Chemokine receptor blockade can diminish the recruitment of host effector cells and prolong allograft survival, but little is known of the role of chemokine receptors in promoting host sensitization. We engrafted fully allogeneic islets into streptozotocin-treated normal mice or mice with the autosomal recessive paucity of lymph node T cell (plt) mutation; the latter lack secondary lymphoid expression 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 allografts in 12 days (p < 0.001), and consistent with this, plt mice had normal allogeneic T cell responses, but deficient migration of donor dendritic cell to draining lymph nodes. Peritransplant i.v. injection of donor splenocytes caused plt recipients to reject their allografts by 12 days, and sensitization at 60 days posttransplant of plt mice with well-functioning allografts restored acute rejection. Finally, islet allografts transplanted intrahepatically in plt mice were rejected ∼12 days posttransplant, like controls, as were primarily revascularized cardiac allografts. These data show that the chemokine-directed homing of donor dendritic cell to secondary lymphoid tissues is essential for host sensitization and allograft rejection. Interruption of such homing can prevent T cell priming and islet allograft rejection despite normal T and B cell functions of the recipient, with potential clinical implications.

Although it has been known for decades that lymphocyte recirculation is important for adaptive immune responses (1), only recently have the underlying mechanisms 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 (secondary lymphoid organ chemokine (SLC)), which are expressed by lymphatics and on high endothelial venules (HEV) in lymph nodes and mediate the homing of naive CCR7+ T cells to secondary lymphoid tissues (2, 3, 4). If specific foreign Ag is not encountered there, lymphocytes re-enter the bloodstream and recirculate through other lymph nodes. Upon activation, dendritic cells (DC) undergo maturation, express CCR7, and migrate via lymphatics to secondary lymphoid organs, where they present foreign Ags to naive lymphocytes (5, 6). Hence, expression of CCR7 by naive T cells and mature DC plays a key role in initiation of immune responses.

Both direct and indirect recognition of foreign Ags may occur, with direct recognition most likely being especially important for the initiation of allograft rejection because of naive T cell exposure to donor MHC-rich DC (7). Recently, mice with the alymphoplasia mutation were shown, after splenectomy, to accept cardiac allografts long-term despite having normal numbers of circulating T cells, indicating the importance of events occurring within secondary lymphoid organs in initiating allograft rejection (8). With relevance to the current study, mice with the paucity of lymph node T cell (plt) mutation lack SLC and ELC expression on HEV in lymph nodes, although some SLC is still detected within lymphatics, leading to markedly decreased T cell and DC homing to lymph nodes (9, 10, 11, 12, 13). Although T cell migration via the blood to the spleens of plt mice is unimpaired, these cells cannot efficiently home to T-dependent areas in the spleens, whereas B cells display normal migration to all secondary lymphoid tissues (9, 10, 11, 12, 13).

Our lab has documented the significant effects on allograft survival of targeting chemokine receptors expressed by effector T cells, such as CXCR3 (14), CX3CR1 (15), CCR1 (16), CCR2 (17), and CCR5 (18). Targeting of effector cell recruitment diminishes intragraft leukocyte accumulation and associated expression of cytokines and various inflammatory mediators (19). The current study examined, using plt mice, whether interruption of the afferent pathways leading to allosensitization might also be of potential therapeutic importance. The data generated show that such targeting can have profound biologic effects.

We purchased 6- to 8-wk-old BALB/c (H-2d), C57BL/6 (H-2b), and C57BL/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 generations, housed in specific pathogen-free conditions, and studied using a protocol approved by the Institutional Animal Care and Use Committee of the Children’s Hospital of Philadelphia.

Diabetes was induced with a single i.p. injection of streptozotocin (225 mg/kg; Sigma-Aldrich). Mice with two consecutive nonfasting blood glucose levels of >300 mg/dl were used as recipients. Murine islets were isolated, as described (20). Briefly, after injection of collagenase via the common bile duct, the pancreas was removed and digested at 37°C, and islets were separated over discontinuous Ficoll gradients. Islets were cultured for 24 h, as described (17), in the presence of LPS (1 μg/ml) or one of the following recombinant murine cytokines (R&D Systems): IFN-γ (100 U/ml), IL-1 (100 ng/ml), or TNF-α (100 ng/ml); they were then harvested and used for real-time quantitative RT-PCR (qPCR). Approximately 300 islets were transplanted into the liver via the portal vein insertion or under the kidney capsule. Primary graft function was defined as a decrease in nonfasting blood glucose levels to <200 mg/dl, and graft rejection was determined when blood glucose levels climbed to >300 mg/dl.

Heterotopic abdominal cardiac allografts with end-to-side anastomosis of aorta to aorta and pulmonary artery to vena cava (21) were undertaken using B6 donors and plt or BALB/c recipients; survival data were determined using six allografts per group. Graft function was monitored daily by palpation, and rejection was confirmed by laparotomy and histology. At rejection or at the time indicated, grafts were fixed in Formalin for light microscopy or snap frozen in liquid nitrogen and stored at −80°C for qPCR and immunohistology.

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 into C57BL/6 × DBA F1 (H-2b/d) recipients, a parent→F1 MHC mismatch in which only donor cells respond (22). Spleens were harvested from F1 recipients after 3 days, and splenocytes were incubated with CD69-PE, CD4-PE-Texas Red, CD44-PE-cy5, CD8-PE-cy7, CD62L-allophycocyanin, and biotin-conjugated anti-H-2Kd and anti-H-2Dd mAb, followed by streptavidin-allophycocyanin-cy7 (BD Pharmingen). Donor alloreactive T cells were distinguished from recipient T cells by gating on H-2kd- and H-2dd-negative cells (Cyan; DakoCytomation), and T cell proliferation was assessed using CFSE division profiles. Responder frequencies were calculated, as previously described (22). For intracellular cytokine staining, splenocytes (3 × 106 cells/ml) were treated with Golgi-stop (BD Pharmingen), stimulated for 4 h with PMA (3 ng/ml) and ionomycin (1 μM), stained with cell markers (CD4-PE-cy5, CD8-PE-cy7, biotin-conjugated H-2kd or H-2dd, followed by streptavidin-allophycocyanin-cy7), fixed in 1% formaldehyde, permeabilized, and stained with anti-IFN-γ PE and anti-IL-2 allophycocyanin mAbs.

Bone marrow cells flushed from the femur and tibia were cultured in RPMI 1640 plus 10% FBS, GM-CSF (20 ng/ml), and IL-4 (20 ng/ml). Adherent cells were cultured for 3 days, harvested, and labeled with CFSE (5 mM). CFSE-labeled DC or freshly harvested and CFSE-labeled splenocytes were injected (in 30 μl of PBS) into the left footpads of mice. At 24 h, draining and nondraining lymph nodes and spleens were harvested for flow cytometric analysis of T cell subsets, or in the case of DC transfer, fixed for 1 h in 10% formaldehyde and sectioned (50 μm), and FITC-positive cells were identified by confocal microscopy (Olympus Fluoview FV1000 Confocal Microsystem).

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

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

Donor MHC class II-positive cells were detected by immunoperoxidase staining of cryostat sections of draining lymph nodes, nondraining lymph nodes, and spleen with anti-C57BL/6 MHC II mAb (BD Pharmingen). For imaging of CFSE-labeled cells, 40-μm cryostat sections were evaluated by confocal microscopy.

Graft survival was evaluated by Kaplan-Meier; a p value <0.05 was considered significant.

C57BL/6 islets cultured in vitro showed up-regulation of CCR7 mRNA expression on exposure to inflammatory stimuli such as microbial products and cytokines, consistent with a potential role for CCR7 in early intragraft events postislet transplantation (Fig. 1,a). This was analyzed in vivo by rendering plt and wild-type BALB/c mice diabetic by injection of streptozotocin, and engrafting them in each case under the recipient’s renal capsule with fully MHC-disparate islet allografts from C57BL/6 donors. Induction of diabetes, restoration of euglycemia, and islet allograft survival were monitored by serial blood glucose measurements. In both groups, islet allografts restored euglycemia within 24–48 h, but islet allografts in wild-type recipients were rejected by 14 days, whereas those in plt recipients functioned long-term (>120 days, p < 0.001) (Fig. 1,b). Comparison of islet allografts in the two groups at 14 days posttransplant showed dense mononuclear cell infiltration and an absence of islet production in wild-type recipients, but well-preserved islets, free of leukocyte infiltration and with dense staining for insulin, in plt recipients (Fig. 1,c). Similarly, allografts in wild-type recipients had elevated levels of expression of key cytokines, chemokines, and chemokine receptors typical of acute islet allograft rejection (17), whereas these were lacking in allografts harvested from plt recipients (Fig. 1 d). These data indicate that compared with the fulminant inflammatory and immune responses in wild-type islet allograft recipients, plt recipients display negligible intragraft antidonor immune responses.

FIGURE 1.

Islet activation in vitro and allografting in vivo. a, qPCR analysis of CCR7 mRNA expression by islets cultured in vitro for 24 h with medium alone or LPS (1 mg/ml), TNF-α (100 ng/ml), IL-1β (100 ng/ml), or IFN-γ (100 U/ml); data shown as CCR7 mRNA expression relative to medium alone and are representative of six experiments. b, C57BL/6 islets transplanted into wild-type BALB/c recipients were rejected within 14 days of engraftment, whereas allografts in plt recipients were accepted indefinitely (>120 days) (eight allografts per group). c, Destruction of islet allografts under the kidney capsule and absence of insulin production at 14 days posttransplant in wild-type recipients, whereas plt/plt recipients showed preservation of islet allografts and dense insulin production (H & E and immunoperoxidase-stained paraffin sections, ×200). d, Cytokine, chemokine, and chemokine receptor gene expression in islet allografts harvested at 14 days posttransplant; duplicate samples shown and representative of three separate studies.

FIGURE 1.

Islet activation in vitro and allografting in vivo. a, qPCR analysis of CCR7 mRNA expression by islets cultured in vitro for 24 h with medium alone or LPS (1 mg/ml), TNF-α (100 ng/ml), IL-1β (100 ng/ml), or IFN-γ (100 U/ml); data shown as CCR7 mRNA expression relative to medium alone and are representative of six experiments. b, C57BL/6 islets transplanted into wild-type BALB/c recipients were rejected within 14 days of engraftment, whereas allografts in plt recipients were accepted indefinitely (>120 days) (eight allografts per group). c, Destruction of islet allografts under the kidney capsule and absence of insulin production at 14 days posttransplant in wild-type recipients, whereas plt/plt recipients showed preservation of islet allografts and dense insulin production (H & E and immunoperoxidase-stained paraffin sections, ×200). d, Cytokine, chemokine, and chemokine receptor gene expression in islet allografts harvested at 14 days posttransplant; duplicate samples shown and representative of three separate studies.

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The minimal alloresponses seen at allograft sites in plt vs wild-type recipients led us to check by flow cytometry for the presence in secondary 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, and spleen samples were harvested at 14 days posttransplant from plt and wild-type islet allograft recipients. plt and wild-type recipients had comparable levels of CD25+CD4+ and CD25+CD8+ T cells in nondraining lymph nodes, but compared with wild-type mice, plt recipients had reduced activation of both T cell subsets, as reflected by reduced levels of CD25, CD44high, and CD62Llow in draining lymph node and spleen samples (Fig. 2). Hence, the lack of host responses at the graft site in plt recipients is associated with evidence of decreased T cell activation in relevant secondary lymphoid tissues.

FIGURE 2.

Flow cytometric analysis of surface expression of cellular activation markers by CD4 and CD8 T cells within allograft draining and nondraining 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.

FIGURE 2.

Flow cytometric analysis of surface expression of cellular activation markers by CD4 and CD8 T cells within allograft draining and nondraining 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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Although plt mice can mount T cell-dependent contact hypersensitivity responses that, albeit delayed in tempo, become equal or greater than those of wild-type mice (10), we are unaware of data as to thymic development and T cell-dependent alloresponses in plt recipients. We therefore tested whether T cells from plt mice develop normally and are capable of alloantigen-induced activation, proliferation, and cytokine production (Fig. 3).

FIGURE 3.

Flow cytometric study of T cell populations and proliferative responses in plt mice. a, plt mice had similar proportions of thymic and splenic T 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 vitro MLR by CD4 and CD8 T cells of plt mice and wild-type controls to irradiated C57BL/6 splenocytes. c, Comparable in vivo allogeneic activation and proliferation of CFSE-labeled plt and wild-type controls upon adoptive transfer to F1 recipients; analysis at 3 days posttransfer and inset shows equal responder 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-γ production by plt T cells vs wild-type controls. Data in b–d are representative of three independent experiments.

FIGURE 3.

Flow cytometric study of T cell populations and proliferative responses in plt mice. a, plt mice had similar proportions of thymic and splenic T 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 vitro MLR by CD4 and CD8 T cells of plt mice and wild-type controls to irradiated C57BL/6 splenocytes. c, Comparable in vivo allogeneic activation and proliferation of CFSE-labeled plt and wild-type controls upon adoptive transfer to F1 recipients; analysis at 3 days posttransfer and inset shows equal responder 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-γ production by plt T cells vs wild-type controls. Data in b–d are representative of three independent experiments.

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Flow cytometric analysis of CD4 and CD8 T cell subsets indicated comparable proportions within each stage of thymic development in plt and wild-type controls, and equal proportions of each T cell subset were also seen using spleen samples (Fig. 3,a). However, as expected from studies of DDD/1-plt mice, the proportions of CD4 and CD8 T cells in plt (BALB/c) were decreased in lymph nodes when compared with wild-type BALB/c controls (Fig. 3,a). The proportions of naturally occurring CD4+CD25+ T regulatory (Treg) cells in spleen and lymph node samples were also comparable (Fig. 3 a). Although clearly by no means exhaustive, these studies suggested normal T cell and Treg development in plt mice, except for the expected diminution of T cells within lymph nodes.

We next evaluated the alloresponses of plt vs wild-type mice. CD4 and CD8 cells from plt mice proliferated in vitro as well, or better, than corresponding wild-type T cell subsets when stimulated with irradiated BALB/c splenocytes (Fig. 3,b). We tested in vivo alloresponses by i.v. injection of CFSE-labeled T cells from plt or wild-type mice into F1 mice. Again, CD4 and CD8 cells from plt mice underwent alloactivation and proliferation similarly to that of wild-type mice (Fig. 3,c). Intracellular cytokine staining by allo-activated T cell subsets from plt mice also showed comparable or greater production of IL-2 (data not shown) and IFN-γ (Fig. 3 d) as compared with wild-type T cells. Hence, in vitro and in vivo MLR studies showed that T cells of plt were perfectly capable of proliferating in response to alloantigens; nor did plt mice appear to possess an expanded population of naturally occurring Treg cells, which might explain the unexpected islet allograft survival data.

Previous studies showed that murine islets contain zero to five DC per islet (23), and in preliminary studies using anti-donor MHC mAb we were unable to localize donor-derived islet DC within sections of draining lymph nodes (data not shown). We therefore assessed 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 resulted in low numbers of T cells being detected by flow cytometry in lymph nodes of wild-type mice at 24 h, whereas none were detected in lymph nodes from plt recipients (Fig. 4,a). After footpad injection of donor CFSE+ DC, CFSE+ DC cells were demonstrable within paracortical areas of draining lymph nodes in wild-type controls, but in plt mice the few CFSE+ cells detected were confined to subcapsular sinus areas of draining lymph nodes (Fig. 4,b). Similar numbers of C57BL/6 CD4 and CD8 cells were detected within recipient spleens of plt and wild-type controls at 72 h after their i.v. injection, whereas their recovery from plt lymph nodes was decreased by >90% compared with wild-type controls (Fig. 4 c). These data indicate impaired donor leukocyte homing to plt recipient lymph nodes, whereas migration to the spleen after i.v. injection was about comparable to that of wild-type mice.

FIGURE 4.

Trafficking of allogeneic CFSE-labeled T cells and DC in plt recipients. a, Decreased trafficking of CFSE-labeled C57BL/6 splenocytes to draining (and nondraining) lymph node (LN) in plt recipients vs wild-type controls, but comparable splenic uptake. b, Confocal microscopy showed numerous CFSE+ donor DC within draining lymph nodes of wild-type mice, whereas only sparse CFSE+ cells were detected within the draining lymph nodes of plt recipients and were predominantly seen within medullary sinuses (lower left scale markers = 50 μm). c, The migration of CFSE-labeled C57BL/6 splenocytes injected into plt recipients was decreased by >90% compared with wild-type recipients, whereas comparable accumulation within spleen was observed. Data from a–c are representative of three separate experiments and were obtained 24 h postadoptive transfer.

FIGURE 4.

Trafficking of allogeneic CFSE-labeled T cells and DC in plt recipients. a, Decreased trafficking of CFSE-labeled C57BL/6 splenocytes to draining (and nondraining) lymph node (LN) in plt recipients vs wild-type controls, but comparable splenic uptake. b, Confocal microscopy showed numerous CFSE+ donor DC within draining lymph nodes of wild-type mice, whereas only sparse CFSE+ cells were detected within the draining lymph nodes of plt recipients and were predominantly seen within medullary sinuses (lower left scale markers = 50 μm). c, The migration of CFSE-labeled C57BL/6 splenocytes injected into plt recipients was decreased by >90% compared with wild-type recipients, whereas comparable accumulation within spleen was observed. Data from a–c are representative of three separate experiments and were obtained 24 h postadoptive transfer.

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Experimentally, different types of donor tissues may be ranked according to the ease by which their rejection by a host may be overcome despite the same degree of MHC disparity (24); e.g., it is usually easier with a given protocol to attain long-term cardiac allograft survival than islet allograft survival, and skin allograft rejection appears particularly difficult to suppress. Given the permanent allograft survival and negligible T cell activation induced by C57BL/6 islet transplantation into plt recipients, long-term survival of C57BL/6 cardiac allografts would be expected. However, we found that cardiac allografts in plt recipients were rejected with the same tempo as observed in wild-type recipients (Fig. 5,a), and histologic examination of both sets of allografts showed a very similar pattern of acute cellular rejection (data not shown). Given the limited ability of T cells to home to lymph nodes in plt mice, we tested whether splenectomy would prolong cardiac allograft survival in plt mice. Splenectomy more than doubled survival of C57BL/6 allografts in plt mice (p < 0.05), whereas the tempo of rejection in wild-type controls was unaffected (Fig. 5 b), indicating a key role for the spleen in mediating T cell-dependent host responses to cardiac allografts in plt mice.

FIGURE 5.

Importance of the route of sensitization on allograft survival in plt mice. a, Unlike their long-term acceptance of C57BL/6 islet allografts, plt mice rejected C57BL/6 cardiac allografts at virtually the same tempo as wild-type controls. b, Survival of C57BL/6 cardiac allografts was modestly prolonged by the splenectomy 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 rejection of islet allografts by plt recipients, as did administration of DST at day 50 posttransplant in plt recipients with previously well-functioning islet allografts. Intrahepatic islet allografts were also acutely rejected. Data in a–c obtained from six to eight allografts/group.

FIGURE 5.

Importance of the route of sensitization on allograft survival in plt mice. a, Unlike their long-term acceptance of C57BL/6 islet allografts, plt mice rejected C57BL/6 cardiac allografts at virtually the same tempo as wild-type controls. b, Survival of C57BL/6 cardiac allografts was modestly prolonged by the splenectomy 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 rejection of islet allografts by plt recipients, as did administration of DST at day 50 posttransplant in plt recipients with previously well-functioning islet allografts. Intrahepatic islet allografts were also acutely rejected. Data in a–c obtained from six to eight allografts/group.

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The findings that cardiac allografts were rejected but islet allografts were accepted in plt recipients suggested differences in priming had occurred. Heterotopic cardiac allografts are primarily revascularized, but lack intact lymphatic drainage, such that donor DC exit via the venous system and are mainly found in recipients’ spleens (25, 26). We showed that systemic injection of donor leukocytes led to comparable splenic accumulation, whereas lymph node uptake in plt recipients was impaired compared with wild-type mice (Fig. 4). Experimentally, islet allografts are most commonly transplanted under the renal capsule, and posttransplant, islet DC up-regulate CCR7 and migrate via recipient lymphatics to draining lymph nodes. However, migration to draining lymph nodes is defective in plt mice (Fig. 4), leading us to hypothesize that islet allografts in plt mice would be rejected if host sensitization to donor MHC was achieved.

The importance of sensitization in explaining permanent islet engraftment 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 the hosts by i.v. injection of 5 million donor splenocytes. Systemic exposure to donor leukocytes led to prompt reversal of graft function and acute rejection within 14 d (Fig. 5,c). Second, we tested the effects of i.v. injection of donor splenocytes at the time of islet transplantation under the kidney capsule. Once again, systemic exposure induced acute rejection within 14 days of islet transplantation (Fig. 5,c). Third, we tested the effects of seeding donor islets via the portal vein into the liver. Intrahepatic islet transplantation restored euglycemia within 12 h, but graft function deteriorated by 10 days and all intrahepatic islet allografts were rejected by 14 days posttransplant (Fig. 5 c). These data indicate that i.v. exposure to donor leukocytes restores the priming of plt recipients and allows development of acute allograft rejection to occur. Similarly, direct access to the vasculature of donor DC in the case of intrahepatic engraftment also promotes splenic homing and sensitization, leading to acute rejection.

Our studies show that survival of islet allografts in unmodified plt recipients depends upon the site of engraftment. Clinically, islets are currently transplanted into the liver via the portal vein (27), although some data suggest this is far from an optimal site for engraftment (28, 29). The current studies indicate that if donor DC or passenger leukocytes are unable to migrate to secondary lymphoid tissues as a result of lack of expression of CCR7 ligands, islet allografts are accepted long-term, whereas if these cells enter the vasculature rather than the lymphatic system they are able to home to the spleen and elicit standard allograft responses. These studies extend work on the role of passenger leukocytes in promoting host alloresponses, as investigated some decades ago by Lafferty and Woolnough (30), by showing remarkable efficacy when a specific chemokine receptor pathway is blocked, although only in the context of tissue transplants whose DC recruitment posttransplant involves host lymphatics. Based upon our data, and the considerations outlined below, we conclude that strategies based on blockade of this pathway may be a useful therapeutic approach clinically for preventing rejection of nonprimarily revascularized islet, cell, or tissue allografts placed at sites for which DC trafficking via lymphatics is likely to be the prime mechanism for host sensitization.

Compared with its human counterpart, regulation of the murine SLC gene is complex. As a result of one or more gene duplication events in mice, multiple genes can encode SLC, depending upon the strain; e.g., BALB/c mice have two genes and C57BL/6 mice have three encoding SLC. The two genes in BALB/c plt mice differ 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 chemoattraction in vitro, SLC-ser is normally mainly expressed by stromal cells within T cell zones of the spleen, lymph nodes, and Peyer’s patches, as well as by the HEV of lymph nodes and Peyer’s patches. SLC-ser is absent in plt mice (31), although these mice do express residual SLC-leu within the lymphatic endothelial cells of most nonlymphoid tissues (13). The second main ligand for CCR7, ELC, is distributed similarly to SLC-ser, but like the latter is also absent in plt mice (11, 32). Given the close proximity of SLC and ELC on mouse chromosome 4, it is speculated that plt mice have a large deletion in the ELC/SLC locus that encompasses both the ELC-atg and SLC-ser genes, but spares the SLC-leu and one or more ELC-related pseudogenes, with the end result of reduced expression SLC-leu within the lymphatic endothelial cells of plt mice and an absence of CCR7 ligands elsewhere (33).

The current study relied on use of plt mice to examine the role of 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, blocking mAbs for CCR7 and its ligands are also unavailable, as are selective CCR7 small molecule antagonists. Hence, use of plt mice provides a practical first approach to tackling the role of CCR7 and its ligands in alloresponses, especially because data from the use of plt or CCR7−/− mice in various models have proven comparable (31, 34). Potential caveats to our data are suggested by the findings that in mice, SLC can also bind to two other receptors, CXCR3 and CCR10 (CCX-CKR). However, SLC binding and recruitment of CXCR3+ cells have only been demonstrated for brain microglial and not lymphoid cells (35), and CXCR3−/− mice or wild-type mice treated with an anti-IFN-γ-inducible protein-10 (CXCL10) mAb reject islet allografts with only a modest delay (<2-fold prolongation of survival) compared with control allograft recipients (36). Similarly, the recently described CCR10 chemokine receptor can bind SLC and ELC, but does not flux calcium or promote the chemotaxis of CCR7 transfectants (37, 38), and is thereby regarded only as a decoy or scavenger receptor (39, 40). Hence, our data are not consistent with significant involvement of either of those pathways.

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

Potential approaches to clinical application of our data are suggested by findings from nontransplant systems. DC expression of CCR7 is normally attenuated by the Runx3 transcription factor, which is a key component of the TGF-β signaling cascade. In the absence of Runx3, DC do not respond to TGF-β, and DC show enhanced CCR7 expression, accelerated migration to draining lymph nodes, and increased hypersensitivity responses to environmental Ags (44). The untoward effects of Runx deficiency can be blocked in vivo by anti-CCR7 Abs, as well as by the drug Ciglitazone (44). Ciglitazone and other selective peroxisome proliferative activated receptor γ (PPARγ) agonists are known to decrease DC expression of CCR7 and inhibit their migratory properties (45). Additional small molecules that modulate DC expression of CCR7 and suppress their migration are under development (46, 47). Hence, in future studies, we will explore the extent to which CCR7 targeting using selective PPARγ agonists or other agents can promote long-term acceptance of subrenal capsule islet allografts in wild-type recipients.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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 (to W.W.H.).

3

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.

1
Gowans, J. L., G. D. McGregor, D. M. Cowen.
1962
. Initiation of immune responses by small lymphocytes.
Nature
196
:
651
.-655.
2
Weninger, W., U. H. von Andrian.
2003
. Chemokine regulation of naive T cell traffic in health and disease.
Semin. Immunol.
15
:
257
.-270.
3
Gunn, M. D..
2003
. Chemokine mediated control of dendritic cell migration and function.
Semin. Immunol.
15
:
271
.-276.
4
Campbell, D. J., G. F. Debes, B. Johnston, E. Wilson, E. C. Butcher.
2003
. Targeting T cell responses by selective chemokine receptor expression.
Semin. Immunol.
15
:
277
.-286.
5
Cyster, J. G..
1999
. Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs.
J. Exp. Med.
189
:
447
.-450.
6
Sallusto, F., C. R. Mackay, A. Lanzavecchia.
2000
. The role of chemokine receptors in primary, effector, and memory immune responses.
Annu. Rev. Immunol.
18
:
593
.-620.
7
Lechler, R. I., J. R. Batchelor.
1982
. Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells.
J. Exp. Med.
155
:
31
.-41.
8
Lakkis, F. G., A. Arakelov, B. T. Konieczny, Y. Inoue.
2000
. Immunologic ‘ignorance’ of vascularized organ transplants in the absence of secondary lymphoid tissue.
Nat. Med.
6
:
686
.-688.
9
Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, H. Nakano.
1999
. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.
J. Exp. Med.
189
:
451
.-460.
10
Mori, S., H. Nakano, K. Aritomi, C. R. Wang, M. D. Gunn, T. Kakiuchi.
2001
. Mice lacking expression of the chemokines CCL21-ser and CCL19 (plt mice) demonstrate delayed but enhanced T cell immune responses.
J. Exp. Med.
193
:
207
.-218.
11
Nakano, H., M. D. Gunn.
2001
. Gene duplications at the chemokine locus on mouse chromosome 4: multiple strain-specific haplotypes and the deletion of secondary lymphoid-organ chemokine and EBI-1 ligand chemokine genes in the plt mutation.
J. Immunol.
166
:
361
.-369.
12
Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, U. H. von Andrian.
2000
. The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules.
J. Exp. Med.
191
:
61
.-76.
13
Vassileva, G., H. Soto, A. Zlotnik, H. Nakano, T. Kakiuchi, J. A. Hedrick, S. A. Lira.
1999
. The reduced expression of 6Ckine in the plt mouse results from the deletion of one of two 6Ckine genes.
J. Exp. Med.
190
:
1183
.-1188.
14
Hancock, W. W., B. Lu, W. Gao, V. Csizmadia, K. Faia, J. A. King, S. T. Smiley, M. Ling, N. P. Gerard, C. Gerard.
2000
. Requirement of the chemokine receptor CXCR3 for acute allograft rejection.
J. Exp. Med.
192
:
1515
.-1520.
15
Haskell, C. A., W. W. Hancock, D. J. Salant, W. Gao, V. Csizmadia, W. Peters, K. Faia, O. Fituri, J. B. Rottman, I. F. Charo.
2001
. Targeted deletion of CX3CR1 reveals a role for fractalkine in cardiac allograft rejection.
J. Clin. Invest.
108
:
679
.-688.
16
Gao, W., P. S. Topham, J. A. King, S. T. Smiley, V. Csizmadia, B. Lu, C. J. Gerard, W. W. Hancock.
2000
. Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection.
J. Clin. Invest.
105
:
35
.-44.
17
Lee, I., L. Wang, A. D. Wells, Q. Ye, R. Han, M. E. Dorf, W. A. Kuziel, B. J. Rollins, L. Chen, W. W. Hancock.
2003
. Blocking the monocyte chemoattractant protein-1/CCR2 chemokine pathway induces permanent survival of islet allografts through a programmed death-1 ligand-1-dependent mechanism.
J. Immunol.
171
:
6929
.-6935.
18
Fischer, F. R., L. Santambrogio, Y. Luo, M. A. Berman, W. W. Hancock, M. E. Dorf.
2000
. Modulation of experimental autoimmune encephalomyelitis: effect of altered peptide ligand on chemokine and chemokine receptor expression.
J. Neuroimmunol.
110
:
195
.-208.
19
Hancock, W. W., L. Wang, Q. Ye, R. Han, I. Lee.
2003
. Chemokines and their receptors as markers of allograft rejection and targets for immunosuppression.
Curr. Opin. Immunol.
15
:
479
.-486.
20
Yang, H., D. Thomas, D. J. Boffa, R. Ding, B. Li, T. Muthukumar, V. K. Sharma, M. Lagman, G. X. Luo, S. Kapur, et al
2002
. Enforced c-REL deficiency prolongs survival of islet allografts 1.
Transplantation
74
:
291
.-298.
21
Corry, R. J., S. E. Kelley.
1975
. Survival of cardiac xenografts: effect of antithymocyte serum and enhancing heteroantiserum.
Arch. Surg.
110
:
1143
.-1145.
22
Suchin, E. J., P. B. Langmuir, E. Palmer, M. H. Sayegh, A. D. Wells, L. A. Turka.
2001
. Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question.
J. Immunol.
166
:
973
.-981.
23
Faustman, D. L., R. M. Steinman, H. M. Gebel, V. Hauptfeld, J. M. Davie, P. E. Lacy.
1984
. Prevention of rejection of murine islet allografts by pretreatment with anti-dendritic cell antibody.
Proc. Natl. Acad. Sci. USA
81
:
3864
.-3868.
24
Jones, N. D., S. E. Turvey, A. VanMaurik, M. Hara, C. I. Kingsley, C. H. Smith, A. L. Mellor, P. J. Morris, K. J. Wood.
2001
. Differential susceptibility of heart, skin, and islet allografts to T cell-mediated rejection.
J. Immunol.
166
:
2824
.-2830.
25
Larsen, C. P., P. J. Morris, J. M. Austyn.
1990
. Migration of dendritic leukocytes from cardiac allografts into host spleens: a novel pathway for initiation of rejection.
J. Exp. Med.
171
:
307
.-314.
26
Saiki, T., T. Ezaki, M. Ogawa, K. Matsuno.
2001
. Trafficking of host- and donor-derived dendritic cells in rat cardiac transplantation: allosensitization in the spleen and hepatic nodes.
Transplantation
71
:
1806
.-1815.
27
Shapiro, A. M., J. R. Lakey, E. A. Ryan, G. S. Korbutt, E. Toth, G. L. Warnock, N. M. Kneteman, R. V. Rajotte.
2000
. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.
N. Engl. J. Med.
343
:
230
.-238.
28
Mattsson, G., L. Jansson, A. Nordin, A. Andersson, P. O. Carlsson.
2004
. Evidence of functional impairment of syngeneically transplanted mouse pancreatic islets retrieved from the liver.
Diabetes
53
:
948
.-954.
29
Andersson, A., P. O. Carlsson, C. Carlsson, R. Olsson, A. Nordin, M. Johansson, F. Palm, B. Tyrberg, O. Kallskog, L. Tillmar, et al
2004
. Promoting islet cell function after transplantation.
Cell Biochem. Biophys.
40
:
55
.-64.
30
Lafferty, K. J., J. Woolnough.
1977
. The origin and mechanism of the allograft reaction.
Immunol. Rev.
35
:
231
.-262.
31
Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, H. Nakano.
1999
. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.
J. Exp. Med.
189
:
451
.-460.
32
Luther, S. A., H. L. Tang, P. L. Hyman, A. G. Farr, J. G. Cyster.
2000
. Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse.
Proc. Natl. Acad. Sci. USA
97
:
12694
.-12699.
33
Muller, D. N., E. M. A. Mervaala, R. Dechend, A. Fiebeler, J. K. Park, F. Schmidt, J. Theuer, V. Breu, N. Mackman, T. Luther, et al
2000
. Angiotensin ii (at(1)) receptor blockade reduces vascular tissue factor in angiotensin ii-induced cardiac vasculopathy.
Am. J. Pathol.
157
:
111
.-122.
34
Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp.
1999
. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.
Cell
99
:
23
.-33.
35
Rappert, A., K. Biber, C. Nolte, M. Lipp, A. Schubel, B. Lu, N. P. Gerard, C. Gerard, H. W. Boddeke, H. Kettenmann.
2002
. Secondary lymphoid tissue chemokine (CCL21) activates CXCR3 to trigger a Cl current and chemotaxis in murine microglia.
J. Immunol.
168
:
3221
.-3226.
36
Baker, M. S., X. Chen, A. R. Rotramel, J. J. Nelson, B. Lu, C. Gerard, Y. Kanwar, D. B. Kaufman.
2003
. Genetic deletion of chemokine receptor CXCR3 or antibody blockade of its ligand IP-10 modulates posttransplantation graft-site lymphocytic infiltrates and prolongs functional graft survival in pancreatic islet allograft recipients.
Surgery
134
:
126
.-133.
37
Gosling, J., D. J. Dairaghi, Y. Wang, M. Hanley, D. Talbot, Z. Miao, T. J. Schall.
2000
. Cutting edge: identification of a novel chemokine receptor that binds dendritic cell- and T cell-active chemokines including ELC, SLC, and TECK.
J. Immunol.
164
:
2851
.-2856.
38
Townson, J. R., R. J. Nibbs.
2002
. Characterization of mouse CCX-CKR, a receptor for the lymphocyte-attracting chemokines TECK/mCCL25, SLC/mCCL21 and MIP-3β/mCCL19: comparison to human CCX-CKR.
Eur. J. Immunol.
32
:
1230
.-1241.
39
Comerford, I., R. J. Nibbs.
2005
. Post-translational control of chemokines: a role for decoy receptors?.
Immunol. Lett.
96
:
163
.-174.
40
Locati, M., Y. M. Torre, E. Galliera, R. Bonecchi, G. Vago, A. Vecchi, and A. Mantovani. 2005. Silent chemoattractant receptors: D6 as a decoy and scavenger receptor for inflammatory CC chemokines. Cytokine Growth Factor Rev. In press.
41
Hopken, U. E., J. Droese, J. P. Li, J. Joergensen, D. Breitfeld, H. G. Zerwes, M. Lipp.
2004
. The chemokine receptor CCR7 controls lymph node-dependent cytotoxic T cell priming in alloimmune responses.
Eur. J. Immunol.
34
:
461
.-470.
42
Beckmann, J. H., S. Yan, H. Luhrs, B. Heid, S. Skubich, R. Forster, M. W. Hoffmann.
2004
. Prolongation of allograft survival in CCR7-deficient mice.
Transplantation
77
:
1809
.-1814.
43
Colvin, B. L., Z. Wang, H. Nakano, W. Wu, T. Kakiuchi, R. L. Fairchild, A. W. Thomson.
2005
. CXCL9 antagonism further extends prolonged cardiac allograft survival in CCL19/CCL21-deficient mice.
Am. J. Transplant.
5
:
2104
.-2113.
44
Fainaru, O., D. Shseyov, S. Hantisteanu, Y. Groner.
2005
. Accelerated chemokine receptor 7-mediated dendritic cell migration in Runx3 knockout mice and the spontaneous development of asthma-like disease.
Proc. Natl. Acad. Sci. USA
102
:
10598
.-10603.
45
Hammad, H., H. J. de Heer, T. Soullie, V. Angeli, F. Trottein, H. C. Hoogsteden, B. N. Lambrecht.
2004
. Activation of peroxisome proliferator-activated receptor-γ in dendritic cells inhibits the development of eosinophilic airway inflammation in a mouse model of asthma.
Am. J. Pathol.
164
:
263
.-271.
46
First, M. R., W. E. Fitzsimmons.
2004
. New drugs to improve transplant outcomes.
Transplantation
77
:
S88
.-S92.
47
Chen, X., T. Murakami, J. J. Oppenheim, Z. Howard.
2005
. Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of denritic cell maturation and trafficking.
Blood
106
:
2409
.-2416.