The authors report on six pediatric liver transplant recipients for whom allograft tolerance occurred shortly after transplantation (ie, less than 1.5 years). All the patients had associated life-threatening viral complications. They are currently immmunocompetent. The tolerant state may be related to the development of a TH2 cytokine pattern. © 1994

Adrianna Zeevi

Andreas G. Tzakis

Bakr Nour

Calne

Hector Ramos

Hoffman

Jorge Reyes

Keenan

Murase

Nancy Reinsmoen

Powrie

Reinsmoen

Reyes

Rolides

Romagnani

Satoru Todo

Starzl

Takeuchi

Thomas E. Starzl

Tzakis

English

PubMed

Tzakis, AG

Reyes, J

Zeevi, A

Ramos, H

Nour, B

Reinsmoen, N

Todo, S

Starzl, TE

D-Scholarship@Pitt

Early Tolerance in Pediatric Liver Allograft Recipients By Andreas G. Tzakis, Jorge Reyes, Adrianna Zeevi, Hector Ramos, Bakr Nour, Nancy Reinsmoen, Satoru Todo, and Thomas E. Starzl Pittsburgh. Pennsylvania and Minneapolis, Minnesota • The authors report on six pediatric liver transplant recipients for whom allograft tolerance occurred shortly after transplantation (ie, lesl than 1.5 yearl,. All the patients had aSlociated life-threatening viral complications. They are cur-rently immunocompetent. The tolerant state may be related to the development of a TH2 cytokine pattern. Copyright CI 7994 by W.B. Saunders Company INDEX WORDS: Uver tranlplantation, allograft. DEVELOPMENT of a drug-free state has long been an aspiration in transplantation. It is particularly desirable in children because they are subject to complications secondary to the lifelong administration of immunosuppressive agents. We report on a small group of pediatric patients who. in the presence of life-threatening complications. had allograft tolerance shortly after transplantation. The immune unresponsiveness appeared to be donor-specific. They were shown to be otherwise immuno-competent. MATERIALS AND METHODS Case Material Among our pediatnc transplant population (liver. kidney. intes-tinal. and pancreatic islet recipients). there were six patients (Table I) who had life-threatemng vlfal infections not long after transplantation but also had tolerance to their grafts. All the patients were liver transplant recIpients. treated with ch-RM~ hased immunosuppression. I The age range at the time of transplan-tation was 0.3 to 1.5 vears. The pnmary disease was biliarv atresia (3). neonatal hepatitIS (I). fulminant hepatitis C (I). or ~at"s eve syndrome (I). Posttransplant. three of them had one or mo~e episodes of acute rejection that requlfed treatment with sterOids. and three had no rejection at all. The complications that necessitated discontinuation of the immunosuppressIOn were Iymphoproliferative disease (PTLD) From the Trump/antallon Illstitule. UlliversrlV Heallh Celller at Pittsbul1?h. UniversrlV ol Pillsbul1?h. and the setlD~ans Adminislratll~ I(edica/ ('('lIIU. PwsbUll(h. PA: and The Unil't'rsllV of Millnesota. .Hinneapo/IS. MN. l'rl'sellled at Ih,' ~lfth Annual 11Jll'matlOna/ COfII(T'l'SS of the Bntislr .4ssocwllun o( Paed,arne SUl1?eolls. ,\fanehester. Lng/and. Jlliv 21 ·23. 1<)93. Supponl'd hv Rt'Search Grams trom tire Veterans Administration <llld ProWCl uralll So. DK 29961 from tire NallOnai InslIlUles oj' Heallh. Bethesda. ,\ID. .4ddress repnm requesls to Thomas E. SlanL .\lD. PhD. Depan· mem of SUll(erv. ]()()I Fifth Al'e. Clln'ersrlV oj' PillSbull(h. PillSbull(h. PA 15213. Copynght..: 1<N4 bl' W. B. Saunders Compallv U()22-301bli 1901 I 29()6..00/OS03. 00 I 0 754 (n = 5) and severe hepatitis C (n = 2). Patient no. 4 had both complications .. Hepatitis was recurrent in one case and acquired after transplantation in the other. Immunosuppression was discon-tinued 0.5 to \.3 years posttransplantation. and. to date (1.3 to 2.8 years later). it has not been resumed. One patient died after retransplantation for hepatitis C. All others are alive with the same grafts. PTLD and hepatitis C have been controlled successfully in al\ surviving patients. There have been no other severe infections. All patients are rejection-free; this was demonstrated clinically. biochemically. and histologically. Another patient (no. 7. Table I). a l-year-old girl who received a liver allograft under FK-506 for biliary atresia. had severe hepatitis C 2 years after transplantation. and the immunosuppression was withdrawn. She remained rejection-free for 0.9 years. Rejection then developed. which required resumption of FK-506. The patient still has evidence of active hepatitis C. In Vitro Immunologic Studies These tests were done to evaluate immune competence. Func-tional evaluation of T helper cells was performed by measuring the ability of peripheral blood lymphocytes (PBL) to proliferate in response to nonspecific plant lectins: phytohemagglutinin (PHA). concanavalin A (Can A). and alloantigens. Although the "normal" range for each test may vary. the response is considered significant if the test value is at least 5- to IO-fold higher than the background value obtained in the absence of any stimuli. Patients who are immunocompromlsed and at high risk for frequent opportunistic infections exhibit very low responses to mitogens and alloantigen. These low responders may include transplant recipients on high doses of immunosuppressive drugs or patients with the human immunodeficiency virus. 2 . .1 The specllic response of recipient PBL to donor alloantigens can he tested with one-wav mixed lymphoc'Yte reaction (MLR). whereby t:qual numbers of recipient PBL and irradiated donor cells are mixed. and proliferation I, assessed hy the incorporation of 'H·thymidine at day 6 of coculture. Donor-specific hyporeactivity is considered when the patient's MLR response to the donor de-creases in comparison to the pretransplant values. while the proliferative responses to other HLA-incompatible donors remain high. When donor cells are not avaIlable. we use a panel of stimulators in the MLR. whIch are homozygous tor class II HlAs. These homozygous tvpmg cells (HTC) are selected to represent cells that express "self' HlAs. "donor" HLAs. and other HlAs that are unrelated to reCipient or donor antigens. Donor-specific hyporeac-tivity occurs when the proliferative responses of patIent PBL to self and donor HTC are similar. and both responses are SIgnificantly I,)wer than those ohserved toward unrelated HTCs.' Helper T cclls respond to stimuli in vitro and in vivo by producing vanous cvtokines. These mediators have an important role in the regulation of cell· mediated and humoral immunity. T helper cells can be categOrized according to the l'Ytokine profile they secrete. It:. THI or TH,,' Cytokrnes produced by the ru l subset are involved in allograft rejection and cellular responses to viral infections. whereas those secreted by TH2 cells are mainly elevated in allen!!c responses and helminth infections. Cytokines produced bv one subset mav modulate and antagonize the etfector (unction 01 the other suhset. For instance. TH2·like cYlOkines (ie. Journal of PedlBtriC Surgery, 1101 29. No 6 (June). 1994: pp 754-756 EARLY TOLERANCE IN LIVER ALLOGRAFT RECIPIENTS 755 Table 1. Uver Transplant Recipients Who Had Developed urty Tolerance to Grafts Patient Primary Age at No. Sex Diagnosis TranSD4ant8tion Immunosuppression 1 M Biliary atresia 0.6 FK·506. prednisone 2 F Neonatal hepatitis 0.3 FK·506. prednisone 3 F Biliary atresia O.S FK·506. prednisone 4 M Cat's eye 1.5 FK·506. prednisone syndrome 5 M Biliary atresia FK·506. prednisone 6 F Fulminant FK·506. prednisone hepatitis 7 F Biliary atresia 1.3 FK·50S. prednisone Abbreviations: ACR, acute cellular rejection; HCV. hepatitis C virus. "Died after retransplantation for hepatitis C. elevated levels of interleukin [IL] 4 or 10) may downregulate the rn 1 function (secretion of interferon and activation of macro-phages).b Circulating PBL may already express elevated levels of cytokine genes. which could be detected by using sensitive molecular probes. In addition. PBL respond to in vitro stimuli by a transient upregulation of cytokine genes. We evaluated the T helper pheno-type (rn I \. TH2) in pediatric liver transplant recipients by testing cytokine mRNA expression before PHA stimulation (spontane· ous) and after 2 hours' exposure to PHA. The studies performed in our patients were as follows. Mitogen·mduced proliferotion. Mononuclear peripheral blood cells from these patients were isolated with Ficoll gradient and cultured in tissue-culture media in the presence of either Con A or PHA. Proliferation was measured by JH-thymidine uptake. Mixed /\mphocyte reaction (MLR). Unidirectional MLR cui· tures were set up with normal. unrelated. irradiated lymphocytes In J tissue·culture medium. Proliferation was assessed with .1H· thymidine. Homozvgous rvping assay. In the absence of donor lymphocytes. IITC were used as stimulators of the pal1ents' PBL. using the MLR protocol described above. Seven different Ow specifics were tested with 2 to J HTC. for specificity.-DetectIOn ot' cwokine gene expression /Jv reverse transcriptase po/v· //Ierase cham reaction (RT·PCR). Total RNA was extracted from PBL. A known quantity of total RNA was used for cDNA synthesis. The latter was used as a template for PCR amplification. The PCR products were analyzed by gel electrophoresis. The presence of I L-2 .. t, n. and 10 mRNA in the pallents' PBL was tested before and 2 hours alter PHA stimulation. RESULTS Five of the tested patients (nos. L 3, 4. 5, and 7) had normal Can A- (12.491 to 41.979 cpm) and PHA·induced (58.385 to 188.434 cpm) proliferation l)f PBL O.l) to 2.6 years after discontinuation of immunosuppression. They also had normal ratio of MLR to third-party lymphocytes (23.035:88.l11 cpm). These results suggest that pediatric liver transplant recipients who are off immunosuppression arc immu-nocompetent. In two patients (nos. 1 and 3) in whom HTC assay could be performed. the proliferative response to Time Posttransplant Total Time Off Posttran.plant Until Off Medication Currant ACR Medication Iv') Iv') Complication BioPSV None 1.2 2.8 PTLO NoACR Mild 0.8 2.1 PTLO NoACR ACRX2 0.8 2.5 PTLO NoACR Multiple 1.3 PTLO.HCV HCV· None 1.3 1.3 PTLO NoACR None 0.5 2.8 HCV No ACR. noHCV ACRX2 2 0.9 HCV Mild ACR. HCV· HTC that presents donor antigens was in the same range as those towards stimulators that present self HLA-DR antigens. Both self and donor-specific re-sponses were less than 50% of the responses toward an unrelated third-party stimulator. These results suggest that donor-specific hyporeactivity was achieved in these two patients. Three of four tested patients (nos. 2, 3, and 6) showed the presence of IL-4, IL-lO and IL-6 mRNA before PHA stimulation. After activation, all PBL samples (from patients 1, 2, 3, and 6) showed IL-2, 4, 6, and 10 mRNA. The spontaneous cytokine profile is of the TH2 phenotype. An increase of IL-2 mRNA in the stimulated samples confirmed the patients' immu-nocompetence. DISCUSSION The description of the two-way cell traffic between donor and recipient and the development of mixed chimerism;·s have exposed previously unrecognized mechanisms of tolerance induction in human recipi-ents of whole organs. particularly of livers. It also led to a controlled attempt at reduction of immunosup-pression at our center. with total withdrawal as the goai~ Long-term survivors have been selected for this trial. To date. three kidney recipients (of living-related I-HLA haplotype-matched grafts) and 52 liver recipi-ents (mean survival time after transplantation, 8 years) have been enrolled in this study.t') Fifteen liver (29%) and two kidney recipients are currently off immunosuppression. The remaining patients are gradually being weaned. All patients are alive. and there have been no graft losses. Eleven patients (21 t;f). all liver recipients. have sustained one episode of rejection. which responded to resumption of immu-nosuppression. In three patients. mild rejection was not treated and subsided spontaneously. 756 The fact that immunosuppression can be re-duced 1.11 or stopped8 after transplantation is best exemplified in pediatric patients. In them. immuno-suppression is automatically down-regulated because no increase is made to compensate for growth. Discontinuation of immunosuppression has been practiced in the treatment of life-threatening compli-cations after transplantation. 12 The treatment usually is resumed because of the fear or occurrence of rejection. All but one of our seven patients (no. 7) exhibited graft tolerance and were immunocompe-tent. The potential drug independence of liver recipi-ents has been noted previously,S but it did not occur as after transplantation: in our study, the average time until drug independence was 0.93 years. The early timing of tolerance and the association with life-threatening viral infections differentiate these patients from those who had the immunosuppression withdrawn systematically. All the patients were treated with FK-506. Al-though in experimental animals there was a higher incidence of drug-free tolerance induction with FK-506 than with conventional immunosuppression in-cluding cyclosporine.13·14 the end result was not drug-specific; 15.16 in humans. tolerances has been achieved with azathioprine or cyclosporine-based cocktail regi-mens.s We are currently observing a patient treated with cyclosporine who is tolerant to her combined liver-kidney graft 8 years after transplantation. TZAKIS ET AL All patients in the present series were solitary liver recipients. Although the liver is known to be more tolerogenic than the other solid organs.S.16-18 rejection remains a threat. as exemplified by case 7. If drug weaning is undertaken. the need for careful supervi-sion and long-term follow-up cannot be overempha-sized. The resting PBL cytokine pattern was negative for IL-2 mRNA and positive for IL-4, 6, and 10, suggest-ing a TH2 cytokine pattern. This dominant TH2-like immune response may have facilitated donor-specific hyporeactivity and graft acceptance in a drug-free environment. 19 A similar TH2 cytokine pattern has been seen in liver biopsy specimens from patients with PTLD.20 The emergence of T helper phenotype 2 in liver transplant recipients after PTLD or other infections requires further consideration. TH2-like cytokines (IL-4 and IL-lO) may produce a noninflammatory environment by down-regulating the immune re-sponse to the allograft. promoting anergy and acceler-ating graft acceptance. In these cases. tolerance can be maintained in a drug-free environment. We conclude that resumption of immunosuppres-sion is not needed in some cases after life-threatening PTLD and HeV. This phenomenon may be more common in FK-506-treated liver recipients. The de-velopment of a tolerant state may be related to an alteration of cytokine patterns. REFERENCES I. Tzakis AG. Reyes J. Todo S. e:t al: FK 506 versus cyclosporine In rediatnc liver transplantation. Transplant Proc 23:3010-3015. 1991 2. Rolides M. C1erici M. DePalma L. e:t al: Helper T cell responses in children Infected with human Immunodeficiencvvirus type I.J Pediatr II tl:724-730. 1991 3. Ke:enan R. Uknis M. Pham S. et al: Mitogen responses of Iymphocvtes from lung transplant n:cipients. correlation with rejection and infection. Transplantation 54:241-245.1992 -I. Reinsmoen NL. Matas AJ. Kaufman D. et al: Acquired donor-specific hyporesponsiveness in long-term kidney allograft recipients. Transplant Proc 23:141-142.1991 5. Romagnam S: Human THI and qe~ subsets: Doubt no more. Immunol Today 12:156-257. 1991 n. Powrie F. Coffman RL: Cytokine regulallon of T-cell func-tion: Potential for therapeutic intervenllon. Immunol Today 14:270-~T4K I'N3 ~K Starzl TE. Demetns AJ. Murase N. et al: Cell migrallon. chimerISm and graft acceptance. Lancet 339:1579-1582.1992 K~ Starzl TE. Dl!metris AJ. Trucco M. et al: Cell migration and chimerISm alter whole-organ transplantation: The baSIS of graft .Icceptance. Hepatology 17:1127-1152.1993 'I. Reves J. Tzakis A. Ramos He. et al: The frequent achieve-ment of a drug free state alter orthotopIC liver transplantallon. Transplant Proc 15:3315-3319. 1993 IU. Ramos H (University of Pittsburgh): Personal communica-lion. Julv 14'1.' II. Starzl TE. Iwatsuki S. Malatack JJ. c:t al: Liver and kidnev Iransplantallon In children receiving cycl0psofln A and steroids. J Pediatr J(X):oXI-oxo. 19t12 12. Starzl TE. Nalesnik MA. Porter KA. ct al: Reversibility of lymphomas and Ivmphoproliferallve lesions developing under cyclo-'porin-steroid therapy. Lancet 1:583-5117. 1984 13. Lee KKW. Stangl MJ. Todo S. t:l al: Comparison of short-term Immunosuppression therapy with cyclosporme and FK 506 in small-bowel transplantation. Transplant Proc 22:24tl5-2486. 1<)90 14. Hoffman AL. Makowka L. Banner B. et al: The use of FK 506 for small inlestine allotransplantation: Inhihition of acute rejection and prevention of fatal grafl-ve:rsus-host disease. Trans-plantation 4<):4113-490. 1990 15. Murase N. Kim DG. Todo S. ct al: FK SOo immunosuppres-sion of heart and liver allograft rejecllon 11: The mduction of graft acceptance in ral. Transplantation 50:739-744. 1<)40 10. Starzl TE. Demetrls AJ. Murase: N. et al: Cell chimerism permitted hy Immunosuppressive drugs IS the baSIS of organ transplant acceptance and tolerance. Immunol Todav 14:326-332. 1993 17. Starzl TE. Kaupp HA Jr. Brock DR. ct ;11: Ilomotransplanta-lion of muluple visceral organs. Am J Surg 111.1:219-229. 1962 Itl. Caine RY. Sells RA. Pena JR. et al: Inducllon of immuno-logical tolerance bv porcine liver allogratts. Nature 223:472-474. 1969 14. Takeuchi T. Lowry RP. Konieczny 13: f kart allografts in murine systems: The dlfferenllal aClivalion ot TII:-Iike: elfector (.:lIs in penpheraltoll!rance:. Transplantation ~K;·1Op 1-1294. 1992 20. Nalesntk MA. Frve C. Zcevi A. ct al: (",tokine protiles in Epstein Barr Virus aSSOCiated post-transplant Ivmphoprollferatlvc disorders. (submitted) 

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Early tolerance in pediatric liver allograft recipients

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