To date, about 150 vascularized composite allotransplants (VCAs) including over 40 face and 120 extremity transplantations, have been performed worldwide. Approximately 40 VCA programs across five continents have been established to date, and more VCA centers are anticipated to be established in the future (1,2).

In the field of reconstructive surgery, the geometric uniqueness and often resulting functional deficit of a composite tissue defect are major challenges. Vascularized composite allotransplantation (e.g., facial VCA) has revolutionized restoration of form and function of the most complex defects. For example, facial transplantation can now be offered to selected patients at experienced centers with reproducible results (3-5). However, acute and chronic rejection as well as the resulting need for life-long multidrug immunosuppression (IS) limit the more widespread use of this revolutionary biotechnology.

Side-effects related to these IS protocols (e.g., increased susceptibility to infection, malignancy, and organ dysfunction) continue to adversely affect the risk-benefit ratio transplants, particularly in case of VCA which is not a lifesaving but rather a life-giving procedure.

In this context, tolerance induction, the long-term immunosuppression-free graft acceptance without clinical or histological evidence of rejection, has become the topic of many preclinical and clinical research endeavors. The strategy of tolerance induction is often viewed as the holy grail of achieving improved transplant outcomes for example by preventing or slowing the development of chronic rejection, which is still an important long-term cause of graft loss in solid organ transplantation and presumably also in VCA (6,7).

Given the inherent complexity and diversity of VCA procedures, different animal models have been established and evaluated, while additional models are being developed to help answer specific VCA related immunological questions. We present a brief review of the most commonly used preclinical approaches of tolerance induction in the field of VCA.

Tolerance induction strategies can be broadly categorized into cell- and pharmaceutical based strategies. The goal is to achieve graft acceptance and minimize the need for less specific and highly toxic systemic immunosuppression such as tacrolimus and MMF. The interest in cell-based strategies has been increasing over the past 10 years. Cell-based strategies include the use of stem (e.g.,: bone marrow transplant-derived, mesenchymal stem cells) and/or immune cells (e.g.,: dendritic cells, T reg cells) plus usually a co-administration of a drug therapy regimen (8). Figure 1 illustrates standard tolerance induction strategies that have been used in preclinical models of VCA. The goal of stem cell-based therapies is the establishment of mixed chimerism which was described in detail in 1988 by Sykes and Sachs as a phenomenon of host and donor bone marrow-derived cell coexistance (9). For example, mixed chimerism can be induced by transfer of donor hematopoietic stem cells (HSCs) from bone marrow or cytokine-mobilized peripheral stem cells. Typically, the recipient is conditioned in order to control alloreactivity and generate space for donor HSCs engraftment. The co-existence of donor and recipients HSC is thought to promote donor-specific tolerance while maintaining immunocompetence (10,11).

Mixed chimerism alone was shown to not consistently achieve tolerance induction and thus Leonard et al. described different approaches achieving mixed chimerism combined with adjuvant cellular therapies leading to tolerance induction in pigs (3,10). The longest tolerance induction through establishment of mixed chimerism was demonstrated in a pig model by Leonard et al. (12,13) in 2015 (14) after performing VCA in stable mixed chimerism or concurrent with induction of mixed chimerism following hematopoietic stem cell transplantation. Both groups showed no signs of rejection up to 504 days after transplantation.

To achieve a continuous supply of donor derived bone marrow elements, Barth et al. further analyzed the role of vascularized bone in a heterotopic partial face transplantat model in NHPs (15). VCA containing vascularized bone not only led to prolonged, but rejection free graft survival (430 days) compared to VCA without vascularized bone (7 days). Schneeberger et al. developed the “Pittsburg Protocol” as an approach in humans following upper-extremity transplantation. Bone marrow cell-based treatment allowed a maintenance low-dose immunosuppression protocol compared to conventional protocols (16). However, long-term results have not been published and further studies are needed to evaluate the safety and reliability of this approach.

Conditioning of the recipient entails toxic side effects, thus approaches of bone marrow infusion-mediated immunomodulation without the necessity of conditioning are studied at the time (17). In this context other immune cells have been studied. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into mesenchymal cell lineages. MSCs are derived from the bone marrow but can also be extracted from for example adipose tissue (= adipose derived MSCs) and have shown promising immunomodulatory effects by regulation of T-cell proliferation and inhibition of dendritic cell differentiation as they interact with the innate and adaptive immune system (18). Not only can MSCs induce mixed chimerism in the recipient, but they can also modulate cytokine expression in VCAs and hereby may be able to prolong graft survival(19). Promising results have been shown in small and large animal models, and clinical trials using mesenchymal stem cells have been established in solid organ transplants with promising results (8).

Ongoing clinical trials in humans evaluate the potential of dendritic cells as major regulator of the human immune system. An injection of immature dendritic cells was shown to have an immunosuppressant effect and to achieve inhibition of memory T cells (20). Another promising approach is the use regulatory T-cells. Tregs help maintain and regulate self-tolerance, antimicrobial resistance, tumor immunity and transplant rejection. Tregs exert their immunosuppressive effects via cell-to-cell interaction with target immune cells, via removal of IL-2 (potent factor in T cell survival and growth), via anti-inflammatory molecules (TGF-beta) or through costimulatory pathways such as binding to cytotoxic T-lymphocyte-associated protein 4. Nevertheless, irregularity in Tregs can lead to autoimmune disorders, reduced disease tolerance or higher risk for cancer(21). Recent studies have investigated the potential therapeutic value of genetically modified patient-derived Tregs, such as antigen-specific Chimeric Antigen Receptor (CAR)-Tregs, targeting for example MHC-class I expressed by donor cells. Tregs can be isolated from patient´s blood samples, cultured and expanded to produce for example CAR-Treg cells. Those conditioned T cells can be infused into the patient. Clinical trials did show the safety, efficacy, and proof of concept of Treg therapy in kidney and liver transplantation (22). The engineering of Tcells (e.g.: CAR-Tregs) might be a promising supplementary treatment of rejection in the future. CAR-T cells are typically designed to recognize donor MHC molecules, thus localizing to donor tissues and exerting their regulatory effects in a precise and targeted manner (21).

Pharmaceutical therapy regimens are the current gold standard in VCA and are mostly modifications of regimens that have already been established in SOT (23). Usually, an induction therapy (e.g.,: Anti-Thymoglobulin, Alemtuzumab) is administered first and a maintenance immunosuppressive regimen (Tacrolimus, Mycophenolate Mofetil (MMF), Prednisone) is given (8). Anti-Thymocyte globulin (ATG) is used as induction agent in most cases and leads to decreased T-cell mediated rejection (10). The maintaining therapy of the calcineurin inhibitor (CNI) Tacrolimus, Corticosteroids (Prednisone) and MMF (inhibits proliferation of lymphocytes) often involves side effects such as myopathy, diabetes mellitus, impaired kidney function, abdominal pain or diarrhea. Aside from the standard immunosuppressive regiments, other pharmaceutical approaches have been tested, for example tolerance inducing medications.

Cell mediated graft rejection in VCA is fueled by the CD28 and CD80/86 co-stimulatory pathway of T-cells (Figure 2). Preventing or interfering with co-stimulation of T cells is an approach for improved allograft survival and a more specific pharmaceutical approach that may cause less severe side effects compared to the conventional approaches (11). CD28 surface T-cell receptors communicate with antigen-presenting cells through CD80 and CD86 ligands and evokes both T-cell proliferation and pro-inflammatory pathways. Drugs like Belatacept or Abatacept are already established anti-rejection medications used in humans to interfere with co-stimulatory pathways. Both inhibit the costimulatory pathway through CTLA4-Ig or CD 125 (Fusion protein of cytotoxic T-lymphocyte-associated antigen 4 Immunoglobulin and IgG1 Fc) by inhibiting the linking of CD28 (T-cell surface) and CD80/86 (APC surface) (15,17). Belatacept can serve as replacement for CNI after kidney transplantation as side effects appear to be less than with CNI. Belatacept is already in clinical application following hand and/or face transplantation along with low dose of CNI/MMF/prednisone. Subsidiary, topical administration of immunosuppression might also reduce the systematically dosage of immunosuppressants. Tacrolimus and steroids are available as creams or mouthwashes as “transplant-targeted therapy” and show promising results in first approaches (8).

Rodent models are of great value especially in VCA research and are most popular for preclinical studies as they allow precise genetic breeding, are easy to reproduce and handle, produce low costs in housing and breeding and enable therefore larger study groups. The majority of the described animal models in this article used rat models and most commonly the hindlimb model was used. Mice allow more precise genetic manipulation which makes analysis of immunological mechanisms easier when compared to other small animal models. However, disadvantages of rat and mice models are related to their smaller size in terms of anatomic structures and the lack of similarity to the human immune system. Furthermore, the results of rodent models have often failed to be successfully transferred to large animal models or human trials (8). In addition, their short lifespan can make it difficult to truly assess long-term graft survival. Most importantly, the development of a naive immune system in largely germ-free laboratory environments and the lack of class II antigen expression on rodent vascular endothelium suggests a fundamentally different immune system than that of humans (11, 24-27).

Large animals are suited as preclinical models as they allow evaluation of a hypothesis in a complex system that has strong similarities to humans in terms of anatomical structures, size, and immune responses, making data potentially more reliable for translation to humans (28). Swine models also allow genetic manipulation to control for example the degree of MHC mismatch in transplantation and share great similarity with humans in structure, cellular and antigenic attributes in skin (8). NHP models are also of great value as they are even more similar in human anatomy than pigs and showed similar transplant rejection to humans. Hand transplantation is feasible and did show good motor function in NHPs (29). Disadvantages are cost and for example with primates’ ethical concerns, slower reproductive cycles. Especially the skin of humans and pigs show a great similarity which is even more important with skin being a crucial part of VCA and complicating factor on the way to long-term graft survival and tolerance induction. Having a great similarity with pig skin makes pig animal model most valuable for research. Finally, the high costs of livestock breeding and research in large animals, the ethical status in NHP models on the one hand and the simplicity of reproducibility, handling of rodents and low costs on the other hand may explain why most research groups are using rodents.

Both small and large animal models are relevant for preclinical VCA research. Small animal models are advantageous because of low costs in breeding and housing and especially mice are well suited for analysis of immunological mechanisms. Large animal models offer greater similarity to human immune system and human anatomy, but models tend to be more complex and cost intensive. Tolerance induction in preclinical models was achieved using both cellular based and pharmaceutical based strategies. Clinical translation of such strategies to human trials has yet to be done successfully, however, the induction of donor-specific tolerance may ultimately help improve the risk benefit ratio of VCA transplantation.