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T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation

Summary Limb transplantation is a life-changing procedure for amputees. However, limb recipients have a 6-fold greater rejection rate than solid organ transplant recipients, related in part to greater immunogenicity of the skin. Here, we report a detailed immunological and molecular characterization of individuals who underwent bilateral limb transplantation at our institution. Circulating Th17 cells are increased in limb transplant recipients over time. Molecular characterization of 770 genes in skin biopsies reveals upregulation of T cell effector immune molecules and chemokines, particularly CCL18. Skin antigen-presenting cells primarily express the chemokine CCL18, which binds to the CCR8 receptor. CCL18 treatment recruits more allo-T cells to the skin xenograft in a humanized skin transplantation model, leading to signs of accelerated graft rejection. Blockade of CCR8 remarkedly decreases CCL18-induced allo-T cell infiltration. Our results suggest that targeting the CCL18:CCR8 pathway could be a promising immunosuppressive approach in transplantation.


INTRODUCTION
Vascularized composite allotransplantation (VCA), including limb transplantation, is a life-changing procedure for individuals who have suffered severe traumatic injuries. Unlike solid organ transplantation, VCA involves transplantation of multiple tissues with different immunogenicity levels, including skin, muscle, bones, and nerves. Among these, the skin has the highest immunogenicity of all 1 because of the presence of the following components: a dense population of antigen-presenting cells (APCs) and resident T cells, a rich microbiota, and continuous exposure to environmental threats, both physical and chemical. These unique skin features may explain the 6-fold greater rejection rate of VCA recipients compared with solid organ transplant recipients. 2 More than 85% of individuals undergoing VCA experience acute cellular rejection in the first year after transplantation, and many have multiple episodes of rejection, leading to a higher burden of immunosuppressive therapy over time. 3 It is reported that the number of rejection episodes may portend forthcoming chronic rejection events and graft loss. 2 A better understanding of key distinctions between VCA and solid organ transplantation immune responses is essential for discovery of novel markers of rejection and predictive biomarkers.
Here we identified unique molecular signatures in skin graft biopsies at times of rejection that were dominated by T-effector molecules and chemokines, particularly CCL18. The CCL18 chemokine is present only in primates, with no murine ortholog, and has been identified as a key chemokine in the skin. CCL18 has been described to bind to the CCR8 receptor 4 and induce homing of T cells in inflammatory skin conditions. [5][6][7] Based on the unique rejection signal on graft biopsies, we sought to investigate the role of CCL18 in skin transplantation. By using a humanized skin transplantation model, we observed that CCL18 treatment recruited more allo-T cells to the skin and led to accelerated damage of the xenograft. This effect was abrogated markedly by treating the recipients with an anti-CCR8 blocking antibody. We characterized immunological changes in the graft microenvironment compared with proximal native skin tissue. Our data suggest an increase in T cell infiltration and action in the graft microenvironment compared with native tissue. We demonstrated a role of the chemokine CCL18 in attracting inflammatory allo-T cells to skin grafts, identifying the CCL18-CCR8 pathway as a potential therapeutic target in transplantation.

Expansion of circulating Th17 cells after transplantation
Th17 cells have a pathogenic role in different skin inflammatory disorders. 8,9 We reasoned that Th17 cells would be enriched over time upon limb transplantation. Three individuals who received limb transplants in our institution between October 2011 and August 2016 were included in this analysis, with a mean follow-up of 5.2 years. The clinical characteristics of these individuals are detailed in Table 1. The individuals' pre-and post-operative appearance is shown in Figure S1. Detailed information about the individuals' immunosuppression is given in the STAR methods. We initially characterized T cell subsets from peripheral blood collected over time after transplantation according to our protocol (pre-transplantation, 24 h, 1 week, and 3, 6, and 12 months; Figure 1A). Analyses of the effector and memory T cell subsets ( Figure S2) revealed that CD4 + central memory T cells (TCM cells; CD45RA -CCR7 + ) were the predominant T cell phenotype in the pool of CD4 + T cells, although they decreased after transplantation, whereas CD4 + effector memory T cells (TEMs; CD45RA -CCR7 -) increased over time ( Figure 1B). TEMs and effector memory CD45RA + T cells (TEMRA cells; CD45RA + CCR7 -) were the main subsets presented in the pool of CD8 + T cells with a decrease in naive CD8 + T cells (CD45RA + CCR7 + ) after transplantation ( Figure 1C). Next, we as-sessed the T helper (Th) phenotypes based on CXCR3 and CCR6 expression ( Figure 1D). Circulating Th2 cells were the predominant phenotype in individuals with upper extremity transplantation over time ( Figure 1E). Th17 cells were increased markedly after transplantation ( Figure 1F), whereas Th1 cells were stable over time (Figure 1E). The percentage of circulating regulatory T (Treg) cells (CD4 + CD25 + CD127 À/low cells; Figure 1G) and T follicular helper (Tfh) cells (CD4 + CXCR5 + PD-1 + cells; Figure 1H) had no significant expansion after transplantation, other than transient changes early after transplantation, likely related to use of depletion induction therapy. These findings indicate that Th2 cells were the dominant phenotype over time after transplantation, with a significant expansion of circulating Th17 cells.
Increased T cell infiltration and activation in the nonrejecting graft microenvironment compared with native skin In transplantation, characterization of the T cells infiltrating human allografts compared with native tissues is technically limited by the small sample sizes collected in punch biopsies. We took advantage of debulking surgeries performed in our limb transplant recipients to examine T cells in the graft microenvironment at nonrejection time points and compared it with recipients' adjacent native skin removed during the procedure. Allografts and native skins were processed, and infiltrating cells were isolated and stained by flow cytometry ( Figure S3). Allografts had higher frequencies of activated CD4 + T cells, including CD4 + TEM cells (Figure 2A), Th1 cells (CD4 + CXCR3 + CCR6 À ; Figure 2B), and Th17 cells (CD4 + CXCR3 À CCR6 + ; Figure 2C) compared with native skin. Similarly, total CD8 + cells ( Figure 2D), CD8 + TEM ( Figure 2E) and TEMRA cells ( Figure 2F) were also increased markedly compared with native skin. Immunofluorescence

Expansion of circulating Th1 and TEM cells during rejection
In our cohort, all individuals developed at least one episode of acute cellular rejection within the first 3 years of transplantation (a total of 9 episodes; range, 2-4); about half occurred during the first 3 months after transplantation, whereas the remaining occurred later (>1 year after transplantation). Clinical aspects of the rejection included a maculopapular rash and edema ( Figure 3A). Acute cellular rejection was assessed using Banff grading of skin-containing composite tissue, 10 and most clinical rejection episodes were classified as between grades 2 and 3 ( Figure 3B). There were no graft failures or recipient deaths. To characterize the unique features of rejection compared with nonrejection time points in limb transplantation, we characterized circulating T cells during those time points. For the rejection events, we selected blood samples corresponding to a respective Banff grading of 2, 2/3, or 3 skin biopsies from all three individuals. For nonrejection time points, the samples revealed grade 0 or 1 (grade 1 biopsy findings are regarded as non-specific for rejection and are not treated at our institution but monitored closely over time 11 Figure 3I), whereas absolute numbers tended to be decreased at rejection time points compared with nonrejection ( Figure 3J). In the peripheral blood, we observed an increase in interferon (IFN)-g-producing CD4 + T cells ( Figure 3K) and a decrease in interleukin-4 (IL-4) production by CD4 + T cells during rejection ( Figure 3L). No changes were observed in production of IL-17 by circulating CD4 + T cells ( Figure 3M) or IFN-g production by CD8 + T cells ( Figure 3N) during rejection events compared with nonrejection.

Molecular characterization of the graft microenvironment during rejection
To determine the molecular characteristics of the skin tissue associated with rejection, we compared the gene expression profiles of rejection (grades 2-3) and nonrejection (grade 0) events using NanoString technology. Among 770 genes analyzed, 57 genes were differentially expressed during rejection (log2 fold change > 2; unadjusted p < 0.01). The differentially expressed genes (DEGs) are displayed in Figure 4A. A principal-component analysis (PCA) was performed for the top 57 DEGs and demonstrated separate clustering of samples with rejection compared with nonrejection events, except for one resolving rejection that clustered with the nonrejection samples ( Figure 4B). We used Gene Ontology analysis (GO) to better assess the biological processes that occurred during rejection in the allograft microenvironment. Immune, inflammatory, and chemokine-related responses were strongly associated with the DEGs during the rejection events ( Figure 4C). Skin biopsies with rejection had a distinct gene signature compared with nonrejection biopsies.

Rejection is characterized by expression of T cellrecruiting chemokines
From the distinctive gene signature during rejection ( Figure 5A), the single most upregulated gene was CXCL13 ( Figure 5B; log2 fold change = 6.1 compared with nonrejection). Following this, many of the top upregulated genes encoded for proteins associated with chemokines and chemokine-mediated signaling (CCL18, CCL17, CXCL9, and CCL5; Figure 5B), with CCL18 having a 2-fold increase during rejection. We also found increased gene expression in association with T cell co-stimulation (TNFRSF4 and CD28; Figure 5C) and effector immune molecules (GZMB, GZMA, KLRK1, and GNLY; Figure 5E). On the other hand, rejection biopsies also showed increased expression of inhibitory immune checkpoints (LAG3, CTLA4, and CD274; Figure 5D), suggesting that regulatory pathways may be triggered during rejection to counterbalance strong inflammatory responses.
CCL18 enhances recruitment of allogeneic T cells to human skin xenografts Among the chemokines upregulated during rejection, CCL18 was particularly interesting, based on its primary expression in skin dendritic cells, Langerhans cells, and macrophages [12][13][14] and its role in recruiting T cells in inflammatory skin conditions such as atopic dermatitis. 5 Therefore, we evaluated the in vivo effect of CCL18 and its receptor CCR8 in a humanized skin transplantation model because CCL18 has no murine ortholog (Figure 6A). In this model, we transplanted human foreskins onto genetically immunosuppressed NSG mice and then administered allogeneic PBMCs 4 weeks after the skin transplantation. One week later, animals received daily subcutaneous injections of recombinant human CCL18 or PBS 13 into human skin xenografts for 10 days ( Figure 6A). In a subgroup, CCL18-treated animals were administered an anti-CCR8 blocking antibody or isotype control daily starting on the day of PBMCs transfer (Fig-ure 6A). Among the NSG recipients that had received PBMCs, subcutaneous injections of CCL18 into human skin xenografts led to significant macroscopic changes, including tissue shrinkage, discoloration, and dry appearance ( Figure 6B). Anti-CCR8 treatment reduced the macroscopic signs of graft rejection induced by CCL18 ( Figure 6B). To evaluate whether CCL18 deleterious effects on skin xenografts were dependent on the recipient's immune system, a subgroup of animals was treated with subcutaneous injections of CCL18 or PBS 13 in the absence of PBMCs ( Figure 6A). Interestingly, CCL18 treatment did not induce macroscopic changes in skin xenografts of animals that did not receive PBMCs, suggesting that CCL18 effects are dependent on the recipient's immune system ( Figure 6C). Histologically, xenografts treated with CCL18 demonstrated reduced CD31 + (vascular endothelium) staining ( Figures 6B and 6D) and increased presence of CD3 + cells ( Figures 6B and 6E) compared with PBS-injected animals. The anti-CCR8 treatment restored the presence of CD31 + structures ( Figures 6B and 6D) and decreased CD3 + cells ( Figures 6B and 6E). NSG mice that did not receive PBMCs presented the highest levels of CD31 + vessels that were not affected by CCL18 treatment (Figures 6C  and 6D). Xenograft-resident human CD45 + cells were unchanged in CCL18-treated animals that did not receive PBMCs ( Figures  6C and 6F). Thus, our data suggest that CCL18 can have major deleterious effects on skin xenografts, which is dependent on the presence of human immune cells.
We next assessed and quantified recruitment of T cells to the skin using T cell extraction protocols and flow cytometry. CCL18 significantly increased the numbers of CD4 + and CD8 + T cells per skin area ( Figure 6G and S4). The anti-CCR8 treatment markedly decreased CCL18-induced recruitment of CD4 + and CD8 + T cells ( Figure 6G). CCL18 has been shown to recruit cutaneous lymphocyte-associated antigen (CLA) + T cells to the skin microenvironment. 5 We observed enhanced numbers of CLA + CD4 + and CLA + CD8 + T cells ( Figure 6H) as well as CCR8 + CD4 + and CCR8 + CD8 + T cells ( Figure 6I) in skin allografts treated with CCL18. In contrast, CCL18-induced recruitment of CLA + and CCR8 + T cells was reduced markedly in anti-CCR8-treated animals ( Figures 6H and 6I). Last, we found that CCL18-treated skin was associated with an increased number of Th1, Th2, and Th17 CD4 + T cells compared with vehicle-treated skin, whereas anti-CCR8 treatment inhibited this recruitment ( Figure 6J). Our data suggest that CCL18 is an important local chemokine that can increase recruitment of CLA + and CCR8 + allo-T cells to human skin xenografts and accelerate graft rejection. CCR8 blockade can substantially abrogate CCL18-induced T cell recruitment and pathogenic effects in skin xenografts.

DISCUSSION
This study demonstrates that circulating immune cells from limb transplant recipients with no significant graft rejection have predominant Th2 and Treg cell phenotypes that are shifted to Th1 and CD8 responses during rejection. A similar peripheral immune profile was observed in face transplant recipients, as described previously by our group. 15 However, despite the increased circulating Th17 cells over time, we did not observe an increase in Th17 cells infiltrating the allograft during rejection, as demonstrated in face transplant recipients. 15 On one hand, these data suggest that, although skin is the main organ targeted by the immune system in both cohorts of individuals, the dominant effector response appears to differ. On the other hand, chronic rejection has been reported for VCA patients, 16 and this increase in circulating Th17 cells has been associated with chronic graft injury in kidney transplant recipients, whereas reduced Th17 cells have been linked to allograft tolerance. 17,18 In our previous work, focused on face transplant rejection, 19 we found that effector cells represented contributions from Article ll recipient and donor immune pools. Moreover, donor T cells in rejecting grafts exhibited resident memory phenotypes, implicating local expansion in the transplanted tissue. Targeting events appeared to involve primarily cutaneous venules as well as epithelial domains in the epidermis and hair follicles, where keratinocyte stem cells normally reside. 19 Whether key differences will also emerge in the evolutionary immunopathology between limb and face transplant rejection is a topic in need of further study. However, it is intriguing that Th17 cell pathways are implicated in limb transplantation in the context of data showing that IL-17 can target and activate skin epithelial stem cells through the TRAF4-ERK5 axis. 20 The skin is an immunologically rich tissue with more than $1 3 10 6 resident T cells/cm 2 21 and a diverse and dynamic population of APCs. 22 Seminal studies by Murray 1 have suggested that the skin is the most immunogenic organ. Different skin locations are exposed to diverse external physical and chemical insults that may affect the skin microbiome and the local immune response. 2,23,24 This exposure could cause local non-specific inflammation and mimic alloimmune injury. Through debulking surgeries, we were able to evaluate the local immune profile in the allograft microenvironment and compared it with the adja-cent native skin, demonstrating higher numbers of activated T cells in non-rejecting allograft skin. The comparison with adjacent native tissue is crucial because skin from different body areas has significant variation in its immunological content. 25 In agreement with our data, an independent study demonstrated that the cellular infiltrates in skin biopsies from hand transplant recipients were predominantly composed of T cells. 26 Because allograft and adjacent native skin tissues were exposed to the same external factors, our data suggest a more immunologically active environment in the allograft, likely related to continuous local shedding of alloantigens that primarily trigger the adaptive immunity and potentially the innate memory alloresponse. 27 Alternatively, it is possible that leukocyte-endothelium interactions responsible for T cell trafficking and accumulation are altered even in a homograft setting, and this important control situation requires further scrutiny to define this issue.
CCL18 is a chemokine produced by APCs from the dermis and epidermis as well as by keratinocytes, and skin-homing human T cells express CCR8. 28 Increased levels of CCL18 have been linked to atopic dermatitis, 5 psoriasis, 29 allergic contact hypersensitivity, 30 and other human chronic inflammatory diseases. 31 In this report, we found an increase in CCL18 in skin tissue during Article ll OPEN ACCESS rejection of VCA. In a humanized skin transplantation model, we demonstrated that local CCL18 injection led to a higher infiltration of T cells in the xenografted human skin. Our results suggest that CCL18 may contribute to VCA rejection by promoting binding of CLA + T cells and increasing homing of human memory T cells to the skin. 5 Supporting this possibility, we also observed an increase in skin-infiltrating CLA + T cells following CCL18 treatment. These cells have been described to mainly have a memory Th1 cell phenotype 28,32 and collaborate in immune surveillance of healthy skin. When activated T cells infiltrate the skin Figure 6. CCL18 attracts human allo-T cells to human skin xenografts (A and B) Discarded human skin was transplanted into NSG recipient mice. Mice were injected weekly with anti-Gr1 to reduce local inflammation and establish an intact vasculature (A). Twenty-eight days after transplantation, recipients were injected with 20 3 10 6 allo-PBMCs. Seven days thereafter, 300 ng of CCL18 or PBS 13 was injected subcutaneously into the skin xenografts for 10 consecutive days. Recipient animals were treated with anti-CCR8 or isotype control daily. Skin xenografts and peripheral blood were analyzed by histology and flow cytometry on day 45 after transplantation. (B-D) Representative macroscopic images of skin xenografts, H&E graft staining, and immunohistochemical staining for human CD31, human CD3, or human CD45 after the CCL18 injections into animals (A) injected (B) or not injected (C) with human PBMCs. Shown are absolute counts of human CD31 + vessels. (E and F) Human CD3 + cells (E) and human CD45 + cells (F) in the skin xenografts, 1003. The number of positive cells was quantified from two representative 4003 fields from each transplanted xenograft. Statistics by one-way ANOVA with Tukey's post-test. (G-J) Representative contour plot and absolute numbers of skin-infiltrating (G) CD4 + and CD8 + cells, (H) skin CD8 + CLA + and CD4 + CLA + cells, (I) skin CD8 + CCR8 + and CD4 + CCR8 + cells, and (J) Th1, Th17, and Th2 in PBS-, CCL18 À , or CCL18 + anti-CCR8-treated groups that received human PBMCs. All data were normalized by square centimeter of tissue. Data represent a pool of two independent experiments (n = 4-6 animals per group) and are represented as mean ± SD. Statistics by one-way ANOVA with Tukey's post-test. See also Figure S4 Identification of potential biomarkers of rejection is of paramount importance to provide additional tools to diagnose rejection, help physicians in their decision-making about treatment, and develop new therapies. Our group has previously identified serum MMP3 protein as a potential biomarker to stratify VCA recipients according to the severity of rejection. 33,34 Here, besides the increase in CCL18, we also observed an increase in different chemokines, including CXCL9 and CCL5. Along the same lines as our findings, Hautz et al. 26 have demonstrated that markers related to lymphocyte trafficking correlated with the severity of skin rejection in a cohort of five limb transplant recipients. Thus, limb transplant rejection is characterized by upregulation of lymphocyte-attracting chemokines and trafficking markers. These markers comprise potential targets for immunosuppressive drugs. Different chemokines have been reported to be upregulated and have a role during skin allograft rejection responses. CXCL9 and CCL5 have been demonstrated to be upregulated in skin allografts, but not isografts, a few days before rejection. 35 CXCL9, CXCL10, and CCL5 are associated with kidney rejection, and their presence in the urine of transplant recipients is being explored as a rejection biomarker. [36][37][38] This highlights the potential of chemokines to be used as biomarkers.
Our small number of individuals reflects, in part, the novelty and challenges of extremity transplantation in humans. Despite this, our study employed prospective blood and skin graft collection in combination with high-throughput technologies like Nano-String to undercover unique aspects of the rejection process in extremity transplant recipients that may account for its relatively high rejection rate. This comprehensive report of limb transplant recipients is a result of the assembly and curation of a unique biobank with more than 45 time points involving surveillance and rejection episodes. It is now important to validate the pathogenic role of CCL18 and other potential T cell-attracting chemokines in other limb and VCA transplantation cohorts.
Limb transplantation is a clinically feasible procedure for amputees, and use of a solid organ transplantation-based immunosuppressive regimen has yielded good short/medium-term graft outcomes. Nonetheless, the high frequency of cellular rejection is a concerning long-term barrier. Development of novel biomarkers in larger cohorts and less toxic, narrowly targeted skin-specific immunosuppression strategies are critical to advance the field.
Limitations of the study Limitations of our study include its single-center nature and the small number of individuals evaluated, limiting major extrapolations. Our findings need to be validated in other limb and VCA cohorts from different centers. We also acknowledged that, although NanoString is a useful tool, its detection capacity is limited to fewer than 1,000 transcripts. More comprehensive analyses, like single-cell RNA sequencing, could have identified other transcriptional pathways unique to the rejection process in limb transplant recipients. Finally, the results from the humanized skin transplantation are limited by the variability found in this model and the inability to fully recapitulate the complexity of the human immune system.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact (lriella@ mgh.harvard.edu).

Materials availability
This study did not generate new unique reagents.

OPEN ACCESS
Data and code availability All data reported in this paper will be shared by the lead contact upon request. This paper does not report the original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Upper extremities transplant subjects and study approval Three patients who received upper extremities transplants at the Brigham and Women's Hospital were included in the study. All patients provided written informed consent to participate in the clinical trial (ClinicalTrials.gov NCT01293214) for upper extremities transplantation, as approved by the Human Research Committee at Brigham and Women's Hospital (2009P001719). Before participation, patients were evaluated by our multidisciplinary team. Donors and recipients were matched according to sex, skin color, and ABO compatibility, in addition to a negative T and B cell cytotoxic crossmatch. Demographic details are displayed in Table 1. Patients were followed weekly during the first 4-6 weeks after transplantation and if stable, clinical visits were further spaced to every 2 weeks, every month, and then every 3 months. After the first year of transplantation, the clinical visits were scheduled semiannually.

Patients' immunosuppression
All patients received mycophenolate mofetil (1,000 mg), methylprednisolone (500 mg), and rabbit anti-thymocyte globulin (1.5 mg/kg/ day 3 4 days) for induction therapy starting at the time of transplantation. Maintenance immunosuppression consisted of mycophenolate mofetil (initially 1,000 mg twice daily, and reduced to 500-750 mg twice daily in long-term), tacrolimus (adjusted to achieve target levels of 10-15 ng/mL in the first 6 months, followed by 8-12 ng/mL up to 1 year and 6-10 ng/mL thereafter), and prednisone taper (down to 20 mg on day 5, and 5-7.5 mg in long-term) ( Table 1). Prednisone withdrawal was attempted in Patient 1, but due to the higher occurrence of acute rejection episodes during winter months, low dose prednisone (5 mg) was seasonally reinitiated. 24 Perioperative antibacterial prophylaxis consisted of vancomycin and cefazolin and was modified according to perioperative findings. All patients received trimethoprim-sulfamethoxazole and valganciclovir prophylaxis against Pneumocystis jirovecii and cytomegalovirus, respectively, for R6 months. In the presence of clinical acute cellular rejection, patients were treated with pulse solumedrol 500 mg/day for 3 days, followed by a taper. Acute rejection episodes with no clinical signs were treated with an increase in maintenance immunosuppression and closely followed. Topical steroids or tacrolimus were also used in a few cases as adjuvant therapy. In case of no response, further T cell-depletion therapy (rabbit anti-thymocyte globulin, alemtuzumab) was attempted.