Thymic Rejuvenation and the Induction of Tolerance by Adult Thymic Grafts

Thymic rejuvenation and the induction of tolerance by adult thymic grafts

Shuji Nobori, Akira Shimizu, Masayoshi Okumi, Emma Samelson-Jones, Adam Griesemer, Atsushi Hirakata, David H. Sachs, and Kazuhiko Yamada*

Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129

Edited by Ralph M. Steinman, The Rockefeller University, New York, NY, and approved October 25, 2006 (received for review June 22, 2006) The thymus, the site of origin of T cell immunity, shapes the repertoire procedure and then facilitated the induction of tolerance, suggest- of T cell reactivity through positive selection of developing T cells and ing that thymic function is largely controlled by the host environ- prevents autoimmunity through negative selection of autoreactive T ment rather than by the local thymic milieu. cells. Previous studies have demonstrated an important role for the thymus not only in central deletional tolerance, but also in the Results induction of peripheral tolerance by vascularized renal allografts in Thymic Involution with Age In Miniature Swine. We first examined juvenile miniature swine recipients. The same protocol did not induce the natural course of thymic involution with aging in naive minia- tolerance in thymectomized recipients nor in recipients beyond the ture swine (9). Right cervical thymic lobes were biopsied from 53 age of thymic involution. We subsequently reported that vascularized naive Massachusetts General Hospital (MGH) miniature swine of thymic lobe grafts from juvenile donors were capable of inducing different ages. The cortex and medulla in whole biopsy specimens tolerance in thymectomized juvenile hosts. However, the important containing at least 10 lobules were subjected to morphometric question remained whether aged, involuted thymus could also in- analysis by using NIH Image (Scion Image) software (10). duce tolerance if transplanted into thymectomized hosts, which, if Fig. 1A shows a plot of the ratio of cortical to medullary areas true, would imply that thymic involution is not an intrinsic property (c/m ratio) as a function of age in miniature swine. Fig. 1 B and C of thymic tissue but is rather determined by host factors extrinsic to shows representative histologic findings of naive thymic tissue at 4 the thymus. We report here that aged, involuted thymus transplanted months and 20 months of age, respectively. As shown in this figure, as a vascularized graft into juvenile recipients leads to rejuvenation of thymi from 4-month-old miniature swine had well defined, thick both thymic structure and function, suggesting that factors extrinsic cortical thymic areas, whereas thymi from 20-month-old animals to the thymus are capable of restoring juvenile thymic function to were consistently involuted. At 4 months of age, at which point aged recipients. We show furthermore that rejuvenated aged thymus peripheral tolerance can readily be induced (11), pigs had a c/m has the ability to induce transplant tolerance across class I MHC ratio between 3 and 5, whereas pigs older than 20 months, when barriers. These findings indicate that it may be possible to manipulate such peripheral tolerance could no longer be induced (12), had c/m thymic function in adults to induce transplantation tolerance after the ratios of Ϸ0.8 (Fig. 1A). The correlation coefficient between c/m age of thymic involution. ratio and age (Ϫ0.82) was highly significant (P Ͻ 0.0004). No difference was seen between the thymi of female and male animals. aging ͉ miniature swine ͉ thymus Rejuvenation of Aged Thymus Grafts in Juvenile Recipients. We next he thymus plays a central role in the development of immuno- assessed the ability of involuted thymus grafts to rejuvenate and to Tlogic tolerance. Thymic epithelial cells and dendritic cells in the support thymopoiesis after transplantation into juvenile animals. thymus are responsible for the induction of central tolerance to self VTL grafts were harvested from aged pigs (20–21 months of age) among developing T cells through negative selection. A similar and transplanted into MHC-matched, 4-month-old juvenile ani- process has been shown to lead to the development of central mals (recipients 1–3) that had been thymectomized 3 weeks earlier. transplantation tolerance to alloantigens after allogeneic hemato- A 28-day course of FK506 (tacrolimus) was administered to assure poietic stem cell (HSC) transplantation, because of negative selec- that the grafts would not be rejected because of minor antigen tion by HSC-derived donor dendritic cells that migrate to the host differences. FK506 has been found previously not to have measur- thymus (1–4). Finally, the thymus has been shown to be required able effects on thymus structure or involution in miniature swine for the rapid induction of the stable peripheral tolerance that occurs (13). The morphometric analysis of aged thymus grafts transplanted under the influence of a short course of calcineurin inhibitors after into juvenile recipients revealed marked rejuvenation of these allogeneic renal transplantation in miniature swine (5). grafts within two months of transplantation (Fig. 2). Aged thymic Ϯ Ϯ Thymic structure and function decline with age, however, leading grafts with an initial c/m ratio of 1 0.3 showed c/m ratios of 3.1 MEDICAL SCIENCES by adolescence to a significant reduction in thymic mass and an 0.8 by 60 days after transplant and 4.1 Ϯ 0.5 by 100 days after impaired ability to regenerate normal T cell numbers after T cell transplant. After day 180, the thymic architecture began to rein- depletion (6, 7). It is not clear whether this thymic involution is an intrinsic property of thymic tissue or is due to the effects of factors extrinsic to the thymus. Likewise, it remains unclear which Author contributions: K.Y. designed research; S.N., A.S., and K.Y. performed research; S.N., M.O., E.S.-J., A.G., A.H., D.H.S., and K.Y. analyzed data; and K.Y. wrote the paper. tolerance-inducing properties of the thymus are affected by its involution. The authors declare no conflict of interest. We have recently established a procedure for vascularized thymic This article is a PNAS direct submission. transplantation in MHC inbred miniature swine that permits a Abbreviations: CML, cell-mediated lympholysis; CTL, cytotoxic T lymphocyte; CyA, cyclo- sporine A; DP, double positive; FK506, tacrolimus; MLR, mixed lymphocyte response; PAA, transplanted thymus to function immediately after revasculariza- porcine allele-specific antibody; SLA, swine leukocyte antigen; PBMC, peripheral blood tion (8). We have now used this technique to study the basis of the mononuclear cells; Treg; regulatory T cell; VTL, vascularized thymic lobe; PCNA, prolifer- immunologic consequences of aging on the thymus. For this ating cell nuclear antigen; c/m, cortical/medullary. purpose, we transplanted vascularized thymic lobe (VTL) grafts *To whom correspondence should be addressed. E-mail: [email protected]. from aged miniature swine to young animals of the same swine This article contains supporting information online at www.pnas.org/cgi/content/full/ leukocyte antigen (SLA) inbred line (9). As we report here, these 0605159103/DC1. aged, involuted VTL grafts underwent rejuvenation after this © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605159103 PNAS ͉ December 12, 2006 ͉ vol. 103 ͉ no. 50 ͉ 19081–19086 Downloaded by guest on September 26, 2021 Fig. 3. Representative histology of VTL grafts. H&E staining (ϫ40) of biopsy samples from an aged VTL graft (from a 20-month-old donor) in a juvenile Fig. 1. Morphometric analysis and histology of naive thymus at various 4-month-old recipient pig on day 0 (c/m ratio 0.79) (A), day 60 (c/m ratio 2.68) stages. (A) Morphometric analysis showing c/m areas of porcine thymus at (B), and day 100 (c/m ratio 3.62) (C). various times during the aging process. Histologic ﬁndings of porcine thymus at 4 months (H&E, ϫ100) (B) and 20 months (H&E, ϫ100) (C) of age. Cortical regions contain small, dense thymocytes showing strong hematophilic stain- PCNA on cytokeratin-positive cells in both the cortical and med- ing (c) compared with medullary regions (m). ullary regions increased to a maximum on day 100 (SI Fig. 6 D and F), indicating that active proliferation of thymic epithelial cells occurred. After day 100, the expression of PCNA decreased in a volute, with the c/m ratios on day 240 resembling those of recipient- pattern similar to the expression of PCNA in a naive, age-matched age-matched naive animals at these time points (Fig. 2). Fig. 3 shows thymus. the histology of biopsy specimens from one representative thymec- Cells dispersed from thymic biopsy tissues were also analyzed tomized juvenile animal (recipient 1) that received an aged thymic by flow cytometry by using allele-specific antibodies. Whereas all graft (c/m ratio of 0.79). By 60 days after transplant, the cortical of the thymocytes on day 0 were donor-type cells [negative for thymus had markedly expanded with larger lobular structures and pig allele-specific antigen (PAA)], most of the thymocytes in the well defined cortical and medullary regions (Fig. 3B). The animal day 60 VTL graft were recipient-type cells (PAA-positive) (SI maintained a thymic architecture that resembled that of a juvenile Fig. 7a). All thymocytes in the VTL graft were recipient type by animal until day 180 [Fig. 3C and supporting information (SI) Fig. day 100. The day 100 analysis revealed a phenotypic pattern 6A]. By day 240 (SI Fig. 6B), the cortical thymus of the grafts similar to that of a naive 4-month-old thymus (SI Fig. 7a). seemed to be thinner. This reinvolution was likely due to the Furthermore, the FACS profile of the thymocytes from aged recipient’s age, not to immunologic causes, because the thymi of thymic grafts on day 60 and thereafter revealed that a majority naive pigs also involute at this age (Fig. 1). of the cells were CD4/CD8 double-positive, CD1 highly positive We stained the thymus biopsy tissues for proliferating cell nuclear cells that expressed CD3 poorly (SI Fig. 7 b–d). This profile antigen (PCNA, a marker of proliferation) and cytokeratin (a indicated that the cells in the VTL on day 60 and thereafter were marker of thymic epithelial cells) (SI Fig. 6 C–F), to assess the host-type thymocytes derived by thymopoiesis, and not graft proliferation of epithelial cells in the VTL graft. The expression of infiltrating cells.

Fig. 2. Morphometric analysis showing comparison of c/m ratios of aged porcine thymus grafts after transplantation to juvenile recipients versus recipient age-matched naive thymuses. Shown are results for recipient-age-matched naive thymus (average) (ࡗ) and for recipients 1 (‚),2(छ), and 3 (ᮀ).

19082 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605159103 Nobori et al. Downloaded by guest on September 26, 2021 Fig. 4. Histology of aged VTL grafts transplanted across a class I-mismatched barrier with a 28-day course of FK506. (A) Results for day 60 (H&E, ϫ100) (Aa), day 60 (cytokeratin, ϫ100) (Ab), day 100 (H&E, ϫ100) (Ac), day 180 (H&E, ϫ100) (Ad), and day 260 (H&E, ϫ100) (Ae). (B) Immunohistochemistry of VTL grafts stained with anti-recipient MHC class I at day 100 (ϫ400) (Ba) and day 180 (ϫ400) (Bb) and with anti-donor MHC class I antibody at day 180 (ϫ400) (Bc). Arrows in Bc indicate donor-type cells with dendritic cell morphology at the corticomedullary junction. (Bd and Be) Double-staining with donor-type class I phycoerythrin-conjugated antibody and class II pig-speciﬁc FITC-conjugated antibody (ϫ800) at day 100 (Bd) and day 180 (Be). Double-stained cells (donor class I-positive/pig class II-positive) appear yellow. Morphologically dendritic-like cells double-positive for donor class I and pig class II were detected at days 100 and 180 (white arrows in Bd and Be). Class II-positive recipient-type cells with dendritic cell-like morphology (green cells in Bd and Be) increased between days 100 and 180.

Aged VTLs Induce Transplant Tolerance Across MHC Disparate Barriers (recipient 4) that did not receive a renal transplant, underwent but Require a Longer Period of Immunosuppression than Juvenile serial thymic biopsies on days 150, 180, and 260. The VTL graft VTLs. We subsequently assessed the ability of older thymus grafts to biopsies at day 150 and 180 showed markedly expanded thymic induce tolerance across MHC disparate barriers with the same lobules with a thick cortical thymic area and well defined medullar 28-day course of FK506 that was used across MHC-matched structure (Fig. 4Ad). Recipient-type MHC class I staining showed barriers. Based on our previous experience with mouse and porcine that the majority of thymocytes in the medullary region of the VTL thymic transplantations (13, 14), recipients (recipients 4–6) were grafts were of recipient-type (dull brown cells) (Fig. 4Bb). SI Fig. 8 completely thymectomized before the transplantation of two hap- shows the FACS profile of the VTL graft biopsy on day 180. lotype, class I-mismatched VTL grafts. Frequent VTL biopsies Consistent with the immunohistochemistry, the majority of cells in were performed on recipient 4 to examine the development of the VTL grafts were recipient-type cells as assessed by porcine thymopoiesis in the VTL graft. The other two animals received allele-specific antibody (PAA) (SI Fig. 8b). FSC/SCC, CD4/CD8, donor-matched kidney grafts on day 100 and third-party kidney and CD1/CD3 profiles confirmed that these cells were thymocytes, grafts Ͼ250 days after VTL transplantation to test in vivo tolerance. indicating that rejuvenation with active thymopoiesis occurred in Because of previous evidence (15) that thymic biopsies during the the aged VTL graft (SI Fig. 8 a and c–e). However, in a manner induction period may interfere with the induction of transplant similar to that seen in MHC-matched grafts, the thymus began tolerance, we did not perform VTL biopsies on these recipients reinvoluting without evidence of vasculitis after day 260 (Fig. 4Ae). after kidney transplantation until the end of the experimental VTL grafts in the two animals that received kidney transplants were

period. examined when either the third-party kidney was removed or the MEDICAL SCIENCES Thymopoiesis. Thymopoiesis was markedly delayed in MHC- experiment was terminated (see next section). The VTL graft on mismatched VTL grafts as compared with either MHC- day 259 in recipient 5 maintained thymic structure with slight matched grafts (see Figs. 2 and 3 and SI Fig. 7) or juvenile involution, comparable with the animal that did not receive a MHC-mismatched VTLs (13). Aged MHC-mismatched VTL kidney transplant (recipient 4). Biopsies of VTL grafts on day 310 grafts were still hypocellular on day 60, but thymic stromal cells (recipient 6) and 315 (recipient 5) revealed more obvious involution were present without vasculitis (Fig. 4 Aa and Ab). However, with fatty degeneration. by day 100, the thymus seemed to have essentially normal, In vivo donor-specific tolerance. Two animals, including the animal juvenile architecture. The cortex was well developed with that had anti-donor CTL responses at day 60 (see below), received dense mononuclear cell migration. The medullary thymus was donor-matched kidneys without immunosuppression on days 95 not as well defined as the cortex, probably because of the stage (recipient 5) and 105 (recipient 6), respectively. Both animals of thymic development at this time point (Fig. 4Ac). Immu- maintained their grafts for longer than 5 months without any signs nohistochemistry revealed a small number of recipient-type of rejection (Fig. 5A, solid lines). There was no endothelialitis, class I-positive cells in the medullary area (dull brown stained hemorrhage, or mononuclear cell infiltrate in the kidney grafts at cells), indicating that thymopoiesis occurred between day 60 the time of biopsies (days 30, 60, and 153/163 after kidney trans- and 100 (Fig. 4Ba). plantation) (Fig. 5B). These two animals received third-party To assess thymic function at later time points, one animal kidneys 163 (recipient 5) and 153 (recipient 6) days after donor-

Nobori et al. PNAS ͉ December 12, 2006 ͉ vol. 103 ͉ no. 50 ͉ 19083 Downloaded by guest on September 26, 2021 double-stained morphologically dendritic-like cells (Fig. 4Bd, yellow cells indicated by white arrows) were seen at the corticomedullary junction. In addition, recipient-type morphologically dendritic cells (Fig. 4Bd, green cells that were donor class I-negative but pig class II-positive) migrated to this area by this time point. Donor-type class I-positive, morphologically dendritic-like cells remained at the corticomedullary junction at day 180 (Fig. 4Bc, dense brown cells with arrows). These cells were confirmed to be class II-positive (Fig. 4Be, yellow cells indicated by white arrows). Class II-positive recipient-type cells with dendritic cell-like morphology (green cells in Fig. 4 Bd and Be) increased between day 100 and day 180. The coexistence of these donor and recipient cells suggests the potential for host and donor negative selection of T cells in the VTL graft (16). Inferior ability of aged VTL relative to juvenile VTL to induce tolerance. We have previously shown that VTL grafts from juvenile donors induce tolerance with a 12-day course of FK506 across MHC- mismatched barriers in miniature swine. In contrast to a 28-day FK506 regimen, biopsies on day 60 of the VTLs of three animals (recipients 7–9) receiving only 12 days of FK506 revealed mononuclear cells with vasculitis (SI Fig. 10a). FACS data indicated that a majority of these cells were CD3-positive with very few CD1-positive cells, a phenotypic pattern similar to that Fig. 5. Plasma creatinine levels after donor-matched kidney transplant in of peripheral blood and indicating the presence of graft infil- recipients of aged VTLs with 28 days of FK506 across a class I-mismatched barrier trating cells (SI Fig. 10 b and c). Two of the animals had very few (A), donor-matched kidney biopsy at day 153 after kidney transplant (H&E, ϫ100) cytokeratin-positive cells in the VTL (SI Fig. 10d); the VTL in (B), and third-party kidney biopsy rejected on day 7 (H&E, ϫ100) (C). the remaining animal (recipient 9) had slightly more cytokeratin- positive epithelial cells (SI Fig. 10e). All recipients maintained anti-donor CTL responses after VTL transplants (SI Fig. 10f). matched kidney transplantation (days 259 and 258 after VTL), To evaluate the in vivo immunologic status of these recipients, respectively, to evaluate immunocompetence. The ureter of the we transplanted donor-matched kidney grafts without immuno- tolerated kidney was tied to permit evaluation of function of the suppression to all three animals on day 100. Two animals second kidney. Third-party grafts were rejected within 10 days (Fig. rejected their renal grafts acutely on days 7 and 15, respectively 5A, dashed line) with histologic evidence of both cellular and (SI Fig. 10g). The third animal, which had a slightly higher humoral rejection (Fig. 5C), whereas no rejection was noted in the number of cytokeratin-positive epithelial cells on day 60, main- tolerated kidneys (Fig. 5B), indicating that aged VTL and 28 days tained its graft with unstable renal function (Crea 3–6 mg/dl) for of FK506 had donor-specific tolerance. Both animals were kept 102 days, at which point it was euthanized because of weight loss alive by untying the ureter of the tolerated kidney when the and renal dysfunction (SI Fig. 10g). This animal showed signs of third-party graft was nephrectomized for in vitro study. The creat- acute rejection in the day 30 renal graft biopsy specimen, with inine levels immediately returned to normal, where they remained diffuse mononuclear cell infiltrates, vasculitis and glomerulitis. until euthanization on days 315 and 310 after VTL transplantation. A renal biopsy on day 60 revealed chronic glomerulopathy, In vitro assays to determine mechanisms of tolerance. All animals had moderate mononuclear cell infiltrate, and mild fibrosis (SI Fig. anti-donor CTL responses Ͼ40% at a 100:1 effector to target 10h). At the time of euthanization, the renal graft showed ratio before VTL transplantation. Two (recipients 4 and 5) of the moderate interstitial fibrosis, with increased chronic glomeru- three animals developed donor-specific unresponsiveness by day lopathy. The VTL graft at this time point was totally fibrotic. 60 (SI Fig. 9 a and b). The other animal (recipient 6) maintained Thus, VTL grafts transplanted from an aged donor to a juvenile anti-donor responses at day 60 (SI Fig. 9c), thereafter becoming recipient with a 12-day course of FK506 prolonged the survival donor-unresponsive by day 120 (SI Fig. 9d). We further assessed of donor-matched kidney grafts but failed to induce stable the mechanisms of tolerance in this model by using coculture tolerance. CTL assays to examine the regulatory mechanisms. Coculture assays were set up on day 60 in recipient 5 and on days Ͼ250 in Discussion all three recipients. Donor-specific suppression of the naive T The thymus plays a central role in the development of T cells and cell response was seen in all of these assays, indicating the the induction of immunologic tolerance. However, thymic struc- presence of regulatory cells in the peripheral blood (SI Fig. 9e ture and function decline with age, leading by adolescence to a from recipient 5 at day 315). In addition, we assessed whether significant reduction in thymic mass and an impaired ability to anti-donor CTLs were restored by removal of CD25-positive regenerate normal T cell numbers after T cell depletion (6, 7). cells from PBLs from a long-term acceptor on day 315 (recipient Whereas the age related changes of the thymus are well estab- 5). The anti-donor CTL response was restored only minimally in lished, it is not clear whether the involution and decline in the CD25-depleted CML culture (blue solid line compared with function is more dependent on the host environment (extrinsic blue dashed line in SI Fig. 9f). Because the CD25 depletion was factors) or on the local thymic milieu (intrinsic factors). Using confirmed by FACS (see Materials and Methods), these data our model of vascularized thymic lobe transplantation, we suggest that deletional tolerance mechanisms are also likely to be transplanted aged thymus into juvenile animals to study the operative. effect of the juvenile milieu on aged thymus (13). The data To further characterize deletional mechanisms, we performed presented demonstrate the rejuvenation of aged VTL grafts double-staining with donor-type class I phycoerythrin (PE)- within juvenile recipients, suggesting that thymic structure and conjugated antibody and class II pig-specific FITC-conjugated function are more dependent on extrinsic factors than on antibody. At day 100, donor-type class I-positive/class II-positive intrinsic ones.

19084 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605159103 Nobori et al. Downloaded by guest on September 26, 2021 Age-related changes that may affect T cell development occur during the induction period may have damaged the thymic simultaneously within the bone marrow, the extrathymic milieu, epithelial and stromal cells either in number or function, inhib- and the thymus (17, 18). Mackall et al. (19) have reported that iting thymopoiesis at early time points despite the prolonged the administration of young T cell-depleted bone marrow to course of immunosuppression. lethally irradiated, aged mice failed to reverse thymic involution The present study, involving the transplantation of old thymi into or histologic abnormalities (including a loss of cortical epithe- young recipients, may provide a model for isolating and examining lium and a disruption of the normal cortical architecture) individual external factors that control the regeneration of thymic associated with the aged recipient thymus. Although young capacity. Further decreases in the number of thymic stromal cells marrow restored the ability to negatively select autoreactive T by more formidable immunologic barriers (i.e., full MHC mismatch cell clones, it did not rejuvenate the aged thymi. These studies are or xenotransplantation) or aged VTL into older recipients, could consistent with our conclusion that host factors extrinsic to the potentially result in a negative cytokine milieu for the aged VTLs thymus control its involution and suggest that these factors are and limit the ability of the VTLs to induce T cell tolerance to donor not provided by the bone marrow. antigens. We are therefore currently investigating ways to acceler- The effects of the young vs. old milieu on liver and muscle stem ate the regeneration process of aged thymus to facilitate more rapid cells and on progenitor cells has recently been studied by using engraftment and function in these grafts. Preliminary data using parabiotic pairings (20). The normal decline in tissue regener- Lupron, an LH-RH agonist, suggest that the regeneration of the ative potential with age was shown to be reversed in the older aged thymus may be possible (K.Y., unpublished data). Medical paired mice through exposure to serum factors from young mice. thymic regeneration, combined with VTL transplantation, could The authors did not, however, examine the involution of the move our tolerance induction regimen one step closer to the clinic thymi in these studies, possibly because of the potentially con- by regenerating aged thymus either before or after VTL transplan- founding effects of having old and young thymi present in the tation into an aged recipient. This strategy could have practical same system. Our thymus transplantation model between MHC- implications for extending thymus-dependent tolerance-inducing matched recipients provides a model that eliminates this variable protocols (5, 12, 13, 30–32) to adult patient populations. while exposing aged thymus to a young milieu. There have been several studies attempting to characterize the Materials and Methods factors in the microenvironment that cause thymic involution. Animals. Animals were cared for according to the guidelines of Surgical castration and chemical castration using luteinizing Massachusetts General Hospital Institutional Animal Care and hormone-releasing hormone (LH-RH) have been shown to have Use Committee. MGH miniature swine represent one of the few a profound rejuvenating effect on the thymus in mammalian large animal species in which breeding characteristics make models (17, 21–23). Additionally, IL-7, produced by MHC class genetic experiments possible (9). Fifty-three naive animals eu- II-positive thymic epithelial cells, has been linked to the survival thanized as organ donors for other experiments were used for a and proliferation of thymocytes (24). IL-7 levels decrease with morphometric study to assess natural thymic involution. Six age (25), but whereas thymopoiesis increased in young mice that juveniles (Ͻ6 months of age) and six adults (20–21 months of received IL-7, treatment of aged mice did not augment thymo- age) served as donors of VTL transplants in either MHC- poiesis (26, 27). Although the present study demonstrates that matched or class I MHC-mismatched combinations. extrinsic factors can facilitate thymic regeneration, thus far, no factor has definitively been shown to be directly involved in the Surgical Procedures. Complete thymectomy in the recipient. As described involution or regeneration of the thymus. (5), the pretracheal muscles were retracted, exposing the cervical Data in the present study have also demonstrated that reju- thymus and trachea from the cervicothoracic junction to the venated aged VTLs could induce tolerance but require a longer mandibular area. The cervical thymus was excised, after which course of immunosuppression to establish tolerance than do the mediastinal thymus was removed through a sternotomy. recipients of juvenile VTLs. We have previously shown that a VTL transplantation. The procedure for VTL graft transplantation 12-day course of FK506 facilitated the induction of tolerance by has been reported (13). Briefly, the right cervical thymic lobe juvenile VTL grafts across even a full MHC mismatch (13). Aged was carefully harvested, perfused with cold Eurocollins solution, VTL grafts required a 28-day course of FK506 to induce and transplanted into the retroperitoneum of recipients, by using tolerance in the class I MHC disparate model studied here. the abdominal aorta and the inferior vena cava. This technique Plausible explanations for the requirement of prolonged immu- led to perfusion of the VTL grafts with an immediate blood nosuppression include: (i) in aged VTL grafts, there are fewer supply. Six animals received a 28-day course of FK506, and three natural regulatory cells to inhibit alloreactive T cells in a animals received a 12-day course of FK506, starting at 0.15 mg/kg nonspecific manner during the induction period, and (ii) the per day and adjusted to maintain blood levels between 30 and 40 aged thymic grafts are slower in generating donor-specific ng/ml (31).

regulatory cells and in deleting/anergizing new alloreactive Thymus biopsy. VTL biopsies were carried out at 0, 60, 100, 180, MEDICAL SCIENCES thymocytes. Because T cells were not depleted in the present and Ͼ240 days after VTL transplantation. Renal transplant study, preexisting alloreactive precursor T cells circulating in the recipients had thymus biopsies when the experiment was termi- blood could enter the VTL graft (28, 29) during the induction nated. Approximately 1 ϫ 0.5 ϫ 0.5 cm of thymic tissue was period and damage the thymic epithelial and stromal cells. This biopsied for histology (formalin and frozen samples) and flow damage could further inhibit the graft’s ability to generate cytometric analysis. The sections of naive thymus used for donor-specific antigen-dependent regulatory cells and to delete morphometric analysis were taken from naive pigs that served as or anergize alloreactive cells in the graft. Aged VTL grafts that organ donors for other studies. Tissue samples were fixed in 1% were protected by a 28-day course of FK506 in the induction formaldehyde solution (Denison Pharmaceuticals, Pawtucket, period had no evidence of vasculitis, indicating that this immu- RI), embedded in paraffin, and subsequently sectioned. Tissues nosuppression was sufficient to protect the aged, involuted VTL were stained by using hematoxylin/eosin (H&E). graft from alloreactive circulating T cells. Thymopoiesis was Kidney transplantation. The kidney transplantation procedure has further delayed by transplanting aged VTLs across a class I been reported (5). Both native kidneys were removed on the day mismatch barrier (thymopoiesis occurred after day 60) as com- of kidney transplantation. pared with across a minor MHC mismatch (near complete Catheter placement. Two semipermanent, indwelling, Hickman conversion to recipient-type thymopoiesis by day 60). The in- silastic central venous catheters were placed in the external creased immunological response caused by the class I disparity jugular veins to facilitate drug administration and frequent blood

Nobori et al. PNAS ͉ December 12, 2006 ͉ vol. 103 ͉ no. 50 ͉ 19085 Downloaded by guest on September 26, 2021 sampling for in vitro assays and laboratory tests including com- Morphometric analysis. Morphometric studies were performed on plete blood count and blood chemistry, and for monitoring of thymic biopsy samples by using NIH Image (Scion Image, whole-blood FK506 levels. Frederick, MD) software (10). Biopsy samples with at least 10 lobules were stained with H&E, and the areas of the cortex and Evaluation of Thymic Rejuvenation/Involution. Preparation of thymo- medulla were measured to obtain a c/m ratio in the cross-section cytes. Biopsied tissue from thymic grafts (100–200 mg) was finely of the thymus. Increases in this ratio were taken as a measure of minced in Hanks’ balanced salt solution (HBSS) buffer; the cell rejuvenation of the thymus. suspension was then filtered twice through a 200-␮m nylon mesh. Statistical analysis. Student’s t test was used to compare c/m ratios. Flow cytometry. FACS analysis of PBMCs was performed by using a Becton Dickinson FACScan (San Jose, CA) with CellQuest In Vitro Cellular Assays. Cell-mediated lymphocytotoxicity (CML) assays. FACStation software (Becton Dickinson) as reported (13). CML assays were set up before VTL transplantation, and days Phenotypic analysis of cells was attained by three-color staining 30, 60, 90, 150, 180, and Ͼ250. Methods have been described (5). with directly conjugated murine anti-swine mAbs. CD25 depletion CML assays. To test whether anti-donor CTL activity Monoclonal antibodies (mAbs) used for phenotypic characterization of cell was restored by removal of CD25-positive cells from responders, populations in VTL grafts. Thymocyte development was assessed by a CD25 depletion CML was set up with PBL from a long-term immunohistochemistry and FACS analyses by using the murine acceptor (recipient 6). CD25-positive cells were depleted by anti-swine mAbs 74-12-4 (IgG2b, anti-swine CD4), 76-2-11 (IgG2a, magnetic cell separation (MACS; Miltenyi Biotec, Auburn, CA). anti-swine CD8), 76-7-4 (IgG2a, anti-swine CD1), BB23-8E6 CD25-negative cells were used as responders and cultured with (IgG2b, anti-swine CD3), 231.3B2 (IgG1, anti-swine CD25), 10-9 stimulators in the same manner as in a primary CML. Comple- (anti-PAA), 2.12.3a (IgM, class Id), 16.7.E4.2 (IgM, class Ic), and tion of CD25 depletion was confirmed by FACS analysis. TH16 (IgG2a, anti-swine class II DQ) as reported (8). Coculture assays. Peripheral regulatory mechanisms were investi- Histological analysis. Formalin-embedded tissue was stained by using gated by in vitro coculture assays (33). The primary culture was H&E. Coded samples were examined through a light microscope by set up as in CML assays. The primed responder cells were then a pathologist. Frozen samples were prepared for immunohisto- harvested and rested overnight at 4°C, after which they were chemical analysis with the avidin–biotin horseradish-peroxidase coincubated with naive SLA-matched PBMCs and irradiated complex technique (30). Thymopoiesis was assessed by immuno- donor-type or third-party PBMCs for 5 additional days. histochemistry by using the swine-specific murine mAbs described above. Double immunostaining with PCNA and cytokeratin (Z622; We thank Norwood Immunology (Victoria, Australia) for partial support of these studies, Fujisawa Healthcare, Inc. (Deerfield, IL) for Dako, Carpinteria, CA) for the identification of the proliferating generously providing FK506, Dr. Isabel McMorrow for helpful review of thymic epithelial cells was performed in formalin-fixed paraffin the manuscript, and Maria Doherty for editorial assistance. This work sections by using a combination of immunoalkaline phosphatase was supported by National Institutes of Health Program Project Grants and standard avidin–biotin complex (ABC) technique. 5P01-A145897 and 5P01-A139755 and by Fujisawa Health Care, Inc.

1. Sharabi Y, Sachs DH (1989) J Exp Med 169:493–502. 17. Kappler JW, Roehm N, Marrack P (1987) Cell 49:273–280. 2. Sachs DH, Sharabi Y, Sykes M (1990) Progress in Immunology: Proceedings of 18. Marchetti B, Guarcello V, Morale MC, Bartoloni G, Raiti F, Palumbo G, Jr, the 7th International Congress of Immunology Berlin 1989, eds Melchers F, Farinella Z, Cordaro S, Scapagnini U (1989) Endocrinology 125:1037–1045. Nicklin L (Springer, Berlin), Vol VII, pp 1171–1176. 19. Mackall CL, Punt JA, Morgan P, Farr AG, Gress RE (1998) Eur J Immunol 3. Khan A, Tomita Y, Sykes M (1996) Transplantation 62:380–387. 28:1886–1893. 4. Manilay JO, Pearson DA, Sergio JJ, Swenson KG, Sykes M (1998) Transplan- 20. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA tation 66:96–102. (2005) Nature 433:760–764. 5. Yamada K, Gianello PR, Ierino FL, Lorf T, Shimizu A, Meehan S, Colvin RB, 21. Oner H, Ozan E (2002) Arch Androl 48:115–126. Sachs DH (1997) J Exp Med 186:497–506. 22. Azad N, LaPaglia N, Agrawal L, Steiner J, Uddin S, Williams DW, Lawrence 6. Tyan ML (1977) J Immunol 118:846–851. AM, Emanuele NV (1998) J Endocrinol 158:229–235. 7. Mackall CL, Gress RE (1997) Immunol Rev 160:91–102. 23. Aspinall R, Andrew D (2000) Vaccine 18:1629–1637. 8. LaMattina JC, Kumagai N, Barth RN, Yamamoto S, Kitamura H, Moran SG, 24. Moore TA, Freeden-Jeffry U, Murray R, Zlotnik A (1996) J Immunol Mezrich JD, Sachs DH, Yamada K (2002) Transplantation 73:826–831. 157:2366–2373. 9. Sachs DH (1992) in Swine as Models in Biomed Res, eds Swindle MM, Moody 25. Aspinall R, Andrew D, Pido-Lopez J (2002) Springer Semin Immunopathol DC, Phillips LD (Iowa State Univ Press, Ames, IA), pp 3–15. 24:87–101. 10. Rasband W, Bright D (1995) Microbeam Analysis 4:137–149. 26. Fry TJ, Mackall CL (2002) Springer Semin Immunopathol 24:7–22. 11. Rosengard BR, Ojikutu CA, Guzzetta PC, Smith CV, Sundt TM, III, Nakajima 27. Sempowski GD, Gooding ME, Liao HX, Le PT, Haynes BF (2002) Mol K, Boorstein SM, Hill GS, Fishbein JM, Sachs DH (1992) Transplantation Immunol 38:841–848. 54:490–497. 12. Yamada K, Gianello PR, Ierino FL, Fishbein J, Lorf T, Shimizu A, Colvin RB, 28. Gopinathan R, DePaz HA, Oluwole OO, Ali AO, Garrovillo M, Engelstad K, Sachs DH (1999) Transplantation 67:458–467. Hardy MA, Oluwole SF (2001) Transplantation 72:1533–1541. 13. Kamano C, Vagefi PA, Kumagai N, Yamamoto S, Barth RN, LaMattina JC, 29. Agus DB, Surh CD, Sprent J (1991) J Exp Med 173:1039–1046. Moran SG, Sachs DH, Yamada K (2004) Proc Natl Acad Sci USA 101:3827– 30. Yamada K, Shimizu A, Utsugi R, Ierino FL, Gargollo P, Haller GW, Colvin 3832. RB, Sachs DH (2000) J Immunol 164:3079–3086. 14. Rodriguez-Barbosa JI, Haller GW, Zhao G, Sachs DH, Sykes M (2005) Transpl 31. Utsugi R, Barth RN, Lee RS, Kitamura H, LaMattina JC, Ambroz J, Sachs H, Immunol 15:25–33. Yamada K (2001) Transplantation 71:1368–1379. 15. Yamada K, Ierino FL, Gianello PR, Shimizu A, Colvin RB, Sachs DH (1999) 32. Yamada K, Yazawa K, Shimizu A, Iwanaga T, Hisashi Y, Nuhn M, O’Malley Transplantation 67:1112–1119. P, Nobori S, Vagefi PA, Patience, C, et al. (2005) Nat Med 11:32–34. 16. Zhao Y, Sergio JJ, Swenson K, Arn JS, Sachs DH, Sykes M (1997) J Immunol 33. Ierino FL, Yamada K, Hatch T, Rembert J, Sachs DH (1999) J Immunol 159:2100–2107. 162:550–559.

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