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PathwayTargeting a Single Chemokine ReceptorMHC-Mismatched Islet Allografts by Permanent Survival of Fully

Guoxiang Xiong, Michael D. Gunn and Wayne W. HancockLiqing Wang, Rongxiang Han, Iris Lee, Aidan S. Hancock,

http://www.jimmunol.org/content/175/10/6311doi: 10.4049/jimmunol.175.10.6311

2005; 175:6311-6318; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Permanent Survival of Fully MHC-Mismatched Islet Allograftsby Targeting a Single Chemokine Receptor Pathway1

Liqing Wang,* Rongxiang Han,* Iris Lee,* Aidan S. Hancock,* Guoxiang Xiong,†

Michael D. Gunn,‡ and Wayne W. Hancock2*

Chemokine receptor blockade can diminish the recruitment of host effector cells and prolong allograft survival, but little is knownof the role of chemokine receptors in promoting host sensitization. We engrafted fully allogeneic islets into streptozotocin-treatednormal mice or mice with the autosomal recessive paucity of lymph node T cell (plt) mutation; the latter lack secondary lymphoidexpression of the CCR7 ligands, secondary lymphoid organ chemokine (CCL21) and EBV-induced molecule-1 ligand chemokine(CCL19). plt mice showed permanent survival of islets engrafted under the kidney capsule, whereas controls rejected islet allo-grafts in 12 days (p < 0.001), and consistent with this, plt mice had normal allogeneic T cell responses, but deficient migration ofdonor dendritic cell to draining lymph nodes. Peritransplant i.v. injection of donor splenocytes caused plt recipients to reject theirallografts by 12 days, and sensitization at 60 days posttransplant of plt mice with well-functioning allografts restored acuterejection. Finally, islet allografts transplanted intrahepatically in plt mice were rejected �12 days posttransplant, like controls, aswere primarily revascularized cardiac allografts. These data show that the chemokine-directed homing of donor dendritic cell tosecondary lymphoid tissues is essential for host sensitization and allograft rejection. Interruption of such homing can prevent Tcell priming and islet allograft rejection despite normal T and B cell functions of the recipient, with potential clinicalimplications. The Journal of Immunology, 2005, 175: 6311–6318.

A lthough it has been known for decades that lymphocyterecirculation is important for adaptive immune re-sponses (1), only recently have the underlying mecha-

nisms been elucidated. Expression of the chemokine receptor,CCR7, promotes binding to the chemokine ligands CCL19 (EBV-induced molecule-1 ligand chemokine (ELC))3 and CCL21 (sec-ondary lymphoid organ chemokine (SLC)), which are expressedby lymphatics and on high endothelial venules (HEV) in lymphnodes and mediate the homing of naive CCR7� T cells to second-ary lymphoid tissues (2–4). If specific foreign Ag is not encoun-tered there, lymphocytes re-enter the bloodstream and recirculatethrough other lymph nodes. Upon activation, dendritic cells (DC)undergo maturation, express CCR7, and migrate via lymphatics tosecondary lymphoid organs, where they present foreign Ags tonaive lymphocytes (5, 6). Hence, expression of CCR7 by naive Tcells and mature DC plays a key role in initiation of immuneresponses.

Both direct and indirect recognition of foreign Ags may occur,with direct recognition most likely being especially important forthe initiation of allograft rejection because of naive T cell exposureto donor MHC-rich DC (7). Recently, mice with the alymphoplasiamutation were shown, after splenectomy, to accept cardiac allo-grafts long-term despite having normal numbers of circulating Tcells, indicating the importance of events occurring within second-ary lymphoid organs in initiating allograft rejection (8). With rel-evance to the current study, mice with the paucity of lymph nodeT cell ( plt) mutation lack SLC and ELC expression on HEV inlymph nodes, although some SLC is still detected within lymphat-ics, leading to markedly decreased T cell and DC homing to lymphnodes (9–13). Although T cell migration via the blood to thespleens of plt mice is unimpaired, these cells cannot efficientlyhome to T-dependent areas in the spleens, whereas B cells displaynormal migration to all secondary lymphoid tissues (9–13).

Our lab has documented the significant effects on allograft sur-vival of targeting chemokine receptors expressed by effector Tcells, such as CXCR3 (14), CX3CR1 (15), CCR1 (16), CCR2 (17),and CCR5 (18). Targeting of effector cell recruitment diminishesintragraft leukocyte accumulation and associated expression of cy-tokines and various inflammatory mediators (19). The currentstudy examined, using plt mice, whether interruption of the affer-ent pathways leading to allosensitization might also be of potentialtherapeutic importance. The data generated show that such target-ing can have profound biologic effects.

Materials and MethodsAnimals

We purchased 6- to 8-wk-old BALB/c (H-2d), C57BL/6 (H-2b), andC57BL/6 � DBA F1 (H-2b/d) mice (The Jackson Laboratory). DDD/1-plt/plt (hereafter plt) mice were backcrossed with BALB/c mice for 10 gen-erations, housed in specific pathogen-free conditions, and studied using aprotocol approved by the Institutional Animal Care and Use Committee ofthe Children’s Hospital of Philadelphia.

*Division of Transplantation Immunology, Department of Pathology and LaboratoryMedicine, Joseph Stokes Jr. Research Institute and Biesecker Pediatric Liver Center,and †Department of Neurology, The Children’s Hospital of Philadelphia and Univer-sity of Pennsylvania School of Medicine, Philadelphia, PA 19104; and ‡Departmentof Medicine, Duke University Medical Center, Durham, NC 27710

Received for publication July 22, 2005. Accepted for publication September 1, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grant AI40152 (toW.W.H.).2 Address correspondence and reprint requests to Dr. Wayne W. Hancock, Division ofTransplantation Immunobiology, Department of Pathology and Laboratory Medicine,916E Abramson Research Center, The Children’s Hospital of Philadelphia, 3615Civic Center Boulevard, Philadelphia, PA 19104-4318. E-mail address:whancock@mail.med.upenn.edu3 Abbreviations used in this paper: ELC, EBV-induced molecule-1 ligand chemokine;DC, dendritic cell; HEV, high endothelial venule; qPCR, quantitative RT-PCR; SLC,secondary lymphoid organ chemokine; Treg, T regulatory.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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Islet isolation, culture, and transplantation

Diabetes was induced with a single i.p. injection of streptozotocin (225mg/kg; Sigma-Aldrich). Mice with two consecutive nonfasting blood glu-cose levels of �300 mg/dl were used as recipients. Murine islets wereisolated, as described (20). Briefly, after injection of collagenase via thecommon bile duct, the pancreas was removed and digested at 37°C, andislets were separated over discontinuous Ficoll gradients. Islets were cul-tured for 24 h, as described (17), in the presence of LPS (1 �g/ml) or oneof the following recombinant murine cytokines (R&D Systems): IFN-�(100 U/ml), IL-1 (100 ng/ml), or TNF-� (100 ng/ml); they were then har-vested and used for real-time quantitative RT-PCR (qPCR). Approximately300 islets were transplanted into the liver via the portal vein insertion orunder the kidney capsule. Primary graft function was defined as a decreasein nonfasting blood glucose levels to �200 mg/dl, and graft rejection wasdetermined when blood glucose levels climbed to �300 mg/dl.

Cardiac transplantation

Heterotopic abdominal cardiac allografts with end-to-side anastomosis ofaorta to aorta and pulmonary artery to vena cava (21) were undertakenusing B6 donors and plt or BALB/c recipients; survival data were deter-mined using six allografts per group. Graft function was monitored daily bypalpation, and rejection was confirmed by laparotomy and histology. Atrejection or at the time indicated, grafts were fixed in Formalin for lightmicroscopy or snap frozen in liquid nitrogen and stored at �80°C forqPCR and immunohistology.

Flow cytometry

Alloreactive T cell responses were generated by i.v. injection of 40 � 106

CFSE-labeled pooled wild-type or plt spleen and lymph node cells intoC57BL/6 � DBA F1 (H-2b/d) recipients, a parent3F1 MHC mismatch inwhich only donor cells respond (22). Spleens were harvested from F1 re-cipients after 3 days, and splenocytes were incubated with CD69-PE, CD4-PE-Texas Red, CD44-PE-cy5, CD8-PE-cy7, CD62L-allophycocyanin, andbiotin-conjugated anti-H-2Kd and anti-H-2Dd mAb, followed by strepta-vidin-allophycocyanin-cy7 (BD Pharmingen). Donor alloreactive T cellswere distinguished from recipient T cells by gating on H-2kd- and H-2dd-negative cells (Cyan; DakoCytomation), and T cell proliferation was as-sessed using CFSE division profiles. Responder frequencies were calcu-lated, as previously described (22). For intracellular cytokine staining,splenocytes (3 � 106 cells/ml) were treated with Golgi-stop (BD Pharm-ingen), stimulated for 4 h with PMA (3 ng/ml) and ionomycin (1 �M),stained with cell markers (CD4-PE-cy5, CD8-PE-cy7, biotin-conjugatedH-2kd or H-2dd, followed by streptavidin-allophycocyanin-cy7), fixed in1% formaldehyde, permeabilized, and stained with anti-IFN-� PE and anti-IL-2 allophycocyanin mAbs.

In vivo migration of bone marrow-derived DC and T cells

Bone marrow cells flushed from the femur and tibia were cultured in RPMI1640 plus 10% FBS, GM-CSF (20 ng/ml), and IL-4 (20 ng/ml). Adherentcells were cultured for 3 days, harvested, and labeled with CFSE (5 mM).CFSE-labeled DC or freshly harvested and CFSE-labeled splenocytes wereinjected (in 30 �l of PBS) into the left footpads of mice. At 24 h, drainingand nondraining lymph nodes and spleens were harvested for flow cyto-metric analysis of T cell subsets, or in the case of DC transfer, fixed for 1 hin 10% formaldehyde and sectioned (50 �m), and FITC-positive cells wereidentified by confocal microscopy (Olympus Fluoview FV1000 ConfocalMicrosystem).

Mixed leukoocyte reaction

Various numbers of irradiated (2000 rad) splenocytes from B6 mice (H-2b)were cultured in 96-well plate with 1–2 � 105 plt or BALB/c splenocytes(H-2d) in RPMI 1640 supplemented with 10% FCS (Sigma-Aldrich), pen-icillin, streptomycin, and 2-ME (50 �M) for 72 h. Cells were pulsed with1 mM BrdU for 4–5 h before harvesting and stained with mAbs against cellsurface markers. Cells were then washed, fixed with 2% formaldehyde,permeabilized with Perm/Wash solution (BD Pharmingen), and stainedwith PE anti-BrdU Ab; cell proliferation was assessed by flow cytometry(Cyan) based on the extent of BrdU labeling.

qPCR

Primers and probes for IL-2, IL-10, IFN-�, IFN-�-inducible protein-10,CCR2, CCR5, and CXCR3 were purchased from Applied Biosystems, andgene profiles were quantitated using an ABI-7000 (Applied Biosystems);data were expressed as fold increase.

Immunopathology

Donor MHC class II-positive cells were detected by immunoperoxidasestaining of cryostat sections of draining lymph nodes, nondraining lymphnodes, and spleen with anti-C57BL/6 MHC II mAb (BD Pharmingen). Forimaging of CFSE-labeled cells, 40-�m cryostat sections were evaluated byconfocal microscopy.

Statistics

Graft survival was evaluated by Kaplan-Meier; a p value �0.05 was con-sidered significant.

ResultsLong-term islet allograft survival in plt recipients

C57BL/6 islets cultured in vitro showed up-regulation of CCR7mRNA expression on exposure to inflammatory stimuli such asmicrobial products and cytokines, consistent with a potential rolefor CCR7 in early intragraft events postislet transplantation (Fig.1a). This was analyzed in vivo by rendering plt and wild-typeBALB/c mice diabetic by injection of streptozotocin, and engraft-ing them in each case under the recipient’s renal capsule with fullyMHC-disparate islet allografts from C57BL/6 donors. Induction of

FIGURE 1. Islet activation in vitro and allografting in vivo. a, qPCRanalysis of CCR7 mRNA expression by islets cultured in vitro for 24 hwith medium alone or LPS (1 mg/ml), TNF-� (100 ng/ml), IL-1� (100ng/ml), or IFN-� (100 U/ml); data shown as CCR7 mRNA expressionrelative to medium alone and are representative of six experiments. b,C57BL/6 islets transplanted into wild-type BALB/c recipients were re-jected within 14 days of engraftment, whereas allografts in plt recipientswere accepted indefinitely (�120 days) (eight allografts per group). c,Destruction of islet allografts under the kidney capsule and absence ofinsulin production at 14 days posttransplant in wild-type recipients,whereas plt/plt recipients showed preservation of islet allografts and denseinsulin production (H&E and immunoperoxidase-stained paraffin sections,�200). d, Cytokine, chemokine, and chemokine receptor gene expressionin islet allografts harvested at 14 days posttransplant; duplicate samplesshown and representative of three separate studies.

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diabetes, restoration of euglycemia, and islet allograft survivalwere monitored by serial blood glucose measurements. In bothgroups, islet allografts restored euglycemia within 24–48 h, butislet allografts in wild-type recipients were rejected by 14 days,whereas those in plt recipients functioned long-term (�120 days,p � 0.001) (Fig. 1b). Comparison of islet allografts in the twogroups at 14 days posttransplant showed dense mononuclear cellinfiltration and an absence of islet production in wild-type recip-ients, but well-preserved islets, free of leukocyte infiltration andwith dense staining for insulin, in plt recipients (Fig. 1c). Simi-larly, allografts in wild-type recipients had elevated levels of ex-pression of key cytokines, chemokines, and chemokine receptorstypical of acute islet allograft rejection (17), whereas these werelacking in allografts harvested from plt recipients (Fig. 1d). These

data indicate that compared with the fulminant inflammatory andimmune responses in wild-type islet allograft recipients, plt recip-ients display negligible intragraft antidonor immune responses.

Impaired T cell activation after islet allografting in plt mice

The minimal alloresponses seen at allograft sites in plt vs wild-typerecipients led us to check by flow cytometry for the presence in sec-ondary lymphoid tissues of features characteristic of T cell activation.We compared the levels of activated T cells expressing CD25,CD44high, and CD62Llow in draining or nondraining lymph node, andspleen samples were harvested at 14 days posttransplant from plt andwild-type islet allograft recipients. plt and wild-type recipients hadcomparable levels of CD25�CD4� and CD25�CD8� T cells in

FIGURE 2. Flow cytometric analysis of surface expression of cellular activation markers by CD4 and CD8 T cells within allograft draining andnondraining lymph nodes and spleen at 14 days posttransplant in plt and wild-type islet allograft recipients. Expression of CD25, CD44high, and CD62Llow

was markedly decreased in plt recipients as compared with wild-type mice; data are representative of three separate experiments.

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nondraining lymph nodes, but compared with wild-type mice, plt re-cipients had reduced activation of both T cell subsets, as reflected byreduced levels of CD25, CD44high, and CD62Llow in draining lymphnode and spleen samples (Fig. 2). Hence, the lack of host responses atthe graft site in plt recipients is associated with evidence of decreasedT cell activation in relevant secondary lymphoid tissues.

plt mice have normal T cell alloresponses

Although plt mice can mount T cell-dependent contact hypersen-sitivity responses that, albeit delayed in tempo, become equal orgreater than those of wild-type mice (10), we are unaware of dataas to thymic development and T cell-dependent alloresponses inplt recipients. We therefore tested whether T cells from plt mice

FIGURE 3. Flow cytometric study of T cell populations and proliferative responses in plt mice. a, plt mice had similar proportions of thymic and splenicT cell subsets, and CD4�CD25� Treg cells, to wild-type mice, but had decreased proportions of CD4 and CD8 lymph node T cells. b, Comparable in vitroMLR by CD4 and CD8 T cells of plt mice and wild-type controls to irradiated C57BL/6 splenocytes. c, Comparable in vivo allogeneic activation andproliferation of CFSE-labeled plt and wild-type controls upon adoptive transfer to F1 recipients; analysis at 3 days posttransfer and inset shows equalresponder frequencies by CD4 and CD8 T cells of both groups. d, Intracellular cytokine staining of cell populations shown in c showed comparable (CD4)or enhanced (CD8) IFN-� production by plt T cells vs wild-type controls. Data in b–d are representative of three independent experiments.

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develop normally and are capable of alloantigen-induced activa-tion, proliferation, and cytokine production (Fig. 3).

Flow cytometric analysis of CD4 and CD8 T cell subsets indi-cated comparable proportions within each stage of thymic devel-opment in plt and wild-type controls, and equal proportions of eachT cell subset were also seen using spleen samples (Fig. 3a).However, as expected from studies of DDD/1-plt mice, the pro-portions of CD4 and CD8 T cells in plt (BALB/c) were decreasedin lymph nodes when compared with wild-type BALB/c controls(Fig. 3a). The proportions of naturally occurring CD4�CD25� Tregulatory (Treg) cells in spleen and lymph node samples werealso comparable (Fig. 3a). Although clearly by no means exhaus-tive, these studies suggested normal T cell and Treg developmentin plt mice, except for the expected diminution of T cells withinlymph nodes.

We next evaluated the alloresponses of plt vs wild-type mice.CD4 and CD8 cells from plt mice proliferated in vitro as well, orbetter, than corresponding wild-type T cell subsets when stimu-lated with irradiated BALB/c splenocytes (Fig. 3b). We tested invivo alloresponses by i.v. injection of CFSE-labeled T cells fromplt or wild-type mice into F1 mice. Again, CD4 and CD8 cellsfrom plt mice underwent alloactivation and proliferation similarlyto that of wild-type mice (Fig. 3c). Intracellular cytokine stainingby allo-activated T cell subsets from plt mice also showed com-parable or greater production of IL-2 (data not shown) and IFN-�(Fig. 3d) as compared with wild-type T cells. Hence, in vitro andin vivo MLR studies showed that T cells of plt were perfectlycapable of proliferating in response to alloantigens; nor did pltmice appear to possess an expanded population of naturally oc-curring Treg cells, which might explain the unexpected islet allo-graft survival data.

Decreased donor DC and T cell homing to plt recipient lymphnodes, but not spleen

Previous studies showed that murine islets contain zero to five DCper islet (23), and in preliminary studies using anti-donor MHCmAb we were unable to localize donor-derived islet DC withinsections of draining lymph nodes (data not shown). We thereforeassessed the migration of allogeneic CFSE-labeled cells, including

DC, into secondary lymph nodes following their footpad or i.v.injection. Footpad injection of CFSE-labeled splenocytes resultedin low numbers of T cells being detected by flow cytometry inlymph nodes of wild-type mice at 24 h, whereas none were de-tected in lymph nodes from plt recipients (Fig. 4a). After footpadinjection of donor CFSE� DC, CFSE� DC cells were demonstra-ble within paracortical areas of draining lymph nodes in wild-typecontrols, but in plt mice the few CFSE� cells detected were con-fined to subcapsular sinus areas of draining lymph nodes (Fig. 4b).Similar numbers of C57BL/6 CD4 and CD8 cells were detectedwithin recipient spleens of plt and wild-type controls at 72 h aftertheir i.v. injection, whereas their recovery from plt lymph nodeswas decreased by �90% compared with wild-type controls (Fig.4c). These data indicate impaired donor leukocyte homing to pltrecipient lymph nodes, whereas migration to the spleen after i.v.injection was about comparable to that of wild-type mice.

plt mice acutely reject cardiac allografts

Experimentally, different types of donor tissues may be rankedaccording to the ease by which their rejection by a host may beovercome despite the same degree of MHC disparity (24); e.g., itis usually easier with a given protocol to attain long-term cardiacallograft survival than islet allograft survival, and skin allograftrejection appears particularly difficult to suppress. Given the per-manent allograft survival and negligible T cell activation inducedby C57BL/6 islet transplantation into plt recipients, long-term sur-vival of C57BL/6 cardiac allografts would be expected. However,we found that cardiac allografts in plt recipients were rejected withthe same tempo as observed in wild-type recipients (Fig. 5a), andhistologic examination of both sets of allografts showed a verysimilar pattern of acute cellular rejection (data not shown). Giventhe limited ability of T cells to home to lymph nodes in plt mice,we tested whether splenectomy would prolong cardiac allograftsurvival in plt mice. Splenectomy more than doubled survival ofC57BL/6 allografts in plt mice ( p � 0.05), whereas the tempo ofrejection in wild-type controls was unaffected (Fig. 5b), indicatinga key role for the spleen in mediating T cell-dependent host re-sponses to cardiac allografts in plt mice.

FIGURE 4. Trafficking of allogeneicCFSE-labeled T cells and DC in plt recipi-ents. a, Decreased trafficking of CFSE-la-beled C57BL/6 splenocytes to draining (andnondraining) lymph node (LN) in plt recipi-ents vs wild-type controls, but comparablesplenic uptake. b, Confocal microscopyshowed numerous CFSE� donor DC withindraining lymph nodes of wild-type mice,whereas only sparse CFSE� cells were de-tected within the draining lymph nodes of pltrecipients and were predominantly seenwithin medullary sinuses (lower left scalemarkers � 50 �m). c, The migration ofCFSE-labeled C57BL/6 splenocytes injectedinto plt recipients was decreased by �90%compared with wild-type recipients, whereascomparable accumulation within spleen wasobserved. Data from a–c are representativeof three separate experiments and were ob-tained 24 h postadoptive transfer.

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Sensitization induces acute rejection of islet allografts in pltrecipients

The findings that cardiac allografts were rejected but islet allo-grafts were accepted in plt recipients suggested differences inpriming had occurred. Heterotopic cardiac allografts are primarilyrevascularized, but lack intact lymphatic drainage, such that donorDC exit via the venous system and are mainly found in recipients’spleens (25, 26). We showed that systemic injection of donor leu-kocytes led to comparable splenic accumulation, whereas lymphnode uptake in plt recipients was impaired compared with wild-type mice (Fig. 4). Experimentally, islet allografts are most com-

monly transplanted under the renal capsule, and posttransplant,islet DC up-regulate CCR7 and migrate via recipient lymphatics todraining lymph nodes. However, migration to draining lymphnodes is defective in plt mice (Fig. 4), leading us to hypothesizethat islet allografts in plt mice would be rejected if host sensitiza-tion to donor MHC was achieved.

The importance of sensitization in explaining permanent isletengraftment in untreated plt mice was evaluated in three ways.First, at 50 d posttransplant, we took plt recipients bearing well-functioning C57BL/6 islet allografts and sought to sensitize thehosts by i.v. injection of 5 million donor splenocytes. Systemic

FIGURE 5. Importance of the route of sensitizationon allograft survival in plt mice. a, Unlike their long-term acceptance of C57BL/6 islet allografts, plt micerejected C57BL/6 cardiac allografts at virtually the sametempo as wild-type controls. b, Survival of C57BL/6cardiac allografts was modestly prolonged by the sple-nectomy of plt (p � 0.05), but not wild-type (p � 0.05)recipients. c, Intravenous donor splenocyte transfusion(DST) at the time of transplantation led to acute rejec-tion of islet allografts by plt recipients, as did adminis-tration of DST at day 50 posttransplant in plt recipientswith previously well-functioning islet allografts. Intra-hepatic islet allografts were also acutely rejected. Datain a–c obtained from six to eight allografts/group.

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exposure to donor leukocytes led to prompt reversal of graft func-tion and acute rejection within 14 d (Fig. 5c). Second, we tested theeffects of i.v. injection of donor splenocytes at the time of islettransplantation under the kidney capsule. Once again, systemicexposure induced acute rejection within 14 days of islet transplan-tation (Fig. 5c). Third, we tested the effects of seeding donor isletsvia the portal vein into the liver. Intrahepatic islet transplantationrestored euglycemia within 12 h, but graft function deteriorated by10 days and all intrahepatic islet allografts were rejected by 14days posttransplant (Fig. 5c). These data indicate that i.v. exposureto donor leukocytes restores the priming of plt recipients and al-lows development of acute allograft rejection to occur. Similarly,direct access to the vasculature of donor DC in the case of intra-hepatic engraftment also promotes splenic homing and sensitiza-tion, leading to acute rejection.

DiscussionOur studies show that survival of islet allografts in unmodified pltrecipients depends upon the site of engraftment. Clinically, isletsare currently transplanted into the liver via the portal vein (27),although some data suggest this is far from an optimal site forengraftment (28, 29). The current studies indicate that if donor DCor passenger leukocytes are unable to migrate to secondary lym-phoid tissues as a result of lack of expression of CCR7 ligands,islet allografts are accepted long-term, whereas if these cells enterthe vasculature rather than the lymphatic system they are able tohome to the spleen and elicit standard allograft responses. Thesestudies extend work on the role of passenger leukocytes in pro-moting host alloresponses, as investigated some decades ago byLafferty and Woolnough (30), by showing remarkable efficacywhen a specific chemokine receptor pathway is blocked, althoughonly in the context of tissue transplants whose DC recruitmentposttransplant involves host lymphatics. Based upon our data, andthe considerations outlined below, we conclude that strategiesbased on blockade of this pathway may be a useful therapeuticapproach clinically for preventing rejection of nonprimarily revas-cularized islet, cell, or tissue allografts placed at sites for whichDC trafficking via lymphatics is likely to be the prime mechanismfor host sensitization.

Compared with its human counterpart, regulation of the murineSLC gene is complex. As a result of one or more gene duplicationevents in mice, multiple genes can encode SLC, depending uponthe strain; e.g., BALB/c mice have two genes and C57BL/6 micehave three encoding SLC. The two genes in BALB/c plt micediffer by a single nucleotide, leading to either a leucine (SLC-leu,CCL21a) or serine (SLC-ser, CCL21b) at position 65 (11, 13).Although the differing forms of SLC are equally capable of che-moattraction in vitro, SLC-ser is normally mainly expressed bystromal cells within T cell zones of the spleen, lymph nodes, andPeyer’s patches, as well as by the HEV of lymph nodes and Pey-er’s patches. SLC-ser is absent in plt mice (31), although thesemice do express residual SLC-leu within the lymphatic endothelialcells of most nonlymphoid tissues (13). The second main ligandfor CCR7, ELC, is distributed similarly to SLC-ser, but like thelatter is also absent in plt mice (11, 32). Given the close proximityof SLC and ELC on mouse chromosome 4, it is speculated that pltmice have a large deletion in the ELC/SLC locus that encompassesboth the ELC-atg and SLC-ser genes, but spares the SLC-leu andone or more ELC-related pseudogenes, with the end result of re-duced expression SLC-leu within the lymphatic endothelial cellsof plt mice and an absence of CCR7 ligands elsewhere (33).

The current study relied on use of plt mice to examine the roleof CCR7 and its ligands in alloresponses for several reasons. First,knockout mice lacking CCR7 are not widely available, and mice

selectively lacking ELC or SLC are unknown. Second, blockingmAbs for CCR7 and its ligands are also unavailable, as are selec-tive CCR7 small molecule antagonists. Hence, use of plt miceprovides a practical first approach to tackling the role of CCR7 andits ligands in alloresponses, especially because data from the use ofplt or CCR7�/� mice in various models have proven comparable(31, 34). Potential caveats to our data are suggested by the findingsthat in mice, SLC can also bind to two other receptors, CXCR3 andCCR10 (CCX-CKR). However, SLC binding and recruitment ofCXCR3� cells have only been demonstrated for brain microglialand not lymphoid cells (35), and CXCR3�/� mice or wild-typemice treated with an anti-IFN-�-inducible protein-10 (CXCL10)mAb reject islet allografts with only a modest delay (�2-fold pro-longation of survival) compared with control allograft recipients(36). Similarly, the recently described CCR10 chemokine receptorcan bind SLC and ELC, but does not flux calcium or promote thechemotaxis of CCR7 transfectants (37, 38), and is thereby re-garded only as a decoy or scavenger receptor (39, 40). Hence, ourdata are not consistent with significant involvement of either ofthose pathways.

Although no data have previously been reported on the role ofthe CCR7 pathway in islet allograft rejection, cardiac allograftingacross fully MHC-disparate strain combinations was shown to pro-long allograft survival by only a few days, whether CCR7�/� micewere used as allograft recipients or as donors (41, 42). Similarly,use of plt (BALB/c) mice as recipients of C57BL/6 cardiac or skinallografts showed only up to 3–4 days of prolongation of allograftsurvival (43). Given these findings and our own data using cardiacallografts, initial results involving attempts to target the CCR7pathway as a means to prolong primarily revascularized allografts,as well as skin allografts whose donor DC also drain from the skinvia recipients’ blood vessels, are not encouraging. However, ourinfrarenal capsule islet allograft data offer a remarkable contrastwith such data and emphasize the potential value of allografts forwhich donor DC drainage is likely to occur predominantly via recip-ient lymphatics. Nevertheless, because normal recipient micepromptly reject islet allograft under the renal capsule, the questionarises as to how might our data ever translate to clinical application?

Potential approaches to clinical application of our data are sug-gested by findings from nontransplant systems. DC expression ofCCR7 is normally attenuated by the Runx3 transcription factor,which is a key component of the TGF-� signaling cascade. In theabsence of Runx3, DC do not respond to TGF-�, and DC showenhanced CCR7 expression, accelerated migration to draininglymph nodes, and increased hypersensitivity responses to environ-mental Ags (44). The untoward effects of Runx deficiency can beblocked in vivo by anti-CCR7 Abs, as well as by the drug Cigli-tazone (44). Ciglitazone and other selective peroxisome prolifer-ative activated receptor � (PPAR�) agonists are known to decreaseDC expression of CCR7 and inhibit their migratory properties(45). Additional small molecules that modulate DC expression ofCCR7 and suppress their migration are under development (46,47). Hence, in future studies, we will explore the extent to whichCCR7 targeting using selective PPAR� agonists or other agentscan promote long-term acceptance of subrenal capsule islet allo-grafts in wild-type recipients.

DisclosuresThe authors have no financial conflict of interest.

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