Two Months Delayed Induction of Mixed Chimerism After Heart and Kidney Cotransplantation in Nonhuman Primates

S192

The Journal of Heart and Lung Transplantation, Vol 36, No 4S, April 2017

neogenesis. These data suggest that early graft injury may trigger specific forms of chronic alloimmunity.

of transplant that transfer pro-inflammatory properties leading to TA into a humanized mouse model. TA remains sensitive to inhibition by autologous regulatory T cells, suggesting a cell therapy-based approach for the prevention and treatment of CLAD after LTX.

Table

Number of animals Average graft density at day 2-3 (CT) Average graft density at day 27 (CT) Acute rejection score (Pathology) Presence of airway obliteration (Animals with airway obliteration / total animals) (Pathology) Parenchymal fibrosis (% of total area) (Pathology)

4( 93)

CITm+WITm ( Control, CITm+WITm CITm+WIT60 CIT6+WIT60 Allografts) (Allografts) (Allografts) (Allografts)

CIT6+WIT60 (Isografts)

20

6

5

14

6

-437 HU

-435 HU

-466 HU

-300 HU (p< 0.01)

-294 HU

-526 HU

-396 HU

-442 HU

-430 HU

-305 HU (p< 0.05)

2.0

2.0

2.2

2.7 (p< 0.01) 0.17

0/20

0/6

0/5

5/14 (p< 0.005)

0/6

0%

0%

0.4%

13.9% (p< 0.05)

0%

4( 92) Linking Chronic Lung Allograft Dysfunction with Early Immunologic Events in a Humanized Mouse Model T. Siemeni ,1 A. Knöfel,1 F. Ius,1 K. Jansson,1 J. Salman,1 W. Sommer,1 M. Avsar,1 C. Kühn,1 C. Falk,2 A. Haverich,1 G. Warnecke.1  1Hannover HTTG, Hannover, Germany; 2Institute of Transplant Immunology, Hannover, Germany. Purpose: Chronic lung allograft dysfunction (CLAD) is a severe complication of lung transplantation limiting long term survival. Here, we studied leukocyte-mediated development of transplant arteriosclerosis (TA) in a humanized mouse model and correlated the results with the later onset of CLAD in the respective lung transplantation (LTX) recipients. Methods: The pericardiophrenic artery was procured from surplus tissue of donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient NRG mice. Experiments from seventeen lung recipients (LR) were divided into two groups. Six patients (35%) developed CLAD 22±5 months after LTX. The remaining eleven patients (65%) did not develop CLAD within 26±5 months after LTX. Experimental mouse cohorts were divided into three treatments. Negative control mice received no human leukocyte reconstitution (neg. co). PBMC group mice received 5x106 allogeneic human peripheral blood mononuclear cells (PBMC) procured the respective LR, whereas a further group was reconstituted with the respective PBMC enriched with autologous CD4+CD25high cells (putative regulatory T cells, Treg; PBMC+Treg group). Human leukocyte engraftment was monitored by FACS and development of TA was histologically assessed. In the LR, onset of CLAD was monitored. Results: The neg. co group showed only mild thickening of the intima (9.3±9%). In the PBMC group from CLAD+ LR, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC group from CLAD- recipients (37.9 ±11% vs. 15.6±4%, p= 0.015). Then, intimal thickening was significantly inhibited by Treg in the PBMC+Treg group from CLAD+ LR as compared to the respective PBMC group (0.3±4% vs 37.9 ±11%, p= 0.01). In the setting using PBMC from LR without CLAD, enriching Treg also further suppressed the development of TA (0.6±7% PBMC+Treg vs. 15.6±4% PBMC, p= 0.007).Cytokine levels showed no significant differences among all groups. Conclusion: Lung transplant recipients, who later develop CLAD, have peripheral leukocytes and/or donor vessel endothelium already at the time

Selective Reprogramming and Recapitulation of CD4 and CD8 T Cells in the Neonate to Induce Tolerance to Heart Transplants R.A. Bascom , K. Tao, L.J. West.  University of Alberta, Edmonton, AB, Canada. Purpose: Allogeneic spleen and bone marrow cells (allo-SC/BMC) from adult mice induce acute graft-versus-host disease (aGVHD) in non-conditioned neonates. To prevent aGVHD and induce transplant tolerance with allo-SC/BMC we reprogrammed donor/host CD4 T cells by co-stimulation blockade to preserve regulatory CD4 T cells while recapitulating depleted donor/host CD8 T cells. Methods: C3H (H-2k) neonatal mice were injected iv with total or depleted B6 (H-2b) GFP+ SC/BMC. CD8 T and CD49b NK cells were depleted from donor inocula using StemCell Technologies kits. In vivo neonatal host CD8 T cells were depleted (Mab 53-6.7) and donor/host CD4 T cells reprogrammed (CD154 Mab MR1). Trafficking and interactions of injected cells were monitored by microscopy. Tolerance induction was assessed by transplanting treated mice as adults with donor-type hearts. Results: Neonatal mice injected with GFP+ allo-SC/BMC developed aGVHD, with diarrhea, reduced growth and early death. GFP+ cells proliferated and spread throughout injected mice indicating systemic inflammation. Depletion of donor/host CD8 T cells together with co-stimulation blockade of donor/host CD4 T cells (CD154) resulted in GFP signal being reduced and restricted to lymphoid organs (day 6). High resolution microscopy showed donor T and B cells positioned respectively in PALS and follicular regions of host spleen; lack of Ki67 immunostaining indicated many donor cells were not proliferating. Donor DC were detected in host thymus (day 6) suggesting a possible role in central tolerance. Donor-type B6 hearts transplanted into neonatally-treated adult C3H mice (n= 5) (CD8 depleted/CD4 co-stimulation blockade) all continued to beat at 100 days post-transplant, 3 grafts with maximal strength and 2 grafts with diminished strength. In untreated control mice (n= 3) donor-type hearts stopped beating by day 10. H&E staining of a strongly beating B6 heart graft at 100 days showed undamaged cardiomyocytes with little if any cellular infiltrate and absence of macrophages and T cells by immunostaining. Conclusion: Reprograming CD4 T cells while depleting/recapitulating CD8 T cells in allo-SC/BMC treated neonates leads to 100% cardiac allograft survival at 100days; low beat scores in 2/5 grafts suggests further optimization is required. These findings provide insight into robust tolerance induction in neonates. 4( 94) Two Months Delayed Induction of Mixed Chimerism After Heart and Kidney Cotransplantation in Nonhuman Primates K. Huh ,1 W. Sommer,1 K.A. Robinson,1 X. Wu,1 J.T. Paster,1 I. Hanekamp,1 A. Dehnadi,1 T. Kawai,1 R.N. Smith,2 R. Colvin,3 G. Benichou,1 J. Madsen.1  1Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA; 2Department of Pathology, Massachusetts General Hospital, Boston, MA; 3Center for Transplantation Sciences, Department of Pathology, Massachusetts General Hospital, Boston, MA. Purpose: We have previously achieved transplant tolerance in cynomologous recipients cotransplanted with heart and kidney allografts by treating recipients with standard triple drug immunosuppression for 4 months followed by mixed chimerism induction consisting of conditioning and bone marrow transplantation (BMT). Reducing the period of maintenance immunosuppression before BMT may diminish risks of immunosuppression-associated complications, post-transplant proliferative disorder (PTLD) and graft sensitization when applied to the clinic. Here we tested a more clinically relevant 2 month-delayed BMT protocol in heart plus kidney recipients. Methods: Following heart and kidney cotransplantation, recipients received tacrolimus, mycophenolate mofetil and methylprednisolone for 2 months

Abstracts S193 after which they underwent nonmyeloablative conditioning and bone marrow transplantation (BMT). We divided recipients into 2 groups according to the conditioning regimens. Group A consisted of horse anti-thymocyte globulin, anti-CD154 mAb, and anti-CD8 mAb (n= 3). Group B consisted of rabbit antithymocyte globulin and belatacept (n= 4). Three Gy total body irradiation, 7 Gy local thymic irradiation, and 28 day course of cyclosporine were used in both groups. Allo-heart function was monitored by palpation, and serial protocol biopsies of allo-heart and kidney were performed to monitor graft rejection. Results: In group A, one recipient is currently day 462 post-BMT without rejection. Two recipients were euthanized due to post-transplant proliferative disorder (PTLD) and urologic complication before BMT, respectively. In group B, one recipient is surviving day 168 post-BMT without cardiac allograft rejection. Another recipient had acute AMR in allo-heart and allokidney at day 76 post-BMT. Two recipients were euthanized due to PTLD and severe anemia detected day 74 and day 12 post-BMT, respectively. Conclusion: Shortening the interval between organ and BM transplantation to 2 months permits tolerance induction. However, the efficacy and safety of including rabbit anti-thymoglobulin plus belatacept in the conditioning regimens requires further evaluation. 4( 95) Generating Hypoimmunogenic Murine Induced Pluripotent Stem Cells by the Disruption of beta-2-Microglobulin X. Hu ,1 T. Deuse,2 N. Kooreman,3 M. Alawi,4 G. Tediashvili,1 H. Reichenspurner,5 S. Schrepfer.1  1Surgery, TSI Lab, UCSF, San Francisco, CA; 2CT Surgery, UCSF, San Francisco, CA; 3Cardiovascular Medicine, Stanford University, Stanford, CA; 4Heinrich Pette, UKE, Hamburg, Germany; 5Cardiovascular Surgery, UHZ, Hamburg, Germany. Purpose: Induced pluripotent stem cells (iPSCs) are promising candidates for truly regenerative cell-based therapies. Because autogenic iPSCs are not available for acute treatment procedures, allogeneic strategies have to be developed which evade immune rejection. Since major histocompatibility complex (MHC) antigens are the major immune antigens, we sought to generate MHC I-deficient murine iPSCs (miPSCs) via disruption of beta-2-microglobulin. Methods: IPSCs were generated from C57BL/6 (H2b) mouse fibroblasts by non-viral minicircle transfection of the transcription factors c-Myc, Sox-2, Klf4 and Oct4. CRIPSR/Cas9 technology was used to generate β 2m knockout miPSCs (β 2m-/- miPSCs). Unmodified and β 2m-/- miPSCs were injected into syngeneic C57BL/6 or allogeneic BALB/c (H2d) mice. The cellular and antibody response was quantified and teratoma formation assays were performed to assess cell survival. Results: FACS analysis showed normal expression of MHC I on unmodified miPSCs and depleted MHC I on β 2m-/- miPSCs. Our results indicate a strongly decreased immunogenicity of β 2m-/- miPSC compared to unmodified counterparts. After allogeneic transplantation into BALB/c mice, β 2m-/miPSCs generated a significantly weaker response in both IFNγ  (p< 0.01) and IL-4 (p< 0.01) ELISPOT assays. Also, the level of donor-specific antobodies was significant lower for β 2m-/- miPSC (p< 0.01). Overall, the level of allogeneic immune activation by β 2m-/- miPSCs in both assays was so low that it was even similar to that of syngeneic transplants. The reduced immunogenicity also translated into higher cell survival rates. Teratomas developed out of β 2m-/- miPSCs in 60% of the allogeneic,recipients whereas no teratoma formation occurred with unmodified miPSCs. Conclusion: Our results clearly demonstrate that the main burden of immunogenicity is from MHC I antigens and that MHC I depletion generated largely hypoimmunogenic cells. Thus, MHC I-deficient iPSCs might serve an unlimited cell source for the generation of universally-compatible “offthe-shelf” cells grafts or tissues in future clinical applications. Subsequent research has to assess how the immunogenicity changes with cell differentiation into derivatives of β 2m-/- iPSCs. 4( 96) Advanced Therapy Utilization and Survival in Ambulatory Patients with Advanced Heart Failure: Results from the Medical Arm of Mechanically Assisted Circulatory Support (MedaMACS) Registry V. Ambardekar ,1 M. Kittleson,2 M. Palardy,3 M. Mountis,4 R. FordeMcLean,5 A. DeVore,6 S. Pamboukian,7 J. Thibodeau,8 J. Teuteberg,9 L. Cadaret,10 R. Xie,7 L. Stevenson,11 G. Stewart.11  1U Colorado, Aurora,

CO; 2Cedars, Los Angeles, CA; 3U Michigan, Ann Arbor, MI; 4Cleveland Clinic, Cleveland, OH; 5U Penn, Philadelphia, PA; 6Duke, Durham, NC; 7UAB, Birmingham, AL; 8UTSW, Dallas, TX; 9U Pitt, Pittsburgh, PA; 10U Iowa, Iowa City, IA; 11Brigham, Boston, MA. Summary of Objectives: The outlook for ambulatory patients on optimal oral medical therapy for advanced heart failure (HF) and the appropriate timing of transplant or ventricular assist device (VAD) implant remain uncertain. The goal of the Medical Arm of Mechanically Assisted Circulatory Support (MedaMACS) Registry was to identify and study a multi-center cohort of these patients to better understand and address their disease trajectory. Methods: Ambulatory patients with advanced HF were prospectively enrolled from 11 VAD/transplant centers across the U.S. from May 2013 to October 2015. Inclusion criteria included HF for at least one year, LVEF≤ 35%, NYHA class III-IV, INTERMACS Profile 4-7, at least one HF hospitalization in the last year, plus one additional high risk feature (additional HF hospitalizations within one year, peak oxygen uptake < 55% predicted, 6-minute walk distance < 300 meters, BNP> 1000 or NT-proBNP> 4000 pg/mL, or a Seattle HF Model 1 year survival of ≤ 83%). Patients were excluded if they were inotrope dependent, listed for transplant, or had a non-cardiac diagnosis limiting functional status or survival. At the time of enrollment information was collected on demographics, clinical characteristics, functional capacity, and quality-of-life. Subjects were followed for 1-2 years for primary endpoints of survival, transplantation, and LVAD placement, and secondary endpoints of hospitalization, inotrope utilization, functional status, health-related quality of life, and satisfaction with therapy. A total of 161 patients were enrolled. Baseline characteristics included a mean age of 59±11 years, LVEF of 21±7%, and NT-proBNP of 5365±5065 pg/ml with 31% of patients of nonCaucasian race and 36% female gender. INTERMACS profiles at enrollment were: Profile 4 (12%), Profile 5 (32%) Profile 6 (49%), and Profile 7 (7%). Endpoints: The baseline therapies and severity of disease will be presented with the 12 month actuarial survival on medical therapy and the rates of transplant and VAD placement using cumulative incidence methods, as all patients will have one year of follow-up by October 31, 2016. In addition, data on secondary end-points of hospitalization frequency, inotrope use, functional capacity, and quality of life will be presented. This large, multicenter, real world registry enrolled a diverse patient population with ambulatory advanced HF predominantly in INTERMACS profiles 5-6, an understudied cohort of HF patients. The MedaMACS one year follow up data will provide unique insights into the profiles and prognosis of patients with advanced HF for whom the optimal timing of VAD implant and transplant have not been established. 4( 97) The Effects of Patient Blood Pressure on the Occurrence of Serious Adverse Events in the ENDURANCE Trial J.A. Cowger ,1 J.G. Rogers,2 C.A. Milano,2 J.J. Teuteberg,3 N.A. Mokadam,4 E.J. Birks,5 S.W. Boyce,6 K.D. Aaronson,7 F.D. Pagani.7  1St. Vincent Heart Center, Indianapolis, IN; 2Duke University Medical Center, Durham, NC; 3University of Pittsburgh Medical Center, Pittsburgh, PA; 4University of Washington, Seattle, WA; 5University of Louisville, Louisville, KY; 6MedStar Health, Washington, DC; 7University of Michigan Medical Center, Ann Arbor, IL. Purpose: Current literature has reported ventricular assist device (VAD) patients with elevated blood pressure are at a higher risk for experiencing adverse events such as pump thrombosis and cerebrovascular accidents. As a result, mechanical circulatory support guidelines for patient management have generally suggested keeping mean arterial blood pressure (MAP) below 90mmHg. Further investigation into the relationship between blood pressure and VAD patient outcomes is necessary to determine if the risk continuum for blood pressure is more complex than a binary threshold. Methods: In the prospective, randomized ENDURANCE trial, 296 patients were supported by a HeartWare ventricular assist device system (HVAD). The HVAD cohort was divided into quartiles based on MAP as well as systolic blood pressure (SBP) measured at discharge post VAD implant. The patient quartiles were assessed for occurrence of pump thrombosis, ischemic, and hemorrhagic cerebrovascular accidents (ICVA and HCVA respectively). Logistic regression analyses with continuous predictors were used to test association.