Infectious tolerance develops after intrathymic alloantigen–induced acceptance of rat heart allografts can be adoptively transferred

Infectious tolerance develops after intrathymic alloantigen–induced acceptance of rat heart allografts can be adoptively transferred Tsukasa Takayashiki, MD, Hiroyuki Asakura, MD, Grace Ku, PhD, Masaaki Kataoka, MD, and M. Wayne Flye, MD, PhD, St. Louis, Mo

Background. We have shown that intrathymic (IT) injection of alloantigen with antirat lymphocyte serum (ALS) treatment can induce donor-specific allograft acceptance. The purpose of this study was to investigate whether T-regulatory (T-reg) cells play a role in the maintenance of donor-specific heart graft tolerance that develops after IT injection of Lewis (LEW, RT1l) alloantigen into a Dark Agouti (DA, RT1a). Methods. Naı¨ve DA rats were injected IT with 2.5 3107 LEW donor splenocytes and injected intraperitoneally with 1 mL ALS. Twenty-one days after pretreatment, a LEW or Brown Norway (BN, RT1n) heart was transplanted into a treated DA recipient. Splenocytes (1 3 108 or 5 3 107) from a LEW heart–tolerant long-term survivor (LTS; >60 days) DA recipient were harvested and adoptively transferred (AT) into an irradiated (450 rad) naı¨ve DA rat 24 hours before transplanting a LEW heart. Results. All LEW heart allografts were rejected by untreated DA rats in a mean survival time (MST) of 7.4 ± 1.7 days (n = 7). In contrast, 66.7% of LEW heart grafts into IT + ALS–pretreated DA recipients were accepted indefinitely (n = 24). When either 1 3 108 (n = 5) or 5 3 107 (n = 5) splenocytes from a LEW heart graft–tolerant LTS (>60 days) DA recipient were AT into a new naı¨ve DA rat, all new LEW heart grafts were accepted indefinitely. Conclusions. The donor-specific tolerance that develops after IT + ALS–induced LEW heart acceptance by DA recipients can be transferred adoptively to new naı¨ve DA recipients, thus indicating that it is infectious tolerance. (Surgery 2005;138:254-60.) From the Department of Surgery, Washington University School of Medicine, St. Louis, Mo

THE THYMUS plays an important central role in the acquisition of tolerance to self-antigens during the development of the immune system. During thymic maturation, low-affinity thymocytes become restricted to self–major histocompatibility complex (MHC, positive selection), while T cells with high affinity for self-antigens are deleted (negative selection). This deletion avoids the risk that devel-

Presented at the 66th Annual Meeting of the Society of University Surgeons, Nashville, Tennessee, February 9-12, 2005. Reprint requests: M. Wayne Flye, MD, PhD, Department of Surgery, Washington University School of Medicine, 5103 Queeny Tower, Box 8109, St. Louis, MO 63110. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2005.06.012

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oping antiself T cells may induce autoimmune diseases.1 It also is recognized that other mechanisms operate in the development of peripheral tolerance including deletion, ignorance, clonal anergy, exhaustion, and deletion of antigenspecific T cells, and regulation by T-reg cells.2 There has been interest in the findings that the injection of alloantigens directly into the thymus of adult rodents induces allograft tolerance. Posselt et al3 showed that the IT inoculation of islets of Langerhans in conjunction with a single dose of ALS induced the acceptance of peripheral islet allografts from the same rat donor strain. We4-8 and others9-12 have shown that alloantigen IT injection could induce donor-specific tolerance to rat heart, liver, kidney, and smallbowel allografts. However, the mechanisms responsible for the induction and maintenance of this tolerance after IT injection of alloantigen remain unclear.

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There appears to be 2 major mechanisms of IT tolerance: a mechanism that occurs centrally within the thymic microenvironment and a postthymic peripheral deletion and/or regulation of the mature antigen reactive T cells.13 Several studies have reported that thymocyte deletion and apoptosis are important in tolerance induction.14,15 Conversely, nondeletional mechanisms, such as peripheral T-cell anergy and regulation, also may play any important role in modulating the long-term immune response and may account for the persistence of IT tolerance.15,16 Earlier studies showed that populations of T-reg cells that can regulate a state of immune unresponsiveness specific to the donor antigens can be generated in mice treated with anti--T-cell monoclonal antibody (mAb).17 The prominent feature of this phenomenon is that, once tolerance is established, it can be perpetuated in subsequent recipients by the AT of T-reg cells without any additional immunosuppressive treatment. This condition has been termed infectious tolerance.18 We have recently reported that a donor-specific transfusion (DST)–induced LEW heart or liver allograft tolerance can be AT to subsequent naı¨ve DA recipients by tolerant recipient DA splenocytes without any additional immunosuppression.19,20 This infectious nature suggests that T-reg cells play a pivotal role in the development and transfer of donor-specific transplantation tolerance. In this study, we also demonstrated that LEW donor--specific tolerance established with IT alloantigen injection + ALS pretreatment also can be uniformly AT to new naı¨ve DA recipients of a new LEW heart allograft. This finding indicates that IT alloantigen acts both centrally to induce tolerance and peripherally to generate and maintain infectious regulatory cells that, in turn, can transfer adoptively the specific state of tolerance to new recipients. MATERIAL AND METHODS Animals. Inbred male rats, weighing 200 to 250 g were purchased from Harlan Sprague-Dawley (Indianapolis, Ind). The animals were maintained under standard conditions and given rodent food and water ad libitum throughout all studies. LEW (RT1l) and DA (RT1a) rats were used as donor and recipient, respectively, and BN (RT1n) rats were used as third-party controls. The care and use of laboratory animals conformed to the National Institutes of Health and Washington University guidelines. Preparation of donor antigen. A single-cell suspension of LEW splenocytes was prepared in

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ice-cold RPMI 1640 (Sigma, St. Louis, Mo) supplemented with 10 mmol/L HEPES and 100 U penicillin and 100 lg/mL streptomycin (Invitrogen, Carlsbad, Calif). The erythrocytes were lysed by resuspension of the cell pellet with H2O, followed by the immediate addition of an equal volume of 23 phosphate-buffered saline (PBS) at pH 7.4. The remaining spleen cells were washed twice with RPMI 1640 and counted. Trypan blue exclusion was used to determine cell viability. Intrathymic alloantigen injection and systemic immunosuppression. Under isoflurane inhalation anesthesia, the thymus of a DA rat was exposed by a partial median sternotomy, and 1.25 3 107 LEW splenocytes in 75 lL of 13 PBS at pH 7.4 were injected into each lobe of the thymus with a 30gauge needle. Immediately after IT injection, 1 mL of rabbit antirat ALS (Accurate Chemical, Westbury, NY) was administered intraperitoneally. Control DA recipients received either no IT injection or no ALS treatment. Heart transplantation. A heterotopic heart transplantation was performed by using the modified technique of Ono and Lindsey.21 Under inhalation anesthesia of 1% isoflurane + 99% O2, the donor thoracic aorta and pulmonary artery were anastomosed to the infrarenal aorta and inferior vena cava, respectively, with 9-0 proline (a gift provided by Ethicon, Somerville, NJ). Graft survival was assessed by the cessation of a heartbeat and rejection was confirmed histologically. Histologic studies. For histologic assessment, rejected LEW heart grafts and LTS (>60 days) LEW heart grafts were fixed in formalin and then embedded in paraffin. Morphology of the histologic sections was evaluated by examination of the hematoxylin-eosin (H&E)--stained sections with light microscopy. Flow cytometric analysis. Splenocytes were incubated with Cy-Chrome (Cy-C)--conjugated antirat CD4 mAb (mouse IgG2a, clone OX35), fluorescein isothiocyanate (FITC)–conjugated antirat T-cell receptor (TCR) ab (mouse IgG1, clone R73), FITC-conjugated antirat CD25 (mouse IgG1, clone OX39), phycoerytherin (PE)-conjugated antirat CD8a mAb (mouse IgG1, clone OX8), or PEconjugated antirat CD3 mAb (mouse IgG3, clone G4.18) (all antibodies were purchased from BD Pharmingen, San Diego, Calif) for 30 minutes at 4°C. The cells were washed, resuspended in Hanks’ balanced saline solution + 1% fetal bovine serum + 0.1% (w/v) sodium azide and analyzed by FACScan (BD Biosciences, Mountain View, Calif) with the use of CellQuest (BD Biosciences) software.

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Fig 1. Sixteen of 24 (66.7%) LEW cardiac allografts transplanted 21 days after IT injection of 2.5 3 107 LEW splenocytes + ALS were accepted greater than 60days (d, n = 24), whereas untreated control DA recipients rejected LEW cardiac allografts in a MST of 7.4 ± 1.7 days (:, n = 7), and third-party BN heart grafts transplanted into IT + ALS–treated DA recipients were rejected in a MST of 10.7 ± 2.5 (n, n = 3). IT + ALS, Intrathymic + antirat lymphocyte serum.

Experimental design. Donor-specific LEW heart grafts were transplanted into DA recipients 21 days after IT injection of LEW splenocytes + ALS. We demonstrated that, by 21 days, recipient lymphocytes had recovered from the nonspecific immunosuppression of ALS. To confirm donor-strain specificity, third-party BN heart grafts were transplanted into LEW splenocyte IT– and ALS-treated DA recipients. As a control group, LEW hearts were transplanted into untreated naı¨ve DA recipients. To test for the presence of T-reg cells, splenocytes were harvested from an IT + ALS–treated LEW to DA heart recipient on day 30 or greater than 60 days post-transplantation. DA splenocytes (1 3 108 or 5 3 107 cells) suspended in 1 mL of 13 PBS at pH 7.4 were injected intravenously into a lightly c-irradiated (450 rad) naı¨ve DA recipient, which was then transplanted 24 hours later with a donor LEW heart. Irradiation was performed with a 137-cesium irradiator (Model GC-40, Type B; Atomic Energy of Canada Ltd, Ottawa, Canada). Statistical analysis. The survival curves of the heart grafts were analyzed by the Kaplan-Meier test. Statistical significance in graft survival between the experimental groups was determined by the log-rank test. A value of P less than .05 was considered significant.

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Fig 2. The survival of new LEW cardiac allografts was measured in lightly irradiated (450 rad) naı¨ve DA rats after the AT of different numbers of splenocytes harvested from IT + ALS–induced cardiac–tolerant LTS (>60 days) DA recipients. LEW cardiac allografts survived indefinitely when 1 3 108 (d, n = 5) or 5 x 107 (:, n = 5) splenocytes were AT from LTS (>60 days) DA recipients (P = .0015, compared with irradiation without cell transfer controls), whereas 2 of 3 LEW cardiac allografts were rejected in a MST of 13.0 ± 7.0 days when 1 3 108 splenocytes were AT from a day-30 LEW heart–tolerant DA recipient (n, n = 3). A MST of control, lightly irradiated (450 rad) naı¨ve DA rats without the transfer of splenocytes was 24.2 ± 12.9 days; only 1 graft survived more than 60 days (s, n = 13). AT, Adoptively transferred; HTx, heart transplant.

RESULTS Induction of allograft acceptance by IT + ALS. All DA rats receiving no pretreatment acutely rejected a LEW heart allograft in a MST of 7.4 ± 1.7 days (n = 7); however, 16 of 24 (66.7%) DA recipients pretreated with IT + ALS accepted LEW heart allografts for greater than 60 days (Fig 1). Allograft survival in the IT + ALS–pretreated group, compared with that of the untreated control group, was significant at P = .0001. A thirdparty BN heart transplanted into an LEW IT + ALS–pretreated DA recipient was rejected acutely in a MST of 10.7 ± 2.5 days (n = 3, Fig 1), indicating donor antigen specificity. Survival of LEW heart allografts after adoptive transfer. When a LEW heart was transplanted 24 hours after the AT of splenocytes harvested from a LEW heart--tolerant LTS (>60 days) DA rat into a lightly irradiated (450 rad) naı¨ve DA rat, all allografts were accepted (n = 5, P = .0015 vs controls without AT). In contrast, when 1 3 108 cells harvested from a DA recipient accepting a LEW heart graft for only 30 days were AT, only 1 of 3 allografts were accepted (Fig 2). In addition, in the control group given only 450 rad irradiation

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Fig 3. A representative histologic section of a rejected LEW heart removed from untreated DA recipients at the time of the cessation of a heartbeat. Note the presence of the dense mononuclear cellular infiltration. (H&E staining; original magnification: A, 3100; B, 3400.)

without AT of LTS (>60 days) DA splenocytes, 12 of 13 LEW heart grafts were rejected with a prolonged MST of 24.2 ± 12.9 days (vs 7.4 ± 1.7 days in the nonirradiated group) as a result of the irradiation. A decrease in the number of LTS (>60 days) DA splenocytes AT from 1 3 108 to 5 3 107 cells also resulted in all LEW hearts being accepted indefinitely (n = 5, Fig 2). Histologic analysis of LEW heart allografts. The rejected LEW heart allografts removed from untreated control DA recipients at the time of heartbeat cessation demonstrated a dense mononuclear cellular infiltration (Figs 3, A and B). In contrast, the accepted LTS (>60 days) LEW heart allografts removed from DA recipients that were previously treated by IT + ALS or AT from a heart-tolerant LTS (>60 days) DA recipient demonstrated healthy

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Fig 4. A representative histologic section of an accepted LTS (>60 days) LEW heart allograft, removed from DA recipients that were previously treated by IT + ALS or AT from a cardiac allograft tolerant LTS (>60 days) DA recipient. Of note, the section shows healthy cardiac myocytes with no mononuclear cellular infiltration or tissue damage (H&E staining; original magnification: A, 3100; B, 3400.)

cardiac myocytes without mononuclear cell infiltration or tissue damage (Fig 4, A and B). Phenotype of LTS (>60 days) DA splenic T cells AT into naı¨ve DA heart recipients. The LTS (>60 days) DA splenocytes injected into irradiated LEW heart recipients were approximately 30.9% CD3+ T cells that consisted of 11.0% CD4+CD25+, 10.9% CD4+CD25ÿ, and 9.0% CD8+ T cells (Fig 5, A and C). In contrast, splenic T cells (42.8%) from a representative naı¨ve DA rat comprising 7.9% CD4+CD25+, 24.8% CD4+CD25ÿ, and 10.1% CD8+ T cells (Fig 5, B and D). DISCUSSION IT alloantigen injection into the adult thymus is well-known to be a powerful method to induce the

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Fig 5. A representative flow cytometric analysis of splenic T cells from a naı¨ve DA rat and a cardiac allograft–tolerant LTS (>60 days) DA recipient. The percentage cells of the gated population is given in each quadrant. Cy-C, Cy-Chrome; LTS, long-term survivor; DA, Dark Agouti; PE, phycoerytherin; FITC, fluorescein isothiocyanate.

donor-specific tolerance.3-13 We have reported that IT injection of LEW splenocytes + ALS pretreatment resulted in 67% to 86% indefinite LEW cardiac allograft survival in the Buffalo (BUF, RT1b) strain recipient with this treatment.5,6 The administration of only ALS or the IT injection did not result in tolerance.5,6 We have now shown that LEW to DA IT splenocytes also promote donorspecific LEW heart allograft acceptance. Histologic analysis of accepted heart allografts from IT + ALS–treated DA recipients demonstrates healthy cardiac myocyte morphology without mononuclear cell infiltration or tissue damage, indicating a downregulation of the recipient DA anti-LEW donor allograft response. This tolerance can be uniformly adoptively transferred by splenocytes from a LTS (>60 days) LEW heart–tolerant DA recipient to a new naı¨ve DA recipient, implying the presence of IT + ALS–induced T-reg cells. This finding is consistent with a state of infectious tolerance. Several studies of TCR transgenic mice have shown that the deletion by apoptosis of thymocytes is involved in the induction of donor-specific tolerance after IT injection of alloantigen.14,15 Jones et al14 showed that, in BM3.6 mice (H-2k, expressing a transgenic CD8-dependent H-2Kb– specific TCR), a substantial deletion of both CD8+

Surgery August 2005 single–positive and CD4+CD8+ double–positive anti-Kb–specific TCR-expressing thymocytes (76% and 70%, reduction respectively) compared with naı¨ve controls occurred after the IT injection of C57BL/10 (H-2b) splenocytes and depletion of peripheral T cells by anti-CD4 mAb. They concluded that clonal deletion of donor reactive thymocytes had occurred. Chen et al15 also showed that the CD4+CD8+ double–positive antiOVA323-329–specific TCR-expressing thymocytes were significantly decreased after IT injection of chicken OVA into transgenic mice bearing a TCR specific for the chicken OVA peptide 323-339. However, the exact mechanisms responsible for the induction and maintenance of the tolerance after IT alloantigen injection remain unclear. Recently, increased attention has been focused on the possible role of T-reg cells in the maintenance of tolerance after its induction by IT inoculation of the antigen. The functional and phenotypic characterization of T cells indicates that CD4+CD25+ T-reg cells may develop as a result of pre-exposure to donor alloantigen within the ITinjected thymus and may be responsible for the development of peripheral donor allograft tolerance.22 Trani et al23 showed that the percentage of CD4+ CD25+ T-reg cells increased in the thymus and the periphery of transgenic mice with T cells specific for MHC class II–restricted peptide (S1) of the influenza PR8 hemagglutinin molecule 1 week after IT injection with S1 peptide, compared with untreated controls. This study suggested that S1 peptide–specific CD4+CD25+ T-reg cells migrated from the thymus to the periphery to regulate S1 peptide tolerance. Similar T-reg cells also may play a role in the generation of IT tolerance. While the initial IT + ALS pretreatment of DA rats incompletely induced the development of the donor-specific tolerance (66.7% heart allografts were accepted indefinitely, n = 24; Fig 1) the AT of greater than 60 days LEW-tolerant DA cells into a new recipient uniformly resulted in LEW heart tolerance. The initial IT alloantigen injection most likely results in direct alloantigen contact with thymocytes, which then leads to the formation of alloantigen-specific T-reg cells, similar to the development of naturally occurring self-antigen– specific T-reg cells within the thymus. Incomplete IT tolerance may result when inadequate alloantigen distribution does not delete completely all thymocytes that express a low-affinity donorreactive TCR. Our group recently has shown that DST-induced T-reg cells play a crucial role in the maintenance and perpetuation (over multiple generations) of

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the donor-specific tolerance of a LEW to DA heart or liver transplant. Kataoka et al19,20 demonstrated that the AT of splenocytes from the DST-treated LEW cardiac allograft–tolerant LTS (>60 days) DA recipients into naı¨ve lightly irradiated (450 rad) DA rats induced indefinite acceptance to new LEW cardiac allografts. Asakura et al24 also reported that the donor-specific tolerance that develops after the spontaneous acceptance of a liver transplant from a LEW to a DA rat can be similarly AT to new naı¨ve DA recipients. In this study, we have further demonstrated that IT injection of LEW alloantigen + ALS pretreatment also can promote the development of regulatory cells that can transfer and maintain infectious tolerance in new DA recipients. The establishment of this ability to reliably and uniformly transfer tolerance requires acceptance of the LEW heart by DA recipients for more than 60 days. When recipient splenocytes were AT from recipients only 30 days after LEW heart transplantation, 2 of 3 LEW cardiac allografts were rejected at 8 and 18 days. Odorico et al10 have shown that the AT of 2.0 to 2.5 3 108 splenocytes from LEW cardiac allograft– tolerant LTS (>100 days) Wistar Furth (WF, RT1u) rats, given a single IT injection of 2.0 to 2.5 3 107 donor LEW bone marrow cells with the intraperitoneal injection of ALS, led to the permanent survival (>100 days) of all LEW cardiac allografts in new 450-rad irradiated WF recipients. Reemtsen et al25 confirmed that IT injection of 2.5 3 107 donor LEW splenocytes + ALS-induced LEW to BUF cardiac allograft tolerance can be transferred by 2.5 3 107 LTS (>60 days) BUF splenocytes in a LEW donor–specific and infectious manner to another 450-rad irradiated BUF rat. Niimi et al16 also showed that the AT of 2.0 3 107 splenocytes from CBA (H-2k) mice with LTS primary C57BL/ 10 (H-2b) cardiac allografts induced by IT donor resting B cells, but not IT donor splenocytes, with anti-CD4 mAb indefinitely prolonged (>100 days) allograft survival in a naı¨ve nonirradiated CBA recipient. These studies differ in the animal strains used and the inducing regimen; however, the mechanisms of this mode of tolerance remain poorly defined. Phenotypic flow cytometric analysis of the AT LTS DA splenocytes demonstrates an increase in the numbers of CD4+CD25+ T cells, compared with the numbers found in a naı¨ve DA spleen. These findings suggest that LTS DA CD4+CD25+ T-reg cells mediate cardiac allograft tolerance. However, we have demonstrated previously that CD4+ and CD8+ T cells from DST-induced LEW allograft– tolerant rats can transfer tolerance to new naı¨ve

DA recipients.19 It is yet unknown whether CD4+25+, CD4+CD25ÿ and CD8+ T-reg cells will function similarly in IT + ALS–induced cardiac allograft acceptance. Further studies of the ITinduced DA T-reg cells will correlate T-cell surface phenotype with the cells’ regulatory function to mediate cardiac allograft acceptance.

CONCLUSION We have confirmed that the donor-specific tolerance induced by pretreatment with IT + ALS can be transferred in an infectious manner in the LEW to DA rat cardiac allograft transplantation. Our result suggests that T-reg cells play a pivotal role in the maintenance of this donor-specific IT tolerance.

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