ACR

H

FtssTC

©3

T

llogeneic Hematopoietic Stem-Cell Transplantation, Mixedhimerism, and Tolerance in Living Related Donor Renal Allograftecipients

.L. Trivedi, A.V. Vanikar, P.R. Modi, V.R. Shah, J.M. Vakil, V.B. Trivedi, and S.I. Khemchandani

ABSTRACT

Objective. We designed a prospective, randomized, and controlled clinical trial toevaluate the efficacy and safety of achieving a mixed chimerism-associated toleranceprotocol for recipients of living related donor (LRD) renal allografts.Patients and methods. Sixty-six consecutive patients were divided into two equal groupsof 33 patients with end-stage renal disease. They were enrolled for transplantation afternegative lymphocytotoxicity cross-matching (LCM). Both groups (treated [Tn] and control[Cn]) showed similar clinical and laboratory parameters and donor HLA match profiles.The Tn group underwent thymic transplantation of donor renal tissue, two donor-specifictransfusions, low-intensity conditioning, and high-dose hematopoietic stem-cell transplan-tation (HSCT) before renal transplantation. The conditioning regimen included low-dose,target-specific irradiation (to abdominal and inguinal lymph nodes, bone marrow [BM]from thoracolumbar vertebrae and part of the pelvis on alternate days, 100 rad � 4),anti-T-cell antibodies (1.5 mg/kg body weight [BW]), cyclophosphamide (10 mg/kg BW �2 consecutive days), and cyclosporine (CyA; �3 mg/kg BW/d). Unfractionated HSCTprocured from the donor marrow was administered into the BM, portal and peripheralcirculations, within 24 hours of achieving CD 4�/CD 8� T-cell count less than 10% ofnormal. This infusion was supplemented with a dose of peripherally mobilized stem cells(mean total dose of 20 � 108 cells/kg recipient BW) administered peripherally. Renaltransplantation was performed after negative LCM. Donor-specific cytotoxic antibodieswere eliminated with intravenous immunoglobulins and plasmapheresis before renaltransplantation. Mixed chimerism was evaluated before and after transplantation atmonthly intervals in patients with donors of opposite gender by the FISH technique. Bothgroups received CyA and prednisolone for immunosuppression; Cn subjects also receivedmycophenolate mofetil/azathioprine. Rejection was treated with standard treatment.Immunosuppression was withdrawn 6 months after renal transplantation for patients withconsistently positive chimerism. Clinical tolerance was defined as stable allograft functionfor more than 100 days without immunosuppression and confirmed by allograft biopsy.Results. Over a mean follow-up of 210 days, all Tn patients showed stable allograftfunction with mean serum creatinines (SCr) of 1.20 mg/dL, no rejection/CMV infections/graft or patient loss. A low-level donor-specific cytotoxic antibody was observed in all Tn

rom the Department of Nephrology and Clinical Transplanta-ion; Department of Pathology, Laboratory Medicine, Transfu-ion Services and Immuno-Hematology; Department of Anesthe-iology and Critical Care, and Department of Urology andransplantation Surgery, Institute of Kidney Diseases & Research

Address reprint requests to Prof. Hargovind L. Trivedi, Insti-tute of Kidney Diseases & Research Centre and Institute ofTransplantation Sciences, Civil Hospital Campus, Asarwa,Ahmedabad—380 016, Gujarat, India. E-mail: ikdrcad1@sancharnet.in

entre and Institute of Transplantation Sciences, Gujarat, India.

2005 by Elsevier Inc. All rights reserved. 0041-1345/05/$–see front matter60 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2005.01.028

ransplantation Proceedings, 37, 737–742 (2005) 737

Taolgahld

P

Swlri(crco

S

Tuprpdt

T

Patldagt

738 TRIVEDI, VANIKAR, MODI ET AL

patients. The CyA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoi-etic chimerism was seen in all 21 patients irrespective of donor-recipient HLA matching(mean 0.5% before and 1 � 0.3% after transplantation). All four patients on drugwithdrawal have shown donor-specific tolerance at a mean follow-up of 129.8 days. OtherTn patients are in the process of being weaned off immunosuppression. Mean SCr ofcontrols was 1.45 mg/dL over a mean follow-up of 216 days. Acute rejection was observedin 17 (51.5%) patients; no CMV infection/patient loss was noted and one (3.03%) graft waslost in controls. No patient was lost in controls. No graft-versus-host disease was observedin Tn patients.Conclusion. We have achieved mixed hematopoietic chimerism-associated tolerancewith high-dose HSCT, intrathymic donor renal tissue transplantation, and minimal

conditioning without any adverse effects.

tddsmBaatCnc1tGdcmtscDsibo

HC

Lgcmcam(co

OLERANCE is the dream of every transplanter. Do-nor-specific immunologic tolerance would protect an

llograft from chronic rejection and allow discontinuationf chronic immunosuppressive therapy to avoid side effects

ike infections and malignancies. Experimental models sug-est that mixed hematopoietic chimerism leads to toler-nce. However, it is not easy to validate this concept inumans. We implemented a protocol to induce mixed

ymphohematopoietic chimerism (MLHC) in living relatedonor (LRD) renal allograft recipients.

ATIENTS AND METHODS

ixty-six consecutive patients with end-stage renal disease (ESRD)ere enrolled for this prospective, randomized, open-label, paral-

el-control, and single-center clinical trial in LRD renal allograftecipients from May 2003 to December 2003 after gaining patients’nformed consent. Thirty-three patients were included in treatedTn) versus control (Cn) groups. Patients in both groups wereomparable for demographic features, ESRD etiology, and donor–ecipient HLA match profiles (Tables 1 and 2). The three mostommon etiologies of ESRD were chronic glomerulonephritis,bstructive uropathy, and diabetic nephropathy.

TUDY DESIGN

his prospective, open label, randomized clinical trial eval-ated the efficacy and safety of a tolerance inductionrotocol associated with MLHC in LRD renal allograftecipients. Group Tn underwent the tolerance inductionrotocol before renal transplantation. The Cn groups un-erwent transplantation directly after a negative lymphocy-otoxicity cross-match (LCM).

olerance Induction Protocol

atients were screened for the presence of lymphocytotoxicntibodies by LCM serologically before and at the end ofhe tolerance induction protocol, which included donoreukocyte infusions on the 1st and 3rd days to stimulateonor-specific T-cell clones (Fig. 1). Minimal non-myelo-blative conditioning, including fractionated low-dose, tar-et-specific irradiation (125 cGy � 4 on alternate days) to

he abdominal and the inguinal lymph nodes, as well as the a

horacolumbar vertebrae and pelvis, was delivered fromays 4 to 10, to create space for stem-cell grafting byeleting resident stem, B, and T cells as well as “cobble-tone appearance” colony-forming stem cells residing in thearrow. Administration of cyclophosphamide (10 mg/kgW/d) occurred on days 11 and 12 as a myelosuppressivegent to create space in the thymus. Polyclonal anti-T-cellntibody (1.5 mg/kg BW) was delivered on day 13 to achieveransient abrogation of alloresistance offered by CD4�/D8� cells by reducing their counts to below 10% oformal. To prevent graft-versus-host disease (GVHD),yclosporine (CyA; 3 mg/kg BW/d) was prescribed on day1 and continued posttransplant, as an immunosuppressant,o protect chimeric cells and for prophylaxis againstVHD. Administration of treosulfan (1.3 mg/kg BW) on

ays 11 to 13 was used to delete “cobblestone appearance”olony-forming stem cells residing in the marrow. Intrathy-ic donor renal tissue transplantation was done on day 11

o present MHC I/II-rich renal tissue to the thymic medullao that developing double-positive (CD4�/CD8�) thymo-ytes would register the presence of donor alloantigens.onor marrow infusion into the marrow, and portal and

ystemic circulations was performed on day 14 followed bynfusion of cytokine-stimulated and mobilized peripherallood stem cells (PBSC) on days 17 and 20, to reach a targetf 20 � 108 cells/kg BW.

ematopoietic Stem-Cell (HSC) Stimulation, Mobilization,ollection, and Infusion Techniques

iving related donors (LRD) for the patients receivedranulocyte colony-stimulating factor (15 �g/kg BW) sub-utaneously for 2 consecutive days followed by a bonearrow (BM) aspiration from the posterior superior iliac

rest under general anesthesia. About 400 mL of thisspirated unmodified marrow was infused into the recipientarrow (80 mL), the portal (200 mL), and the peripheral

120 mL) circulation. The procedure was followed by PBSCollection twice to reach the target of 20 � 108 cells/kg BWf the recipient. Cobe Spectra version 7 (Gambro, China)

nd MCS 3p (Hemonetics, Braintree, Mass) were used for

lc

H

ApLtcptia

P

Awnmfp(Baiwj

I

Bms(wmm

MMGAC

ECODARSI

s

STEM-CELL TRANSPLANT 739

eukophoresis. The nucleated cell count and CD34� cellounts were performed after each collection.

LA and LCM Technique

ll patients and donors were tested for HLA. The LCM waserformed using conventional serological techniques (one-ambda predot trays were used for HLA-A, -B, -DR

yping). An auto-cross-match, dithiothreitol, and standardytotoxicity cross-match tests were performed on mixed-cellopulations and on separated T and B lymphocytes. Renalransplantation was performed at 1 week after the last HSCnfusion following a negative LCM in the Tn and immedi-tely after a negative LCM in the Cn cohort.

Table 1. Patient Demographics

Group (N � 66)Treated (Tn)

(n � 33)Control (Cn)

(n � 33)

ean follow-up (days) 372 378ean age in years (range) 33 (8–57) 34 (10–56)ender (M:F) 30:3 30:3verage third-party infusion 21 20MV seropositivityRecipients 22 (66.7%) 23 (69.7%)Donors 32 (97%) 32 (97%)

tiology of ESRDhronic glomerulonephritis 15 (45.5%) 16 (48.5%)bstructive uropathy 8 (24.2%) 7 (21.1%)iabetic nephropathy 5 (15.1%) 4 (12%)DPKD 2 (6.2%) 2 (6.2%)eflux nephropathy 1 (3%) 2 (6.2%)LE 1 (3%) 1 (3%)

gA nephropathy 1 (3%) 1 (3%)

Abbreviations: ADPKD, autosomal dominant polycystic kidney disease; SLE,ystemic lupus erythematosus.

Fig 1. Ahmedabad Toleran

osttransplant Follow-up

ll patients were followed at the same outpatient clinic, ateekly intervals for the first 3 months, fortnightly for theext 3 months, monthly for the next 6 months, and 3onthly intervals thereafter. At each visit, renal and liver

unction status was monitored, complete blood counts wereerformed, and cyclosporine (CyA) levels were measuredEMIT 2000 CyA specific assay system; Syva Co., Dadeehring, Cupertino, Calif, USA). Patients were monitoredt monthly intervals for HIV, HBsAg, HCV, and CMVnfections using an ELISA technique. The Tn recipientsere monitored for the development of a skin rash, fever,

aundice, or gastrointestinal symptoms of GVHD.

mmunosuppression

oth groups received CyA (5 mg/kg BW/d) for the firstonth along with prednisolone (0.5 mg/kg BW/d). The Cn

ubjects received azathioprine/mycophenolate mofetilMMF) as additional immunosuppressants. The CyA doseas tapered (3 mg/kg BW/d) in Tn subjects at the 2ndonth posttransplant, and prednisolone was tapered (0.2g/kg BW/d) in both groups from the 2nd month posttrans-

Table 2. Donor–Recipient HLA Match Profile

Group(N � 66)

Treated (Tn)(n � 33)

Control (Cn)(n � 33)

0/6 5 (15.2%) 6 (18.2%)1/6 10 (30.3%) 11 (33.3%)2/6 3 (9.1%) 3 (9.1%)3/6 12 (36.4%) 12 (36.4%)4/6 1 (3%) 1 (3%)5/6 1 (3%) 06/6 1 (3%) 0

ce Induction Protocol

pnj(ndTt

R

Rtna

C

Petaa

su

bbG(n

R

TB0tipiss3rtappaf

F

T

740 TRIVEDI, VANIKAR, MODI ET AL

lant. The Cn subjects were continued on the same immu-osuppressive regime thereafter. The CyA dose was ad-

usted to maintain trough levels of 50 to 100 ng/mLrecommended trough values: 76 to 150 ng/mL). Pred-isolone was weaned off at 6 months’ posttransplant to beiscontinued at the end of 9 months’ posttransplant. Thus,n patients were kept on low dose CyA monotherapy (0.5

o 2 mg/kg BW/d) from 9 months onwards (Table 3).

ejection and Treatment

ejection diagnosed as per the Banff classification wasreated with three doses of intravenous (IV) methylpred-isolone (500 mg/d).1 Tacrolimus was used as rescue ther-py for steroid-resistant rejections.

himerism Studies

atients with cross-gender donors were subjected to periph-ral blood chimerism studies before stem-cell transplanta-ion (for Cn), a day before renal transplantation, 100 daysfter the last PBSC infusion, and at 6 monthly intervalsfterwards to look for MLHC.

Peripheral blood (5 mL in heparin) was collected from aubset of patients in both groups. The red cells were lysedsing RBC lysis buffer following spinning with phosphate

Table 3. Immuno

TimeScale

(months) Cyclosporine (per day)

0–1 Tn � Cn (5 mg/kg BW) Tn �

2–5 Tn � Cn (3 mg/kg BW) Tn (6–9 Tn � Cn (2 mg/kg BW) Cn (3 mg/kg BW) Tn (10� Tn (1 mg/kg BW) Cn (3 mg/kg BW) Cn (

Abbreviations: Cn, control group; Tn, treated group.

ig 2. Allograft function in terms of mean SCr level at different

ime Interval (Months)

36912

Present

uffered saline at 4–10°C for 10 minutes. The lysed cellutton was subjected to Cytospin; slides were stained withiemsa stain, and some were destained for XX/XY probes

Vysis probes) to study chimerism (the Skyvision Cytoge-etics Workstation).

ESULTS

he mean total nucleated cell count was 21 � 108 cells/kgW in the recipient with a mean CD34� count of 0.8 �.2%. Serum creatinine (SCr) values were used as indica-ors of allograft function. A comparative study of SCr valuesn both groups was undertaken at 3, 6, 9, and 12 months’osttransplant. The mean SCr level was significantly better

n the Tn than the Cn group Student’s t test of the twoamples, assuming equal variances, determined statisticalignificance (P � .001) (Fig. 2). Over a mean follow-up of72 days, the mean SCr of Tn was 1.23 mg/dL, with noejection/CMV infection/graft or patient loss. There was aransient rise in donor-specific antibodies (mean, 25%) inll patients, which declined to less than 5% over a meaneriod of 2 weeks. Three (10%) patients required threelasmaphoresis procedures on alternate days to reach anntibody level of 5%. No adverse effects on allograftunction were observed among the Tn group. Fourteen

ession Scheme

Prednisolone (per day)Azathioprine/Mycophenolate

Mofetil (per day) �

(0.5 mg/kg BW) Cn (2 mg/kg BW)g/kg BW) Cn (0.5 mg/kg BW) Cn (2 mg/kg BW)g/kg BW) Cn (0.5 mg/kg BW) Cn (2 mg/kg BW)g/kg BW) Cn (2 mg/kg BW)

intervals

p Value

�0.001�0.001�0.001�0.001

supr

Cn0.2 m0.1 m0.5 m

time

�0.001

(fttssPb0pt

dtumnunl

trtt(ptrptron(e1ow

D

ObtOpMbtBBlsb

b

padZdmasthddeistTc(esaua

1lip

R

c

m

t

oc

taa

aT

Pr

ns6

tpr

STEM-CELL TRANSPLANT 741

42.4%) recipients were biopsied at a mean posttransplantollow-up time of 47.3 days for a rise in SCr above 10% ofhe baseline level; 13 (92.9%) biopsies showed acute CyAoxicity in the form of subintimal segmental hyalinosis in themall arteries and toxic tubulopathy. One (7.1%) biopsyhowed acute tubular necrosis with acute CyA toxicity.ersistent MLHC was noted in all 21 patients across MHCarriers. Mean MLHC before stem-cell transplantation was.05% in the 15 subjects’ who were mother-to-child trans-lants, and absent in the others at 100 days after transplan-ation, the value was 0.7 � 0.3% in all of them.

Immunosuppression has thus far been completely with-rawn in four patients who were mother-to-child transplan-ation recipients (with micro- and macro-MLHC) with anneventful mean follow-up of 210 days. By the end of 9onths’ posttransplant in the remaining 29 patients, pred-

isolone has been discontinued. Cyclosporine was contin-ed with an intent to maintain trough levels of 70 � 10g/mL at a required dose of 1.5 � 0.5 mg/kg BW/d,

ow-dose CyA monotherapy.Over a mean follow-up of 378 days, the mean SCr among

he Cn group was 1.52 mg/dL. Twenty-two (66.6%) patientsequired biopsies at a mean follow-up of 24.4 days’ post-ransplant, to evaluate the cause of rises in SCr. Acuteubulointerstitial rejection (type IA) was observed in 1254.6%) patients and 5 (22.7%) had acute tubulointerstitiallus acute vascular rejection (types IB � IIA). Acute CyAoxicity was observed in 7 (31.8%) biopsies. Two patientsequired re-biopsy at a mean follow-up of 352.5 days’osttransplant, both showing acute tubulointerstitial rejec-ion on a chronic tubulointerstitial process plus vascularejection with chronic transplant glomerulopathy. More-ver, CMV-infection-related graft and patient loss wasoted in 1 (3%) patient at 345 days’ posttransplant and 13%) graft loss was observed owing to repeated rejectionpisodes. The CyA trough levels were maintained between00 to 150 ng/mL in the Cn patients with the required dosef 3.5 � 0.5 mg/kg BW/d in all of them. In addition, theyere all continued on prednisolone (1 mg/kg BW/d).

ISCUSSION

ver the last five decades, transplantation biologists haveeen trying to define the mechanisms of donor-specificolerance in solid-organ transplantation. Fifty years agowen observed that freemartin cattle sharing common

lacenta exhibited erythrocytic chimerism.2 Billingham andedawar3,4 were surprised to observe acceptance of grafts

etween genetically nonidentical chimeric twins with rejec-ion of third-party grafts. This link was further confirmed byillingham, Brent, and Medawar3 where adult spleen andM-derived stem cells were infused into neonatal mice who

ater accepted donor skin grafts without immunosuppres-ion. Slavin et al5 demonstrated that such tolerance cannote induced in adult mice without irradiation.We have reported successful implementation of donor

one marrow derived cells infusion protocol to inducems

roper tolerance in the clinic.6–9 We used mega doses ofllo-HSCs to achieve the benefits of activation-induced celleath leading to depletion of the T-cell repertoire utilizinginkernagel’s concept of MHC restriction.10 Gorczynski’semonstration of persistent donor-specific tolerance in aouse model after portal rather than systemic infusion of

llogeneic stem cells encouraged us to implement thistrategy in our patients.11 We successfully induced properolerance by effectively controlling the peripheral arm;owever, the central arm (thymus) was yet to be ad-ressed.12 Posselt’s work encouraged us to inoculate theonor renal tissue into the host thymus to expose donorndothelial cells rich in MHC-II expression to the develop-ng thymocytes.13 We observed the presence of donor-pecific regulatory cells immediately after thymic inocula-ion of donor antigen by performing an ELISPOT assay.he T-cell repertoire was rich in cells secreting IL-10 asompared to a smaller population of cells secreting IL-2unpublished data). The presence of maternal hematopoi-tic cells in children known as “feto-maternal chimerism”uggests that immunologic tolerance exists between mothernd child.14 Our data support this observation. We havesed MLHC in a human model for four (12%) recipientscross MHC barriers.

In conclusion, our data suggest that low levels (0.7% to%) of MLHC achieved across MHC barriers using aow-intensity conditioning regime may be associated withnduction of donor-specific tolerance in LRD renal trans-lantation.

EFERENCES

1. Racusen LC, Solez K, Colvin RB, et al: The Banff 97 workinglassification of renal allograft pathology. Kidney Int 55:713, 1999

2. Owen RD: Immunogenetic consequences of vascular anasto-oses between bovine twins. Science 102:400, 19453. Billingham RE, Brent L, Medawar PB: ‘Actively acquired

olerance’ of foreign cells. Nature 172:603, 19534. Anderson D, Billingham RE, Lampkin GH, et al: Tolerance

f homografts, twin diagnosis and the freemartin conditions inattle. Heredity 6:201, 1952

5. Slavin S, Strober S, Fukes Z, et al: Induction of specific tissueransplantation tolerance using fractionated total lymphoid irradi-tion in adult mice: long-term survival of allogeneic bone marrownd skin grafts. J Exp Med 146:34, 1977

6. Trivedi HL, Shah VR, Shah PR, et al: High-dose PBSCssociated tolerance in live-related renal allograft recipients.ransplant Proc 32:2001, 20007. Trivedi HL, Shah VR, Shah PR, et al: Mega-dose approach to

BSC infusion-induced allograft hypo-responsiveness in living-elated renal allograft recipients. Transplant Proc 33:71, 2001

8. Trivedi HL, Shah VR, Vanikar AV, et al: High dose alloge-eic PBSC infusion—a strategy to induce allograft hypo-respon-iveness in pediatric renal transplant recipients. Pediatr Transplant:63, 20029. Trivedi HL, Vanikar AV, Shah VR, et al: Mega-dose unfrac-

ionated donor bone marrow derived cell infusion in thymus anderiphery—an integrated clinical approach for tolerance in liveelated renal allograft. Transplant Proc 35:203, 2003

10. Zinkernagel RM, Doherty PC: Restriction of in vitro T-cell-

ediated cytotoxicity in lymphocytic choriomeningitis within a

yngeneic or semiallogeneic system. Nature 248:701, 1974

gdt

pi

dt

br

742 TRIVEDI, VANIKAR, MODI ET AL

11. Gorczynski R, Chen Z, Zeng H, et al: Specificity for in vivoraft prolongation in gamma delta T-cell receptor hybridomaerived from mice given portal vein donor-specific pre-immuniza-ion and skin allografts. J Immunol 159:3698, 1997

12. Calne RY, Friend PJ, Moffatt S, et al: Proper tolerance,erioperative campath 1H, and low-dose cyclosporin monotherapy

n renal allograft recipients (letter). Lancet 351:1701, 1998 2

13. Posselt AM, Barker CF, Tomaszewski JE, et al: Induction ofonor-specific unresponsiveness by intrathymic islet transplanta-ion. Science 249:1293, 1990

14. Tamaki S, Ichinohe T, Matsuo K, et al: Superior survival oflood and marrow cell transplants given maternal groups overecipients given paternal groups. Bone Marrow Transplant 28:375,

001