Acute Rejection Rates in Vascularized Composite Allografts: A Systematic Review of Case Reports.

INTRODUCTION: Vascularized Composite Allografts (VCA) are usually performed in a full major histocompatibility complex mismatch setting, with a risk of acute rejection depending on factors such as the type of immunosuppression therapy and the quality of graft preservation. In this systematic review, we present the different immunosuppression protocols used in VCA and point out relationships between acute rejection rates and possible factors that might influence it. METHODS: This systematic review was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We systematically searched Medline (PubMed), Embase, and The Cochrane Library between November 2022 and February 2023, using following Mesh Terms: Transplant, Transplantation, Hand, Face, Uterus, Penis, Abdominal Wall, Larynx, and Composite Tissue Allografts. All VCA case reports and reviews describing multiple case reports were included. RESULTS: We discovered 211 VCA cases reported. The preferred treatment was a combination of antithymocyte globulins, mycophenolate mofetil (MMF), tacrolimus, and steroids; and a combination of MMF, tacrolimus, and steroids for induction and maintenance treatment, respectively. Burn patients showed a higher acute rejection rate (P = 0.073) and were administered higher MMF doses (P = 0.020). CONCLUSIONS: In contrast to previous statements, the field of VCA is not rapidly evolving, as it has encountered challenges in addressing immune-related concerns. This is highlighted by the absence of a standardized immunosuppression regimen. Consequently, more substantial data are required to draw more conclusive results regarding the immunogenicity of VCAs and the potential superiority of one immunosuppressive treatment over another. Future efforts should be made to report the VCA surgeries comprehensively, and muti-institutional long-term prospective follow-up studies should be performed to compare the number of acute rejections with influencing factors.

OPTN/SRTR 2022 Annual Data Report: Vascularized Composite Allograft.

This year's chapter on vascularized composite allograft (VCA) encompasses reviews of data collected from 2014 (when VCA was included in the Final Rule) through 2022. The present Annual Data Report shows that the number of VCA recipients in the United States continues to be small and has remained consistent from the prior report. The data continue to be limited by sample size, with trends persistently demonstrating a predominance of White males in the young/middle-aged population as both donors and recipients for nonuterus VCA transplants, and White women younger than 35 years as the predominant recipients of uterus transplant. Similar to the 2021 report, there were only eight failed uterus grafts and one failed nonuterus VCA graft reported from 2014 through 2022. Standardization of definitions of success and failure as well as outcome measures for the different VCA types remain unmet needs in VCA transplantation.

OPTN/SRTR 2021 Annual Data Report: Vascularized Composite Allograft.

Year 2020 marked the first OPTN/SRTR Annual Data Report that included a chapter on vascularized composite allograft (VCA), which encompassed reviews of data collected between 2014 (when VCA was included in the Final Rule) and 2020. The present Annual Data Report shows that the number of VCA recipients in the United States continues to be small and trended downward in 2021. While data continue to be limited by sample size, trends continue to show a predominance in White, young/middle-aged, male recipients. Similar to the 2020 report, eight uterus and one non-uterus VCA graft failures were reported from 2014 through 2021. Critical to advancement of VCA transplantation will be the standardization of definitions, protocols, and outcome measures for the different VCA types. Like intestinal transplants, it is likely that VCA transplants will be concentrated and performed at referral transplant centers.

Insights from immunoproteomic profiling of a rejected full-face transplant.

Vascularized composite allografts (VCAs) of faces and extremities are subject to chronic rejection that is incompletely understood. Here we report on immunoproteomic evaluation of a full facial VCA removed 88 months after transplantation due to chronic rejection. CD8-positive T cells of donor (graft) origin infiltrate deep intragraft arteries in apposition to degenerating endothelium of chimeric recipient origin in association with arteriosclerotic alterations. Digital spatial proteomic profiling highlighted proteins expressed by activated cytotoxic T cells and macrophages as well as pathway components involved in atherogenic responses, including Indoleamine 2,3-Dioxygenase 1 (IDO1) and Stimulator of Interferon Response CGAMP Interactor (STING). Chronic facial VCA rejection thus involves T cell/macrophage-mediated accelerated arteriosclerosis not normally represented in punch biopsies and potentially driven by persistent graft-resident effector T cells and recipient target endothelium that chimerically repopulates graft arteries.

Tolerance of a Vascularized Composite Allograft Achieved in MHC Class-I-mismatch Swine via Mixed Chimerism.

Background: Vascularized composite allografts (VCAs) allow reconstruction of devastating injuries and amputations, yet require lifelong immunosuppression that is associated with significant morbidity. Induction of immune tolerance of VCAs would permit widespread use of these procedures. VCAs are acquired from deceased donors most likely to be fully-MHC-mismatched (in contrast to living-related renal transplant donor-recipient pairs matched at one MHC haplotype). After achieving VCA tolerance in a swine model equivalent to clinical living-related renal transplants (single-haplotype MHC mismatches: e.g., "mother-daughter"/haploidentical), we tested our protocol in MHC class I, class II, and fully-MHC-mismatched pairs. Although class II mismatched swine demonstrated similar results as the haploidentical scenario (stable mixed chimerism and tolerance), our protocol failed to prevent rejection of class I and full mismatch VCAs. Here, we describe a new adapted conditioning protocol that successfully achieved tolerance across MHC class-I-mismatch barriers in swine. Methods: Swine were treated with non-myeloablative total body and thymic irradiation two days prior to infusion of bone marrow cells from an MHC class I-mismatched donor. They also received a short-term treatment with CTLA4-Ig (Belatacept®) and anti-IL6R mAb (Tociluzimab®) and were transplanted with an osteomyocutaneous VCA from the same donor. Results: Stable mixed chimerism and tolerance of MHC class-I-mismatched VCAs was achieved in 3 recipients. Allograft tolerance was associated with a sustained lack of anti-donor T cell response and a concomitant expansion of double negative CD4-CD8- T cells producing IL-10. Conclusions: This study demonstrates the first successful mixed chimerism-induced VCA tolerance in a large animal model across a MHC class-I-mismatch. Future studies aimed at fully-mismatched donor-recipient pairs are under investigation with this protocol.

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies.

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, failed by the first steps of the reconstructive ladder. Despite promising aesthetic and functional outcomes, the main limiting factor remains the need for a drastically applied lifelong immunosuppression and its well-known medical risks, preventing broader indications. Therefore, lifting the immune barrier in VCA is essential to tip the ethical scale and improve patients' quality of life using the most advanced surgical techniques. De novo creation of a patient-specific graft is the upcoming breakthrough in reconstructive transplantation. Using tissue engineering techniques, VCAs can be freed of donor cells and customized for the recipient through perfusion-decellularization-recellularization. To develop these new technologies, a large-scale animal VCA model is necessary. Hence, swine fascio-cutaneous flaps, composed of skin, fat, fascia, and vessels, represent an ideal model for preliminary studies in VCA. Nevertheless, most VCA models described in the literature include muscle and bone. This work reports a reliable and reproducible technique for saphenous fascio-cutaneous flap harvest in swine, a practical tool for various research fields, especially vascularized composite tissue engineering.

OPTN/SRTR 2020 Annual Data Report: VCA.

The first vascularized composite allograft (VCA) transplant in the United States was performed in 1998 in a 40-year-old man who received a laryn-geal transplant after experiencing severe trauma to the throat 20 years before. The following VCA was a hand transplant in 1999 in a 37-year-old man who lost his left hand 13 years before. Since then, the field of VCA transplantation has made significant strides. On July 3, 2014, the Or gan Procurement and Transplantation Network (OPTN)/United Network for Organ Sharing (UNOS) oversight of VCA procurement and transplant in the United States went into effect. In the last decade, the number of candidates listed for and transplanted with VCA has increased. While patient demographic data, whether listed candidates or patients undergoing VCA transplant, is limited by sample size, the trend is a predominance toward a young/middle-aged, White population. Overall outcomes data have been promising, with the vast majority of VCA transplants resulting in functioning grafts.

The MHC-characterized Miniature Swine: Lessons Learned From a 40-Year Experience in Transplantation.

Over the last 40 y, a specialized herd of miniature swine has been intentionally bred to develop lines of animals homozygous for the swine major histocompatibility complex (MHC), which have facilitated transplantation studies across reproducible MHC and minor antigen mismatch barriers. These MHC-characterized miniature swine (Mc-MS) have been used for the study of novel surgical techniques, various approaches to tolerance induction of solid organ and vascularized composite allografts, as well as studies of the immunobiology of allografts and xenografts. Mc-MS possess characteristics that are highly advantageous to these studies, and their continued use will likely continue to play an important role in bridging "bench-to-cage-to bedside" therapies in the field of transplantation. In this review, we highlight the seminal contributions of the Mc-MS model to the field and analyze their role in the broader context of large animal models in transplantation research.

A meta-analysis of the efficacy of vascularised lymph node transfer in reducing limb volume and cellulitis episodes in patients with cancer treatment-related lymphoedema.

BACKGROUND: Lymphoedema after cancer treatment is a chronic and disabling complication that presents a significant health care burden during survivorship with limited treatment options. Vascularised lymph node transfer (VLNT) can reconstruct lymphatic flow to reduce limb volumes, but limited higher-order evidence exists to support its effectiveness. AIM: The aim of the study was to systematically review and meta-analyse the effectiveness of VLNT in reducing upper limb (UL) or lower limb (LL) volume and cellulitis episodes in patients with cancer treatment-related lymphoedema (CTRL). METHODS: PubMed, Medline (Ovid) and Embase databases were searched between January 1974 and December 2019. Full-length articles where VLNT was the sole therapeutic procedure for CTRL, reporting volumetric limb, frequency of infection episodes and/or lymphoedema-specific quality-of-life data, were included in a random-effects meta-analysis of circumferential reduction rate (CRR). Methodological quality was assessed using STROBE/CONSORT, and a novel, lymphoedema-specific scoring tool was used to assess lymphoedema-specific methodological reporting. Sensitivity analyses on the site of VLNT harvest and recipient location were performed. RESULTS: Thirty-one studies (581 patients) were eligible for inclusion. VLNT led to significant limb volume reductions in UL (above elbow pooled CRRs [CRRP] = 42.7% [95% confidence interval (CI): 36.5-48.8]; below elbow CRRP = 34.1% [95% CI: 33.0-35.1]) and LL (above knee CRRP = 46.8% [95% CI: 43.2-50.4]; below knee CRRP = 54.6% [95% CI: 39.0-70.2]) CTRL. VLNT flaps from extra-abdominal donor sites were associated with greater volume reductions (CRRP = 49.5% [95% CI: 46.5-52.5]) than those from intra-abdominal donor sites (CRRP = 39.6% [95% CI: 37.2-42.0]) and synchronous autologous breast reconstruction/VLNT flaps (CRRP = 32.7% [95% CI: 11.1-54.4]) (p < 0.05). VLNT was also found to reduce the mean number of cellulitis episodes by 2.1 episodes per year (95% CI: -2.7- -1.4) and increased lymphoedema-specific quality-of-life scores (mean difference in Lymphoedema-Specific Quality of Life (LYMQOL) "overall domain" = +4.26). CONCLUSIONS: VLNT is effective in reducing excess limb volume and cellulitis episodes in both UL and LL lymphoedema after cancer treatment. However, significant heterogeneity exists in outcome reporting, and standardisation of reporting processes is recommended.

Donor Recipient Chimeric Cells Induce Chimerism and Extend Survival of Vascularized Composite Allografts.

This study evaluated the efficacy of donor recipient chimeric cell (DRCC) therapy created by fusion of donor and recipient derived bone marrow cells (BMC) in chimerism and tolerance induction in a rat vascularized composite allograft (VCA) model. Twenty-four VCA (groin flaps) from MHC-mismatched ACI (RT1a) donors were transplanted to Lewis (RT1l) recipients. Rats were randomly divided into (n = 6/group): Group 1-untreated controls, Groups 2-7-day immunosuppression controls, Group 3-DRCC, and Group 4-DRCC with 7-day anti-αßTCR monoclonal antibody and cyclosporine A protocol. DRCC created by polyethylene glycol-mediated fusion of ACI and Lewis BMC were cultured and transplanted (2-4 × 106) to VCA recipients via intraosseous delivery route. Flow cytometry assessed peripheral blood chimerism while fluorescent microscopy and PCR tested the presence of DRCC in the recipient's blood, bone marrow (BM), and lymphoid organs at the study endpoint (VCA rejection). No complications were observed after DRCC intraosseous delivery. Group 4 presented the longest average VCA survival (79.3 ± 30.9 days) followed by Group 2 (53.3 ± 13.6 days), Group 3 (18 ± 7.5 days), and Group 1 (8.5 ± 1 days). The highest chimerism level was detected in Group 4 (57.9 ± 6.2%) at day 7 post-transplant. The chimerism declined at day 21 post-transplant and remained at 10% level during the entire follow-up period. Single dose of DRCC therapy induced long-term multilineage chimerism and extended VCA survival. DRCC introduces a novel concept of customized donor-recipient cell-based therapy supporting solid organ and VCA transplants.

Biomarker and surrogate development in vascularised composite allograft transplantation: Current progress and future challenges.

Vascularised composite allograft (VCA) transplantation is now a feasible reconstructive option for patients who have suffered significant soft tissue injuries. However, despite numerous technical advances in the field over two decades, a number of challenges remain, not least the management of transplant rejection. Part of the difficulty faced by clinicians is the early recognition and prevention of acute rejection episodes. Whilst this is potentially easier in VCAs than solid organ transplants, due to their visible skin component, at present the only validated method for the diagnosis of acute rejection is histological examination of a tissue biopsy. The aim of this review article is to provide an evidence-based overview of progress in the field of VCA biomarker discovery, including immune cell subsets, immune cell effector pathways, and circulating markers of allograft damage, and to discuss future challenges in the field.

Unraveling the Crucial Roles of FoxP3+ Regulatory T Cells in Vascularized Composite Allograft Tolerance Induction and Maintenance.

BACKGROUND: The role of regulatory T cells (Treg) in tolerance induction of vascularized composite allotransplantation (VCA) remains unclear. This study was designed to examine characteristics of Treg after VCA and their capacity to rescue allografts from rejection. METHODS: Osteomyocutaneous allografts were transplanted from Balb/c to C57BL/6 mice. All mice received costimulatory blockade and a short course of rapamycin. To elucidate the role of Treg for tolerance induction, Treg depletion was performed at postoperative day (POD) 0, 30, or 90. To assess capacity of Treg to rescue allografts from rejection, an injection of 2 × 106 Treg isolated from tolerant mice was applied. RESULTS: Eighty percent of VCA recipient mice using costimulatory blockade and rapamycin regimen developed tolerance. The tolerant recipients had a higher ratio of circulating Treg to effector T cells and elevated interleukin-10 at POD 30. A significantly higher rejection rate was observed when Treg were depleted at POD 30. But Treg depletion at POD 90 had no effect on tolerance. Treg from tolerant recipients showed stronger suppressive potential and the ability to rescue allografts from rejection. Furthermore, transplanted Treg-containing skin grafts from tolerant mice delayed rejection elicited by adoptively transferred effector T cells to Rag2-/- mice. CONCLUSIONS: Circulating Treg are crucial for inducing VCA tolerance in the early posttransplant phase, and allograft-residing Treg may maintain tolerance. Treg may, therefore, serve as a potential cellular therapeutic to improve VCA outcomes.

Combination of a CD26 Inhibitor, G-CSF, and Short-term Immunosuppressants Modulates Allotransplant Survival and Immunoregulation in a Rodent Hindlimb Allotransplant Model.

BACKGROUND: Recent studies have demonstrated that inhibition of CD26 potentiates stromal cell-derived factor-1α (SDF-1α), promotes tissue regeneration, and suppresses the rejection of organ transplants. This study investigated whether the combination of a CD26 inhibitor (CD26i) with granulocyte colony-stimulating factor (G-CSF) and short-term immunosuppressants modulates vascularized composite tissue allotransplant survival in a rodent orthotopic hindlimb allotransplant model. METHODS: The hindlimb allotransplantation from Brown-Norway to Lewis rats was divided into 4 groups. Group 1 (controls) did not receive any treatment. Group 2 was treated with short-term antilymphocyte serum (ALS) and cyclosporine-A (CsA). Group 3 was administrated CD26i and G-CSF. Group 4 received a combination of CD26i/G-CSF/ALS/CsA. Each subgroup comprised 10 rats. Peripheral blood and sampling of transplanted tissues were collected for immunological and histological analysis. RESULTS: The results revealed that allotransplant survival was found to be significantly prolonged in group 4 with CD26i/G-CSF/ALS/CsA treatment compared with those in the other groups. The interleukin-10 and transforming growth factor-ßl levels, the percentage of CD4+/CD25+/FoxP3+ T cells, as well as the levels of SDF-1α expressions were significantly increased in group 4 compared with those in the other groups. Group 4 revealed a statistical increase in the percentage of donor cells (RT1n) expression in the recipient peripheral blood, and the mixed lymphocyte reaction showed hyporesponsiveness of the T cells to donor alloantigens. CONCLUSION: The combination of CD26i/G-CSF and short-term immunosuppressants prolongs allotransplant survival by inducing immunoregulatory effects and enhancing the percentage of SDF-1α expression. This immunomodulatory approach has great potential as a strategy to increase vascularized composite allotransplantation survival.

Pharmacokinetics and Biodistribution of Tacrolimus after Topical Administration: Implications for Vascularized Composite Allotransplantation.

AIM: The high doses of oral tacrolimus (TAC) (1,2) necessary to prevent acute rejection (AR) after vascularized composite allotransplantation (VCA) are associated with systemic adverse effects. The skin is the most antigenic tissue in VCA and the primary target of AR. However, the short-term use of topical TAC (Protopic®), as an off-label adjunct to oral TAC, to treat AR episodes pro re nata (PRN), has yielded inconsistent results. There is lack of data on the pharmacokinetics and tissue distribution of topical TAC in VCA, that hampers our understanding of the reasons for unreliable efficacy. Toward this goal, we evaluated the ability of topical TAC to achieve high local tissue concentrations at the site of application with low systemic concentrations. MATERIALS AND METHODS: We assessed the pharmacokinetics and tissue distribution of topical TAC (Protopic®, 0.03%) after single or repeated topical application in comparison to those after systemic delivery in rats. Animals received a single topical application of TAC ointment (Group 1) or an intravenous (IV) injection of TAC (Group 2) at a dose of 0.5 mg/kg. In another experiment, animals received daily topical application of TAC ointment (Group 3), or daily intraperitoneal (IP) injection of TAC (Group 4) at a dose of 0.5 mg/kg for 7 days. TAC concentrations in blood and tissues were analyzed by Liquid Chromatography-Mass Spectrometry (LC/MS-MS). RESULTS: Following single topical administration, TAC was absorbed slowly with a Tmax of 4 h and an absolute bioavailability of 11%. The concentrations of TAC in skin and muscle were several folds higher than whole blood concentrations. Systemic levels remained subtherapeutic (< 3 ng/ml) with repeated once daily applications. CONCLUSION: Topical application of TAC ointment (Protopic®, 0.03%) at a dose of 0.5 mg/kg/day provided high concentrations in the local tissues with low systemic exposure. Repeated topical administration of TAC is well tolerated with no local or systemic adverse effects. This study confirms the feasibility of topical application of TAC for site specific graft immunosuppression and enables future applications in VCA.

A Case Report of Bilateral Navicular Osteonecrosis Successfully Treated With Medial Femoral Condyle Vascularized Autografts: A Case Report.

CASE: A 17-year-old boy with a history of chronic bilateral navicular osteonecrosis with fragmentation was treated with 6-month staged bilateral open reduction and internal fixation of tarsal navicular with debridement of the necrotic bone and ipsilateral medial femoral condyle vascularized bone grafting. CONCLUSION: The patient progressed to full painless weight-bearing on each extremity by 4 months postoperatively with osseous union of both chronic fracture sites and incorporation of vascularized bone grafts. Patient-Reported Outcomes Measurement Information System (PROMIS) scores were improved from preoperative levels at 6 months from each operation. This patient's atypical presentation of a rare disease was successfully treated with the utilization of vascularized bone grafting to salvage the tarsal navicular and preserve the talonavicular joint, enabling return of function and avoidance of early arthrodesis procedure.

Infectious complications of vascularized composite allograft transplantation.

PURPOSE OF REVIEW: Vascularized composite allograft (VCA) transplants constitute multiple tissues transplanted together as one functional unit. These procedures are increasing in frequency and complexity, yet data about graft survival, quality of life, and infection risk remain limited. RECENT FINDINGS: Informative guidance for this patient population is often inferred from the solid organ transplantation literature. Yet, it is important to understand that VCA transplantation additionally carries its own significant and distinctive risk factors for infection. SUMMARY: In this review, we give an overview of previously described infectious complications of VCA transplantation in the literature, discuss risk factors for future infection in these patients, and discuss how to manage such obstacles.

Local Immunosuppression for Vascularized Composite Allografts: Application of Topical FK506-TyroSpheres in a Nonhuman Primate Model.

Transplantation of vascularized composite allografts (VCAs) provides a means of restoring complex anatomical and functional units following burns and other disfigurement otherwise not amenable to conventional autologous reconstructive surgery. While short- to intermediate-term VCA survival is largely dependent on patient compliance with medication, the myriad of side effects resulting from lifelong systemic immunosuppression continue to pose a significant challenge. Topical immunosuppression is therefore a logical and attractive alternative for VCA. Current formulations are limited though, by poor skin penetration but this may be mitigated by conjugation of immunosuppressive drugs to TyroSpheres for enhanced delivery. Therefore, we investigated the topical application of FK506-TyroSpheres (in the form of a gel dressing) in a clinically relevant nonhuman primate VCA model to determine if allograft survival could be prolonged at reduced levels of maintenance systemic immunosuppression. Six Major Histocompatibility Complex (MHC)-mismatched cynomolgus macaques (Macaca fascicularis) served as reciprocal donors and recipients of radial forearm fasciocutaneous flaps. Standard Bacitracin ointment and FK506-TyroSpheres were applied every other day to the VCAs of animals in groups 1 (controls, n = 2) and 2 (experimental, n = 4), respectively, before gradual taper of systemic FK506. Clinical features of VCA rejection still developed when systemic FK506 fell below 10 ng/ml despite application of FK506-TyroSpheres and prolonged VCA survival was not achieved. However, unwanted systemic FK506 absorption was avoided with TyroSphere technology. Further refinement to optimize local drug delivery profiles to achieve and maintain therapeutic delivery of FK506 with TyroSpheres is underway, leveraging significant experience in controlled drug delivery to mitigate acute rejection of VCAs.

Allotransplantation of adult spinal cord tissues after complete transected spinal cord injury: Long-term survival and functional recovery in canines.

Spinal cord injury (SCI), especially complete transected SCI, leads to loss of cells and extracellular matrix and functional impairments. In a previous study, we transplanted adult spinal cord tissues (aSCTs) to replace lost tissues and facilitate recovery in a rat SCI model. However, rodents display considerable differences from human patients in the scale, anatomy and functions of spinal cord systems, and responses after injury. Thus, use of a large animal SCI model is required to examine the repair efficiency of potential therapeutic approaches. In this study, we transplanted allogenic aSCTs from adult dogs to the lesion area of canines after complete transection of the thoracic spinal cord, and investigated the long-term cell survival and functional recovery. To enhance repair efficiency, a growth factor cocktail was added during aSCT transplantation, providing a favorable microenvironment. The results showed that transplantation of aSCTs, in particular with the addition of growth factors, significantly improves locomotor function restoration and increases the number of neurofilament-, microtubule-associated protein 2-, 5-hydroxytryptamine-, choline acetyltransferase- and tyrosine hydroxylase-positive neurons in the lesion area at 6 months post-surgery. In addition, we demonstrated that donor neurons in aSCTs can survive for a long period after transplantation. This study showed for the first time that transplanting aSCTs combined with growth factor supplementation facilitates reconstruction of injured spinal cords, and consequently promotes long lasting motor function recovery in a large animal complete transected SCI model, and therefore could be considered as a possible therapeutic strategy in humans.

Toward Development of the Delayed Tolerance Induction Protocol for Vascularized Composite Allografts in Nonhuman Primates.

BACKGROUND: Transplantation of vascularized composite allografts is limited mainly by the need for life-long immunosuppression. The consequent side effects and looming specter of chronic rejection portend eventual allograft loss. Development of tolerogenic protocols is thus of utmost importance to the field of vascularized composite allograft transplantation. METHODS: With a modified delayed tolerance induction protocol, 10 cynomolgus macaques received hand (n = 2) or face vascularized composite allografts across both full and haploidentical major histocompatibility complex barriers before donor bone marrow transplantation at a later date. Protocol and for-cause allograft skin biopsies were performed for immunohistochemical analysis and analysis of donor-recipient leukocyte contribution; mixed chimerism in peripheral blood and in vitro immune responses were assessed serially. RESULTS: Before bone marrow transplantation, maintenance immunosuppression for 4 months led to lethal complications, including posttransplant lymphoproliferative disorder (in two of four recipients), which necessitated early study termination. Shortening the maintenance period to 2 months was clinically relevant and allowed all subsequent subjects (n = 6) to complete the delayed tolerance induction protocol. Acute rejection developed within the first 2 to 4 weeks after transplantation, with corresponding near-complete turnover of allograft leukocytes from donor to recipient origin, but donor-specific antibodies remained negative. After bone marrow transplantation, mixed chimerism failed to develop, although carboxyfluorescein succinimidyl ester mixed lymphocyte reaction demonstrated generalized unresponsiveness. However, the accrual of subsequent rejection episodes eventually culminated in graft vasculopathy and irreversible allograft loss. CONCLUSIONS: Despite the various advantages of the delayed tolerance induction protocol, it failed to reliably induce mixed chimerism and thus immunologic tolerance to vascularized composite allografts, given currently available immunosuppression treatment options. Ongoing work shows promise in overcoming these limitations.