Over 65 y after the first human kidney transplant in 1954 by Dr Murray, irreversible tissue loss remains a challenging clinical problem despite distinctive advancements in translational surgery research. Nowadays, the transplantation of hearts, livers, kidneys, and even faces between unrelated human beings is possible.1,2 Although transplantation represents the most complete option for functional restoration of form and function, it requires the patient to be on lifelong multidrug immunosuppression. This entails a wide array of possible side effects, such as increased risk of infection, metabolic complications, and malignancies.3 Furthermore‚ the long-term survival of the allograft can be subject to acute and chronic graft rejection in solid organ transplantation (SOT) and vascularized composite allotransplantation (VCA), such as face, hand, penile, or abdominal wall transplants.1,4–10 Given the higher rate of diagnosed acute rejection episodes in up to 85% of VCA recipients, the link between long-term allograft survival and rejection episodes could theoretically even be more pronounced in VCA.11
One approach to optimizing this risk–benefit ratio is the development of therapies allowing for safe reduction of immunosuppression. In this setting, the engineering of tolerance promoting immune cells has gained popularity. This approach aims to exploit immune system functions and mechanisms that naturally ensure unresponsiveness toward self-antigens. These cell lines can be harnessed to promote unresponsiveness to foreign antigens. One such approach is the bioengineering of antigen-specific regulatory T cells (Treg).12
DEFINITION AND FUNCTIONING OF TREG
Treg represent a subpopulation of T helper cells. Treg comprise different subsets and were initially defined as FoxP3+CD25+CD4+ T cells‚ which represent the central subpopulation discussed in this review.13 Currently, Foxp3+CD25+CD127-CD4+ T cells represent the most common Treg phenotype.14 Regarding the CD4+ Treg subset, natural Treg develop in the thymus during positive and negative selection process, whereas induced Treg are found in the periphery emerging from conventional CD4+ T cells following antigenic stimulation.15 Of note, specific human markers for both subsets remain to be ascertained‚ whereas fluctuations of FoxP3 stability in induced Treg mark a distinct difference. In terms of differentiation, Treg are subdivided into naive, central memory, effector memory, and effector cells displaying various expression combinations of CD127, FoxP3, CD45-RA, CCR7, or CD62L.13 For example, CCR7 and CD62L molecules represent homing factors navigating Treg into the secondary lymphoid organs.16 CD8+ Treg have been identified by Gershon and Kondo in 1970, but the body of evidence regarding their biology and overall function is comparably limited.17 CD8+ Treg will be addressed in the section CAR-Treg in Transplantation.
Treg play a key role in maintaining homeostatic immune processes such as regulation of self-tolerance, antimicrobial resistance, tumor immunity, and transplant rejection.18
Tang and Bluestone have outlined 4 key areas of Treg as living drugs‚ including suppression, specificity, stability, and survival.19
In terms of suppression, Treg exert their immunosuppressive potential either via direct cell-to-cell interaction with target immune cells or via the deprivation of interleukin-2 (IL-2; a potent factor in overall T-cell survival and growth),20 via the release of anti-inflammatory molecules such as transforming growth factor beta (TGF-β),21,22 or through the costimulatory molecules from antigen-presenting cells (APCs) via high-affinity binding to cytotoxic T-lymphocyte–associated protein 4.23 Hence, Treg are considered potent suppressive cells because of the intrinsic properties of broad T-cell suppression (bystander suppression) and induction of other suppressive cells by infection tolerance.24–26 Currently, the suppressive potency of Treg is quantified by measuring the degree to which they inhibit the proliferation of T effector cells in vitro or prevent graft-versus-host disease (GvHD) in immunodeficient mice.19 Antigen-activated Treg can suppress nearby conventional or naive T cells with differential antigen specificity via linked suppression when APCs simultaneous express both antigens on their surface.27 Via infectious tolerance, Treg can transform conventional T cells into induced Treg by secreting the immunosuppressive cytokines TGF-β, IL-10, or IL-35 or by interfering with dendritic cells (DCs).28
Yet, this mode of action is very sensitive because any irregularity in Treg function and count leads to reduced disease tolerance or autoimmune disorders. In addition, they can hinder the immune detection of malignancies in healthy individuals‚ as well as prevent the progression of implicit antitumor immunity in individuals with tumors.
Nonetheless, it is subject to current scientific work whether and via which pathways Treg help in shielding hosts against the manifestation of autoimmune diseases and allergies. Therefore, Treg have been used to modulate immune responses in transplantation, autoimmune diseases, and gene therapy.29–32
Consequently, restoring immune homeostasis and tolerance by providing, enhancing, or activating Treg has recently become an emerging focus for novel adoptive cell therapies.
In the field of SOT, preclinical studies suggested that the infusion of Treg holds the potential to regulate alloimmune responses in a targeted manner and to promote sustained immune acceptance of transplants.33–36
The general safety for adoptive transfer of polyclonal Treg to regulate antigen-independent bystander suppression has been verified in early clinical research studies to protect against graft rejection.37,38 Nevertheless, the use of these polyclonal Treg tends to impair sufficient immune responses, for example, in response to life-threatening pathogens, and increase the risk of cancer in patients.39,40 Similarly, the nonspecific antigen recognition of polyclonal Treg has counteracted their therapeutic efficacy in preliminary clinical findings. Nonengineered Treg may also convert into proinflammatory T helper cells under specific immunological conditions, such as various autoimmune diseases.41
It is important to differentiate between cellular (T-cell mediated) and antibody-mediated (humoral) allograft rejection (AMR). T cells are the protagonists of cellular rejection that primarily target the epithelium (epithelial targeting) in VCAs.3,42 On the other hand, B cells and plasma cells are considered mediators of AMR leading to mostly endothelial damage in transplantation.3,43 Treg have been studied for regulation of both pathways of allograft rejection with Liao et al‚44 demonstrating the potency of Treg in controlling AMRs in immunocompetent mice receiving renal allografts. The authors found Treg to influence the levels of circulating DSAs, as well as the presence of antibody deposition within allografts. Furthermore‚ Treg hindered the infiltration of AMR-driving cells, such as natural killer cells, B cells, and plasma cells.44
METABOLIC PATHWAYS AND IMMUNOMETABOLISM IN TREG
For Treg stability and survival, Foxp3 expression is considered a hallmark of the Treg population and can be modulated by various metabolites and vitamins. All-trans retinoic acid promotes activation of extracellular-related kinase signaling‚ which drives Foxp3 expression. Interestingly, all-trans retinoic acid may preserve Foxp3 expression during Treg expansion and inflammatory processes‚ yielding stronger effects than rapamycin (ie, an mechanistic target of rapamycin complex 1 inhibitor; known to stabilize Foxp3 expression).45 Vitamin C yields similar effects in preventing the loss of Foxp3 expression but acts via demethylation of the CNS2 region‚ which is orchestrated by the Tet enzyme family.46 Although Foxp3 occupies a key position in Treg function, Treg-specific epigenetic changes (eg, CpG demethylation and histone modification) ensure consistent patterns of Treg gene expression also in a Foxp3-independent manner. Of note, single nucleotide polymorphisms in Treg-specific DNA demethylated regions associated with Treg signature genes like CD25 have been linked to altered function in naive Treg.47
Treg preferentially utilize mitochondrial metabolism and oxidative phosphorylation for cellular energy production and to exert their immunosuppressive activity. The AMP-activated protein kinase pathway is one of the central signaling routes in Treg because it mediates their response to cell stress by increasing oxidative phosphorylation.48 Other pathways, such as the PI3K/Akt/Foxo signaling, have been identified to regulate Treg plasticity besides influencing metabolic programming.49 In the tumor microenvironment, Treg compete with cancer cells for extracellular glucose, glutamine, or leucin with the latter being drivers of Treg differentiation.50 Furthermore‚ anaerobic and facultative aerobic gut organisms, such as Clostridium perfringens, Bacteroides fragilis, and Lactobacillus spp., have been shown to promote Treg induction.51 Serena et al52 demonstrated that butyrate-producing bacterial flora increased Treg effects in a celiac disease model.
TREG, T-CELL RECEPTOR, AND CHIMERIC ANTIGEN RECEPTOR
Acute and ultimately chronic transplant rejection is thought to be driven by recognition of donor non-self HLA antigens (major histocompatibility complex(MHC) I and II) by recipient immune cells. Hence, designing Treg that recognize a unique donor HLA molecule ensures accumulation of Treg in the graft tissue‚ which can then tame alloreactive recipient T effector cells (or donor resident memory T-cell–induced aggression). Antigen specificity significantly increases effectiveness of Treg‚ and thus‚ engineering of a chimeric antigen receptor (CAR) has revolutionized the field of targeted cellular therapy.19 A clinical example is the design of (CAR)-T-cells targeting CD19 antigen for the treatment of B-cell acute lymphoblastic leukemia and large B-cell lymphoma.19
In the following, we discuss isolation methods of Treg and review basic principles of CAR design. In the scientific literature, CD25 is considered the standard marker for Treg population and is thus deployed for their isolation via various isolation methods.14 Magnet-based Treg isolation has shown promising outputs.53 Flow cytometric sorting allows for increased purity because of the inclusion of additional markers, such as CD127 and CD45RA,54,55 but sorters fulfilling the good manufacturing practice guidelines are not widely accessible to this date.56 In general, Treg need to be expanded in vitro to produce clinical dosages because they account for <1% of white blood cells.30 To this end, anti-CD3/CD28 beads, IL-2, or artificial APCs are added to the cell suspension following the isolation process.29,57,58 Polyclonal Treg exhibit multiple T-cell receptor (TCR) specificities and thus execute their therapeutic impact via bystander immunosuppression.59 This population interferes with a plethora of antigens, eventually resulting in severe off-target side effects, whereas antigen-specific Treg exert more potent and targeted immunosuppression.59
Regarding (antigen) specificity, Treg can be equipped with a TCR or CAR or harnessed with predetermined antigen specificity via viral transfection encoding a specific TCR or CAR (Figure 1).59 In their research work on T-cell therapy in transplantation, Tang and Bluestone presented a detailed comparison of both approaches: TCR-Treg yielded higher sensitivity recognizing even 1 molecule per target cell versus CAR-Treg not requiring co-receptors to exert immunosuppressive effects.19,60 TCR-Treg address peptide-HLA complexes, whereas CAR-Treg can theoretically target any surface or soluble antigen. In contrast to synthetic modular signaling domain in CAR-Treg, the signaling chain of TCR-Treg is based on a CD3 complex. Of note, specificity can be tailored by utilizing synthetic Notch receptors.19 Desreumaux et al61 proposed an alternative pathway toward antigen specificity of Treg. They generated ovalbumin-specific Treg ex vivo by expanding and exposing peripheral blood mononuclear cells (PBMCs) using artificial APCs to preferentially select Treg that expressed TCR with ovalbumin specificity. Adoptive transfer of these Treg led to significant signs of remission in 8 of 20 Crohn’s disease patients.61 Yet, as highlighted by Boardman et al,62 this clinical trial has remained the only one to investigate the potential benefits of Treg in Crohn’s disease.
On the other hand, CARs can serve as a navigation system in the human body. CARs are artificial fusion proteins composed of 3 elements: an intracellular transduction domain, an extracellular antigen-binding recognition domain (adaptable), and a connecting transmembrane domain. Currently, there are 4 different generations of CAR constructs with the first-generation signaling domain containing CD3ζ as a stimulatory domain. In contrast, the second and third generations include ≥1 costimulatory domains (eg, CD28 and/or 4-1BB). The fourth generation is referred to as TRUCK T cells armed with immune stimulatory cytokines.63 Lamarthée et al64 found that 4-1BB CAR-Treg display impaired lineage stability and in vivo suppressive potential. However, mammalian target of rapamycin inhibitors and vitamin C administration rescued 4-1BB CAR-Treg from such dysfunctional states.64 Mohseni et al65 have outlined the evolving process of CAR-T therapy from cancer research to CAR-Treg with the second-generation CAR-T cells considered a breakthrough in cancer therapy because of their costimulatory domain. Following the Food and Drug Administration approval of the first clinical products, tisagenlecleucel and axicabtagene ciloleuce, for the treatment of for the treatment of B-cell acute lymphoblastic leukemia and B-cell lymphoma in 2017, the research has shown the potential benefits of chimeric auto-antibody receptor T cells in autoimmune diseases.65 Although such constructs yielded positive outcomes in an animal model of pemphigus vulgaris, CAR-Treg are considered even more predestinated to control autoimmunity given the high baseline immunosuppressive potential of Treg.31 Unlike TCR-Treg, CAR-Treg show no MHC dependency and are less dependent on the cytokine IL-2.59,66 Still, they are able to uphold the phenotype and function and colonize in the target tissue while exerting a more effective suppression than polyclonal Treg. Viral vector systems (eg, lentivirus and adeno-associated vectors), viral-free systems (eg, Sleeping Beauty or piggyBac transposon), as well as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) gene-editing platform‚ are utilized for engineering CAR-Treg.66 m viral vectors‚ as well as nuclease systems‚ pose potential immunogenic triggers and eventual safety risks. Furthermore‚ most human adults have preexisting adaptive immunity toward Cas9 proteins‚ and viral vectors and nuclease systems pose potential immunogenic triggers; CRISPR/Cas9-edited T cells did not provoke immune responses and displayed as stable in the patients.67 Advanced CAR-T-cell design investigates the potency of multiantigen targeting CAR-T cells. This approach involves administering CAR-T cells with different antigen specificities, expression of 2 antigen-binding domains in tandem, or bidirectional vectors to co-transduce 2 CAR constructs and initiate immune response up on each.68 Although these innovations have shown promising antitumoral effects and specific immune responses up on each antigen, their principles have yet to be translated to CAR-Treg.69 However, Spence et al70 demonstrated the protective effects of Treg with multiple specificity for insulin and other islet-derived antigens against diabetes in a murine model. In short, CAR-Treg may represent the next-generation immunomodulative cellular treatment of rejection.
EFFICACY OF CAR-TREG
The efficacy of CAR-Treg has been well studied both in vitro and in vivo. For example, MacDonald et al and Bézie et al have gathered evidence in murine models that CAR-Treg are effective in preventing xenogenic GvHD and/or in controlling alloimmune-mediated rejection of human skin grafts.71,72
Of note, the human skin model is a well-established method to evaluate the alloimmune response of human cells. Further studies using this approach documented the potency in reducing posttransplantation inflammation. However, because of the limited experiment duration, the long-term effects of CAR-Treg application, such as overly strong signaling of CAR and excessive Treg stimulation, remain subject to future studies.59 On the one hand, the increase of CAR-Treg specificity relies on the determination of self- and alloantigens, which are‚ more generally put, target antigens. To this end, CRISPR/Cas9 genome-editing has shown to be effective in identifying further target antigens.73 On the other hand, the ability of CAR-Treg to specifically home to target tissue could be improved by engineering CAR-Treg that are controllably activated by monoclonal antibodies.74
CAR-TREG IN TRANSPLANTATION
Allotransplantation requires lifelong immunosuppression to prevent rejection of the graft. Yet, the immunosuppression carries varying side effects because it has been shown to increase the risk of infections and trigger carcinogenesis. Therefore, current approaches aim to support immune tolerance (as an essential prevention of graft rejection) and diminish general immunosuppression. Ultimately, the scientific efforts are driven by the claim to which transplantation medicine has been dedicated ever since its beginnings: minimize the patients’ morbidity and mortality.
The mismatch of HLA receptors between donor and recipient is considered to be the key reason for graft rejection. These glycoproteins, anchored in the cell membrane, are therefore worthwhile targets for CAR-Treg. Researchers have primarily focused on anti-HLA-A2-CAR (A2-CAR) as a navigational modification for Treg. For example, MacDonald et al created the A2-CAR by using the scFv of the mouse anti-HLA A2 clone BB7.2, which was reactively mirrored by Bézie et al.71,72 Both studies investigated human A2-CAR-Treg in vitro and in vivo with humanized mouse models using a GvHD model with Bézie et al75 further elucidating skin transplantation rejection involving HLA-A2+ donor tissue. In the GvHD model of Bézie et al,75 immunodeficient (NSG) mice were irradiated and then injected with PBMCs with or without CAR-Treg added. GvDH disease was assessed via body weight, whereas MacDonald et al72 additionally included fur texture, posture, activity level, and skin integrity. For skin graft rejection model, human skin was placed on a previously exposed graft bed in NOD scid gamma(NSG) mice‚ and 1 mo later‚ PBMCs with or without CAR-Treg were injected. Graft rejection reaction was scored via macroscopic observation‚ and histological analysis of the skin graft was conducted at day 100 posttransplantation.75
Both groups found that A2-CAR-Treg exhibit greater in vitro suppression following stimulation by HLA-A2 antigen versus polyclonal Treg.71,72 In addition, using A2-CAR-Treg (compared with TCR stimulation), the researchers found upregulation of proteins essential for cell function, including cytotoxic T-lymphocyte–associated protein 4, latency-associated peptide, and glycoprotein A repetitions predominant. They further reported that A2-CAR-Treg remained phenotypically unaffected. Of note, Bézie et al71 utilized CD8+ CAR-Treg in their experimental setup. CD8+ Treg represent a heterologous cell set that is often defined as CD122hiLy49+CD8+ or Foxp3+CD8+ Treg.76 CD8+ Treg have demonstrated potent effects in suppressing autoimmunity (especially by reducing IL-2 and interferon-γ secretion in conventional T cells).77 When compared with the well-established CD4+ Treg, the CD8+ counterparts tend to possess both regulatory and proinflammatory capacities.78 Thus, the overlap and differences in CD8+ versus CD4+ CAR-Treg still have to be ascertained.
Sicard et al79 could demonstrate the beneficial use of CAR-Treg in immunocompetent murine recipients. To this end, they infused Treg expressing an anti-HLA-A2–specific CAR to Bl/6 mice that received an HLA-A2+ Bl/6 skin graft. Anti-HLA-A2–specific CAR-Treg significantly delayed skin rejection and diminished DSAs and frequencies of DSA-secreting B cells. The CAR-Treg also displayed a stable phenotype in vivo without any signs of antigraft cytotoxicity. Yet, when administered to HLA-A2–sensitized immunocompetent mice, CAR-Treg were unable to significantly delay allograft rejection.79 These results remain to be corroborated in other specifically vascularized transplant models but overall suggest that donor-specific CAR-Treg functions may be inhibited in sensitized recipients who may produce DSAs that mitigate CAR-Treg-target binding. This may limit the use of HLA-antigen–specific CAR-Treg in sensitized patients; however‚ the limitation must be further explored in composite tissue transplants.
In summary, the current body of studies describes points toward a superior function of A2-CAR-Treg with regard to prevention of allogeneic skin graft rejection and xenogeneic GvHD. Thus, this evidence documents the practicability of piloting CAR-Treg to alloantigens and underscores their potential in the field of transplantation.
This strategy is still largely unexplored in clinical setting of transplantation, specifically in VCA. The ONE Study included polyclonal Treg in kidney transplants but did not find preventive effects regarding rejection reaction.5 Several issues have to be addressed, such as antigen specificity, ease of use, and cell manufacturing. In the long run, “off-the-shelf” designer Treg may become available while maintaining high specificity for a specific antigen.
Muller et al80 engineered Treg with an anti-HLA-A2-CAR and deleted the endogenous TCR via CRISPR/Cas9 technology. This way, they generated A2-CAR+TCRdeficient Treg‚ which displayed a stable phenotype (ie, stable expression of FoxP3) and remained fully functional in vitro. In a GvHD model utilizing NSG mice, the authors showed that the bioengineered Treg can effectively mitigate GvHD by suppressing alloreactivity of infused immune cells (HLA-A2+) in an antigen-dependent manner. This was reproducible in NSG mice expressing HLA-A2 and/or following infusion of human HLA-A2+ PBMC. Vice versa, A2-CAR+TCRdeficient Treg failed to control GvHD in HLA-A2- NSG mice‚ which had been reconstituted infusing HLA-A2- PBMCs. Additionally, the authors found that engineered Treg selectively traffic to HLA-A2 expressing transplanted islet cells, thereby underscoring their ability to recognize their cognate antigen in vivo.80
Their study outlined the need for engineering highly specific Treg without disrupting their functionality by introduction of a single HLA-antigen–specific CAR. Removal of the endogenous TCR and demonstration of stability of Treg function in vivo encourage the potential use of “off-the-shelf” Treg for the treatment of rejection reaction in transplant patients. The study also underlines the potential integration of genome modifying tools such as CRISPR/Cas9.
TREG IN SOT
Given promising findings in in vitro and murine models, recent clinical studies have focused on further investigating the in vivo relevance of Treg mechanisms in humans and specifically SOT.
To elicit potential therapeutic benefits of immune cell-based therapy, Sawitzki et al81 conducted the ONE Study. In these single-arm trials, researchers investigated the benefits and utility of Treg therapy in living donor kidney transplant recipients (including a 60-wk follow-up scheme) in a multicentric prospective fashion.81 The researchers compared a standard-of-care group (ie, treatment with basiliximab, tapered prednisolone, mycophenolate mofetil, and tacrolimus) versus a cell-therapy group (ie, treatment with cell-based medicinal products, tapered prednisolone, mycophenolate mofetil, and tacrolimus). Same dosing and immunosuppression were used in both groups with regard to the following exceptions: In the cell-therapy group, the option to taper away mycophenolate mofetil was added‚ and basiliximab therapy was replaced by cell product administration. For this purpose, either monocyte-derived cell products (ie, regulatory macrophages or autologous tolerogenic DCs) were given before renal transplantation or T-cell–derived products (ie, polyclonal Treg or donor-antigen reactive Treg) were administered following renal transplantation.
Regarding the (biopsy-confirmed) rejection rate, similar results were reported for both groups‚ yielding a 12% rejection rate in the standard-of-care group versus 16% in the cell-therapy group. Of note, 40% of the patients treated with cell-based products were effectively discontinued from mycophenolate mofetil and went on to receive tacrolimus monotherapy. Although the incidence of most medical disorders remained similar, the rate of infections and infestations was almost 6-fold higher in the reference group compared with the cell-therapy group. Primarily, viral infections, such as cytomegalovirus, herpes virus‚ and polyoma virus, were significantly less frequent among patients in the therapy group.
The impact of Treg infusion on the alloimmune response of liver transplant recipients has also been under investigation in clinical trials. Sánchez-Fueyo et al82 described the safety and well tolerability of polyclonal Treg immunotherapy in their phase 1 study. They included 9 patients undergoing deceased liver transplantation: Although 3 participants received a dose of 0.5–1 × 106 Treg/kg before transplantation, 6 probands were given an infusion of 3 to 4.5 × 106 Treg/kg at a time point 6 to 12 mo posttransplant. The researchers also outlined a potential dose-effect correlation. Although the infusion of the lower Treg dose was not associated with a diminution in donor-specific T-cell responses, the T-cell activity directed against donor cells was demonstrably decreased following injection of 3 to 4.5 × 106 Treg/kg.
Todo et al83 exemplified the potential of cell-based therapy in organ transplantation by conducting a pilot study. On day 13 after living donor liver transplantation, 10 probands were given a Treg-enriched cell product (averagely 3.39 ± 106 Treg/kg) as a bulk infusion. In 70% of patients, immunosuppression could be successfully tapered and then completely discontinued. The remaining 3 participants suffered from autoimmune liver diseases and experienced mild rejection episodes during weaning and were subsequently readministered conventional low-dose immunotherapy. Although the 7 liver transplant recipients showed no signs of rejection in the long-term follow-up, 3 of them developed a significant immune response to the donor cells in the mixed lymphocyte reaction analysis several years after transplantation.84 This fact underlines the need for further trials to observe long-term patient outcome after immediate therapy.
Currently, there is a plethora of ongoing studies that aim to confirm the potential of Treg therapy, verify its safety, and further explore its efficacy. For example, the TWO trial is being conducted as a randomized, controlled, single-center phase IIb trial in which 30 living donor kidney transplant recipients will be infused with 5 to 10 × 106 Treg/kg.85 Throughout an 18-mo follow-up posttransplant period, patient-related clinical course and outcome will be compared with a control group receiving standard basiliximab-based immunosuppression with tacrolimus and mycophenolate mofetil. The occurrence of acute rejection reactions and the practicability and utility of cellular therapy in kidney transplantation are to be assessed in detail.
To gain a comprehensive understanding of cell-based therapy and to explore its definite benefit and clinical applicability in organ transplantation, a potpourri of studies is currently underway.86–90 Their results are expected in the coming years and will provide profound insights into the research field of Treg in transplantation.
TREG AND VCA
VCA is a relatively novel field of transplant surgery that has revolutionized restoration of form and function of catastrophic limb and facial soft tissue defects. Specifically, the transplantation of facial VCAs (fVCAs) has shown promising short- and long-term functional and aesthetic outcomes.1,7
However, one must be aware of the immunological hurdles associated with VCA because this type of transplant is composed of tissues with suspected varying immunogenicity‚ such as skin, mucosa, muscle, vasculature, and lymphatics (Figures 2 and 3).91 We have yet to fully understand the pathophysiology of deep tissue rejection (eg, muscle, vasculature) because most mechanistic investigations on VCA rejection are done using skin samples, the tissue type easiest to biopsy. Additionally, skin is widely regarded as the most immunogenic tissue of skin containing VCAs.11,91,92
In experimental models of VCA, suspected varying levels of immunogenicity led to the discovery of the split tolerance phenomenon in which early rejection of the skin, but not other components such as muscle or bone, was observed.93,94 Transplantation of a rich immune system (including T cells, APCs) with the skin is thought to be at least partially responsible for relatively higher numbers (>85% within the first year) of recorded acute skin rejection episodes in VCAs compared with SOT.95,96 It was hypothesized that this might be due to the high number of T cells residing in the skin compared with the peripheral circulation, as well as the enlarged subset of T cells with an effector memory phenotype featuring the property to assume immune responses independent of chemotactic activity.95,96 In a self-enforcing manner, DCs and other APCs in the skin can present antigens to skin resident memory T cells (and can also be recognized by recipient immune cells via the direct pathway of allorecognition) and initiate their alloreactive response.93 Additionally, specific to VCA may also be both a graft versus host‚ as well as host versus graft‚ reaction inside the skin. Lian et al demonstrated evidence of donor-derived tissue-resident memory (predominantly CD8+) T cells (CD69+/CD103+/CLA+) that are spatially associated with the sites of injury during facial allograft rejection.7 Despite the known proinflammatory components of the skin, the rejection process in other tissues likely differs from skin (Figure 3). Mucosa is now being studied as another highly dynamic and antigenic tissue of facial VCAs.8,91,97 Case studies demonstrate that mucosal rejection, on average, is more severe when compared with skin, with data suggesting that there is rarely skin rejection without concomitant mucosal rejection. To date, the mechanisms of mucosal rejection in comparison to skin have not been studied sufficiently.
In summary, scientific evidence that clearly demonstrates that skin is the most immunogenic and primarily targeted tissue in clinical VCA is lacking‚ and recent studies on mucosa in fVCA suggest that the latter may be more immunogenic and may reject at a higher frequency.91,97–99 Indeed, the discovery of skin being an imprecise indicator of mucosal rejection is relevant because it demonstrates the need for more studies that examine the suitability of skin as a common denominator tissue for diagnosis of whole allograft rejection.
In this context, targeted therapies such as the design of CAR-Treg may be especially relevant. In the future, once tissue-specific mechanistic pathways of rejection are fully understood, tissue targeted therapy with targeted cellular therapeutics may become relevant. Design of therapeutics that target tissue-specific rejection processes may be the ultimate tool to control VCA rejection and limit deleterious systemic side effects while only minimally disrupting physiologic tissue homeostasis.
TREG AND MIXED CHIMERISM
To promote donor-specific tolerance and reduce the intensity of immunosuppressive medication in VCA, different cell- and pharmaceutical-based approaches are under investigation.4
Unique to VCA is the component of a vascularized bone marrow compartment (often included are the jaw, ulna, and radius) that is thought to amplify the presence of donor-derived tolerance promoting chimeric cells by providing niches and a constant source of hematopoietic stem cell (HSC) expansion.100 The strategy of amplifying hematolymphoid chimerism has been the topic of many preclinical and clinical studies.101 For the induction of hematopoietic mixed chimerism, it was shown that Treg are critical in promoting engraftment of donor hematopoietic cells.102 It was shown that patients with mixed chimerism have an increased number of peripheral Treg (both donor and recipient origin) that are thought to suppress the rejection process.103
For VCA specifically given the component of a vascularized bone marrow compartment, this strategy has been proposed as a potential way of reducing the amount of maintenance immunosuppression.104,105 To date, these approaches have not been able to replace the standard triple maintenance immunosuppression regimen‚ nor has any clinical benefit been shown in human trials. Stable chimerism after transplantation of a vascularized bone marrow compartment has not been shown in VCA.
Another example that has been explored in the field of VCA is the posttransplant infusion of donor-derived HSCs. A positive effect of this strategy was shown in preclinical models (including primate models). However, in clinical VCA‚ micro/macrochimerism and a benefit in terms of allowing safe reduction of immunosuppression have not been demonstrated.4
The Pittsburgh group attempted the infusion of donor-derived bone marrow cells following VCA to induce mixed chimerism. To date, no data have been reported regarding the long-term effectiveness of this approach.106 The co-infusion of recipient-derived Treg may need to be explored as a factor to promote engraftment of donor-derived HSCs.
In summary, standard approaches to chimerism induction have not been successful in clinical VCA. A more targeted and effective approach to promote tolerance may be the infusion/injection of Treg to promote donor hematopoietic engraftment. For example, one approach that is under investigation in kidney transplant patients is the infusion of recipient-derived Treg following infusion of donor-derived HSCs with the goal to promote engraftment of the HSCs and thereby amplify the mixed chimerism strategy to minimize systemic pharmaceutical drug-based IS.107
Another VCA-specific therapeutic approach is topical (local) immunosuppression. In clinical practice, topical agents, such as ointments and gels, are frequently used during rejection episodes of facial and extremity allotransplants. Indeed, the direct reachability and accessibility of VCA allow—in contrast to SOT—local administration of immunomodulatory agents through the skin or mucosa. The direct targeting is designed to reduce toxic sequelae of systemic therapy and to effectively suppress rejection via direct immunomodulatory effect on the transplant.6,108,109 The local injection of Treg has already been investigated in several studies with promising results, including the inhibition of inflammation and orchestration of tissue repair.110,111 Yet, this approach remains to be translated to the field of VCA.112
It is important to note that the topical immunomodulatory drugs currently in clinical use can impact the Treg population. For example, tacrolimus has been found to reduce the amount of total and activated Treg in vitro.113 In contrast, Battaglia et al114,115 found sirolimus to promote expansion of Treg. Fisher et al116 have described another method of local immunosuppression by engineering microparticles that release agents (TGF-β1, rapamycin, and IL-2) known to trigger Treg differentiation, with remarkable success in terms of allograft survival in an animal study.
SKIN-SPECIFIC REGULATORY CELLS
Hautz et al117 investigated the role of Treg specifically in the skin rejection of 3 bilateral human hand recipients and found similar kinetics of indoleamine-2,3-dioxygenase (a functional link between DCs and Treg; IDO) and FoxP3 expression with higher expression levels at later time points. Their work points toward a pivotal role of Treg in controlling VCA skin rejection in the long term.117 In combination with the skin as a preferential target of VCA rejection, these observations gave birth to the idea of skin-specific Treg.118 This skin specificity might basically be achieved utilizing mechanisms that are discussed under Definition and functioning of Treg. More precisely, Lian et al95 found CD69+CD103+CLA+ resident memory T cells (Trm) to be the predominant donor T-cell subset (>90%) in skin rejection sites of 5 face transplant patients. Thus, these surface markers may represent promising target structures for bioengineered CAR-Treg. Given the different tissue types in VCA, tissue-overlapping markers/cell types may represent particularly efficient targets candidates. For example, mucosal-associated invariant T cells populate different potential VCA grafts‚ such as the skin or the nasal/oral cavity, as well as other VCA-associated organs/tissues like the female reproductive tract. They carry distinct metabolic antigens‚ such as 6,7-dimethyl-8-D-ribityllumazine or 7-hydroxy-6-methyl-8-D-ribityllumazine‚ and could therefore be targeted by antigen-specific Treg‚ which would allow shielding the recipient against alloreactive reactions in different tissues.119 In a similar manner, the administration of multispecific Treg could help to control VCA rejection. The beneficial use of multispecific Treg has been demonstrated by Spence et al70 in a murine diabetes model in a murine model but remains to be translated to VCAs. Such approaches would also address the high antigenic potential in mucosa tissue‚ which has resulted in acute VCA rejection preceding and exceeding the skin-based rejection counterpart.8,97,120
In general, VCA recipients receive comparable levels of mycophenolate mofetil/prednisone doses (especially in long-term therapy) when compared with kidney transplants and intensified medication in comparison with liver transplantations but receive less than following heart or lung transplant patients.121 The initial assumption that skin-containing VCA would necessitate extraordinary dosages of immunosuppression has been proven obsolete.122 Yet, the rate of acute rejection is elevated in VCA patients versus SOT, with >85% of VCA recipients experiencing at least 1 episode of acute cellular rejection during their first posttransplant year.123 This may in part be due to the easy accessibility of the transplant and the ease of doing serial surveillance biopsies. However, the exact mechanisms of facial allograft rejection are not yet understood. For example, the mucosa is a common target of rejection and often shows more prominent changes of allograft rejection when compared with the skin. This might be due to an increased density of APCs, vessels, and baseline inflammation in mucosa, but the exact reasons have yet to be determined.91 The finding of higher grades of rejection in mucosa than in skin may indicate that the current immunosuppression may not adequately treat rejection-related mucosal inflammation. Thus, studies are needed to unveil the mechanisms of skin and mucosal rejection because it may be necessary to increase the levels needed for fVCA immunosuppression to adequately cover mucosal rejection.
Leveraging the intrinsic immunoregulatory properties of Treg may therefore be an approach for adapting immunosuppressive therapy in VCA. Resulting from their (to this date) comparative scarcity, the in-clinic use of Treg as a cellular immunotherapy requires ex vivo culture and expansion of patient-derived own Treg, followed by systemic reinfusion. Alternatively, Fisher et al116 engineered microparticles that release TGF-β1, rapamycin, and IL-2 (so-called TRI-MP constellation) to induce Treg differentiation from naive T cells in a rat hindlimb VCA model. More specifically, after microsurgeons transplanted MHC-incongruent hindlimbs, the rodent recipients were administered short-term immunosuppressive therapy (in form of tacrolimus and rabbit antirat antibodies). The researchers found that local administration of this Treg-inducing system extended allograft survival indefinitely without long-term systemic immunosuppression. Additionally, TRI-MP treatment was also reported to reduce the expression of inflammatory mediators while increasing the level of Treg-associated cytokines in allograft tissue and proinflammatory Th1 populations in allograft lymph nodes. Furthermore, by using a VCA model that integrated a short course of typical antirejection agents, Fisher et al provided evidence that TRI-MP can be successfully combined with standard immunosuppressive agents. Importantly, this finding obviates the imperative need for cessation of standard systemic therapy and also allows more variability and leeway in clinical trial design. In a murine model, Balmert et al124 have harnessed these promising effects of the Treg/TRI-MP regimen in the treatment of allergic contact dermatitis and shown that allergy tolerance has been promoted and the dermatitis has been suppressed via induction of Treg. They also pointed out that this approach (engineering microparticles releasing TRI-MP to create an environment that fosters Treg differentiation) may pave the way for new options in the treatment of graft rejection (Figure 2).
As a potential next step, preimplant perfusion of the graft with a high dose of antigen-specific Treg is a potential approach to minimize early alloreactivity and significantly reduce immunosuppression dosages typically needed within the first 12 mo following transplantation (Figure 1).1 Furthermore‚ CRISPR/Cas9-based genome modification, in particular base editing, instead of viral delivery represents a strategy to enhance safety of CAR-Treg engineering.
In summary, this cellular immunotherapy technique may be considered a valuable alternative to conventional side-effect-intensive immunosuppressive drugs in the transplantation of vascularized composite grafts and solid organs and may, in the future, be explored in clinical transplantation of VCAs.
To reduce the use of immunosuppressive drugs in posttransplantation therapy, cell-based treatments, such as Treg, harbor enormous translational potential. CAR-Treg cells seem to be particularly promising given their antigen-specific targeting and the ability to engineer target-specific regulatory cells.
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