American Journal of Transplantation 2008; 8: 1262–1274 C 2008 The Authors Blackwell Munksgaard Journal compilation C 2008 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/j.1600-6143.2008.02230.x

Combined Islet and Hematopoietic Stem Cell : A Clinical Pilot Trial to Induce Chimerism and Tolerance

D. Mineoa,b, C. Ricordia,c,d,X.Xua, A. Pileggia,c, allotransplantation using an ‘Edmonton-like’ immuno- R. Garcia-Moralesc,e, A. Khana, D. A. Baidala, suppression, without ablative conditioning, did not lead to stable chimerism and graft tolerance. D. Hana, K. Monroya, J. Millerc,e, A. Pugliesea,d, T. Frouda,c,f, L. Inverardia,d, N. S. Kenyona,c,d,† a,g,†,∗ Key words: Chimerism, graft tolerance, hematopoietic and R. Alejandro stem cells, islet transplantation, aClinical Islet Transplant Program (CITP), Diabetes Received 31 August 2007, revised 25 February 2008 and Research Institute, L. Miller School of Medicine, accepted for publication 26 February 2008 University of Miami, FL bDepartment of Internal Medicine, University Policlinic, ‘Tor Vergata’ University of Rome, Italy Introduction cDeWitt Daughtry Family Department of Surgery, dDepartment of Microbiology-Immunology, eLillian Jean Kaplan Renal Transplant Center, Division of Recent progress in islet transplantation for brittle Type Transplantation, fDepartment of Radiology, gDepartment 1 Diabetes Mellitus (T1DM) has made this procedure a of Medicine-, L. Miller School of Medicine, feasible, minimally invasive approach to avoid severe hy- University of Miami, FL poglycemia and secondary complications, improving glu- †Co-senior authors. ∗ cose control with variable -independence and im- Corresponding author: Rodolfo Alejandro, proved quality of life (1–4). Long-term results have shown [email protected] immunosuppression-related side-effects and progressive decline in islet graft function, most of the patients requir- To prevent graft rejection and avoid immuno- ing reintroduction of various levels of insulin therapy within suppression-related side-effects, we attempted to in- 4–5 years posttransplant (1,5–7). duce recipient chimerism and graft tolerance in islet transplantation by donor CD34+hematopoietic stem Donor bone marrow cell (BMC) transplantation following cell (HSC) infusion. Six patients with brittle type 1 myelo- or lympho-ablative conditioning has been shown Diabetes Mellitus received a single-donor allogeneic to induce recipient chimerism and graft tolerance in solid islet transplant (8611 ± 2113 IEQ/kg) followed by high , with reduction or discontinuation of doses of donor HSC (4.3 ± 1.9 × 106 HSC/kg), at days 5 and 11 posttransplant, without ablative condi- immunosuppression (8). Similar results were seen in ex- tioning. An ‘Edmonton-like’ immunosuppression was perimental islet transplant models, after minimal or nonab- administered, with a single dose of anti-TNFa anti- lative regimens (9). In animal and clinical studies, infusion body (Infliximab) added to induction. Immunosuppres- of high doses of donor CD34+hematopoietic stem cells sion was weaned per protocol starting 12 months (HSC) using minimal or nonablative conditioning resulted posttransplant. After transplantation, glucose control in successful engraftment, reduced adverse events, im- significantly improved, with 3 recipients achieving munomodulation and increased allograft survival (10–15). insulin-independence for a short time (24 ± 23 days). No severe hypoglycemia or protocol-related adverse In this study, we attempted to induce recipient chimerism events occurred. Graft function was maximal at 3 and graft tolerance in islet transplant recipients with brit- months then declined. Two recipients rejected within 6 months due to low immunosuppressive trough lev- tle T1DM by infusing high doses of donor HSC without els, whereas 4 completed 1-year follow-up with func- ablative conditioning to prevent graft rejection and elim- tioning grafts. Graft failure occurred within 4 months inate life-long immunosuppression. An ‘Edmonton-like’ from weaning (478 ± 25 days posttransplant). Periph- immunosuppression was given, with a single-dose of tu- eral chimerism, as donor leukocytes, was maximal at 1- mor necrosis factor-a (TNFa) monoclonal (In- month (5.92 ± 0.48%), highly reduced at 1-year (0.20 ± fliximab) at induction to limit early posttransplant in- 0.08%), and was undetectable at graft failure. CD25+T- flammation and maximize single-donor islet engraftment lymphocytes significantly decreased at 3 months, but (1,4,16,17). Immunosuppression was weaned per protocol partially recovered thereafter. Combined islet and HSC starting 12 months posttransplant to evaluate islet

1262 Islet Transplant Tolerance by Hematopoietic Stem Cells survival and chimerism (18–21). Clinical status, islet func- Immunosuppressive regimen tion, glucose control, immune reactivity and chimerism Induction included (Zenapax®, Roche-Pharma, 1 mg/kg intra- were monitored to evaluate efficacy and safety of the venously for five doses, starting the day of transplant, then every 14 days) protocol. plus Infliximab (Remicade®, Centocor, 5 mg/kg intravenously 2 h prior to islet infusion) (1). Maintenance consisted of (Rapamune®, Wyeth- Ayerst, 0.2 mg/kg orally pretransplant then 0.15 mg/kg once daily, to attain Materials and Methods trough levels of 12–15 ng/mL the first 3 months and 8–10 ng/mL thereafter) and low-dose (Prograf®, Fujisawa-Astellas, 1 mg orally pretrans- plant then 1 mg twice daily, to maintain trough levels of 3–6 ng/mL) (1,4,16). Study design and population Immunosuppression weaning began by reducing tacrolimus (0.5 mg/week), This prospective, nonrandomized pilot study (NCT00315614) was approved then sirolimus (1 mg/week). by the University of Miami Institutional Review Board (IRB2000/0024) and written informed consent was obtained. The protocol included patients with long-term T1DM receiving a single-donor, freshly isolated allogeneic islet Clinical monitoring transplant followed by two infusions of high doses of cryopreserved donor Metabolic assessments included fasting plasma glucose and C-peptide, HSC at days 5 and 11 posttransplant. No ablative conditioning for HSC en- daily insulin requirement, HbA1c, C-peptide-to-glucose ratio (CPGR) and in- graftment was given to avoid related side-effects and risks. High doses of dexes from the mixed-meal tolerance test (MMTT), namely the 90 minute- donor HSC (or BMC) have been shown to overcome the host HLA barrier, glucose (90 min-glc) and the mixed-meal stimulation index (MMSI), as ratio allowing for successful bone marrow (BM) engraftment, even without ab- of C-peptide and glucose areas under-the-curve (33, 34). lative regimens. Delayed timing of HSC (or BMC) infusions has resulted in greater engraftment and improved allograft survival (11–15). Occurrences of hypoglycemic unawareness and coma were monitored. Re- nal function was evaluated by 24-h albumin urine excretion and estimated An ‘Edmonton-like’ steroid-free immunosuppression was administered, glomerular filtration rate (eGFR), using the modification of diet in renal dis- adding an antiinflammatory agent (Infliximab) at induction (1,4,16). Starting ease equation (35), and the National Kidney Foundation stages for chronic 12 months posttransplant, immunosuppression was weaned. This time- kidney diseases (36). Immunosuppression trough levels were measured frame was considered adequate to allow for stable donor HSC engraft- and drug-related adverse events evaluated. ment (21) and sustained islet function (4). Detectable chimerism was not a prerequisite for weaning of immunosuppression, since occurrence of graft Definitions tolerance has been reported even in absence of measurable donor cells Insulin-independence: C-peptide positive recipients maintaining, without in- (18–20). sulin therapy, an HbA1c <6.5% and a fasting and/or 2-h postprandial finger- stick (capillary) blood glucose (FBG) <140 mg/dL and <180 mg/dL, respec- Recipient selection and tively (34). Islet graft dysfunction: C-peptide positive recipients having, in Inclusion and exclusion criteria for islet transplantation were previously re- the same week, three or more fasting and/or 2-h postprandial FBG >140 ported (1,4,16). Negative cross-match, recipient panel reactive mg/dL and/or >180 mg/dL, respectively, and/or an HbA1c >6.5% in two (PRA) ≤20%, ABO/Rh and CMV serology but not HLA compatibility, were consecutive measurements (33, 34). Islet graft failure: recipients with two required (22). or more consecutive fasting C-peptide <0.15 ng/mL, in the absence of hy- poglycemia, and/or a stimulated peak of C-peptide <0.3 ng/mL during a Islets and HSC were obtained from 15- to 45-year-old, heart-beating de- MMTT within a month (34). ceased donors. Both donor pancreata and vertebral bodies were preserved in specific cold-storage solutions and procurement media, and then shipped Immune monitoring for processing and transplant (16,23,24). Chimerism was assessed monthly as percentage of donor cells circulat- ing in the recipient’s peripheral blood, including leukocytes, CD3+ and Bone marrow cell extraction and infusion CD34+ cells. At 1-year posttransplant, a BM iliac aspirate was obtained Ten to twelve vertebral bodies were processed from each donor using a and chimerism similarly assessed. A combined PCR and flow-cytometry semi-automated system releasing around 3–5 × 1010BMC (25–27). To pre- analysis was used for detection of HLA class II DRb gene and specificCD vent graft-versus-host disease (GvHD), T-cell burden was indirectly reduced cell-surface epitopes on single fixed cells (37). through CD34+ positive selection (Isolex 300i Magnetic Cell Separator; Bax- ter, Deerfield, IL) with a final cell preparation of >80% purity and >80% White blood cell count was routinely measured and immunophenotyp- viability. Percentage of CD34+ and CD3+ cells was assessed by multicolor ing was performed by multicolor flow-cytometry, using fluorochrome la- flow-cytometry (EPIX XL-MCL, Beckman Coulter, Fullerton, CA). Each BMC beled antibodies and isotype matched control (38). Cell-surface markers in- preparation was divided in two aliquots and stored in liquid nitrogen until cluded: total T-lymphocytes (CD45+/3+) and relative subpopulations infusion (28). Patients received a minimum of 2 × 106HSC/kg of recipient (CD45+/3+/4+, CD45+/3+/8+, CD3+/4+/25+, CD3+/8+/25+), B-lympho- body weight (at least 10 × 108nucleated cells/kg), with a minimum of 2- cytes (CD20+/40+/19+) and natural killer (NK) cells (CD56+/16+/3−). logs T-cell reduction. All donors HSC obtained were infused (no maximum) (11–15). Presence of PRA positivity and HLA class I and II alloantibodies was de- tected using a complement-dependent microlymphocytotoxic (CDC) tech- Islet cells isolation and transplantation nique (LCT assay, One Lambda). Recently, stored sera were retested with Pancreata were processed using a modified automated method (29) and more modern and sensitive ELISA and flow-cytometry techniques (LAT as- purified on density gradients (30), with a final islet preparation of purity say and LABScreen assay plus LABScan flow analyzer, One Lambda and >30% and viability >70% (16). Islets were infused by gravity into the portal Luminex) (22). vein (16,31,32) within 4 h from isolation. Patients received a minimum of 5000 IEQ/kg of recipient body weight (at least 300 000 IEQ total) in ≤5mL Recipient ability to respond to donor antigens was evaluated in a one-way of packed tissue. mixed lymphocyte reaction (MLR). Recipient peripheral blood mononuclear

American Journal of Transplantation 2008; 8: 1262–1274 1263 Mineo et al. cells (PBMC) were challenged with c -irradiated donor splenocytes (stored Clinical monitoring frozen cells), third-party cells, and self-PBMC (negative control). Recipients Islet graft failure occurred in recipients 3 and 5 within PBMC were also cocultured with phytohemoagglutinin (PHA) (positive con- 6 months posttransplant, while having low immuno- trol). Data were calculated as mean counts per minute (cpm) of quadrupli- suppressive drugs trough levels due to poor therapeu- cate cultures. Results were expressed as ratio (Stimulation Index) of prolif- tic adherence and severe drug-related psychosis requir- eration (cpm) observed for donor, third-party, or PHA reactions divided by ing reduction of immunosuppression, respectively, and recipient versus self-cpm (39). follow-up was accordingly interrupted. The remaining The cytotoxic T-lymphocyte genes (CLG) Granzyme B (GB), perforin (P) and four recipients maintained graft function up to 1 year Fas-Ligand (FasL) were evaluated by mRNA expression levels using real- (Table 1). time RT-PCR techniques (40). Results were expressed as the percentage ratio of copy number of the target genes to copy number of the control All recipients, except for those with early graft failure, gene b-actin (40). had the greatest islet function and the best improvement in glycemic control in the first 3 months posttransplant Autoantibody levels for GAD65, IA2 and insulin were evaluated for recur- but slowly deteriorated thereafter. Daily insulin require- rence of autoimmunity, using standard radioimmunoassay (41). Results ments significantly decreased within this period (−65% were expressed as the ratio of values of graft recipients to values of age- of pretransplant value), with recipients 2, 3 and 4 achiev- and sex-matched healthy control subjects. ing insulin-independence for a short time (50, 5 and 17 days, respectively). CPGR, 90 min-glc and MMSI signifi- Statistical analysis cantly improved at 3 months posttransplant but worsened ± Demographics and descriptive statistics were expressed as mean stan- thereafter. By 12 months posttransplant, the four recipi- dard deviation (SD). Due to the nonnormal distribution of some variables ents with preserved grafts showed a relevant reduction in and the small sample size, nonparametric tests for paired (intra-group) com- islet function and glycemic control, with increased daily in- parisons were used (Wilcoxon Rank-Sum test, SPSS 14.0, Chicago, IL). A − p-value <0.05 was considered statistically significant. sulin requirements ( 40% of pretransplant value) (Tables 1 and 2, Figure 1).

Results Following the 12-month follow-up visit, weaning of im- munosuppression began for the four recipients with pre- From April through September 2000, 6 patients with T1DM served graft function. Islet graft failure subsequently oc- were transplanted (2 males, 4 females, age 39 ± 6.9 years, curred within 4 months (478 ± 25 days posttransplant). A disease duration 27 ± 11.8 years, weight 67.5 ± 16.9 kg, mild transient amelioration in fasting C-peptide and HbA1c body mass index 23.8 ± 3.9 kg/m2), receiving a total of was seen during weaning, with slight reduction of daily in- 574 475 ± 141 619 IEQ (8611 ± 2113 IEQ/kg) and 283.3 ± sulin requirements and improvement of CPGR, but not of 126 × 106 HSC (4.3 ± 1.9 × 106 HSC/kg). Due to technical 90 min-glc and MMSI. At islet graft failure, the metabolic problems, patient 1 received a whole BMC infusion, a total parameters approximated pretransplant levels in all re- of 3.5 × 1010 nucleated cells, resulting in a total of 227.0 cipients, although lower daily insulin requirements were × 106 HSC (2.3 × 106 HSC/kg) at flow-cytometry (Table 1). maintained (−35% of pretransplant value) (Tables 1 and 2, No procedure-related adverse events occurred. Figure 1).

Table 1: Transplantation characteristics Tacrolimus Sirolimus Graft failure HLA HSCs Days of weaning (Day) weaning (Day) (Day) Weight mismatch IEQ/kg (×106/kg) insulin Pt Sex (kg) (A, B, DR) (Purity%) (Purity%) independence Begun End Begun End Posttransplant Postweaning 1 m 98.0 1, 1, 2 7046 (45) 2.3 (2.2)# – 377 440 478◦ 503◦ 451∞ 74∞ 2 f 54.5 2, 1, 2 12 485 (37) 3.7 (87) 50 387 415 438 480 480 93 3 f 60.6 2, 2, 2 8012 (45) 2.7 (95) 5 –––– 178§ – 4 f 56.0 1, 2, 2 7039 (50) 6.7 (91) 17 388 430 438 465 471 83 5 f 59.5 1, 2, 2 7534 (30) 3.9 (95) – –––– 158§ – 6 m 76.5 1, 1, 2 9547 (40) 6.4 (86) – 395 422 434 463 510 115 M - 67.5 5 8611 (41) 4.3 (91) 24 387 427 437 469 478 91 SD – 16.9 1 2113 (7) 1.9 (4) 23 7 11 2 9 25 18 IEQ = islet equivalent; HSC = hematopoietc stem cells; HLA = human leukocyte antigens; f = female; m = male; M = mean; SD = standard deviation. #Recipient of whole Bone Marrow Cells (purity value not included in the statistics). ◦Variable not included in the statistics because of off-protocol sirolimus weaning. ∞Islet graft failure after tacrolimus weaning only. §Variable not included in the statistics because of early graft rejection.

1264 American Journal of Transplantation 2008; 8: 1262–1274 Islet Transplant Tolerance by Hematopoietic Stem Cells

Table 2A: Metabolic parameters Fasting C-peptide (ng/mL) HbA1c (%) Daily insulin requirements (UI/kg/day) 3 12 Graft 3 12 Graft 3 12 Graft Pt Pretransplant months months failure Pretransplant months months failure Pretransplant months months failure 1 0.13 0.95 0.24 0.12∞ 6.6 5.6 6.1 6.8∞ 0.80 0.38 0.51 0.45∞ 2 0.12 1.21 0.56 0.16 8.1 5.8 6.9 7.3 0.69 0.04 0.17 0.29 3 0.12 0.86§ – 0.15† 9.0 6.4§ – 8.1† 0.58 0.16§ – 0.41† 4 0.12 1.16 0.33 0.14 7.0 5.2 7.0 6.8 0.48 0.05 0.34 0.37 5 0.10 0.55§ – 0.13‡ 6.6 5.6§ – 6.3‡ 0.46 0.26§ – 0.32‡ 6 0.10 1. 11 0.43 0.18 7.1 5.7 6.8 7.1 0.73 0.39 0.46 0.59 M 0.12 1.11∗∗ 0.39 0.15 7.4 5.6∗∗ 6.7 7.1 0.62 0.22∗∗ 0.37 0.40 SD 0.01 0.11 0.14 0.02 0.7 0.3 0.4 0.6 0.14 0.20 0.15 0.11 ∞Islet graft failure after Tacrolimus weaning only. †Value at 6th and ‡ at 5th month after islet transplantation, respectively. §Variable not included in the statistics because of early graft rejection. ∗p < 0.05 and ∗∗p < 0.03 (Wilcoxon Rank-Sum test); M = means; SD = standard deviation.

Table 2B: Renal function and immunosuppression trough levels Albuminuria (mg/24 hr); (eGFR mL/min/1.73 m2) Tacrolimus (ng/mL) Sirolimus (ng/mL) 12 Graft 1 3 12 Graft 1 3 12 Graft Pt Pretransplant months failure◦ month months months failure month months months failure 1 7 (62) 13 (78) 0 (59) 3.41 3.81 3.40 0.30∞ 10.08 11.38 8.81 9.85∞ 2 21(68) 29 (74) 0 (65) 4.10 5.40 5.25 0.00 12.24 12.29 10.80 6.63 3 58 (78) – 65 (86)† 1.62§ 3.58§ – 1.50† 6.93§ 12.01§ – 7.40† 4 26 (46) 80 (43) 19 (47) 3.40 3.53 4.45 0.00 12.59 12.00 10.70 7.55 5 0 (66) – 0 (64)‡ 3.19§ 1.55§ – 1.90‡ 11.91§ 8.19§ – 7.44‡ 6 5 (98) 9 (99) 10 (95) 4.14 3.95 6.10 0.00 14.91 13.05 9.50 7.00 M 19.7 (69.5) 32.9 (73.6) 15.6 (69.3) 3.76 4.17 4.80 0.62 12.46 12.18 9.95 7.64 SD 21.3 (17.3) 32.6 (26.9) 25.2 (17.8) 0.41 0.84 1.15 0.86 1.98 0.69 0.96 1.13 eGFR = estimated glomerular filtration rate; M = mean; SD = standard deviation. ◦Values after graft failure and completion of immunosuppression weaning. ∞Islet graft failure after tacrolimus weaning only. †Value at 6th and ‡ at 5th month after islet transplantation, respectively. §Variable not included in the statistics because of early graft rejection.

Severe hypoglycemic episodes or coma did not recur like’ protocols (5,42,43). The most important were: mu- posttransplant, with all recipients claiming improved hy- cocutaneous (oral ulcers, n = 4 recipients), hematological poglycemia awareness. Renal function was normal pre- (leucopenia, n = 5; anemia, n = 3), infective (respiratory transplant, except for recipient 3 with micro-albuminuria, tract infections, n = 5), metabolic (hyperlipidemia, n = 3) and recipient 4 with reduced eGFR. At 1-year posttrans- gynecological (dysfunctional menstrual bleeding, requiring plant, while on immunosuppression, the remaining four endometrial ablation, n = 1), and neuropsychiatric (depres- recipients remained stable, except for recipient 4 who de- sion and anxiety, requiring reduction of immunosuppres- veloped micro-albuminuria, without changes in eGFR. Re- sion, n = 1). All resolved satisfactorily after specific treat- cipient 3 showed persistent micro-albuminuria even after ments without any sequelae. the end of the study. After islet graft failure and immuno- suppression weaning, no changes were seen in renal pa- Immune monitoring rameters except for recipient 4 who reversed to normo- Peripheral chimerism, measured as donor leukocytes, was albuminuria with unmodified eGFR (Table 2). maximal in the first 3 months posttransplant, then progres- sively decreased, highly reduced at 12 months and was Immunosuppression levels were consistently in the thera- undetectable at islet failure. At 12 months, donors HSC peutic range in the 4 recipients who completed 1 year of were slightly higher in the circulation than in the BM, while follow-up, except for recipient 1, whose sirolimus levels leukocytes and CD3+T-cells showed an opposite trend. Re- were below targeted level in the first 3 months posttrans- cipients 3 and 5 presented higher levels of chimerism at plant (Table 2). During follow-up, all recipients had multiple the time of islet graft failure when compared to the remain- immunosuppression-related side-effects, with similar fre- ing four recipients at 1-year posttransplant, before starting quency and severity previously reported using ‘Edmonton- immunosuppression weaning (Table 3, Figure 2).

American Journal of Transplantation 2008; 8: 1262–1274 1265 Mineo et al.

1.4 8.0 A C-peptide Insulin/kg/day 7.5 1.2 ** ** HbA1c 7.0 1.0 *

6.5 0.8 6.0 ** ** HbA1c% 0.6 * 5.5 ng/mL AND UI/kg/day AND ng/mL 0.4 * 5.0 ** **

0.2 4.5

0.0 4.0 Pre- 136912TacrolimusSirolimus Graft transplant Months weaning weaning failure

B 1.0 CPGR 25

0.9 ** MMSI 90min-glc 0.8 * 20 0.7 ** 0.6 15

0.5 * mmol/L 0.4 ** 10 ng/mL x mg/dL-1 0.3 * 0.2 5

0.1

0.0 0 Pre-transplant 3 6 9 12 IS weaning Graft failure Months

C 14 Tacrolimus

Sirolimus 12

10

8

ng/mL 6

4

2

0 136912IS weaningGraft failure Months

Figure 1: Clinical monitoring. (A) Metabolic parameters (B) Metabolic indexes (CPGR: C-peptide/Glucose Ratio; MMSI = mixed-meal stimulation index; 90 min-glc: 90 minute-glucose). (C) Immunosuppression trough levels. Data are expressed as mean ± SD. IS weaning: Immunosuppression weaning. Solid lines refer to left Y axis, while dotted lines refer to right Y axis; ∗p < 0.05 and ∗∗p < 0.03 (Wilcoxon Rank-Sum test).

1266 American Journal of Transplantation 2008; 8: 1262–1274 Islet Transplant Tolerance by Hematopoietic Stem Cells

Table 3: Chimerism characteristics Circulating donor cells%/Bone marrow donor cells%∞ Leukocytes CD34+cells CD3+cells Pt 1 month 3 months 12 months 1 month 3 months 12 months 1 month 3 months 12 months 1 5.10 3.30 0.32 (0.50) 82.6 72.6 11.1 (1.08) 4.33 2.07 0.19 (0.56) 2 5.92 4.10 0.18 (0.51) 86.0 78.7 10.1 (1.35) 3.64 2.46 0.11 (0.60) 3 5.98 4.40 1.90†§ 85.9 81.9 – 3.79 2.71 – 4 5.80 4.56 0.18 (0.32) 80.3 79.4 11.0 (0.87) 4.90 3.52 0.11 (0.44) 5 6.59 4.70 1.68‡§ 84.1 78.9 – 4.16 3.52 – 6 6.10 4.00 0.12 (0.38) 85.3 80.1 5.3 (0.79) 4.11 2.57 0.08 (0.40) M 5.92 4.18 0.20 (0.43) 84.0 78.6 9.4 (1.02) 4.16 2.81 0.12 (0.50) SD 0.48 0.51 0.08 (0.09) 2.2 3.2 2.8 (0.25) 0.44 0.59 0.05 (0.07) †Value at 6th and ‡ at 5th month after islet transplantation, respectively. §Variable not included in the statistics because of early graft rejection. ∞Bone marrow donor cells from iliac aspirates at 12 months; M = mean; SD = standard deviation.

White blood cell counts presented a significant reduc- tive during the follow-up. All patients developed high tion of leukocytes, mainly neutrophils, within the first PRA positivity after islet graft failure and immuno- trimester posttransplant, with limited recovery at graft fail- suppression weaning, with donor-specific alloantibodies ure. Immunophenotyping showed a similar trend for T- (Table 4). lymphocytes, mainly CD4+cells, in the same period, with increasing levels up to graft failure. The CD25+ subpop- MLR variably decreased in all recipients posttransplant, re- ulations, mainly CD4+cells, significantly decreased in the taining the anti-third-party response, but increased at the first 3 months posttransplant, but partially recovered there- time of reduced graft function and low immunosuppres- after. B-lymphocytes slightly increased at 1-month post- sion levels. Recipient 6, with the highest response during transplant, decreased during follow-up, and normalized at the follow-up, exhibited a prolonged graft survival, similar graft failure. NK-cells were reduced early posttransplant, to 2, the lowest response of all (Figure 4). but tended to increase at immunosuppression weaning (Figure 3). Similarly, in all recipients, analysis of CLG expression re- vealed multiple episodes of elevated and sustained GB PRA was ≤20% pretransplant in all recipients by levels, closely associated with periods of declining islet CDC assay. With ELISA and flow re-testing, recipi- function and reduced immunosuppression levels, mainly ent 1 was slightly positive pretransplant and converted sirolimus, without concomitant infections. Elevations of to negative while on immunosuppression, while re- FasL were seen in recipients 1 and 5 related to graft failure cipients 3, 4 and 5 were well above the inclusion and positivity of anti-IA2 and anti-insulin antibodies, respec- criteria pretransplant and remained persistently posi- tively (Figure 4).

7 Leukocytes 90 CD3+ CD34+ 80 6 70 5 60

4 50 % % 3 40

30 2 20 1 10

0 0 136912 Months

Figure 2: Recipient chimerism. Data are expressed as mean ± SD. Solid lines refer to left Y axis, while dotted lines refer to right Y axis.

American Journal of Transplantation 2008; 8: 1262–1274 1267 Mineo et al.

A 9000 Leukocytes 2800 Neutrophils 8000 Lymphocytes 2400 7000 2000 6000

5000 * 1600 Cell/uL

4000 ** ** Cell/uL ** 1200 3000 ** 800 2000 ** 400 1000

0 0 Pre- 1 3 6 9 12 IS weaning Graft failure transplant Months

B 1200 CD45+/3+/4+ 2000 CD45+/3+/8+ CD20+/40+/19+ 1000 CD56+/16+ 1600 CD45+/3+ 800 * 1200

600 ** Cell/uL Cell/uL * 800 400 ** * 400 200

0 0 Pre- 1 3 6 9 12 IS weaning Graft failure transplant Months

C 140 CD3+/25+ 18 CD3+/4+/25+ 16 120 CD3+/8+/25+ 14 100 12

80 10

8

60 Cell/uL Cell/uL 6 40 * 4 20 * 2

0 ** ** 0 Pre- 136912IS weaningGraft failure transplant Months

Figure 3: Immune monitoring. (A) White blood cell counts, (B) lymphocytes subpopulations and (C) CD25+ T-lymphocytes. Cell-surface markers are specified in the text. Data are expressed as mean ± SD. IS weaning: Immunosuppression weaning. Solid lines refer to left Y axis, while dotted lines refer to right Y axis; ∗p < 0.05 and ∗∗p < 0.03 (Wilcoxon Rank-Sum test).

1268 American Journal of Transplantation 2008; 8: 1262–1274 Islet Transplant Tolerance by Hematopoietic Stem Cells ,51; 16 ,12, 11 ,9,10, ,12,15,16,51; 7 ,8,10,11, ,8,11,12, ,11,12,13,15, ,4, 1,4,8,10, ,16,51 DR1 DR1,4 DR1 DR DR1 DR8,11 15 , ,15,16,51,52,53; DQ4,9 13 12,13,51,53;DQ6,7,8,9 13 DQ6 15,16,51,53; DQ5,6,9 DQ6 66%/DR1,51; DQ8,11 99%/- 72%/DR9; DQ5, 38%/DR11,DR7 91%/- 84%/- 71%/ 77%/ 43%/ 97%/ 86%/ 43%/ – – – – ˆ ˆ – § § § § § § – ˆ ˆ ˆ ˆ ,57,58 , ,58, 51 31 57 , ,45,47, , ; Bw4 ,45,47, 49, 30 44 53 B 44 ; Bw4; Cw8 25,32; B35 , 45, 49 8,13,27,35,37, 8,13,27,35,37 24 ,25,32; B 24, B B44 ,66; 24 ,11,23,24,25, ,24,25,32,66,80; ,23,25; ,45,49 ,23, 3 23, 1,23,24,25, 1, 1,23, 44 A3 A24 A11 A A A A ,45,47,51,52,53,57,58, A24 ,52,53575858,59,63 8,13,26,27,37, 13,27,37,41,44,47,49, 13,27,33,37, 49,51,52,53,57,58,59, 60,61,63,64 B B 51 32,36,80; 44,47,49,51,52, 59,63,64,65; Cw12,17 32,36,80 B49,50,51,52,57,58,63 ,44 59,62,63,64, 65,75,76 49,51,52,53,57,58, 59,60,61,63 32,34,80; B 82%/ 95%/Bw4,6 79%/A1,24;Bw4, 91%/Bw4,6 73%/ 96%/- 87%/ 80% / A1,11,23,24,25,26, 67%/ 84%/ 84%/ 44%/ 22%/B 0% ˆ § § § § § § ˆ ˆ ˆ ˆ ˆ Flow-cytometry assay. ˆ ,4,10,15, DR1 16,51; DQ5,6,9 10,15,16; DQ5,9 ELISA and § 46%/DR1,4,6, 56%/DR51;DQ5 77%/ – – § § ˆ ,45, 44 ; B41,52,72 31 ,11,24,26, ,45,47,53,54,59,60,81 26, A3 A 13,27,37,41, 32,34,36,66,68; B7,44 60,61,67; Cw18 B 47,49,51,52,53,57,58,59,60 61,63 20%/B1379%/A24; Bw4, 25%/DQ9 84%/A11,24; Cw2 31%/A32; B7,13,41,47,49,50, 84%/A1,11,23,24,25,32,80; 44%/ § § § ˆ ˆ ˆ ,10; ,7,9,10, panel reactive antibodies positivity percentage; = DR1 DR1 DQ5,6,8 15,16,51,53; DQ6 6%/DR10 41%/ 29%/DR1,10,15,16; 40%/ § § ˆ ˆ ,45,47,49, Pretransplant PRA% On immunosuppression PRA% Off immunosuppression PRA% 44 c alloantibodies are in bold. fi Antibody response human leukocyte antigens; PRA% 8,13,27,37, B7,13,18,27,37,42,47, 49,50,60; Cw5 B 66;B8,38,39,48,51,52,54, 55,56,59,60,64,65,67,81; Cw8 51,52,53,57,58,59,63,64,65; Cw12 = 29%/B8,13,59 80%/A24,32,66; Bw4 54%/A2,24,29 23%/B13,45 11%/A23,24 36%/A1,23,32,36,80; 73%/A1,23,24,25,32,80; 56%/A2,11,24,25,26,30,34, 20%/A25,29; B57 0% 0% 0% 0%18%/A25; B72; Cw1 21%/ All 0%All 0% 46%/A23,31; B37,41; Cw7 All 0% All 0% 0% All 0% All 0% 21%/A34; B41,46,53,72 ˆ § 0% All 0% All 0% All 0% 43%/A23; All 0%§ All 0%§ All 0%§ All 0% 50%/ ˆ ˆ ˆ ∗ Patient without a previous pregnancy prior to transplantation. 2 4 3 5 6 Table 4: Pt HLA I (A,B,C)1 HLA II (DR, DQ) HLA I (A,B,C) HLA II (DR, DQ) HLA I (A,B,C) HLA II (DR, DQ) HLA ∗ Donor-speci

American Journal of Transplantation 2008; 8: 1262–1274 1269 Mineo et al.

M++LR++ ++ + + +++ Auto-antibodies were positive pretransplant in most of the pt1 * C-peptide recipients (anti-GAD65 in recipients 2 and 3; anti-GAD65 2 GB 140 FasL×10 120 and anti-IA2 in recipient 4; and anti-IA2 in recipient 6) and 1.5 100 persisted with fluctuating levels during follow-up. Antiin- 80 sulin antibodies were negative pretransplant in all recipi- 1 60 ents. Recipients 1 and 5, autoantibodies negative pretrans- ng/mL plant, developed anti-IA2 antibodies just before immuno- 0.5 40

20 (mRNA/Actin)% suppressive weaning and antiinsulin antibodies immedi- 0 0 ately posttransplant, respectively. 0 20 40 60 80 400 300 320 340 380 420 440 460 100 120 140 160 180 200 220 240 260 280 360 Days

MLR+ + -++ + pt2 Discussion * C-peptide 2 GB 140 120 Full or mixed chimerism has been reported in patients 1.5 100 after partially matched BMC transplants for lympho- % 80 hematological malignancies using myelo-ablative condi- 1

ng/mL 60 tioning allowing for the development of tolerance to a solid

0.5 40 organ transplant from the same donor without chronic im-

20 (mRNA/Actin) munosuppression (8). The ability of microchimerism to in- 0 0 duce graft tolerance remains unproven, despite detection 0 20 40 60 80 460 500 320 400 180 200 240 440 100 280 300 340 360 380 480 420 120 140 160 220 260 of donor leukocytes many years after a solid organ trans- Days plant in patients with long-term graft function able to re- duce or discontinue immunosuppression (18). Previous tri- M+++LR++++ + ++ + ++++ * pt4 als using lympho-ablative or no conditioning did not show 2 C-peptide 140 a clear reduction in graft rejection nor immunosuppression GB 120 dosage, despite persistence of <1% of donor cells for over 1.5 100 5 years (8,44–46). 80 1

ng/mL 60 Mismatched BMC transplants in nonhuman primates and 0.5 40 rodents, after nonmyelo-ablative or lympho-depleting reg- 20 (mRNA/Actin)% imens, even nonobese autoimmune diabetic (NOD) mice, 0 0 induced transient or stable chimerism, allowing for islet 0 60 80 20 40 440 480 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 460 graft tolerance and discontinuation of immunosuppression Days (8,9,47–49). MLR++ + + + pt5 2 C-peptide 140 High doses of HSC successfully engraft in animal models, GB FasL×10 120 even without conditioning, allowing for tolerance to donor 1.5 100 islets as well as for diabetes reversal or prevention in NOD % 80 mice (8,10–14,50,51). Similar observations were reported 1

ng/mL 60 in patients with recent T1DM onset after allogeneic BMC 0.5 40 transplant for lympho-hematological malignancies (52) or

20 (mRNA/Actin) autologous HSC infusion for the autoimmune disease itself 0 0 (53). 0 20 40 60 80 120 180 Days 100 140 160

MLR+++ +++++ +++++ +++++ ++ + pt6 ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 2 C-peptide 140 GB 120 1.5 100 Figure 4: Immune reactivity and islet graft function. Cytotoxic × 80 Lymphocyte Genes expression (GB: Granzyme B; FasL 10: Fas- 1 Ligand values multiplied 10 times to match GB scale; ∗ value out of

ng/mL 60 range), fasting C-peptide, and Mixed Lymphocyte Reaction (MLR, 40 0.5 on the top) from recipient 1 to 6 (3 not shown due to lack of follow- 20 (mRNA/Actin)% up exams). Black solid and dotted lines refer to right Y axis, while 0 0 gray solid lines refer to left Y axis. MLR is reported as Stimulation 0 20 80 40 60 + 340 380 500 520 140 180 200 220 240 260 280 300 320 360 400 420 440 460 480 100 120 160 Index (- is less or equal to 1, greater than 1 and less or equal to Days 5, ++ greater than 5 and less or equal to 10, +++ greater than 10 and less or equal to 15, ++++ greater than 15 and less or equal Figure 4: Continued. to 20, +++++ greater than 20).

1270 American Journal of Transplantation 2008; 8: 1262–1274 Islet Transplant Tolerance by Hematopoietic Stem Cells

In the clinical setting, infusion of high doses of donor HSC shorter graft survival, despite moderate immune reactiv- for lympho-hematological malignancies demonstrated sim- ity. Recurrence of autoimmunity at weaning or islet loss ilar efficacy in inducing chimerism, when compared to due to low sirolimus levels in the first trimester may have whole BMC, with fewer complications due to the lower also influenced this outcome. antigenicity and T-cell content (10,15,54,55). Neverthe- less, GvHD incidence remains significant, especially in mis- Peripheral chimerism started to decrease 3 months post- matched transplants, when highly ablative conditioning is transplant, concomitantly with lowering of immunosup- used (56–59). pression. Donor HSC progressively disappeared from the circulation showing poor ability to home to the host BM, The immunomodulatory properties of the HSC remain con- as evidenced by the 12-month iliac aspirate. troversial. Persistent donor cells such as CD34+HSC and CD3+T-cells seem capable of inducing recipient immune Immunophenotyping showed persistent and significant re- unresponsiveness (60). A functional immune homeosta- duction of T-lymphocytes while on immunosuppression. sis, or operational tolerance, deriving from the recipient The CD25+ subsets were almost undetectable in the first chimeric BM, appears able to inhibit the antidonor re- 3 months posttransplant related to Daclizumab infusion, sponse by putative donor suppressor T-cells as well as to but promptly increased thereafter. restore self-tolerance, preventing recurrence of autoimmu- nity (8,18,46,61–63). Conversely, the upregulation of cos- Repetitive episodes of immune reactivity occurred during timulatory molecules on the HSC surface by the early post- follow-up, evidenced by increased CLG levels and MLR transplant inflammatory response, or the differentiation of responses, related to periods of low immunosuppression HSC into antigen-presenting cells by interaction with host levels and declined islet function, probably reducing the T-cells, may be able to trigger alloreactivity, thereby limiting islet graft mass before weaning. the safety and efficacy of their infusion (64,65). Most recipients with negative or borderline-positive pre- This is the first study reporting the attempt to induce transplant PRA at CDC assay were found to be highly recipient hematopoietic chimerism and graft tolerance positive when retested by ELISA or flow-cytometry as- in clinical islet transplantation by infusing high doses of says (higher values in females with a previous pregnancy). donor HSC under a modified ‘Edmonton-like’ immuno- Confirmed negative or low-positive pretransplant PRA re- suppression without ablative conditioning. Recipients with mained negative while under immuno-suppression as pre- 1-year follow-up, despite insulin dependence, showed viously described (22). Allosensitization occurred in all re- C-peptide secretion with significant reduction of insulin re- cipients after weaning (22). Positive PRA has persisted in quirements, normalization of HbA1c, and resolution of hy- the only two recipients (2 and 6) with long-term follow-up poglycemia. Nonetheless, islet graft function started to de- (80% and 40% HLA-I and 10% and 35% HLA-II, 5 and 6 cline 3 months posttransplant, concomitant to completing years posttransplant, respectively). The clinical relevance Daclizumab course and reducing sirolimus levels. of these phenomena needs further evaluation.

No GvHD, malignancy or permanent complications were We hypothesize that infusing highly T-cell-depleted donor recorded, highlighting the relative safety of this protocol. HSC without ablative conditioning could have preserved Renal function did not deteriorate posttransplant, except recipient immune-competence while avoiding GvHD, but transiently in a recipient with a preexisting alteration. might have prevented HSC engraftment in the host BM, limiting achievement of stable chimerism. Adding a sin- Immunosuppressive levels, mainly of sirolimus, seemed to gle dose of TNFa-blocker to a relatively short nonlympo- be critical to preserve islet survival. Graft failure occurred depleting induction could have reduced early HSC acti- in periods of low trough levels or even while in the tar- vation, limiting their antigenicity and allorecognition, but geted ranges, leading us to aim at sirolimus trough levels might not have avoided a late donor-specific immune of 10–12 ng/mL after the third month in subsequent trials response with loss of cell grafts. Chronic immunosup- (66). Recipients 3 and 5 experiencing early graft failure de- pression could have interfered with HSC engraftment and spite high C-peptide at 1-month posttransplant, presented proliferation, and might have impeded the generation of islet dysfunction within 3 months, probably due to low im- regulatory T cells, preventing the induction of tolerance. munosuppressive drug levels, with subsequent graft fail- Transplanting a marginal islet mass from a single-donor ure. Receiving lower IEQ/kg and HSC/kg might have also without adequate lympho-depletion at induction may ex- conditioned their outcome. Indeed, patients 2 and 6 receiv- plain the low rate of insulin-independence in our study ing the highest IEQ/kg had the longest islet graft survival. (67–68). Furthermore, patients 4 and 6 receiving higher HSC/kg, de- spite elevated immune reactivity, had similar or longer islet A limitation of our study is the lack of a control group survival when compared to recipient 2 of lower HSC/kg but receiving an equal islet mass without HSC infusion, to optimal IEQ/kg and mild immune reactivity. Conversely, better evaluate the effect of the HSC on islet sur- patient 1 receiving the lowest HSC/kg and IEQ/kg had a vival and glucose control. Analyses of chimerism and

American Journal of Transplantation 2008; 8: 1262–1274 1271 Mineo et al. immunophenotype, which could have assisted in defin- References ing a possible immunomodulatory role for both HSC and immunosuppressive protocol, were limited. Other T- 1. Froud T, Ricordi C, Baidal DA et al. Islet transplantation in type 1 di- cell subsets, such as CD4+/25+/Foxp3+ regulatory T- abetes mellitus using cultured islets and steroid-free immunosup- lymphocytes were not studied, and their influence on graft pression: Miami experience. Am J Transplant 2005; 5: 2037–2046. tolerance could not be defined. 2. Lee TC, Barshes NR, O’Mahony CA et al. The effect of pancre- atic islet transplantation on progression of diabetic retinopathy and In conclusion, combined islet and HSC allotransplanta- neuropathy. Transplant Proc 2005; 37: 2263–2265. tion under an ‘Edmonton-like’ immunosuppression with- 3. Poggioli R, Faradji RN, Ponte G et al. Quality of life after islet trans- plantation. 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Transplantation 1993; 55: 1272–1277. nologies and Therapies in Surgery’, Department of Surgery, University of 19. Burlingham WJ, Grailer AP, Fechner JH, Jr. et al. Microchimerism Rome ‘Tor Vergata’. linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient. Transplan- We express gratitude to the cGMP Cell Transplant Center and the Tissue tation 1995; 59: 1147–1155. Typing Laboratory, in particular Mr. Joel Schultz and Mrs. Carmen Gomez, 20. Strober S, Benike C, Krishnaswamy S, Engleman EG, Grumet FC. for their support and collaboration. We also deeply thank Mr. John Wilkes Clinical transplantation tolerance twelve years after prospective (regulatory officer) and Ms. Elizabeth Meyer (CITP manager) for reviewing withdrawal of immunosuppressive drugs: Studies of chimerism the manuscript. and anti-donor reactivity. Transplantation 2000; 69: 1549–1554.

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