia, 10% Ketamine (100 mg/kg, Dopalen) and 2% Xylazine (10 mg/kg, Rompun) was used. Metamizol (200 mg/kg) p.o. and Carprofen (5 mg/kg) s.c. served as postoperative analgesia.

Abdominal-heterotopic cardiac transplantation model

Heterotopic intra-abdominal heart transplantation (HTX) from BALB/c or C57BL/6J to C57BL/6J recipient mice was performed as previously described⁶⁴. As video demonstrating the whole process including a detailed method description can be found in our recent publication⁹⁵. Allograft function was evaluated daily by palpation according to Martins et al.⁶⁵. C57BL/6J recipients received injections with plerixafor (1 or 5 mg/kg s.c.) and/or rapamycin (0.4 mg/kg i.p.) two days before, immediately after HTX and every other day for 14 days. The subclinical dosage of rapamycin allowed to early distinguish differences in allograft survival. The treatment groups are depicted in Fig. 1A. Transplantation of C57BL/6J hearts into C57BL/6J recipients served as non-rejection controls. The role of pDCs was investigated by in vivo depletion experiments (groups see Fig. 4A). Therefore, 500 μg of either anti-PDCA-1 or an isotype control antibody was injected every second day starting two days before transplantation. Vehicle controls included 0.9% NaCl (plerixafor) and 0.2% Carboxymethylcellulose + 0.25% Tween-80 (rapamycin). All in vivo applied antibodies were endotoxin-free. The weight of all transplanted animals remained within physiological ranges as depicted in Supplemental Fig. S5.

Reagents and antibodies

Plerixafor (AMD3100, Sigma, Germany) was prepared in 0.9% NaCl (injection volume 150 μl, s.c.). Rapamycin (Wyeth, Münster, Germany) dissolved in EtOH was diluted in 0.2% carboxymethylcellulose + 0.25% Tween80^® (i.p.). Anti-msPDCA-1 (for depletion, clone 927), ratIgG2b isotype control were obtained from Biolegend. Flow cytometry antibodies for CD3 (145-2C11 or eBio500A2), CD4 (RM4-5), CD8 (Clone 53-6.7), CD11b (M1/70), CD11c (HL3 or N418), CD19 (1D3), CD25 (PC61), CD45R/B220 (RA3-6B2), CD86 (GL-1), CD115 (T38-320), CD317/PDCA-1 (927), FoxP3 (MF-14 or MF-23), LAP-1 (TW7-16B4), Ly6C (HK1.4), Ly6G (1A8), Siglec-H (551), and Ly-76/Ter119 (TER-119) were purchased from BioLegend, BD Pharmingen, or eBioscience. Immunohistochemistry antibodies and isotype controls for CD3 (rabbit) and FoxP3 (FJL-16s) were obtained from Dako or eBioscience.

Single cell isolation

Celiac LNs were recovered from paraaortic, pararenal, parapancreatic and mesenteric sites. Celiac LNs, spleen, and transplanted hearts (after flushing with cold DPBS) were teared into small pieces and digested in HBSS + Collagenase IV and DNAse I for 30–45 min as described before⁹⁶. ACK lysis buffer was used to lyse erythrocytes. BM was flushed out with RPMI1640 + 5% FCS (Gibco, Thermo Fisher Scientific). Cell isolation was performed three hours after the last plerixafor injection.

Flow cytometry

Single cells suspensions were blocked with anti-CD16/CD32 (2.4G2) and anti-FcγRIV (9E9) at 4 °C for 10 min. For intracellular staining of FoxP3, cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (Biolegend) according to manufacturer's instructions. Acquisition was performed on a Navio flow cytometer using Caluza 1.3 (Beckman Coulter) or a LSR Fortessa (SORP) using Diva. Data were analyzed using FlowJo 10.6.0 (TreeStar).

Histological evaluation of the heart allografts

The allograft’s left ventricle was fixed in 10% formalin, paraffin-embedded, and sectioned (3 µm). Cellular infiltration and myocyte lesion scores were quantified as previously described⁶⁴. In brief, myocyte lesion score 0: no lesion; 1: myocyte vacuolization; 2: focal myocyte necrosis (irregular border; fragmented sarcoplasm, debris, myocyte dropout); and 3: extensive myocyte necrosis (interstitial hemorrhage/eosinophil infiltration). The heart tissue fibrosis was investigated by Sirius red staining (acquisition: Axio 4.6, Carl Zeiss). Immunohistochemistry was performed after antigen retrieval using Tris–EDTA (pH 9) for 45 min (CD3) or citrate (pH 6) for 60 min followed by permeabilization using 0.25% Triton X-100 in PBS for 10 min (FoxP3). Primary antibody dilutions of 1 µg/ml (CD3) or 10 µg/ml (FoxP3) were applied for 30 min at RT. After washing, goat-anti-rabbit (3 μg/ml, Dianova Jackson ImmunoResearch) or goat-anti-rat (10 μg/ml BD Pharmingen) were stained for 30 min at RT as secondary antibodies. Scores and cells were assessed and calculated as average of 10 high power fields by blinded observers. T_reg/T cell ratio was calculated by dividing FoxP3⁺ cell number/mm² by CD3⁺ cell number/mm² in serial sections. Two blinded observers evaluated five fields of the histological slides and an average scores were calculated.

Statistical analysis

Data in bar graphs and scatter plots are expressed as mean ± SD and evaluated by Mann–Whitney U test (two groups) or One-way ANOVA with Bonferroni post-hoc test (multiple groups) using GraphPad Prism V5.01. Survival data were denoted as median survival days [P25–P75] and were analyzed using the Gehan–Breslow–Wilcoxon test. p values were expressed as *p < 0.05, **p < 0.01 and ***p < 0.001.

Abbreviations

Ag:: Antigen
APC:: Antigen-presenting cell
cDCs:: Conventional dendritic cells
CXCR4:: C-X-C chemokine receptor type 4
FACS:: Fluorescence-activated cell sorting
FoxP3:: Forkhead box P3
HPF:: High power field
i.p.:: Intraperitoneal injection
Iso:: Isotype
MST:: Median survival time
mTOR:: Mammalian target of rapamycin
P:: Plerixafor
P1R:: Plerixafor 1 mg/kg body weight plus rapamycin
P5R:: Plerixafor 5 mg/kg body weight plus rapamycin
pDC:: Plasmacytoid dendritic cells
PDCA-1:: Phosducin A-1
R:: Rapamycin
s.c.:: Subcutaneous injection
Sctrl:: Syngeneic control
SP5R:: Syngeneic control with plerixafor 5 mg/kg body weight plus rapamycin
SR:: Syngeneic vehicle control with rapamycin
SDF1α or CXCL12:: Stromal cell-derived factor 1α
Siglec-H:: Sialic acid binding Ig-like lectin H
Treg:: Regulatory T cells

References

Safinia, N., Scotta, C., Vaikunthanathan, T., Lechler, R. I. & Lombardi, G. Regulatory T cells: Serious contenders in the promise for immunological tolerance in transplantation. Front. Immunol. 6, 438 (2015).
PubMed PubMed Central Google Scholar
Klaeske, K. et al. Identification of the immunological profile in rejection-free heart transplantation. Transpl. Immunol. 59, 101259 (2020).
CAS PubMed Google Scholar
Rogers, N. M., Isenberg, J. S. & Thomson, A. W. Plasmacytoid dendritic cells: No longer an enigma and now key to transplant tolerance?. Am. J. Transplant. 13(5), 1125–1133 (2013).
CAS PubMed PubMed Central Google Scholar
Wood, K. J., Bushell, A. & Hester, J. Regulatory immune cells in transplantation. Nat. Rev. Immunol. 12(6), 417–430 (2012).
CAS PubMed Google Scholar
Riley, J. L., June, C. H. & Blazar, B. R. Human T regulatory cell therapy: Take a billion or so and call me in the morning. Immunity 30(5), 656–665 (2009).
CAS PubMed PubMed Central Google Scholar
Golshayan, D. et al. In vitro-expanded donor alloantigen-specific CD4⁺CD25⁺ regulatory T cells promote experimental transplantation tolerance. Blood 109(2), 827–835 (2007).
CAS PubMed Google Scholar
Ohkura, N., Kitagawa, Y. & Sakaguchi, S. Development and maintenance of regulatory T cells. Immunity 38(3), 414–423 (2013).
CAS PubMed Google Scholar
Edozie, F. C. et al. Regulatory T-cell therapy in the induction of transplant tolerance: The issue of subpopulations. Transplantation 98(4), 370–379 (2014).
CAS PubMed Google Scholar
Singer, B. D., King, L. S. & D’Alessio, F. R. Regulatory T cells as immunotherapy. Front. Immunol. 5, 46 (2014).
PubMed PubMed Central Google Scholar
Presser, D. et al. Differential kinetics of effector and regulatory T cells in patients on calcineurin inhibitor-based drug regimens. Kidney Int. 76(5), 557–566 (2009).
CAS PubMed Google Scholar
Hoerning, A. et al. Cyclosporin but not everolimus inhibits chemokine receptor expression on CD4⁺ T cell subsets circulating in the peripheral blood of renal transplant recipients. Clin. Exp. Immunol. 168(2), 251–259 (2012).
CAS PubMed PubMed Central Google Scholar
Zeiser, R. et al. Inhibition of CD4⁺CD25⁺ regulatory T-cell function by calcineurin-dependent interleukin-2 production. Blood 108(1), 390–399 (2006).
CAS PubMed PubMed Central Google Scholar
Gordon, J. R., Ma, Y., Churchman, L., Gordon, S. A. & Dawicki, W. Regulatory dendritic cells for immunotherapy in immunologic diseases. Front. Immunol. 5, 7 (2014).
PubMed PubMed Central Google Scholar
Gallegos, A. M. & Bevan, M. J. Central tolerance to tissue-specific antigens mediated by direct and indirect antigen presentation. J. Exp. Med. 200(8), 1039–1049 (2004).
CAS PubMed PubMed Central Google Scholar
Yamazaki, S. et al. CD8⁺ CD205⁺ splenic dendritic cells are specialized to induce Foxp3⁺ regulatory T cells. J. Immunol. 181(10), 6923–6933 (2008).
CAS PubMed Google Scholar
Collins, C. B. et al. Flt3 ligand expands CD103(+) dendritic cells and FoxP3(+) T regulatory cells, and attenuates Crohn’s-like murine ileitis. Gut 61(8), 1154–1162 (2012).
CAS PubMed Google Scholar
Kriegel, M. A., Rathinam, C. & Flavell, R. A. Pancreatic islet expression of chemokine CCL2 suppresses autoimmune diabetes via tolerogenic CD11c⁺ CD11b⁺ dendritic cells. Proc. Natl. Acad. Sci. U.S.A. 109(9), 3457–3462 (2012).
ADS CAS PubMed PubMed Central Google Scholar
Park, J. E. et al. DC-based immunotherapy combined with low-dose methotrexate effective in the treatment of advanced CIA in mice. J. Immunol. Res. 2015, 834085 (2015).
PubMed PubMed Central Google Scholar
Escobar, A. et al. Tolerogenic dendritic cells derived from donors with natural rubber latex allergy modulate allergen-specific T-cell responses and IgE production. PLoS One 9(1), e85930 (2014).
ADS PubMed PubMed Central Google Scholar
Scroggins, S. M., Olivier, A. K., Meyerholz, D. K. & Schlueter, A. J. Characterization of regulatory dendritic cells that mitigate acute graft-versus-host disease in older mice following allogeneic bone marrow transplantation. PLoS One 8(9), e75158 (2013).
ADS CAS PubMed PubMed Central Google Scholar
Li, H. & Shi, B. Tolerogenic dendritic cells and their applications in transplantation. Cell. Mol. Immunol. 12(1), 24–30 (2015).
CAS PubMed Google Scholar
Phillips, B. E., Garciafigueroa, Y., Engman, C., Trucco, M. & Giannoukakis, N. Tolerogenic dendritic cells and T-regulatory cells at the clinical trials crossroad for the treatment of autoimmune disease; emphasis on type 1 diabetes therapy. Front. Immunol. 10, 148 (2019).
CAS PubMed PubMed Central Google Scholar
Phillips, B. E., Garciafigueroa, Y., Trucco, M. & Giannoukakis, N. Clinical tolerogenic dendritic cells: Exploring therapeutic impact on human autoimmune disease. Front. Immunol. 8, 1279 (2017).
PubMed PubMed Central Google Scholar
Benham, H. et al. Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients. Sci. Transl. Med. 7(290), 290ra87 (2015).
PubMed Google Scholar
Jauregui-Amezaga, A. et al. Intraperitoneal administration of autologous tolerogenic dendritic cells for refractory Crohn’s disease: A phase I study. J. Crohns Colitis 9(12), 1071–1078 (2015).
PubMed Google Scholar
Ten Brinke, A. et al. Clinical use of tolerogenic dendritic cells-harmonization approach in European collaborative effort. Mediat. Inflamm. 2015, 471719 (2015).
Google Scholar
Ochando, J., Ordikhani, F., Jordan, S., Boros, P. & Thomson, A. W. Tolerogenic dendritic cells in organ transplantation. Transpl. Int. 33(2), 113–127 (2020).
PubMed Google Scholar
Swiecki, M. & Colonna, M. The multifaceted biology of plasmacytoid dendritic cells. Nat. Rev. Immunol. 15(8), 471–485 (2015).
CAS PubMed PubMed Central Google Scholar
Swiecki, M. & Colonna, M. Unraveling the functions of plasmacytoid dendritic cells during viral infections, autoimmunity, and tolerance. Immunol. Rev. 234(1), 142–162 (2010).
CAS PubMed PubMed Central Google Scholar
Reizis, B. Plasmacytoid dendritic cells: Development, regulation, and function. Immunity 50(1), 37–50 (2019).
CAS PubMed PubMed Central Google Scholar
Musumeci, A., Lutz, K., Winheim, E. & Krug, A. B. What makes a pDC: Recent advances in understanding plasmacytoid DC development and heterogeneity. Front. Immunol. 10, 1222 (2019).
CAS PubMed PubMed Central Google Scholar
Webster, B., Assil, S. & Dreux, M. Cell–cell sensing of viral infection by plasmacytoid dendritic cells. J. Virol. 90(22), 10050–10053 (2016).
CAS PubMed PubMed Central Google Scholar
Waisman, A., Lukas, D., Clausen, B. E. & Yogev, N. Dendritic cells as gatekeepers of tolerance. Semin. Immunopathol. 39(2), 153–163 (2017).
CAS PubMed Google Scholar
Podesta, M. A., Cucchiari, D. & Ponticelli, C. The diverging roles of dendritic cells in kidney allotransplantation. Transplant. Rev. (Orlando). 29(3), 114–120 (2015).
PubMed Google Scholar
van Kooten, C. et al. Dendritic cells as a tool to induce transplantation tolerance: Obstacles and opportunities. Transplantation 91(1), 2–7 (2011).
PubMed Google Scholar
Martin, P. et al. Characterization of a new subpopulation of mouse CD8alpha⁺ B220⁺ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential. Blood 100(2), 383–390 (2002).
CAS PubMed Google Scholar
Colonna, M., Trinchieri, G. & Liu, Y. J. Plasmacytoid dendritic cells in immunity. Nat. Immunol. 5(12), 1219–1226 (2004).
CAS PubMed Google Scholar
Boor, P. P. et al. Human plasmacytoid dendritic cells induce CD8(+) LAG-3(+) Foxp3(+) CTLA-4(+) regulatory T cells that suppress allo-reactive memory T cells. Eur. J. Immunol. 41(6), 1663–1674 (2011).
CAS PubMed Google Scholar
Loschko, J. et al. Antigen targeting to plasmacytoid dendritic cells via Siglec-H inhibits Th cell-dependent autoimmunity. J. Immunol. 187(12), 6346–6356 (2011).
CAS PubMed Google Scholar
Bailey-Bucktrout, S. L. et al. Cutting edge: Central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis. J. Immunol. 180(10), 6457–6461 (2008).
CAS PubMed Google Scholar
de Heer, H. J. et al. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen. J. Exp. Med. 200(1), 89–98 (2004).
PubMed PubMed Central Google Scholar
Hadeiba, H. et al. Plasmacytoid dendritic cells transport peripheral antigens to the thymus to promote central tolerance. Immunity 36(3), 438–450 (2012).
CAS PubMed PubMed Central Google Scholar
Hadeiba, H. et al. CCR9 expression defines tolerogenic plasmacytoid dendritic cells able to suppress acute graft-versus-host disease. Nat. Immunol. 9(11), 1253–1260 (2008).
CAS PubMed PubMed Central Google Scholar
Jongbloed, S. L. et al. Plasmacytoid dendritic cells regulate breach of self-tolerance in autoimmune arthritis. J. Immunol. 182(2), 963–968 (2009).
CAS PubMed Google Scholar
Kang, H. K., Liu, M. & Datta, S. K. Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigen-specific regulatory T cells and contraction of inflammatory Th17 cells. J. Immunol. 178(12), 7849–7858 (2007).
CAS PubMed Google Scholar
Nikolic, T., Welzen-Coppens, J. M., Leenen, P. J., Drexhage, H. A. & Versnel, M. A. Plasmacytoid dendritic cells in autoimmune diabetes—Potential tools for immunotherapy. Immunobiology 214(9–10), 791–799 (2009).
CAS PubMed Google Scholar
Ochando, J. C. et al. Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat. Immunol. 7(6), 652–662 (2006).
CAS PubMed Google Scholar
Zeiser, R. et al. Differential impact of mammalian target of rapamycin inhibition on CD4⁺CD25⁺Foxp3⁺ regulatory T cells compared with conventional CD4⁺ T cells. Blood 111(1), 453–462 (2008).
CAS PubMed PubMed Central Google Scholar
Delgoffe, G. M. et al. The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2. Nat. Immunol. 12(4), 295–303 (2011).
CAS PubMed PubMed Central Google Scholar
Hoerning, A. et al. Pharmacodynamic monitoring of mammalian target of rapamycin inhibition by phosphoflow cytometric determination of p70S6 kinase activity. Transplantation 99(1), 210–219 (2015).
CAS PubMed Google Scholar
McMahon, G., Weir, M. R., Li, X. C. & Mandelbrot, D. A. The evolving role of mTOR inhibition in transplantation tolerance. J. Am. Soc. Nephrol. 22(3), 408–415 (2011).
CAS PubMed Google Scholar
Biswas, M. et al. Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell-dependent induction of CD4⁺CD25⁺FoxP3⁺ Treg. Blood 125(19), 2937–2947 (2015).
CAS PubMed PubMed Central Google Scholar
Sukhbaatar, N., Hengstschlager, M. & Weichhart, T. mTOR-mediated regulation of dendritic cell differentiation and function. Trends Immunol. 37(11), 778–789 (2016).
CAS PubMed PubMed Central Google Scholar
Taner, T., Hackstein, H., Wang, Z., Morelli, A. E. & Thomson, A. W. Rapamycin-treated, alloantigen-pulsed host dendritic cells induce Ag-specific T cell regulation and prolong graft survival. Am. J. Transplant. 5(2), 228–236 (2005).
CAS PubMed Google Scholar
Li, X. et al. Tolerance induction by exosomes from immature dendritic cells and rapamycin in a mouse cardiac allograft model. PLoS One 7(8), e44045 (2012).
ADS CAS PubMed PubMed Central Google Scholar
Zhao, E. et al. Bone marrow and the control of immunity. Cell. Mol. Immunol. 9(1), 11–19 (2012).
CAS PubMed Google Scholar
Zou, L. et al. Bone marrow is a reservoir for CD4⁺CD25⁺ regulatory T cells that traffic through CXCL12/CXCR4 signals. Cancer Res. 64(22), 8451–8455 (2004).
CAS PubMed Google Scholar
Fiorina, P. et al. Targeting the CXCR4-CXCL12 axis mobilizes autologous hematopoietic stem cells and prolongs islet allograft survival via programmed death ligand 1. J. Immunol. 186(1), 121–131 (2011).
CAS PubMed Google Scholar
Broxmeyer, H. E. et al. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J. Exp. Med. 201(8), 1307–1318 (2005).
CAS PubMed PubMed Central Google Scholar
Bilgin, Y. M. & de Greef, G. E. Plerixafor for stem cell mobilization: The current status. Curr. Opin. Hematol. 23(1), 67–71 (2016).
CAS PubMed Google Scholar
Loetscher, M. et al. Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes. J. Biol. Chem. 269(1), 232–237 (1994).
CAS PubMed Google Scholar
Couban, S., Wong, P. C. & Schultz, K. R. The case for plerixafor to replace filgrastim as the optimal agent to mobilize peripheral blood donors for allogeneic hematopoietic cell transplantation. Exp. Hematol. 70, 1–9 (2019).
CAS PubMed Google Scholar
Nakano, H., Lyons-Cohen, M. R., Whitehead, G. S., Nakano, K. & Cook, D. N. Distinct functions of CXCR4, CCR2, and CX3CR1 direct dendritic cell precursors from the bone marrow to the lung. J. Leukoc. Biol. 101(5), 1143–1153 (2017).
CAS PubMed PubMed Central Google Scholar
Wu, K. et al. Prolonged cold storage using a new histidine-tryptophan-ketoglutarate-based preservation solution in isogeneic cardiac mouse grafts. Eur. Heart J. 32(4), 509–516 (2011).
CAS PubMed Google Scholar
Martins, P. N. Assessment of graft function in rodent models of heart transplantation. Microsurgery 28(7), 565–570 (2008).
PubMed Google Scholar
Hoerning, A. et al. Subsets of human CD4(+) regulatory T cells express the peripheral homing receptor CXCR3. Eur. J. Immunol. 41(8), 2291–2302 (2011).
CAS PubMed PubMed Central Google Scholar
Fraser, H. et al. A rapamycin-based GMP-compatible process for the isolation and expansion of regulatory T cells for clinical trials. Mol. Ther. Methods Clin. Dev. 8, 198–209 (2018).
CAS PubMed PubMed Central Google Scholar
Strauss, L. et al. Selective survival of naturally occurring human CD4⁺CD25⁺Foxp3⁺ regulatory T cells cultured with rapamycin. J. Immunol. 178(1), 320–329 (2007).
CAS PubMed Google Scholar
Battaglia, M., Stabilini, A. & Roncarolo, M. G. Rapamycin selectively expands CD4⁺CD25⁺FoxP3⁺ regulatory T cells. Blood 105(12), 4743–4748 (2005).
CAS PubMed Google Scholar
Rosborough, B. R., Hackstein, H. & Turnquist, H. R. A window into immunosuppressant immunoregulation: Recipient conversion to rapamycin increases potentially tolerogenic immune cells. Kidney Int. 85(4), 743–745 (2014).
CAS PubMed Google Scholar
Cao, W. et al. Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway. Nat. Immunol. 9(10), 1157–1164 (2008).
CAS PubMed PubMed Central Google Scholar
Boor, P. P., Metselaar, H. J., Mancham, S., van der Laan, L. J. & Kwekkeboom, J. Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions. Clin. Exp. Immunol. 174(3), 389–401 (2013).
CAS PubMed PubMed Central Google Scholar
Melter, M. et al. Expression of the chemokine receptor CXCR3 and its ligand IP-10 during human cardiac allograft rejection. Circulation 104(21), 2558–2564 (2001).
CAS PubMed Google Scholar
Melter, M., McMahon, G., Fang, J., Ganz, P. & Briscoe, D. M. Current understanding of chemokine involvement in allograft transplantation. Pediatr. Transplant. 3(1), 10–21 (1999).
CAS PubMed Google Scholar
Gomperts, B. N. et al. Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury. J. Immunol. 176(3), 1916–1927 (2006).
CAS PubMed Google Scholar
Nourshargh, S. & Alon, R. Leukocyte migration into inflamed tissues. Immunity 41(5), 694–707 (2014).
CAS PubMed Google Scholar
Zhang, N. et al. Regulatory T cells sequentially migrate from inflamed tissues to draining lymph nodes to suppress the alloimmune response. Immunity 30(3), 458–469 (2009).
CAS PubMed PubMed Central Google Scholar
Ziff, O. J., Bromage, D. I., Yellon, D. M. & Davidson, S. M. Therapeutic strategies utilizing SDF-1alpha in ischaemic cardiomyopathy. Cardiovasc. Res. 114(3), 358–367 (2018).
CAS PubMed Google Scholar
Doring, Y., Pawig, L., Weber, C. & Noels, H. The CXCL12/CXCR4 chemokine ligand/receptor axis in cardiovascular disease. Front. Physiol. 5, 212 (2014).
PubMed PubMed Central Google Scholar
Wang, Y. et al. C-X-C motif chemokine receptor 4 blockade promotes tissue repair after myocardial infarction by enhancing regulatory T cell mobilization and immune-regulatory function. Circulation 139(15), 1798–1812 (2019).
PubMed PubMed Central Google Scholar
Proulx, C. et al. Antagonism of stromal cell-derived factor-1alpha reduces infarct size and improves ventricular function after myocardial infarction. Pflugers Arch. 455(2), 241–250 (2007).
CAS PubMed Google Scholar
Okabayashi, T. et al. Mobilization of host stem cells enables long-term liver transplant acceptance in a strongly rejecting rat strain combination. Am. J. Transplant. 11(10), 2046–2056 (2011).
CAS PubMed PubMed Central Google Scholar
Battaglia, M. et al. Rapamycin promotes expansion of functional CD4⁺CD25⁺FOXP3⁺ regulatory T cells of both healthy subjects and type 1 diabetic patients. J. Immunol. 177(12), 8338–8347 (2006).
CAS PubMed Google Scholar
Golovina, T. N. et al. Retinoic acid and rapamycin differentially affect and synergistically promote the ex vivo expansion of natural human T regulatory cells. PLoS One 6(1), e15868 (2011).
ADS CAS PubMed PubMed Central Google Scholar
Singh, K. et al. Regulatory T cells exhibit decreased proliferation but enhanced suppression after pulsing with sirolimus. Am. J. Transplant. 12(6), 1441–1457 (2012).
CAS PubMed PubMed Central Google Scholar
Wang, L. et al. Donor bone-marrow CXCR4⁺ Foxp3⁺ T-regulatory cells are essential for costimulation blockade-induced long-term survival of murine limb transplants. Sci. Rep. 10(1), 9292 (2020).
ADS CAS PubMed PubMed Central Google Scholar
Abe, M., Wang, Z., de Creus, A. & Thomson, A. W. Plasmacytoid dendritic cell precursors induce allogeneic T-cell hyporesponsiveness and prolong heart graft survival. Am. J. Transplant. 5(8), 1808–1819 (2005).
CAS PubMed Google Scholar
Turnquist, H. R. et al. Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4⁺ T cells, but enrich for antigen-specific Foxp3⁺ T regulatory cells and promote organ transplant tolerance. J. Immunol. 178(11), 7018–7031 (2007).
CAS PubMed Google Scholar
Mazariegos, G. V. et al. Dendritic cell subset ratio in peripheral blood correlates with successful withdrawal of immunosuppression in liver transplant patients. Am. J. Transplant. 3(6), 689–696 (2003).
PubMed Google Scholar
Gupta, A. et al. Elevated myeloid: Plasmacytoid dendritic cell ratio associates with early acute cellular rejection in pediatric small bowel transplantation. Transplantation 89(1), 55–60 (2010).
PubMed Google Scholar
Gupta, A. et al. Elevated myeloid: Plasmacytoid dendritic cell ratio associates with late, but not early, liver rejection in children induced with rabbit anti-human thymocyte globulin. Transplantation 88(4), 589–594 (2009).
CAS PubMed PubMed Central Google Scholar
Liu, X. et al. Tolerance induction towards cardiac allografts under costimulation blockade is impaired in CCR7-deficient animals but can be restored by adoptive transfer of syngeneic plasmacytoid dendritic cells. Eur. J. Immunol. 41(3), 611–623 (2011).
CAS PubMed Google Scholar
Hansmann, L. et al. Dominant Th2 differentiation of human regulatory T cells upon loss of FOXP3 expression. J. Immunol. 188(3), 1275–1282 (2012).
CAS PubMed Google Scholar
Waldmann, H., Hilbrands, R., Howie, D. & Cobbold, S. Harnessing FOXP3⁺ regulatory T cells for transplantation tolerance. J. Clin. Investig. 124(4), 1439–1445 (2014).
CAS PubMed PubMed Central Google Scholar
Yin, D. et al. Blood circuit reconstruction in an abdominal mouse heart transplantation model. J. Vis. Exp. https://doi.org/10.3791/62007 (2021).
Article PubMed Google Scholar
Lehmann, C. H. K. et al. DC subset-specific induction of T cell responses upon antigen uptake via Fcgamma receptors in vivo. J. Exp. Med. 214(5), 1509–1528 (2017).
CAS PubMed PubMed Central Google Scholar
Wang, X., Fu, J., Wilde, B., Lu, J., Zhu, J., Kribben, A., Witzke, O. & Hoerning, A. CXCR4-SDF1α blockade reduces the severity of murine heart allograft rejection [abstract #994]. Am. J. Transplant. 15(suppl 3) (2015).
Fu, J. et al. CXCR4-SDF1 alpha blockade reduces the severity of murine heart allograft rejection. Eur. J. Immunol. 47, 16 (2017).
Google Scholar

Download references

Acknowledgements

We would like to thank Tanja Hinkeldein and Barbara Nilewski-Kühl for excellent technical assistance and Professor Dr. Ute Eisenberger for helpful and constructive discussions. Parts of the data reported in this manuscript were presented at the American Transplant Congress in 2015⁹⁷ and at the congress of the German Society of Immunology (DGFI) in 2017⁹⁸.

Funding

Open Access funding enabled and organized by Projekt DEAL. This work was supported in part by the German Research Foundation (DFG) to promote international collaborations (HO2581/4-1 to AH), by the National Science Foundation of China (NSFC; #81760291 to FJ), by the German Research Foundation DFG (/RTG2504-401821119 (B2) to CHKL; /CRC1181-261193037 (A7) and DU548/5-1 to DD), by the Manfred Roth Stiftung (to AH) and intramural funding by the IZKF Erlangen (A87 to CHKL and A80 to DD). The authors have no financial relationships relevant to this study to disclose.

Author information

These authors contributed equally: Jian Fu and Christian H. K. Lehmann.

Authors and Affiliations

Department of Nephrology, University Hospital Essen, University Duisburg-Essen, Essen, Germany
Jian Fu, Xinning Wang, Benjamin Wilde & Andreas Kribben
Department for Pediatric and Adolescent Medicine, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Loschgestrasse 15, 91054, Erlangen, Germany
Jian Fu, Mandy Wahlbuhl, Ida Allabauer, Joachim Woelfle, Wolfgang Rascher & André Hoerning
The Emergency and Trauma Center, The First Affiliated Hospital of Hai Nan Medical University, Haikou, China
Jian Fu
Department of Dermatology, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Research Module II, Hartmannstr. 14, 91052, Erlangen, Germany
Christian H. K. Lehmann, Lukas Amon & Diana Dudziak
Department of Infectious Diseases, West German Centre of Infectious Diseases, Universitätsmedizin Essen, University Duisburg-Essen, Essen, Germany
Sebastian Dolff & Oliver Witzke
Department of Pediatric Cardiac Surgery, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), University Hospital Erlangen, Erlangen, Germany
Robert Cesnjevar
Department of Cardiac Surgery, Universitäts-Kinderspital Zürich, Zurich, Switzerland
Robert Cesnjevar
Department of Pediatrics II, Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, Children’s Hospital Essen, University Duisburg-Essen, Duisburg, Germany
Peter F. Hoyer & André Hoerning
Medical Immunology Campus and German Centre for Immuntherapy (Deutsches Zentrum für Immuntherapie-DZI) Erlangen, FAU Erlangen-Nürnberg, 91054, Erlangen, Germany
Christian H. K. Lehmann & Diana Dudziak
The Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China
Xinning Wang

Authors

Jian Fu
View author publications
You can also search for this author in PubMed Google Scholar
Christian H. K. Lehmann
View author publications
You can also search for this author in PubMed Google Scholar
Xinning Wang
View author publications
You can also search for this author in PubMed Google Scholar
Mandy Wahlbuhl
View author publications
You can also search for this author in PubMed Google Scholar
Ida Allabauer
View author publications
You can also search for this author in PubMed Google Scholar
Benjamin Wilde
View author publications
You can also search for this author in PubMed Google Scholar
Lukas Amon
View author publications
You can also search for this author in PubMed Google Scholar
Sebastian Dolff
View author publications
You can also search for this author in PubMed Google Scholar
Robert Cesnjevar
View author publications
You can also search for this author in PubMed Google Scholar
Andreas Kribben
View author publications
You can also search for this author in PubMed Google Scholar
Joachim Woelfle
View author publications
You can also search for this author in PubMed Google Scholar
Wolfgang Rascher
View author publications
You can also search for this author in PubMed Google Scholar
Peter F. Hoyer
View author publications
You can also search for this author in PubMed Google Scholar
Diana Dudziak
View author publications
You can also search for this author in PubMed Google Scholar
Oliver Witzke
View author publications
You can also search for this author in PubMed Google Scholar
André Hoerning
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

J.F. and C.H.K.L. contributed equally to these studies. J.F. performed allograft transplantation, harvested organs, performed flow cytometry and participated in writing the manuscript. C.H.K.L. recovered organs and performed flow cytometry, analyzed and interpreted the data and participated in writing the manuscript. X.W. performed flow cytometry and immunohistochemistry. I.A. and M.W. performed immunohistochemistry. L.A. participated in performing research and data analysis, B.W., S.D., J.W., A.K., W.R. and P.H. critically discussed the data and reviewed the manuscript. R.C. performed quality assessments of the surgical procedures of the heterotopic HTX model, D.D. and O.W. critically discussed and evaluated the data analyses, and critically reviewed and co-approved the final version of the manuscript. A.H. conceptualized the experiments and designed the study, supervised the data analysis and interpretation, drafted, composed and approved the final version of the manuscript.

Corresponding authors

Correspondence to Christian H. K. Lehmann or André Hoerning.

Ethics declarations

Competing interests

OW has received research grants for clinical studies, speaker’s fees, honoraria and travel expenses from Amgen, Alexion, Astellas, Basilea, Biotest, Bristol-Myers Squibb, Correvio, Chiesi, Gilead, Hexal, Janssen, Dr. F. Köhler Chemie, MSD, Novartis, Roche, Pfizer, Sanofi, TEVA and UCB. AH has received research grants for clinical studies, speaker’s fees, honoraria, and travel expenses from Abbvie, Novartis, Nutricia and Shire/Takeda. All authors declare no conflict of interest.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary Information.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Fu, J., Lehmann, C.H.K., Wang, X. et al. CXCR4 blockade reduces the severity of murine heart allograft rejection by plasmacytoid dendritic cell-mediated immune regulation. Sci Rep 11, 23815 (2021). https://doi.org/10.1038/s41598-021-03115-z

Download citation

Received: 03 December 2020
Accepted: 08 November 2021
Published: 10 December 2021
DOI: https://doi.org/10.1038/s41598-021-03115-z

This article is cited by

Intragraft immune cells: accomplices or antagonists of recipient-derived macrophages in allograft fibrosis?
- Xiaoping Li
- Jing Wu
- Jingtao Chen
Cellular and Molecular Life Sciences (2023)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.