Vascularized Composite Allograft Transplantation Research Articles

Supercooling preservation of vascularized composite allografts through CPA optimization, thermal tracking, and stepwise loading techniques

Vascularized composite allografts (VCAs) present unique challenges in transplant medicine, owing to their complex structure and vulnerability to ischemic injury. Innovative preservation techniques are crucial for extending the viability of these grafts, from procurement to transplantation. This study addresses these challenges by integrating cryoprotectant agent (CPA) optimization, advanced thermal tracking, and stepwise CPA loading strategies within an ex vivo rodent model. CPA optimization focused on various combinations, identifying those that effectively suppress ice nucleation while mitigating cytotoxicity. Thermal dynamics were monitored using invasive thermocouples and non-invasive FLIR imaging, yielding detailed temperature profiles crucial for managing warm ischemia time and optimizing cooling rates. The efficacy of stepwise CPA loading versus conventional flush protocols demonstrated that stepwise (un)loading significantly improved arterial resistance and weight change outcomes. In summary, this study presents comprehensive advancements in VCA preservation strategies, combining CPA optimization, precise thermal monitoring, and stepwise loading techniques. These findings hold potential implications for refining transplantation protocols and improving graft viability in VCA transplantation.

Enhancing Vascularized Composite Allograft Supercooling Preservation: A Multifaceted Approach with CPA Optimization, Thermal Tracking, and Stepwise Loading Techniques.

Vascularized composite allografts (VCAs) present unique challenges in transplant medicine, owing to their complex structure and vulnerability to ischemic injury. Innovative preservation techniques are crucial for extending the viability of these grafts, from procurement to transplantation. This study addresses these challenges by integrating cryoprotectant agent (CPA) optimization, advanced thermal tracking, and stepwise CPA loading strategies within an ex vivo rodent model. CPA optimization focused on various combinations, identifying those that effectively suppress ice nucleation while mitigating cytotoxicity. Thermal dynamics were monitored using invasive thermocouples and non-invasive FLIR imaging, yielding detailed temperature profiles crucial for managing warm ischemia time and optimizing cooling rates. The efficacy of stepwise CPA loading versus conventional flush protocols demonstrated that stepwise (un)loading significantly improved arterial resistance and weight change outcomes. In summary, this study presents comprehensive advancements in VCA preservation strategies, combining CPA optimization, precise thermal monitoring, and stepwise loading techniques. These findings hold potential implications for refining transplantation protocols and improving graft viability in VCA transplantation.

Impact of removing OPTN region from vascularized composite allograft allocation.

On 6/18/2020, the Organ Procurement and Transplantation Network (OPTN) implemented new policy replacing OPTN region with a 500 nautical mile (NM) circle around the donor hospital for the purpose of vascularized composite allograft (VCA) allocation. We used OPTN data to assess deceased donor VCA transplants in the 3 years pre- (6/19/2017-6/17/2020) vs. post-implementation (6/18/2020-6/17/2023). A total of 19 deceased donor VCA transplants were performed pre-policy (10 uterus, 3 bilateral upper limb, 1 unilateral upper limb, 3 face, 1 abdominal wall and 1 penis), and 11 post-policy (4 uterus, 1 bilateral upper limb, 2 face, 1 trachea, 2 abdominal wall, and 1 bilateral upper limb and face). Median distance from donor hospital to transplant hospital increased from 70 NM (range: 0-524 NM) pre-policy to 119 NM (range: 0-464 NM) post-policy. The majority of transplants in both policy eras were within 500 NM of the donor hospital [89.5% (N = 17/19) vs. 100% (N = 11/11)] and most remained within the same OPTN region as the donor hospital [68.4% (N = 13/19) vs. 90.9% (N = 10/11)]. Although it is difficult to draw strong conclusions about the policy's impact due to the low transplant volume and timing of implementation relative to the COVID-19 pandemic, data in the 3 years post-implementation suggest that 500 NM circles were a reasonable replacement for OPTN region in VCA allocation. The OPTN will continue to review data to monitor the policy's impact and inform future changes to VCA allocation, such as the transition to continuous distribution, a points-based framework expected to replace the current framework.

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

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.

Validating a Novel Device to Improve Skin Color Matching for Face Transplants.

A prospective study of 100 individuals was conducted. All participants were photographed and scanned with the Nix Color Sensor. Sixty pairwise comparisons were randomly generated. Skin color analysis was performed using photographs and the Nix Color Sensor. Delta E2000 values were compared to mean evaluator ratings using a Spearman correlation analysis. One hundred patients were included. A Spearman's correlation demonstrated a strong inverse correlation between Delta E2000 values and the mean evaluator ratings. The higher the mean evaluator rating for likeness, the lower the delta. A correlation coefficient of -0.850 demonstrates a statistically significant relationship (P < 0.01). When the Delta E2000 rises above 5 there is a significant drop in the mean evaluator ratings. As mean evaluator ratings of 5 and above would be considered adequate for face transplant amongst most plastic surgeons, an E2000 value of 5 or lower should be targeted when matching donors with recipients for face transplant. The Nix Color Sensor positively correlates to the plastic surgeon's perception of skin color and can serve as an adjunct in donor selection for facial VCAs.

Exceeding the Limits of Static Cold Storage in Limb Transplantation Using Subnormothermic Machine Perfusion.

For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model. Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days. Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure (p = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group (p = 0.01613). To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation.

Video Messaging to Increase Vascularized Composite Allograft Donation Willingness in United States Military Veterans.

Background.Expansion of vascularized composite allograft (VCA) transplantation depends on the public’s willingness to donate VCA organs, including face, extremities, and genitourinary organs. This study evaluated the effectiveness of video messaging on VCA donation willingness in US military veterans, a key stakeholder in VCA transplantation.Methods.Participants (n = 556) were randomized to 1 of 3 VCA video messaging interventions (informational, testimonial, or blended), a general (non-VCA) organ donation video message, or a control (nondonation) video message. Questionnaires were completed at pre- and postintervention and at 3-wk follow-up.Results.Veterans exposed to any VCA video messaging were more likely to express VCA donation willingness (69%, n = 203/296) than those exposed to general donation messaging (53%, n = 47 of 89; P = 0.006) or No Donation Messaging (37%, n = 36 of 97; P < 0.001). A significantly higher proportion of participants who received Blended VCA Messaging were willing to be VCA donors, compared with the Informational VCA Messaging group (79% versus 61%, P = 0.006). Each VCA messaging video resulted in a significant pre- to postintervention increase in the proportion of participants willing to donate their own face, hands, and legs (P < 0.03).Conclusions.Brief educational videos focused on VCA transplantation can have a demonstrable and verifiable impact on rates of VCA donation willingness in veterans.

422.2: Investigating Galectin-3 as a Novel Therapeutic Target To Block Inflammation and Reduce Ischemia Reperfusion Injury During Vascular Composite Allograft Transplantation

Introduction: Vascularized Composite Allograft (VCA) transplantation is a treatment option for complex injuries that leave patients with structural and functional deficits that cannot be adequately reconstructed. During transplantation, grafts are subjected to hypoxic/ischemic injury during procurement, storage, and reperfusion. Skin and muscle containing VCA are highly susceptible to ischemia reperfusion injury. Galectin-3 is an endogenous β-galactoside binding lectin known to play a role in driving inflammatory responses in response to hypoxic/ischemic stress. Blocking galectin-3 using known inhibitors including modified citrus pectin (MCP) has been shown in animal models to reduce inflammation and fibrosis. We hypothesized that galectin-3 significantly contributes to VCA IRI and that blocking galectin-3 can serve as a novel therapeutic approach to prevent or reduce IRI. We aimed to determine the role of galectin-3 in VCA ischemia reperfusion injury and the effect of blocking galectin-3 using MCP in syngeneic VCA models. Method: Male Brown Norway rats underwent syngeneic hind limb transplantation following 0, 6 or 24 hours of cold ischemia time. A group of rats receiving hind limbs subjected to 24 hours of static cold storage were treated with MCP (1% w/v) in the drinking water starting one week prior to transplantation. Recipient serum and donor VCA tissues were collected at end-of-study 6 days post transplantation. Sera and tissues collected from naive Brown Norway rats were included as controls. Sera levels of galectin-3 were determined by sandwich ELISA (Novus Biologics, Inc). Galectin-3 expression in the muscle was determined by Western Blotting. The degree of inflammation in the muscle tissue was determined by H&E. Results: Sera galectin-3 levels averaged 0.854±0.557ng/ml in naïve BN rats (n=7), 4.232±2.134ng/ml in recipients of grafts transplanted immediately without static cold storage (n=3), 12.86±1.292ng/ml in recipients of grafts subjected to 6 hours cold storage without MCP treatment (n=3),20.10±3.185ng/ml in recipients of grafts subjected to 24 hours cold storage without MCP treatment (n=6), and 12.77±2.098ng/ml in recipients of grafts subjected to 24 hours cold storage with MCP treatment (n=9) (Figure 1A). Galectin-3 expression in the VCA muscle increased according to the extent of cold ischemia. MCP treatment decreased muscle galectin-3 expression (Figure 1B) and inflammation in the muscle (Figure 2) post transplantation.Conclusion: Galectin-3 levels significantly increased in recipient sera and donor muscle within a week in rats transplanted with donor VCA grafts subjected to prolonged ischemia. Blocking galectin-3 using MCP can reduce the amount of galectin-3 in circulation and muscle tissue post transplantation and may decrease inflammation associated with VCA IRI. Galectin-3 may serve as a novel therapeutic target to reduce inflammation associated with ischemic injury and improve VCA outcomes. DOD CDMRP RTRP W81XWH1910163 RT180168.

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 laryngeal 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.

FK506-loaded PLGA nanoparticles improve long-term survival of a vascularized composite allograft in a murine model.

BackgroundThe side effects of life-long administration of FK506 limit the clinical practice of vascularized composite allografts (VCAs). This study aimed to evaluate the feasibility of FK506-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (FK506 NPs) for prolonging the long-term survival of VCAs and reducing the side effects of FK506.MethodsPLGA nanoparticles loaded with FK506 were prepared by the solvent evaporation method. The characterization of FK506 NPs was evaluated by electron microscopy. To confirm the function and safety of FK506 NPs, these particles were administrated into rats by intraperitoneal injection. The survival time of the allograft, systemic concentration of FK506, anti-rejection activity, and side-effect of FK506 NPs were evaluated in a Brown Norway (BN)-to-Sprague Dawley (SD) epigastric VCA transplantation model.ResultsCompared with the nontreatment, PLGA control and FK506 groups, the median survival times (MST) of the FK506 NP groups were significantly prolonged. The FK506 NPs could maintain therapeutic drug concentration for 60 days. Moreover, cytokine concentrations, flow cytometry of regulatory T cells (Tregs) and histopathology of allografts revealed significantly prolonged immunosuppression by FK506 NPs. FK506 NPs also ameliorated FK506 nephrotoxicity.ConclusionsFK506 NPs prolong the survival time of VCAs in a murine model with minimal nephrotoxicity, and provide a potential clinical strategy for ameliorating long-term side effects of immunosuppressive therapy.

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-αβTCR 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.

Maximizing donors' gifts: A comparison of actual and expected solid organ yield among VCA donors.

Vascularized Composite Allograft (VCA) transplantation provides life-changing transplants, but VCA adds complexity to the donation process and timing, possibly impeding solid organ donation. Expanding upon descriptive analyses, this study examines risk-adjusted predictions versus the observed number of organs donated by VCA donors. Our cohort included VCA donors in the United States during January 1, 2008-December 31, 2017 (n=51), using OPTN Deceased Donor Registration Form data and the Scientific Registry of Transplant Recipients (SRTR) donor yield models to calculate observed-to-expected (O:E) yield ratios. Almost all VCA donors' livers (48/51; 94.1%) and kidneys (92/102; 90.2%) were transplanted, with fewer hearts (28/51; 54.9%), lungs (46/102; 45.1%), pancreata (15/51; 29.4%), and intestines (3/51; 5.9%) transplanted. O:E ratios for overall organ yield were slightly greater than expected for VCA donors (1.10; 95% CI: 1.02-1.17). Liver (1.17: 1.08-1.27) and lung yields (1.38: 1.07-1.68) were both greater than expected, while kidney, heart, and pancreas yields were similar to expected. Across VCA types, bilateral upper limb and abdominal wall donors had better-than-expected yields while uterus, face, and unilateral upper limb donors all had similar-to-expected yields. Solid organ yield among VCA donors was as good or better than predicted, suggesting that VCA donation does not compromise recovery and transplantation of lifesaving organs.

Infectious complications of vascularized composite allograft transplantation.

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. 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. 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.

The landscape of vascularized composite allograft donation in the United States.

Vascularized composite allograft (VCA) donation and transplantation has now demonstrated feasibility and impact to the lives of patients suffering from devastating and traumatic injury. This review summarizes the current landscape of VCA donation in the United States. VCA donations are nonlife-saving allografts, for nonlife-saving transplants - and therefore, conservative donor selection focused on the optimization of donors for the lowest risk to transplant recipients is paramount. VCA donors in the United States are a demographically and clinically diverse group that largely reflects the characteristics of the VCA candidates waiting for transplants. Public opinion about VCA donation is generally supportive, but has lower support for VCA transplants than for solid organ transplants. As an emerging area of transplantation, VCA donation is evolving in the United States with growing interest by the public, and those suffering from catastrophic injury for which reconstructive transplantation may offer excellent outcomes and a high quality of life.

OPTN Vascularized Composite Allograft Waiting List: Current Status and Trends in the United States.

Vascularized composite allograft (VCA) transplantation is a developing area in the field of transplantation. This study used Organ Procurement and Transplantation Network (OPTN) VCA waiting list and transplant data from July 3, 2014 through February 28, 2018, to characterize the OPTN VCA waiting list in terms of composition, removal patterns, waiting time, resulting transplants, and trends over time. Between implementation of the OPTN VCA waiting list on July 3, 2014 and February 28, 2018, 54 candidates-53.7% were male, 79.6% were white, and 70.4% aged 18 to 44 years-were added to the OPTN VCA waiting list. Of these, 22 received deceased donor VCA transplants (6 bilateral upper limb, 4 unilateral upper limb, 5 craniofacial, 1 scalp, 1 abdominal wall, 1 penile, and 4 uterine), and 6 received living donor uterine transplants. Registrations increased in 2016 after uterine and penile transplants were introduced in the United States, resulting in a large shift in the composition of the VCA waiting list. Waiting times for VCA candidates vary greatly, with some VCA candidates receiving deceased donor transplants quickly and others waiting more than 3 years before transplantation. The field of VCA transplantation and the composition of the OPTN VCA waiting list are evolving rapidly. Additional research is needed to understand these changes and investigate whether differences in need or differences in access have resulted in the variation seen on the VCA waiting list.

Vascularized composite allotransplantation in the United States: A descriptive analysis of the Organ Procurement and Transplantation Network Data.

On July 3, 2014, the Organ Procurement and Transplantation Network/United Network for Organ Sharing was charged with the oversight of vascularized composite allograft (VCA) procurement and transplantation in the United States. As of December 31, 2017, 61 VCA programs at 27 centers were approved in the United States. Fifty candidates have been added to the waiting list at 15 centers. Twenty-eight VCA transplants have been performed at 14 programs (10 upper limb, 10 uterus, 5 craniofacial, 1 scalp, 1 abdominal wall, and 1 penile). Twenty-two VCAs were procured from 21 deceased donors, resulting in 109 non-VCA organs transplanted (15 hearts, 3 intestine, 40 kidney, 20 livers, 24 lungs, and 7 pancreata). Six uterus transplants were performed from living donors. Fourteen candidates were still waiting at 9 centers on December 31, 2017. Two of the 10 uterus recipients had live births and 3 still had viable grafts. Seventeen of 18 nonuterus recipients had functioning grafts. At present, VCA is an emerging field with a small number of patients transplanted. Data on posttransplant survival and functional outcomes continue to be collected to further the understanding of this complex and evolving field. Further systematic data are important for policy refinement and assurance of patient safety.

Research Highlights

Research Highlights

Vascularized Composite Allograft Donation and Transplantation: A Survey of Public Attitudes in the United States.

Vascularized composite allograft (VCA) transplantation has emerged as a groundbreaking surgical intervention to return identity and function following traumatic injury, congenital deformity, or disfigurement. While public attitudes toward traditional organ/tissue donation are favorable, little is known about attitudes toward VCA donation and transplantation. A survey was conducted of 1485 U.S. residents in August 2016 to assess VCA donation attitudes. Participants also completed the Revised Health Care System Distrust Scale. Most respondents were willing to donate hands/forearms (67.4%) and legs (66.8%), and almost half (48.0%) were willing to donate the face. Three-quarters (74.4%) of women were willing to donate the uterus; 54.4% of men were willing to donate the penis. VCA donation willingness was more likely among whites and Hispanics (p < 0.001), registered organ/tissue donors (p < 0.001), and those with less health care system distrust (p < 0.001) and media exposure to VCA transplantation (p = 0.003). Many who opposed VCA donation expressed concerns about psychological discomfort, mutilation, identity loss, and the reaction of others to seeing familiar body parts on a stranger. Attitudes toward VCA donation are favorable overall, despite limited exposure to VCA messaging and confusion about how VCA donation occurs. These findings may help guide the development and implementation of VCA public education campaigns.

Abstract 47: Delayed Tolerance Induction Protocol for Vascularized Composite Allografts in Non-Human Primates: The Immunomodulatory Effect of Donor Bone Marrow Transplantation Does Not Prevent the Development of Chronic Rejection in the Absence of Durable Mixed Chimerism

PURPOSE: Our laboratory is currently developing a delayed tolerance induction protocol for vascularized composite allografts (VCAs) in a clinically relevant non-human primate (NHP) model using donor bone marrow transplantation (DBMT) after VCA to induce durable mixed chimerism and tolerance. DBMT has a demonstrated immunomodulatory effect in our previous experimental work and in clinical VCA, enabling some patients to be maintained on low-dose tacrolimus monotherapy. With longer-term follow-up of clinical VCA, recent reports of chronic rejection have emerged. We sought to investigate in further detail the immunomodulatory effect of DBMT on acute and chronic rejection of VCAs in NHPs. METHODS: Following VCA transplantation and maintenance on standard triple immunosuppression (IS) for 2 months, donor bone marrow cells that were previously harvested from the vertebrae, minimally processed and cryopreserved were thawed and infused into MHC-mismatched recipient NHPs (n=6) conditioned with irradiation (total body and thymic), T cell depletion, co-stimulatory blockade and anti-inflammation (with anti-IL-6 receptor monoclonal antibody). A bridging course of calcineurin inhibition (CNI) was given for 4 weeks before IS withdrawal and assessment of the VCA for tolerance. Observed rejection episodes of the VCA were biopsied and treated with CNI and a tapering course of steroids before IS withdrawal after 2 weeks. Systemic immune function was assessed by CFSE-based mixed lymphocyte reaction (MLR) proliferation assays and allo-sensitization was evaluated by the detection of serum allo-antibody formation. Evidence for mixed chimerism was assayed in peripheral blood. RESULTS: Durable mixed chimerism (only detected transiently at 6 weeks post-DBMT) did not develop but recipients managed to come off all IS for 4–5 weeks before acute rejection (Banff II) developed between POD115-POD126. Rejection could be treated and reversed both clinically and histologically. However, following subsequent re-withdrawal of IS, Banff II rejection developed again within 2 weeks (on POD172), could not be reversed despite treatment (Banff II on POD195) and ultimately culminated in necrosis of the VCA (on POD224). Final histology showed severe acute cell-mediated and antibody-mediated rejection with C4d deposition in subcutaneous arteries and arterioles as well as chronic allograft vasculopathy. Despite these findings, corresponding serial MLR assays demonstrate unresponsiveness after DBMT and no allo-antibody formation was detected. CONCLUSION: While DBMT demonstrated systemic immunomodulation based on MLR, in the absence of durable mixed chimerism, this effect was not enough to prevent acute projection episodes and the development of chronic rejection in this cohort of animals. These rejection episodes may be related to the subsequent development of chronic rejection. The presence of vascular lesions (based on C4d deposition) throughout all time points following DBMT may predate clinical progression to eventual chronic allograft vasculopathy and rejection, and may represent a potential prognostic factor in clinical VCA.