Induction of Chimerism in Rhesus Macaques through Stem Cell Transplant and Costimulation Blockade-Based Immunosuppression
2007; Elsevier BV; Volume: 7; Issue: 2 Linguagem: Inglês
10.1111/j.1600-6143.2006.01622.x
ISSN1600-6143
AutoresLS Kean, Andrew Adams, Elizabeth Strobert, Rose Hendrix, Shivaprakash Gangappa, TR Jones, Nozomu Shirasugi, MR Rigby, Kelly Hamby, J. Jiang, Helia Bello‐Toledo, Douglas J. Anderson, Kenneth Cardona, MM Durham, T. C. Pearson, CP Larsen,
Tópico(s)Organ and Tissue Transplantation Research
ResumoAmerican Journal of TransplantationVolume 7, Issue 2 p. 320-335 Free Access Induction of Chimerism in Rhesus Macaques through Stem Cell Transplant and Costimulation Blockade-Based Immunosuppression L. S. Kean, L. S. Kean The Emory Transplant Center, Department of Surgery Division of Hematology/Oncology/BMT, The Aflac Cancer Center and Blood Disorders Clinic, Department of Pediatrics, Emory University School of Medicine, Atlanta, GeorgiaSearch for more papers by this authorA. B. Adams, A. B. Adams The Emory Transplant Center, Department of SurgerySearch for more papers by this authorE. Strobert, E. Strobert Yerkes National Primate Research Center, Emory University, Atlanta, GeorgiaSearch for more papers by this authorR. Hendrix, R. Hendrix The Emory Transplant Center, Department of SurgerySearch for more papers by this authorS. Gangappa, S. Gangappa The Emory Transplant Center, Department of SurgerySearch for more papers by this authorT. R. Jones, T. R. Jones The Emory Transplant Center, Department of SurgerySearch for more papers by this authorN. Shirasugi, N. Shirasugi The Emory Transplant Center, Department of SurgerySearch for more papers by this authorM. R. Rigby, M. R. Rigby The Emory Transplant Center, Department of Surgery Division of Critical Care Medicine, Department of Pediatrics, Emory University School of Medicine, Atlanta, GeorgiaSearch for more papers by this authorK. Hamby, K. Hamby The Emory Transplant Center, Department of SurgerySearch for more papers by this authorJ. Jiang, J. Jiang The Emory Transplant Center, Department of SurgerySearch for more papers by this authorH. Bello, H. Bello The Emory Transplant Center, Department of SurgerySearch for more papers by this authorD. Anderson, D. Anderson Yerkes National Primate Research Center, Emory University, Atlanta, GeorgiaSearch for more papers by this authorK. Cardona, K. Cardona The Emory Transplant Center, Department of SurgerySearch for more papers by this authorM. M. Durham, M. M. Durham The Emory Transplant Center, Department of SurgerySearch for more papers by this authorT. C. Pearson, T. C. Pearson The Emory Transplant Center, Department of SurgerySearch for more papers by this authorC. P. Larsen, Corresponding Author C. P. Larsen The Emory Transplant Center, Department of Surgery * Corresponding author: Christian P. Larsen, clarsen@emory.orgSearch for more papers by this author L. S. Kean, L. S. Kean The Emory Transplant Center, Department of Surgery Division of Hematology/Oncology/BMT, The Aflac Cancer Center and Blood Disorders Clinic, Department of Pediatrics, Emory University School of Medicine, Atlanta, GeorgiaSearch for more papers by this authorA. B. Adams, A. B. Adams The Emory Transplant Center, Department of SurgerySearch for more papers by this authorE. Strobert, E. Strobert Yerkes National Primate Research Center, Emory University, Atlanta, GeorgiaSearch for more papers by this authorR. Hendrix, R. Hendrix The Emory Transplant Center, Department of SurgerySearch for more papers by this authorS. Gangappa, S. Gangappa The Emory Transplant Center, Department of SurgerySearch for more papers by this authorT. R. Jones, T. R. Jones The Emory Transplant Center, Department of SurgerySearch for more papers by this authorN. Shirasugi, N. Shirasugi The Emory Transplant Center, Department of SurgerySearch for more papers by this authorM. R. Rigby, M. R. Rigby The Emory Transplant Center, Department of Surgery Division of Critical Care Medicine, Department of Pediatrics, Emory University School of Medicine, Atlanta, GeorgiaSearch for more papers by this authorK. Hamby, K. Hamby The Emory Transplant Center, Department of SurgerySearch for more papers by this authorJ. Jiang, J. Jiang The Emory Transplant Center, Department of SurgerySearch for more papers by this authorH. Bello, H. Bello The Emory Transplant Center, Department of SurgerySearch for more papers by this authorD. Anderson, D. Anderson Yerkes National Primate Research Center, Emory University, Atlanta, GeorgiaSearch for more papers by this authorK. Cardona, K. Cardona The Emory Transplant Center, Department of SurgerySearch for more papers by this authorM. M. Durham, M. M. Durham The Emory Transplant Center, Department of SurgerySearch for more papers by this authorT. C. Pearson, T. C. Pearson The Emory Transplant Center, Department of SurgerySearch for more papers by this authorC. P. Larsen, Corresponding Author C. P. Larsen The Emory Transplant Center, Department of Surgery * Corresponding author: Christian P. Larsen, clarsen@emory.orgSearch for more papers by this author First published: 18 January 2007 https://doi.org/10.1111/j.1600-6143.2006.01622.xCitations: 57AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. Mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. Nevertheless, this chimerism-induction regimen induced amongst the longest-lived stem cell chimerism reported to date for non-human primates and thus represents a platform upon which to evaluate emerging tolerance-induction strategies. Introduction In murine transplantation, the induction of mixed hematopoietic chimerism is a potent method of promoting transplant tolerance (1). Early strategies resulted in full donor chimerism, and although tolerance ensued, significant immunodeficiencies occurred when performed between major histocompatibility complex (MHC) disparate transplant pairs (2-4). This incited interest in establishing mixed chimerism, which could potentially increase immunocompetence (4). Initial approaches involved near-complete, non-selective donor T-cell depletion (1, 5). More recently, an emphasis on selective T-cell deletion/inactivation using transient blockade of costimulatory pathways has produced mixed chimerism in mice (1, 6, 7). Costimulation blockade induces selective deletion of peripheral donor-reactive cells (6-9), which, when combined with the maintenance of tolerance by thymic deletion of emerging donor-reactive T cells (6, 7, 10, 11), allows long-term acceptance of MHC disparate grafts. Given its potential importance to clinical transplantation, the translation of sustained chimerism to pre-clinical non-human primate (NHP) models has been actively pursued. Kawai and colleagues used a combination of total body irradiation, local thymic irradiation, anti-thymocyte globulin (ATG), anti-CD154 and cyclosporin to promote acceptance of donor bone marrow (12-18). Additionally, select examples in human transplantation support the potential utility of chimerism-induction-based acceptance of solid organ transplants (19-22). In NHP, these strategies achieved donor chimerism, although its durability was relatively shorter than in mice (13, 14, 16-18). Despite an incomplete understanding of the underlying mechanisms, these studies provide proof of concept that even transient chimerism can lead to tolerance to solid organ allografts. The induction of sustained chimerism after hematopoietic stem cell transplant (HSCT) remains a significant goal after non-myeloablative transplant. Successful induction of sustained chimerism has two major potential clinical applications: first, in organ transplantation, in order to promote robust donor-specific tolerance with freedom from chronic immunosuppression; and second, in non-malignant hematologic disease and inborn errors of metabolism, where such an induction is capable of curing disease (23-26). Correcting hematologic and genetic abnormalities will require durable macrochimerism and thus, these diseases are not amenable to transient chimerism-induction. Herein, we describe a T-cell costimulation blockade-based regimen in NHP that reliably induces high-level chimerism that persists for as many as 196 days post-transplant in the setting of ongoing immunosuppression. While the risk-benefit ratio for selected patients severely affected with hematologic disease may support a strategy requiring chronic immunosuppression, further advances will be required for chimerism and tolerance-induction for organ transplantation. Methods Experimental animals This study utilized rhesus macaques from either the Yerkes National Primate Research Center or the NIH-sponsored NHP colony at Yemassee, SC, managed by Alphagenesis, Inc. All animals were treated in accordance with IACUC regulations. Donor-recipient pairings were based on MHC typing (27-29) to maximize disparity at both class I and class II loci. Sustained chimerism-induction strategy The standard chimerism-induction strategy (abbreviated 'CoBBS') is shown in Figure 1. It consisted of a pre-transplant busulfan infusion (10 mg/kg Busulfex, ESP Pharma, Clinton, NJ), followed by immune modulation with: (i) Two doses of the anti-IL2-receptor antibody basiliximab (Novartis, Basel, Switzerland, 0.3 mg/kg) within the following treatment windows: first, day –1 or day 0; and second, day 3 or day 5. (ii) Blockade of CD28 signaling with the belatacept fusion protein (20 mg/kg/dose, supplied by Bristol Myers Squibb, NY) and CD154 blockade with the H106 monoclonal antibody (20 mg/kg/dose, supplied by Bristol Myers Sqibb), each infused on the following days relative to transplant: Belatacept on day −1, +3, 15, 29, 43, 57, 71, 85, 99, 113, 127, and H106 on day −1, +1, 3, 6, 8, 10, 15, 29, 43, 57, 71, 85, 99, 113, 127. (iii) Sirolimus (Wyeth-Ayerst, Madison, NJ) was dosed daily beginning at day −3 and continued through day 77–100 post-transplant (Table 1), to maintain troughs of 5–15 ng/mL. Figure 1Open in figure viewerPowerPoint Transplant strategy: Our standard chimerism-induction strategy included a single non-myeloablative dose of busulfan given on day −1 relative to the transplant and induction with two peritransplant doses of the IL2-receptor blocking monoclonal antibody basiliximab, and maintenance therapy with anti-CD28/anti-CD154 CoB (belatacept and H106) and sirolimus. This strategy, referred to as 'CoBBS', when accompanied by a stem cell infusion on day 0 routinely produced mixed chimerism in transplant recipient animals. While the treatment with CoB and sirolimus ideally continued until day 100–127 after stem cell transplant, the clinical condition of the transplant often necessitated early withdrawal of one or more of these agents, as indicated in Table 1. Table 1. Characteristics of transplant recipients, hematopoietic stem cell products and transplant outcomes Animal ID Treatment Cell dose/kg (×109) Peak chimerism % Chimerism duration (days) Day of sirolimus d/c Indication of sirolimus d/c Day of CoB d/c Indication for CoB D/C RAf-7 CoBBS 5.4 0 0 90 Diarrhea, dehydration 112 Per protocol RHf-7 CoBBS 4.8 nd nd 9 Herpes B reactivation 9 Herpes B reactivation3 RUn-7 CoBBS 5.3 65 63 9 Herpes B reactivation 112 Per protocol RYj-7 CoBBS 4.7 100 174 62 CMV 112 Per protocol4 RRi-8 CoBBS 7.2 90 189 78 CMV 127 Per protocol RUi-8 CoBBS 2.7 60 161 78 CMV 127 Per protocol RWb-8 CoBBS 5 90 119 77 CMV 127 Per protocol CW7B CoBBS1 2.1 90 133 90 CMV 180 Per protocol RVq-8 CoBBS1,2 2 99 165 161 Animal euthanized 188 Animal euthanized5 RAi-7 CoBBS + CD8 depletion nd 95 196 98 Colitis, dehydration 98 Colitis, dehydration RMf-7 CoBBS + CD8 depletion 5.9 70 175 97 Per protocol 112 Per protocol RUh-7 CoBBS + CD8 depletion 6.8 100 36 36 Herpes B reactivation 28 Herpes B reactivation6 RBg-7 CoBBS + CD8 depletion + DST 2.5 30 133 100 Per protocol 140 Per protocol RTd-7 CoBBS + CD8 depletion + DST 2.7 20 77 100 Per protocol 140 Per protocol RGy-6 CoBBS + CD8 depletion + DST 3.7 20 77 70 Per Protocol 42 Per protocol RSk-7 CoBBS + thymectomy 2.6 60 133 100 Per protocol 112 Per protocol RUu-7 CoBBS + thymectomy 4 20 49 100 Per protocol 112 Per protocol RVu-7 CoBBS + thymectomy 4.9 15 56 38 Acute illness 100 Per protocol CG8B CoBBS + DSG 5.6 60 56 60 Severe diarrhea 55 Severe diarrhea7 RDp-8 CoBBS + DSG 3.5 100 84 86 CMV 71 CMV8 RVf-8 CoBBS + DSG 4.8 90 112 100 Per protocol 85 Per protocol 1For CW7B and RVq-8, chimerism induced after a transplant of peripheral blood stem cells rather than from a bone marrow product. 2RVq-8 initially received a leukopheresis stem cell product that was inadequate to induce chimerism: While the original stem cell product was produced after mobilization with 10 μg/kg GCSF, subsequent leukopheresis products were produced after donor stem cell mobilization with 100 μg/kg GCSF. RVq-9 was thus given a second transplant. All measurements with RVq-8 are relative to the second, successful stem cell infusion. 3Animal was euthanized due to complications of Herpes B reactivation. 4Animal died from acute renal failure and campylobacter sepsis. 5Animal was euthanized in the setting of pancytopenia and a GI bleed after loss of chimerism. 6Animal was euthanized due to complications of Herpes B reactivation. 7Animal died from bacterial typhlitis. 8Animal was euthanized due to complications of CMV reactivation. Modifications to CoBBS CD8 depletion CD8 depletion occurred after OKT8 treatment (5 mg/kg) on days −5, −3, 0, 2, 4, 7, 9, 11, 14 relative to bone marrow transplant (BMT). Donor-specific transfusion A 6 mL/kg whole blood transfusion was given 1 week prior to BMT. After donor-specific transfusion (DST), belatacept and H106 infusions were given on days −6, −4 and −1 and sirolimus was begun on day −6. Thymectomy Recipient animals were surgically thymectomized (as previously described (30)) 14–21 days prior to BMT. Deoxyspergualin Recipient animals were treated with the LF15-0195 analog of deoxyspergualin (DSG) (Richman Chemical, Lower Gwynedd, PA) (31, 32) dosed at 0.3 mg/kg subcutaneously on days −1 through 14, followed by a second 14 day course (0.3 mg/kg/dose) beginning either on day 79 (animal RDp-8) or 91 (animal RVf-8). HSCT Bone marrow Bone marrow was harvested from the vertebral column, long bones and pelvis of terminal donors and then centrifuged at 1500 RPM for 8 min prior to resuspension in RPMI plus 0.2 mg/mL DNAse (Sigma) for 45 min at 37°C. Finally, it was recentrifuged, filtered through a 70μM filter and resuspended in sterile PBS plus heparin (3 u/mL). Peripheral blood stem cell infusion Leukopheresis was performed after donors received 8 days of G-CSF (100 μg/kg, Amgen, Thousand Oaks, CA). The femoral vein was catheterized (9F Duo Flow catheter, Medcomp, Harleysville, PA) prior to leukopheresis with a Cobe Spectra (Gambro BCT, Lakewood, CO) using the auto-PBSC settings and human albumin priming. The leukopheresis circuit was anticoagulated with sodium citrate and the animal concomitantly infused with Calcium Gluconate (2 g per 4 h). iSTAT (Abbott Point-of-Care, East Windsor, NJ) chemistry and hematology analysis was performed to assure calcium homeostasis. Hypotension was treated either with volume or vasopressors (dopamine, maintaining diastolic blood pressure >30 mmHg). Typically, six harvests (using a 5 mL harvest volume and 7 mL chase volume) were collected and infused into the recipient without further processing. Analysis of the hematopoietic stem cell product Both bone marrow and leukopheresis products were analyzed for the following: (i) total nucleated cell dose, (ii) CD34+ cell dose, (iii) CD3+ T-cell dose, (iv) CD4+ T-cell dose, (v) CD4+/CD25+ T-cell dose, (vi) CD8+ T-cell dose and (vii) B-cell dose. This analysis was performed either by automated CBC or by flow cytometric analysis with anti-CD34 (clone 563), anti-CD3 (clone SP34), anti-CD4 (clone SK3), anti-CD8 (clone SK1), anti-CD25 (clone 3G10) and anti-CD20 (clone L27), all antibodies from Pharmingen, San Jose, CA. Chimerism monitoring Donor Mamu A*01, A*03/04, A*08 and/or B*01 MHC alleles were monitored by real-time SybrGreen PCR (ABI, Foster City, CA) (29, 33-35) or for the SRY gene in male-to-female transplantation (36). Sixty nanograms of genomic DNA were used in a 20 μL reaction, containing 200 mM dNTPs, 0.1 U AmpliTaq DNA polymerase and XU-AmpERASE-UNG. Reactions were amplified for 40 cycles using a 7900HT Sequence Detection Systems (Applied Biosystems, Foster City, CA). To construct a standard curve, 60 nanogram mixtures of donor and recipient DNA (representing 5%, 10%, 50% and 100% donor DNA) were prepared and used as PCR templates. Lineage chimerism determination T cell, B cell and granulocyte lineages were sorted using a Miltenyi MACS system (Miltenyi, Auburn, CA) or a FACSAria cell sorter (Becton Dickinson, San Diego, CA) after labeling with CD3 (T cells, clone SP34), CD20 (B cells, clone L27) or CD14 (granulocytes, clone M5E2). Sorted populations were routinely purified to >95% purity (data not shown). After sorting, chimerism was determined as described above. Measuring anti-donor T-cell alloreactivity using CFSE-MLR Recipient peripheral blood lymphocytes (PBL) were enriched for T lymphocytes by depletion of antigen presenting cells by binding to anti-mouse IgG-Dynal Beads (Invitrogen, Carlsbad, CA) after labeling with anti-CD20 and anti-HLA-DR antibodies (Pharmingen). The enriched T-cell fraction was labeled with 5μM CFSE (Molecular Probes, Eugene, OR) and 2 ×105 were incubated for 5 days at 37°C with 2×105 donor PBL. Flow cytometric analysis of APC-labeled anti-CD4 and anti-CD8 antibodies (clones SK3 and SK1, respectively) and CFSE was performed. The CD8-APC antibody labels T cells with a greater fluorescence intensity than does the CD4-APC and, therefore, both populations could be monitored simultaneously. CMV monitoring The number of cytomegalovirus (CMV) copies/mL of whole blood was determined by real-time PCR using TaqMan chemistry. To determine the viral load, 7.5 μL template genomic DNA was placed into a 50 μL PCR reaction using the TaqMan Universal Master Mix (Applied Biosystems). Reactions were amplified for 40 cycles using the default conditions on the ABI 7900HT Sequence Detection System. Sample threshold cycles were converted to quantities per PCR reaction by a standard curve consisting of dilutions of plasmid DNA. Copies of virus/reaction were converted to copies/mL blood by multiplying by the conversion factor of 66.7, the ratio of the amount of DNA analyzed to the amount of DNA in 1 mL of whole blood. The primers used were previously described (28, 29). CMV prophylaxis and treatment regimen Weekly CMV PCR-based surveillance was performed and a prophylaxis strategy was developed including weekly infusions of cidofovir (5 mg/kg i.v.) from day 21 post-transplant until day 90 and oral valganciclovir (12 mg/kg) thereafter. If CMV reactivation was detected, treatment included twice daily ganciclovir (6 mg/kg/dose subcutaneously). Once CMV PCR became negative, ganciclovir treatment was continued twice daily for an additional week, decreased to once daily for 2 weeks, then discontinued and oral valganciclovir was resumed. Results Strategy for the establishment of long-term multilineage chimerism in rhesus macaques Our standard transplant strategy (referred to as CoBBS for Costimulation Blockade/basiliximab/sirolimus) is shown in Figure 1. As shown in the figure, our protocol employed preparation with a single non-myeloablative dose of busulfan. To determine optimal busulfan dosing, a dose-response analysis was performed (Figure 2A–I). While 4 mg/kg and 8 mg/kg produced only modest decreases in the total white blood cell (WBC) count, absolute neutrophil count (ANC) and platelet count (Figure 2A–C), a single 10 mg/kg dose of busulfan produced more substantial cytopenias while still allowing count recovery without stem cell rescue. Figure 2E–I shows that while individual variation existed, 10 mg/kg busulfan resulted in an ANC nadir at ∼21 days, which ranged from 0.171×103/μL–0.608×103/μL. The platelet count nadir exhibited a wider range, from 11×106/μL to 231×106/μL. While one animal (RRq-8) displayed prolonged thrombocytopenia, the other three animals exhibited less extensive decreases in platelet count. Neither the hemoglobin concentration nor the absolute lymphocyte count fell significantly (Figure 2H,I), consistent with the myelospecificity of busulfan. As shown in Figure 2D, the 10 mg/kg busulfan dose cleared rapidly from the peripheral circulation, reaching undetectable levels after 10 h. Figure 3 shows that in a representative animal who received 10 mg/kg busulfan along with HSCT, significantly attenuated cytopenias resulted (nadir ANC was 1.5×103/μL, 14 days post-transplant and nadir platelet count was 163×106/μL, 28 days post-transplant), consistent with rapid hematologic replenishment after HSCT. Figure 2Open in figure viewerPowerPoint Pre-transplant infusion of 10 mg/kg busulfan achieves myelosuppression but not myeloablation and is lymphocyte sparing. (A–C) Effect of dosing with either 4 mg/kg or 8 mg/kg busulfan on WBC count (Figure 2A), ANC (Figure 2B) and platelet count (Figure 2C). Shown is the mean ±SEM (n = 3). (D) A single 10 mg/kg busulfan dose is cleared from the peripheral blood within 10 h after infusion. After the busulfan infusion, serial blood draws were collected and analyzed at the Emory University clinical laboratory for serum busulfan levels. Shown is the clearance of busulfan over time in a single representative animal (of three animals in which this pharmacokinetic analysis was performed). (E–H) Analysis of hematopoiesis in four animals receiving a single 10 mg/kg busulfan infusion. Serial WBC (Figure 2E), ANC (Figure 2F), platelet counts (Figure 2G), hemoglobin concentration (Figure 2H) and absolute lymphocyte counts (Figure 2I) were performed either using a standard veterinary CBC analyzer and manual differential counting (Figure 2E–H) or FACScan flow cytometer (Figure 2I). Shown are the results of these analyses in four animals separately treated with 10 mg/kg busulfan. Units on these measurements are as follows: WBC: ×103/μL, ANC: ×103/μL, platelets: ×106/μL, Hb: g/dL, absolute lymphocyte counts: ×103/μL. Figure 3Open in figure viewerPowerPoint Analysis of hematopoiesis in a representative animal receiving 10 mg/kg busulfan in the context of hematopoietic stem cell transplantation and CoBBS immunomodulation. Shown is the WBC (Figure 3A, ×103/μL), ANC (Figure 3B, ×103/μL), platelet count (Figure 3C, ×106/μL), hemoglobin (Figure 3D, g/dL) and absolute lymphocyte count (Figure 3E, ×103/μL) for a single animal, representative of the eight evaluable animals treated with the CoBBS transplant regimen. CoBBS-mediated chimerism-induction using leukopheresis-derived stem cells In addition to BMT, we developed a leukopheresis-based strategy for harvesting sufficient stem cells from the peripheral blood of living NHP. Living donors potentially increase our ability to perform post-transplant immune monitoring (on freshly isolated donor tissues) and, in the future, solid organ transplants after chimerism-induction. Figure 4A shows that both bone marrow and leukopheresis products yielded significant transplant innocula, with mean nucleated cell doses of 6.7×109 for bone marrow and 8.2×109 for leukopheresis (n = 3). Both had similar CD34+ cell percentages (bone marrow, 7.4 ± 2.3% CD34+ cells; leukopheresis, 10 ± 4.9% CD34+ cells). The stem cell-like CD34+/CD38– fraction was enriched in the bone marrow, (3± 1% CD34+/CD38– cells), while leukopheresis products possessed 0.8 ± 0.2% CD34+/CD38– cells. Both were infused without fractionation, so that recipient animals also received differentiated hematopoietic cells. As shown in Figure 4A, bone marrow had significantly fewer T cells (5.4 ± 1%) than leukopheresis (16.6 ± 5.3%), and CD4 and CD8 T cells were similarly reduced (1.8 ± 0.7% vs. 7.7 ± 2.2% CD4+ T cells and 3.3 ± 0.7% vs. 7.6 ± 2.1% CD8+ T cells, respectively), as were B cells (7 ± 0.5% vs. 13 ± 3.5%, respectively). Leukopheresis products also possessed increased numbers of CD4+CD25+ cells (3.5 ± 2% compared with 0.23 ± 0.1% in bone marrow). Figure 4B shows that the leukopheresis-derived HSCT was as efficient as BMT in producing donor chimerism (peak chimerism levels of 86% compared to 80%, respectively). Figure 4Open in figure viewerPowerPoint Leukopheresis represents a viable alternative to terminal bone marrow harvest for producing mixed chimerism after non-myeloablative transplant in the setting of the CoBBS transplant strategy. (A) Comparison of the components of bone marrow or leukopheresis products. Cell dose: ×10–9. Each parameter is shown as the mean ±SEM, n = 3 separate stem cell donors. (B) Comparison of the peak % peripheral blood donor chimerism from bone marrow (left-hand panel, mean ± SEM n = 3) or leukopheresis (right-hand panel, mean, n = 2) transplants with the CoBBS transplant strategy. Seven recipients received BMT + CoBBS; two received leukopheresis product HSCT + CoBBS. One recipient was not evaluated as he was terminated after 1 week due to Herpes B reactivation. As shown in Figure 5 and Table 1, CoBBS resulted in high-level peripheral blood donor chimerism in seven of the eight evaluable recipients. Recipient RAf-7 (Table 1 and Figure 5A) never achieved chimerism despite adequate bone marrow dose (5.4×109 nucleated cells/kg vs. 5± 1×109 nucleated cells/kg for other transplants). While the cause of this early, isolated lack of engraftment remains unknown, all sixteen subsequent transplants reported in this study did result in chimerism. All seven chimeric animals sustained high levels for the duration of post-transplant treatment with both costimulation blockade (CoB) and sirolimus. Recipient RYj-7 remained chimeric when he died from acute renal failure and campylobacter sepsis on day 200 post-transplant, despite sirolimus discontinuation on day 62 and CoB discontinuation on day 112. The remaining six chimeric animals lost chimerism after either sirolimus discontinuation (Run-7, RWb-8, CW7B, RVq-8, Table 1 and Figure 5B,F–H, respectively) or CoB discontinuation (RRi-8, RUi-8, Table 1 and Figure 5D,E, respectively). As shown in Figure 5I, chimerism existed in multiple sites, including bone marrow, spleen, and lymph nodes. However, despite significant granulocyte chimerism (55%), T-cell chimerism was much reduced (2.9%, Figure 5J). In clinical BMT, a lack of significant T-cell chimerism increases the potential for rejection (37-40), as occurred in these transplants. Despite the lack of indefinite chimerism-induction, CoBBS led to greater chimerism durability than previously reported (13, 17, 18, 41). While other regimens have led to <50 days duration of mixed-chimerism (17), donor cells were present in our transplants for a median of 119 days, and extended as far as 196 days post-transplant. Figure 5Open in figure viewerPowerPoint Peripheral blood donor chimerism in animals transplanted in the context of the CoBBS immunomodulation regimen. (A–H) Percent peripheral blood donor chimerism was determined as described in Methods for animals treated with the CoBBS regimen. As shown in Table 1, all recipients received a bone marrow transplant, except for CW7B and RVq-8, who received leukopheresis transplants. Chimerism is shown for each individual animal over time, with the date of discontinuation of sirolimus (S arrow) or CoB (C arrow) shown superimposed on each chimerism graph. (I) Chimerism in the bone marrow, spleen and lymph nodes was determined for one representative animal after its demise from viral sepsis. Chimerism analysis was performed by real-time PCR, as detailed in Methods. (J) Lineage-specific analysis reveals higher levels of myeloid than lymphoid chimerism. T cells, B cells and granulocytes were sorted as detailed in Methods and then the percent donor chimerism was determined by real-time PCR as detailed in Methods. Shown is the mean ± SEM for six separate lineage-chimerism determinations. Modifications to the standard chimerism-induction strategy did not improve the level or durability of chimerism Based on evidence from murine tolerance-induction models (7, 11, 32, 42, 43), we evaluated four
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