Posttransplant Administration of Donor Leukocytes Induces Long-Term Acceptance of Kidney or Liver Transplants by an Activation-Associated Immune Mechanism This information is current as of September 29, 2021. Yiqun Yan, Suma Shastry, Craig Richards, Chuanmin Wang, David G. Bowen, Alexandra F. Sharland, Dorothy M. Painter, Geoffrey W. McCaughan and G. Alex Bishop J Immunol 2001; 166:5258-5264; ; doi: 10.4049/jimmunol.166.8.5258 Downloaded from http://www.jimmunol.org/content/166/8/5258

References This article cites 34 articles, 7 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/166/8/5258.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Posttransplant Administration of Donor Leukocytes Induces Long-Term Acceptance of Kidney or Liver Transplants by an Activation-Associated Immune Mechanism1

Yiqun Yan,* Suma Shastry,* Craig Richards,* Chuanmin Wang,† David G. Bowen,* Alexandra F. Sharland,* Dorothy M. Painter,‡ Geoffrey W. McCaughan,* and G. Alex Bishop2*

Donor leukocytes play a dual role in rejection and acceptance of transplanted organs. They provide the major stimulus for rejection, and their removal from the transplanted organ prolongs its survival. Paradoxically, administration of donor leukocytes also prolongs allograft survival provided that they are administered 1 wk or more before transplantation. Here we show that administration of donor leukocytes immediately after transplantation induced long-term acceptance of completely MHC-mis-

matched rat kidney or liver transplants. The majority of long-term recipients of kidney transplants were tolerant of donor-strain Downloaded from skin grafts. Acceptance was associated with early activation of recipient T cells in the , demonstrated by a rapid increase in IL-2 and IFN-␥ at that site followed by an early diffuse infiltrate of activated T cells and apoptosis within the tolerant grafts. In contrast, IL-2 and IFN-␥ mRNA were not increased in the of rejecting animals, and the diffuse infiltrate of activated T cells appeared later but resulted in rapid destruction. These results define a mechanism of allograft acceptance induced by donor leukocytes that is associated with activation-induced cell death of recipient T cells. They demonstrate for the first time that posttransplant administration of donor leukocytes leads to organ allograft tolerance across a complete MHC class I plus class http://www.jimmunol.org/ II barrier, a finding with direct clinical application. The Journal of Immunology, 2001, 166: 5258–5264.

onor leukocytes, in particular donor dendritic cells Also, spleen allografts in some completely MHC-mismatched within a transplanted organ, are the major stimulus for strain combinations are accepted without requiring immunosup- D its rejection, and depletion of these passenger leukocytes pression (10). In a model of rat , posttrans- leads to long-term graft acceptance in animal models (1, 2). In plant administration of donor leukocytes slightly prolonged sur- contrast, donor leukocytes can promote survival of transplanted vival although it did not lead to long-term acceptance (11). organs, and infusion of donor leukocytes before transplantation Furthermore, transplantation of a heart together with organs rich in often leads to prolonged survival of organs that would otherwise donor leukocytes such as lung or spleen (12), or of multiple organs by guest on September 29, 2021 have undergone rapid rejection (3, 4). Previous studies in experi- supplemented with donor leukocytes (13), were found to promote mental models have shown that donor leukocytes only prolong long-term allograft acceptance. Posttransplant administration of allograft survival when administered at least 1 wk before transplan- donor leukocytes also led to tolerance of skin grafts across a minor tation (4–6). These findings have a limited application in the clinic, as histocompatibility barrier (14). cadaver donors are the major source of organs for transplantation The aim of the studies presented here was to investigate whether and acceptance of the transplanted organ requires that MHC Ags administration of donor leukocytes at completion of the transplant are shared between the leukocyte donor and the organ donor operation could induce long-term acceptance of rat kidney or liver (reviewed in Ref. 7). Furthermore, pretransplant infusion of donor allografts across a complete major MHC barrier. Kidneys from blood is associated with a risk of presensitization of the recipient, PVG strain donors transplanted to completely MHC-mismatched resulting in hyperacute rejection rather than acceptance (7). DA recipients (PVG 3DA) are rapidly rejected (13) although Despite these initial studies showing the lack of efficacy of do- liver transplants in the same strain combination are spontaneously nor leukocytes when infused at the time of transplantation, some accepted without requiring immunosuppression (15). In contrast, evidence from animal models indicates that they can promote tol- high-responder Lewis strain recipients reject livers from PVG erance when present at the time of transplantation. Acceptance of strain donors (15, 16). The effect of administration of donor leu- liver allografts across complete histocompatibility barriers without kocytes on rejection of PVG3DA kidney allografts and of immunosuppression is dependent on passenger leukocytes (8, 9). PVG3Lewis liver allografts was examined. Previous investiga- tions of the immune mechanism of spontaneous acceptance of liver allografts have shown activation and apoptosis of T cells (16, 17). *Centenary Institute of Cancer Medicine and Cell Biology, and Departments of †Sur- gery and ‡Anatomical Pathology, Royal Prince Alfred Hospital, Sydney, Australia Consequently, these processes were examined in the leukocyte- Received for publication August 8, 2000. Accepted for publication February 5, 2001. induced transplant acceptance reported here. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Animals 1 This work was supported by the National Health and Medical Research Council of Australia Grant 970979, the Wellcome Trust, and the Australian Kidney Foundation. Inbred strains of rats were obtained from the Animal Resources Center 2 (Perth, Western Australia) and were completely mismatched at the rat Address correspondence and reprint requests to Dr. G. Alex Bishop, Centenary c a Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital, Mis- MHC (RT1) locus. The rat strains used were PVG (RT1 ), DA (RT1 ), and l senden Road, Camperdown NSW 2050, Australia. E-mail address: A.Bishop@ Lewis (RT1 ). All experiments were performed with the approval of the centenary.usyd.edu.au Royal Prince Alfred Hospital Animal Care Ethics Committee.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 5259

Transplantation Preparation of spleen leukocytes and leukocyte subsets Orthotopic liver or kidney grafts were performed, and in the case of kidney Spleen leukocytes were aseptically prepared by mashing the spleen and grafts, the contralateral kidney was removed 3 days after transplantation. then lysing the RBC in NH4Cl buffer as described (17). Cells were washed Techniques for liver, kidney, and have been reported previ- and injected at a dose of either 2 ϫ 107 or 6 ϫ 107 i.v. in 1 ml of isotonic ously (13). For some experiments, donors were given 10 Gy of whole body saline. This cell population consisted of a mixture of T cells, B cells, irradiation 1 wk before as described (9). myeloid cells, and small numbers of NK cells, and RBC and platelets were not detectable. In some experiments, spleen cells were fixed by incubation Immunohistochemical staining in 2% paraformaldehyde (BDH, Poole, U.K.) in PBS for 20 min on ice, washed thoroughly with PBS containing FBS, and injected i.v. For sepa- Localization of leukocyte populations was by immunostaining of frozen ration of leukocyte subsets from recipient spleen, aliquots of 1.5 ϫ 107 sections by an indirect immunoperoxidase technique (18). Primary Abs spleen leukocytes were incubated with either OX27, R73, OX35 plus W3/ were obtained from Dr. J. Sedgwick (DNAX Research Institute, Palo Alto 25, or MOPC 21. After incubation for 30 min on ice and washing in PBS, CA). They were OX6, reactive with MHC class II; OX27, reactive with the cells were incubated with 6 ϫ 107 magnetic beads (Dynal, Oslo, Nor- donor (PVG) MHC class I; OX39, reactive with CD25; R73, reactive with ␣ ␤ way), washed, and separated on a magnetic column as described previously / TCR; OX35 plus W3/25 1:1 mixture, reactive with CD4; OX42, re- (13) before RNA extraction. active with CD11b/c expressed by monocytes/macrophages and dendritic cells (19); and MOPC 21 (Sigma, St. Louis, MO), negative control. Apo- Statistical analyses ptotic cells were identified in frozen sections by TUNEL staining as de- scribed previously (20) with reagents obtained from Boehringer Mannheim Comparison of survival data was by log-rank analysis of the product-limit (Mannheim, Germany). estimate of Kaplan and Meier as described previously (9). Cytokine mRNA molecule numbers were analyzed by Mann-Whitney U test, and infiltration Flow cytometry and apoptosis in tissue sections were analyzed by unpaired t test. Error bars for cytokine mRNA expression and infiltration and apoptosis show the Downloaded from Tissues were dissociated by mashing the organs through a 100-mesh steel mean Ϯ SD of three separate animals. sieve. Leukocytes were purified by centrifugation of the washed cell sus- pension on isotonic Percoll (Pharmacia Biotech, Uppsala, Sweden) 1.05 g/ml for 15 min at 840 ϫ g. The leukocyte-containing pellet was washed Results and analyzed by four-color flow cytometry as described (17). Abs to CD4 Posttransplant administration of donor leukocytes induces or PVG MHC class I were detected by anti-mouse Ab conjugated to flu- allograft acceptance orescein (F2266; Sigma). R73-PE and OX39-biotin were obtained from Serotec (Oxford, U.K.); streptavidin-allophycocyanin was obtained from Rat kidney allografts in the strain combination of PVG donor to http://www.jimmunol.org/ Molecular Probes (Eugene, OR). Dead cells were identified with propidium DA recipient (PVG3DA) mismatched at all MHC class I and II iodide at 1 ␮g/ml and excluded from analysis. loci were rejected with a median survival time of 7 days (Table I). Measurement of cytokine mRNA expression Treatment of the recipients with donor leukocytes at completion of transplantation led to long-term acceptance of the transplanted kid- Cytokine mRNA in spleen was measured by quantitative RT-PCR as we neys. All recipients of 6 ϫ 107 spleen leukocytes survived for have described previously (17). Total RNA was isolated and reverse-tran- Ͼ ϫ 7 scribed, and then aliquots of cDNA were measured for cytokine mRNA 300 days, and treatment with 2 10 leukocytes resulted in expression in a quantitative PCR. Quantification was by a noncompetitive long-term acceptance of the majority of kidneys (Table I). Long- method with external standards of known numbers of cytokine cDNA mol- term surviving recipients of PVG kidney allografts plus PVG leu- by guest on September 29, 2021 ecules. In some experiments, the number of cytokine mRNA molecules kocytes were tested 250 days after transplant with a PVG donor was adjusted to account for sample-to-sample variation with a G3PDH- positive internal control. In some experiments, quantitative PCR was per- strain skin graft. In three of four animals so tested, the skin graft formed by using a Model 7700 Sequence Detector (PE Applied Biosys- survived for Ͼ100 days, showing that donor leukocyte treatment tems, Foster City, CA) and a dye-labeled probe. For IL-2, the probe led to tolerance. Donor leukocytes failed to prolong renal allograft sequence was 6FAM-TTG CCC AAG CAG GCC ACA GAA TTG- survival when their viability was impaired by fixation (Table I). TAMRA, and the amplification primers were CCC CAT GAT GCT CAC Unmodified liver transplants were accepted in the PVG3DA GTT TA (forward) and ATT TTC CAG GCA CTG AAG ATG TTT (re- verse). For IFN-␥, the probe sequence was 6FAM-CCT TTT GCC AGT strain combination without a requirement for conventional immu- TCC TCC AGA TAT CCA AGA-TAMRA, and the amplification primers nosuppression or donor leukocyte treatment (Table I). Irradiation were AGT CTG AAG AAC TAT TTT AAC TCA AGT AGC AT (for- of the liver donors abrogated tolerance, and administration of do- ward) and CTG GCT CTC AAG TAT TTT CGT GTT AC (reverse). Dye- nor leukocytes at the time of transplantation of these irradiated labeled primer and probe sets were designed by using Primer Express soft- ware (PE Applied Biosystems). Amplification primers were used at 300 livers resulted in restoration of graft survival (Table I), as we and nM concentration and probes were used at 200 nM in Taqman universal others have shown previously (9, 13, 21). Liver allografts from PCR master mix (PE Applied Biosystems). PVG donors were rejected in the high responder Lewis rat strain,

Table I. Donor leukocytes induce long-term acceptance of kidney and liver transplants

Organ Strain Survival MST p Transplanted Combinationa Treatmentb (days) (days) (vs untreated)

Kidney PVG3DA 6,6,6,7,9,10,13 7 Kidney PVG3DA 2 ϫ 107 PVG spleen cells c168, 253,Ͼ268,Ͼ272 261 0.005 Kidney PVG3DA 6 ϫ 107 PVG spleen cells Ͼ300ϫ 4 Ͼ300 0.005 Kidney PVG3DA 6 ϫ 107 fixed PVG spleen cells 6,8,8,9 8 0.99 Liver PVG3DA Ͼ100ϫ 6 Ͼ100 Liver PVG3DA Donor irradiation (10 Gy) 12,12,12,14,15,16,56,Ͼ100 ϫ 2 15 0.007 Liver PVG3DA Donor irradiation (10 Gy) ϩ 35,97,Ͼ100 ϫ 7 Ͼ100 0.68 6 ϫ 107 PVG spleen cells Liver PVG3Lewis 14,14,15,15,17 15 Liver PVG3Lewis 6 ϫ 107 PVG spleen cells Ͼ100ϫ5 Ͼ100 0.004

a Strains used were PVG (RT1c), DA (RT1a), and Lewis (RT11) and were mismatched at all MHC class I and class II loci. b Treatment involved injection of cells into the recipient or irradiation of the donor or a combination of both. c Pathology analysis showed hydronephrosis, probably due to ureteric obstruction. 5260 DONOR LEUKOCYTE-INDUCED ALLOGRAFT ACCEPTANCE although rejection could be prevented by administration of donor leukocytes to the recipient at the time of transplantation (Table I). Histological examination of the long-term accepted grafts showed little or no evidence of chronic rejection but did reveal occasional small perivascular aggregates of leukocytes that had not damaged the graft or blood vessels within it. The above results showed that large numbers of PVG donor leukocytes administered at the time of transplantation converted rejection of both kidney and liver al- lografts to long-term acceptance.

Rapid migration of donor leukocytes to lymphoid tissues PVG leukocytes administered i.v. to DA recipients of PVG renal allografts at the time of transplantation migrated rapidly to recip- ient lymphoid tissues. Flow cytometric analysis showed that donor cells comprised 4.7 Ϯ 0.5% (n ϭ 3) of recipient spleen leukocytes 1 day after renal transplantation supplemented with 6 ϫ 107 leu- kocytes. This declined rapidly to 1.6 Ϯ 0.6% on day 3. Immuno- histochemical staining demonstrated the presence of large numbers of donor cells (849 Ϯ 97 cells/mm2) in the periarteriolar lymphoid Downloaded from sheaths of recipient spleens on day 1. Many also migrated to the paraaortic lymph nodes draining the graft (288 Ϯ 132 cells/mm2). By contrast, very few donor cells had migrated to the spleen and lymph nodes of recipients of kidney allografts transplanted without 2 additional donor leukocytes, with only 15 Ϯ 8 cells/mm and 10 Ϯ FIGURE 2. Analysis of leukocyte subset expression of IL-2 and IFN-␥ 2 6 cells/mm , respectively, being detected on day 1. mRNA in the spleen of tolerant DA recipients of PVG liver transplants or http://www.jimmunol.org/ in unseparated normal spleen cells. Spleen was removed from DA recipi- Tolerance-associated immune activation ents of PVG liver transplants 1 day after transplantation and donor cells, ϩ ␥ ␣␤ T cells, and CD4 cells were positively selected. The negative (recip- The levels of IL-2 and IFN- mRNA expression were compared in ϩ the spleen of recipients of allografts that were undergoing either rocal) populations contained recipient cells, non-T cells, and non-CD4 cells, respectively. A, IL-2 mRNA expression. B, IFN-␥ mRNA expression rejection or tolerance. Marked activation, as measured by these (n ϭ 3 per group).

parameters, was observed early after transplantation in the spleens by guest on September 29, 2021 from recipients of tolerant but not rejecting grafts (Fig. 1). There was a significant ( p ϭ 0.05) increase of IL-2 mRNA 1 day after transplantation in the tolerant leukocyte-treated kidney or liver re- cipients compared with untreated kidney recipients undergoing re- jection or normal nontransplanted animals (Fig. 1A). The increase in IL-2 mRNA in kidney-tolerant animals was rapid and transient, characterized by a peak on day 1 that had disappeared by day 3, similar to the transient increase in IL-2 mRNA observed in recip- ient lymphoid tissues of animals during liver allograft tolerance (16). No detectable increase in IL-2 expression was observed in the spleen of recipients of untreated kidney allografts undergoing re- jection or in syngeneic controls. IFN-␥ mRNA showed a similar pattern of increase in tolerant kidney or liver allograft recipients that significantly exceeded that observed in untreated recipients or syngeneic controls ( p ϭ 0.05; Fig. 1B).

Cellular source of the IL-2 and IFN-␥ mRNA in tolerant allograft recipients The spleens of recipients of PVG3DA liver allografts were sep- arated into subpopulations of leukocytes on day 1 after transplan- tation. The expression of IL-2 mRNA in these subpopulations is shown in Fig. 2A. Recipient cells expressed significantly ( p ϭ FIGURE 1. Cytokine mRNA expression in recipient spleen after trans- 0.05) more IL-2 than did donor cells. The activation-associated plantation of a PVG kidney or liver to a DA recipient or control DA to DA increase in IL-2 mRNA, compared with cells from a normal syngeneic kidney transplant. A, IL-2 mRNA expression. B, IFN-␥ mRNA spleen, was confined to recipient cells from tolerant animals. Do- expression. Expression in normal DA spleen, in spleen of kidney (KTx) Ϯ ϭ that rejects, or of liver (LTx) allografts or kidney allografts plus donor nor cells, analyzed by flow cytometry, comprised 1.4 0.1% (n Ϯ Kidney or liver 3) of the spleen leukocytes, and produced 3.0 1.3% of the ,ء .leukocytes (KTx ϩ leuk.) that are accepted long term transplant acceptance is associated with significantly (p ϭ 0.05) increased amount of IL-2 mRNA produced by recipient cells. T cells, which cytokine expression in spleen compared with kidney on comprised 37.4 Ϯ 2.7% of the spleen population, produced 97.6 Ϯ day1(n ϭ 3 per group). 35% of the IL-2, whereas non-T cells only yielded 2.4 Ϯ 0.9% of The Journal of Immunology 5261

filtrate of T cells and IL-2R-expressing (IL-2Rϩ) cells in the tol- erant kidney grafts with a high ratio of IL-2Rϩ cells to T cells on day 3 after transplantation (Fig. 3; Table II). However, by day 5 after transplantation comparable numbers of T cells were present in the interstitial areas of both rejecting and tolerant kidney grafts. At the same time, the proportion of IL-2Rϩ cells in these areas had decreased in tolerant vs rejecting grafts (Fig. 3). Thus, the kinetics of the activated T cell response in tolerance and rejection differed markedly, with infiltration of large numbers of T cells and acti- vated cells being seen mainly in the interstitial areas of tolerant grafts at least 2 days before their appearance in rejecting grafts. By contrast, the infiltrate in rejecting grafts was limited mainly to perivascular sites until day 5 after transplantation, when a marked increase in interstitial infiltrate occurred (Table II). Thus, it ap- pears that tolerance is associated with altered patterns of T cell infiltration into the graft. FIGURE 3. Analysis of the graft interstitial infiltrate in PVG3DA kid- Graft-infiltrating leukocytes were stained for markers of T cells, ney allografts. Immunohistochemical staining was used to identify the in- IL-2R, and donor MHC class I to confirm that the infiltrate in day filtrate of IL-2Rϩ cells and T cells in grafts of untreated recipients (rejec- 3 tolerant kidneys was composed of recipient activated T cells and Downloaded from tion) or in recipients that were treated with 6 ϫ 107donor spleen cells to not injected donor cells that had localized to the transplanted kid- become tolerant (tolerance). Results show the ratio of IL-2Rϩ cells to T ney. The majority of T cells in the infiltrate was of recipient origin There was a signifi- (Fig. 4A), donor cells comprising only 4.8 Ϯ 2.6% of the T cell ,ء .cells in the interstitial areas of kidney allografts ϩ cantly (p ϭ 0.02) greater ratio of IL-2R cells in tolerance on day 3 (n ϭ infiltrate. Many of the T cells in the infiltrate were activated as 3 per group). shown by the observation that 32 Ϯ 5% of T cells expressed IL-2R

␣-chain. On further analysis of T cells, the majority (61.7 Ϯ 0.3%) http://www.jimmunol.org/ ϩ the level of IL-2 mRNA expressed by the T cell population. The proved to be CD4 , a considerable proportion of which were IL- ϩ CD4 population, which comprised 31.0 Ϯ 3.6% of spleen leuko- 2R (43.7 Ϯ 4.0%) (Fig. 4B). cytes, was mainly responsible for the increased IL-2 mRNA in The monocyte/macrophage and dendritic cell infiltrate was iden- spleen. Non-CD4 cells, which consisted of CD8 T cells, B cells, tified with the OX42 Ab reactive with CD11b/c expressed on these myeloid cells, and small numbers of NK cells, only expressed cells. Their pattern of infiltration was similar to T cells in that on 3.9 Ϯ 1.7% of the total amount of IL-2. These results show that day 3 there were significantly more ( p ϭ 0.004) monocytes/mac- recipient CD4 T cells produced Ͼ90% of the IL-2 mRNA in the rophages in the interstitial areas of tolerant than rejecting kidneys spleen of tolerant animals. (Table II). By day 5 there were similar numbers of these cells in IFN-␥ mRNA, shown in Fig. 2B, was expressed predominantly the interstitial areas of both tolerant and rejecting kidneys. by guest on September 29, 2021 by recipient compared with donor cells ( p ϭ 0.05), with only 4.4 Ϯ 3% of the total IFN-␥ being produced by donor cells. In contrast to IL-2, IFN-␥ was expressed by both T cells, which ex- Early apoptosis in the lymphoid tissues and graft is associated pressed 71.6 Ϯ 41.5% of the total, and non-T cells, which ex- with tolerance pressed 28.4 Ϯ 16.3% of the total. CD4 and non-CD4 cells pro- To examine whether activation of T cells in tolerant animals led to duced approximately equivalent amounts of IFN-␥ mRNA. Thus, their subsequent death by apoptosis, sections of kidney allografts in quantitative terms, the rapid increase in cytokine mRNA in and of recipient spleen were stained by TUNEL to identify apo- spleens of tolerant animals was largely attributable to activation of ptotic cells. TUNEL staining of tolerant kidney transplants on day recipient CD4 T cells in the case of IL-2 and of recipient T cells 3 identified large numbers of apoptotic cells in the same location and non-T cells in the case of IFN-␥. as the interstitial infiltrate of T cells and IL-2Rϩ cells (Fig. 5A). In contrast, there were few detectable apoptotic cells in the rejecting T cell activation and macrophage infiltration in tolerant kidney kidneys at the same time (Fig. 5B). Examination of the spleen of allografts kidney allograft recipients showed a corresponding large increase The early cytokine increases found in the spleens of kidney or in apoptotic cells in the periarteriolar sheaths of tolerant (Fig. 5C) liver-tolerant animals were followed by an extensive, diffuse in- compared with rejecting animals on day 3 (Fig. 5D).

Table II. Comparison of graft infiltrates during kidney allograft acceptance and rejection

T Cells IL-2Rϩ Cells CD11b/cϩ Cellsa

Perivascular Interstitial Perivascular Interstitial Interstitial

Normal PVG kidney 14 Ϯ 6b 6 Ϯ 40 93Ϯ 13 Day 3 KTxc 865 Ϯ 243*d 71 Ϯ 23** 251 Ϯ 38** 8 Ϯ 5*** 264 Ϯ 80** Day 3 KTx ϩ cells 275 Ϯ 124 206 Ϯ 21 47 Ϯ 11 76 Ϯ 13 634 Ϯ 75 Day 5 KTx 2476 Ϯ 758 604 Ϯ 153 1075 Ϯ 202** 353 Ϯ 129 1594 Ϯ 815 Day 5 KTx ϩ cells 1735 Ϯ 269 546 Ϯ 115 308 Ϯ 47 169 Ϯ 60 2029 Ϯ 756

a Monocytes/macrophages and dendritic cells were identified by expression of CD11b/c. b Number of cells/mm2 of leukocyte subsets was identified by immunohistochemical staining of kidney tissue (mean Ϯ SD of three replicate animals). c PVG to DA kidney transplants (KTx) that reject were compared on day 3 and day 5 after transplantation to PVG to DA kidney transplants injected with 6 ϫ 107 PVG spleen leukocytes (KTx ϩ cells) that are tolerant. d Statistically significant difference between KTx and KTx ϩ cells by unpaired t test (*, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.001). 5262 DONOR LEUKOCYTE-INDUCED ALLOGRAFT ACCEPTANCE

Discussion According to published evidence, donor leukocytes most effec- tively prevent rejection when given at least 1 wk before transplan- tation (5, 6). Such treatment is of little practical value in clinical transplantation programs, which depend on cadaver donors as the major source of organs. There is also the risk of patient presensi- tization by donor leukocyte pretreatment. The results presented here provide the first demonstration of markedly prolonged sur- vival of either liver or kidney allografts when the recipients were given donor leukocytes at the time of transplantation, despite the use of completely MHC-mismatched strain combinations normally associated with rapid rejection. Recipients of kidney allografts treated in this way ultimately developed tolerance to subsequent skin grafts of the same strain as the kidney donor. Presumably, the explanation for the discrepancy between our studies and those of previous investigators is related to the choice of strain combination or organ transplanted. Previous studies have examined heart or skin allografts in mouse or rat models that are likely to present a greater barrier to induction of tolerance than kidney or liver allo- Downloaded from grafts (5, 6, 13). Nevertheless, the transplant models examined here do reflect the strength of the barriers encountered in human transplantation, especially considering that human renal trans- plants, where possible, are matched at the MHC. FIGURE 4. Flow cytometry analysis of the infiltrate on day 3 in Our results show that donor leukocytes rapidly migrated to re- PVG3DA kidney transplants that had been treated with 6 ϫ 107 donor http://www.jimmunol.org/ spleen cells to accept the graft. T cells were gated to identify IL-2Rϩ cells cipient lymphoid tissues, which are the site of initiation of the and donor (RT1c) T cells (A) or IL-2Rϩ and CD4ϩ cells (B). Results are immune response to transplanted organs (22, 23). Within 24 h of representative of three separate experiments. their migration to the recipient spleen, rapid immune activation occurred, accompanied in the case of tolerance, but not rejection, with increased expression of IL-2 and IFN-␥ mRNA. These results Counting of TUNELϩ cells showed that there were significantly for renal allograft tolerance induced by administration of donor more of these cells in the spleen ( p ϭ 0.0003) and transplanted leukocytes at the time of transplantation closely parallel previous kidney (0.006) of tolerant compared with rejecting animals on day findings in spontaneous tolerance of liver allografts that show a 3 (Fig. 6). By day 5 this difference had disappeared and there were rapid increase in splenic IL-2 and IFN-␥ mRNA. This increase was by guest on September 29, 2021 similar numbers of apoptotic cells in both tolerance and rejection not observed during rejection of liver (16), kidney (17), or skin (Fig. 6). It is possible that the increase in TUNELϩ cells in the (24) allografts. rejecting kidney on day 5 might have been attributable to apoptosis Subsequent to immune activation in the recipient lymphoid tis- of kidney parenchymal cells, reflecting damage to these cells as a sues, an extensive diffuse infiltrate of activated recipient T cells, result of rejection, which was usually complete by day 7. These monocytes and macrophages appeared in the tolerant kidney results examining apoptosis in the transplanted kidney and recip- grafts. This diffuse infiltrate differed markedly from the localized ient spleen indicate that early cell death was associated with tol- perivascular infiltrate that was initially observed in the untreated erance rather than rejection. rejecting kidneys. At the same time, programmed cell death was

FIGURE 5. Photomicrographs of apo- ptotic cells in transplanted kidney (A and B) or periarteriolar lymphoid sheaths of recip- ient spleen (the central circular area of C and D)ofPVG3DA kidney allografts on day 3. Apoptotic cells were identified by TUNEL staining of sections of allograft re- cipients that were induced to accept the grafts by treatment with 6 ϫ 107 donor spleen cells (A and C) or were untreated (re- jecting; B and D; magnification, ϫ100). Ap- optotic cells appear as dark dots in the in- terstitial areas of the transplanted kidney (A) and in the periarteriolar lymphoid sheaths (C) of animals in the process of accepting their graft. Few apoptotic cells are observed during rejection (B and D), although apo- ptotic cells are present in splenic red pulp of both treated and untreated animals. The Journal of Immunology 5263

model of renal allograft tolerance induced by treatment with Ab to CD40 ligand, adjunctive immunosuppression with corticosteroids or tacrolimus interfered with induction of tolerance (33). Therapy with mAbs to the IL-2R is effective in preventing transplant re- jection (reviewed in Ref. 34), and their effect on AICD-associated transplantation tolerance, where activated T cells are a prominent early feature, has not been established. In conclusion, evidence is presented that kidney and liver allo- graft acceptance can be induced by donor leukocyte administration at completion of the transplant operation. Such leukocyte-induced allograft acceptance is associated with activation and exhaustive differentiation of recipient alloreactive T cells. This has important implications for management of transplant patients as conventional immunosuppression, which inhibits T cell activation, has the po- tential to inhibit tolerance as well. Therefore, treatment of trans- plant patients might be improved by administration of donor leu- kocytes combined with immunosuppression that complements FIGURE 6. Time course of apoptosis in periarteriolar lymphoid sheaths rather than inhibits tolerance associated with immune activation. 3

of recipient spleens (A) or in transplanted kidneys (B)ofPVG DA kidney Downloaded from transplants. Apoptotic cells were identified by TUNEL staining of sections of untreated recipients (rejection) or of those that were induced to accept Acknowledgments the kidney grafts by treatment with 6 ϫ 107 donor spleen cells (tolerance). We thank Professor A. Basten for helpful comments on the manuscript. Significantly more apoptotic cells were observed in tolerance compared p ϭ 0.0003; n ϭ 3 per group). References ,ءء ;p ϭ 0.006 ,ء) with rejection on day 3 1. Talmage, D. W., G. Dart, J. Radovich, and K. J. Lafferty. 1976. Activation of

transplant immunity: effect of donor leukocytes on thyroid allograft rejection. http://www.jimmunol.org/ Science 191:385. observed in the tolerant kidneys and in the splenic periarteriolar 2. Lechler, R. I., and J. R. Batchelor. 1982. Restoration of immunogenicity to pas- senger cell-depleted kidney allografts by the addition of donor strain dendritic lymphoid sheaths of the recipients of these kidneys, but not in cells. J. Exp. Med. 155:31. those undergoing rejection. Thus, there was a close concordance 3. Guttmann, R. D., and J. B. Aust. 1961. Acquired tolerance to homografts pro- between kidney allograft tolerance induced by donor leukocytes duced by homologous spleen cell injection in adult mice. Nature 192:564. 4. Ockner, S. A., R. D. Guttmann, and R. R. Lindquist. 1970. Renal transplantation and spontaneous acceptance of liver allografts, which show an in the inbred rat. XIII. 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