Role of CXC chemokine ligand 13, CC chemokine ligand (CCL) 19, and CCL21 in the organization and function of nasal-associated lymphoid tissue

Nasal-associated lymphoid tissue (NALT) orchestrates immune responses to Ags in the upper respiratory tract. Unlike other lymphoid organs, NALT develops independently of lymphotoxin-alpha (LTalpha). However, the structure and function of NALT are impaired in Ltalpha(-/-) mice, suggesting a link between LTalpha and chemokine expression. In this study we show that the expression of CXCL13, CCL19, CCL21, and CCL20 is impaired in the NALT of Ltalpha(-/-) mice. We also show that the NALT of Cxcl13(-/-) and plt/plt mice exhibits some, but not all, of the structural and functional defects observed in the NALT of Ltalpha(-/-) mice. Like the NALT of Ltalpha(-/-) mice, the NALT in Cxcl13(-/-) mice lacks follicular dendritic cells, BP3(+) stromal cells, and ERTR7(+) lymphoreticular cells. However, unlike the NALT of Ltalpha(-/-) mice, the NALT of Cxcl13(-/-) mice has peripheral node addressin(+) high endothelial venules (HEVs). In contrast, the NALT of plt/plt mice is nearly normal, with follicular dendritic cells, BP3(+) stromal cells, ERTR7(+) lymphoreticular cells, and peripheral node addressin(+) HEVs. Functionally, germinal center formation and switching to IgA are defective in the NALT of Ltalpha(-/-) and Cxcl13(-/-) mice. In contrast, CD8 T cell responses to influenza are impaired in Ltalpha(-/-) mice and plt/plt mice. Finally, the B and T cell defects in the NALT of Ltalpha(-/-) mice lead to delayed clearance of influenza from the nasal mucosa. Thus, the B and T cell defects in the NALT of Ltalpha(-/-) mice can be attributed to the impaired expression of CXCL13 and CCL19/CCL21, respectively, whereas impaired HEV development is directly due to the loss of LTalpha.     

EBV-induced molecule 1 ligand chemokine (ELC/CCL19) promotes IFN-gamma-dependent antitumor responses in a lung cancer model

The antitumor efficacy of EBV-induced molecule 1 ligand CC chemokine (ELC/CCL19) was evaluated in a murine lung cancer model. The ability of ELC/CCL19 to chemoattract both dendritic cells and T lymphocytes formed the rationale for this study. Compared with diluent-treated tumor-bearing mice, intratumoral injection of recombinant ELC/CCL19 led to significant systemic reduction in tumor volumes (p < 0.01). ELC/CCL19-treated mice exhibited an increased influx of CD4 and CD8 T cell subsets as well as dendritic cells at the tumor sites. These cell infiltrates were accompanied by increases in IFN-gamma, MIG/CXCL9, IP-10/CXCL10, GM-CSF, and IL-12 but a concomitant decrease in the immunosuppressive molecules PGE(2) and TGFbeta. Transfer of T lymphocytes from ELC/CCL19 treated tumor-bearing mice conferred the antitumor therapeutic efficacy of ELC/CCL19 to naive mice. ELC/CCL19 treated tumor-bearing mice showed enhanced frequency of tumor specific T lymphocytes secreting IFN-gamma. In vivo depletion of IFN-gamma, MIG/CXCL9, or IP-10/CXCL10 significantly reduced the antitumor efficacy of ELC/CCL19. These findings provide a strong rationale for further evaluation of ELC/CCL19 in tumor immunity and its use in cancer immunotherapy.     

Cutting edge: JAM-C controls homeostatic chemokine secretion in lymph node fibroblastic reticular cells expressing thrombomodulin

The development and maintenance of secondary lymphoid organs, such as lymph nodes, occur in a highly coordinated manner involving lymphoid chemokine production by stromal cells. Although developmental pathways inducing lymphoid chemokine production during organogenesis are known, signals maintaining cytokine production in adults are still elusive. In this study, we show that thrombomodulin and platelet-derived growth factor receptor α identify a population of fibroblastic reticular cells in which chemokine secretion is controlled by JAM-C. We demonstrate that Jam-C-deficient mice and mice treated with Ab against JAM-C present significant decreases in stromal cell-derived factor 1α (CXCL12), CCL21, and CCL19 intranodal content. This effect is correlated with reduced naive T cell egress from lymph nodes of anti-JAM-C-treated mice.     

Cutting edge: ectopic expression of the chemokine TCA4/SLC is sufficient to trigger lymphoid neogenesis

To test whether accumulation of naive lymphocytes is sufficient to trigger lymphoid development, we generated mice with islet expression of the chemokine TCA4/SLC. This chemokine is specific for naive lymphocytes and mature dendritic cells (DC) which express the CCR7 receptor. Islets initially developed accumulations of T cells with DC, with scattered B cells at the perimeter. These infiltrates consolidated into organized lymphoid tissue, with high endothelial venules and stromal reticulum. Infiltrate lymphocytes showed a naive CD44low CD25- CD69- phenotype, though half were CD62L negative. When backcrossed to RAG-1 knockout, DC were not recruited. Interestingly, islet lymphoid tissue developed in backcrosses to Ikaros knockout mice despite the absence of normal peripheral nodes. Our results indicate that TCA4/SLC can induce the development and organization of lymphoid tissue through diffential recruitment of T and B lymphocytes and secondary effects on stromal cell development.     

Altered antibody production and helper T cell function in mice lacking chemokines CCL19 and CCL21-Ser

The roles of chemokines CCL19 and CCL21 in Ab production were investigated using plt mutant mice, which lack expression of CCL19 and CCL21-ser in their lymphoid organs. In these mice, the Th response has been shown to tend towards the Th1 type because of accumulation of inflammatory dendritic cells. When plt mice were immunized with 100 μg OVA in CFA, the number of Ab-forming cells in the draining LN, and serum concentrations of OVA-specific IgM and IgG Ab, were very close to those of the control, yet IgG2a Ab in plt mice was increased. In vitro IFN-γ production by the draining LN cells of plt mice was increased. In addition, the ability of helper T cells from plt mice to stimulate Ab production in vitro was prolonged. Also, in the plt mice, in vivo challenge with OVA in incomplete Freund's adjuvant elicited a stronger IgG2a response and a weaker IgG1 response, which is suggestive of a Th1-dominant response. Similar findings were obtained when mice were immunized with 100 μg OVA in alum, except that with alum the increases observed in plt mice were IgG1 produced in vivo and IL-4 produced in vitro by draining LN cells. Furthermore, immunization with alum adjuvant also induced a prolonged in vitro recall response of IFN-γ and IL-4. These findings indicate that plt mice mount an anti-OVA Ab response, and suggest that CCL19 and CCL21 induce prompt Ab responses to antigen, and negatively regulate helper T cell responses in vivo.     

Loss of dendritic cell migration and impaired resistance to Leishmania donovani infection in mice deficient in CCL19 and CCL21

The encounter between APC and T cells is crucial for initiating immune responses to infectious microorganisms. In the spleen, interaction between dendritic cells (DC) and T cells occurs in the periarteriolar lymphoid sheath (PALS) into which DC and T cells migrate from the marginal zone (MZ) along chemokine gradients. However, the importance of DC migration from the MZ into the PALS for immune responses and host resistance to microbial infection has not yet been elucidated. In this study, we report that following Leishmania donovani infection of mice, the migration of splenic DC is regulated by the CCR7 ligands CCL19/CCL21. DC in plt/plt mutant mice that lack these chemokines are less activated and produce less IL-12, compared with those in wild-type mice. Similar findings are seen when mice are treated with pertussis toxin, which blocks chemokine signaling in vivo. plt/plt mice had increased susceptibility to L. donovani infection compared with wild-type mice, as determined by spleen and liver parasite burden. Analysis of splenic cytokine profiles at day 14 postinfection demonstrated that IFN-gamma and IL-4 mRNA accumulation was comparable in wild-type and plt/plt mice. In contrast, accumulation of mRNA for IL-10 was elevated in plt/plt mice. In addition, plt/plt mice mounted a delayed hepatic granulomatous response and fewer effector T cells migrated into the liver. Taken together, we conclude that DC migration from the MZ to the PALS is necessary for full activation of DC and the optimal induction of protective immunity against L. donovani.     

Naive T cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.     

Initiation of cellular organization in lymph nodes is regulated by non-B cell-derived signals and is not dependent on CXC chemokine ligand 13

The molecular and cellular events that initiate the formation of T and B cell areas in developing lymph nodes are poorly understood. In this study we show that formation of the lymphoid architecture in murine neonatal lymph nodes evolves through a series of distinct stages. The initial segregation of T and B cells is regulated in a CXCL13-independent manner, characterized by the localization of B cells in a ring-like pattern in the outer cortex on day 4. However, during this CXCL13-independent phase of lymph node modeling, CXCL13 is expressed and regulated in a lymphotoxin-alpha1beta2 (LTalpha1beta2)-dependent manner. Surprisingly, neonatal B cells are unable to respond to this chemokine and also lack surface LTalpha1beta2 expression. At this time, CD45+CD4+CD3- cells are the predominant LTalpha1beta2-expressing cells and are also capable of responding to CXCL13. From day 4 on, architectural changes become CXCL13 dependent, and B cells become fully CXCL13 responsive, express LTalpha1beta2, and cluster in anatomically distinct follicles. Because the initial induction of CXCL13 is dependent on LTalpha1beta2, a role for CD45+CD4+CD3- cells in inducing chemokine expression in the developing lymph nodes is proposed and, as such, a role in initiation of the shaping of the microenvironment.     

Regulatory role of lymphoid chemokine CCL19 and CCL21 in the control of allergic rhinitis

The lymphoid chemokines CCL19 and CCL21 are known to be crucial both for lymphoid cell trafficking and for the structural organization of lymphoid tissues such as nasopharynx-associated lymphoid tissue (NALT). However, their role in allergic responses remains unclear, and so our current study aims to shed light on the role of CCL19/CCL21 in the development of allergic rhinitis. After nasal challenge with OVA, OVA-sensitized plt (paucity of lymph node T cells) mice, which are deficient in CCL19/CCL21, showed more severe allergic symptoms than did identically treated wild-type mice. OVA-specific IgE production, eosinophil infiltration, and Th2 responses were enhanced in the upper airway of plt mice. Moreover, in plt mice, the number of CD4(+)CD25(+) regulatory T cells declined in the secondary lymphoid tissues, whereas the number of Th2-inducer-type CD8alpha(-)CD11b(+) myeloid dendritic cells (m-DCs) increased in cervical lymph nodes and NALT. Nasal administration of the plasmid-encoding DNA of CCL19 resulted in the reduction of m-DCs in the secondary lymphoid tissues and the suppression of allergic responses in plt mice. These results suggest that CCL19/CCL21 act as regulatory chemokines for the control of airway allergic disease and so may offer a new strategy for the control of allergic disease.     

Lymphotoxin-alpha 1 beta 2 and LIGHT induce classical and noncanonical NF-kappa B-dependent proinflammatory gene expression in vascular endothelial cells

Activation of the classical and noncanonical NF-kappaB pathways by ligation of the lymphotoxin (LT)-beta receptor (LTbetaR) plays a crucial role in lymphoid organogenesis and in the generation of ectopic lymphoid tissue at sites of chronic inflammation. Within these microenvironments, LTbetaR signaling regulates the phenotype of the specialized high endothelial cells. However, the direct effects of LTbetaR ligation on endothelial cells remain unclear. We therefore questioned whether LTbetaR ligation could directly activate endothelial cells and regulate classical and noncanonical NF-kappaB-dependent gene expression. We demonstrate that the LTbetaR ligands LIGHT and LTalpha1beta2 activate both NF-kappaB pathways in HUVECs and human dermal microvascular endothelial cells (HDMEC). Classical pathway activation was less robust than TNF-induced signaling; however, only LIGHT and LTalpha1beta2 and not TNF activated the noncanonical pathway. LIGHT and LTalpha1beta2 induced the expression of classical NF-kappaB-dependent genes in HUVEC, including those encoding the adhesion molecules E-selectin, ICAM-1, and VCAM-1. Consistent with this stimulation, LTbetaR ligation up-regulated T cell adhesion to HUVEC. Furthermore, the homeostatic chemokine CXCL12 was up-regulated by LIGHT and LTalpha1beta2 but not TNF in both HUVEC and HDMEC. Using HUVEC retrovirally transduced with dominant negative IkappaB kinase alpha, we demonstrate that CXCL12 expression is regulated by the noncanonical pathway in endothelial cells. Our findings therefore demonstrate that LTbetaR ligation regulates gene expression in endothelial cells via both NF-kappaB pathways and we identify CXCL12 as a bona fide noncanonical NF-kappaB-regulated gene in these cells.     

Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.     

Sphingosine 1-phosphate receptors regulate chemokine-driven transendothelial migration of lymph node but not splenic T cells

Chemokines and chemokine receptors are required for T cell trafficking and migration. Recent evidence shows that sphingosine 1-phosphate (S1P) and S1PRs are also important for some aspects of T cell migration, but how these two important receptor-ligand systems are integrated and coregulated is not known. In this study, we have investigated CCL19-CCR7 and CXCL12-CXCR4-driven migration of both splenic and peripheral lymph node (PLN) nonactivated and naive T cells, and used both S1P and the S1PR ligand, FTY720, to probe these interactions. The results demonstrate that splenic T cell migration to CCL19 or CXCL12 is enhanced by, but does not require, S1PR stimulation. In contrast, PLN T cell migration to CXCL12, but not CCL19, requires both chemokine and S1PR stimulation, and the requirement for dual receptor stimulation is particularly important for steps involving transendothelial migration. The results also demonstrate that: 1) splenic and PLN nonactivated and naive T cells use different molecular migration mechanisms; 2) CCR7 and CXCR4 stimulation engage different migration mechanisms; and 3) S1P and FTY720 have distinct S1PR agonist and antagonist properties. The results have important implications for understanding naive T cell entry into and egress from peripheral lymphoid organs, and we present a model for how S1P and chemokine receptor signaling may be integrated within a T cell.     

Homeostatic lymphoid chemokines synergize with adhesion ligands to trigger T and B lymphocyte chemokinesis

Homeostatic chemokines such as CCL19, CCL21, and CXCL13 are known to elicit chemotaxis from naive T and B cells and play a critical role in lymphocyte homing to appropriate zones within secondary lymphoid organs (SLO). Here we tested whether CCL21 and CXCL13 modulate murine lymphocyte motility in the absence of concentration gradients, using videomicroscopy to directly observe the migration of single cells. CCL21 treatment of T cells induced rapid polarization and sustained random migration with average speeds of 5.16 +/- 2.08 microm/min; B cell migration (average velocity 4.10 +/- 1.58 microm/min) was similarly induced by CXCL13. Migration required the presence of both chemokine and adhesion ligands and was sustained for >24 h. Furthermore, in in vitro assays modeling the relative infrequency of Ag-specific T cell-dendritic cell (DC) encounters during primary immune responses, we found that CCL21 addition to T-DC cocultures accelerated the kinetics of CD69 up-regulation and enhanced by 2-fold the proliferation of Ag-specific T cells in a manner dependent on G-protein-coupled receptor signaling in T cells. These results suggest that homeostatic chemokines could substantially impact the dynamics and priming of lymphocytes within SLO even in the absence of significant concentration gradients.     

A novel model for lymphocytic infiltration of the thyroid gland generated by transgenic expression of the CC chemokine CCL21

Lymphocytic infiltrates and lymphoid follicles with germinal centers are often detected in autoimmune thyroid disease (AITD), but the mechanisms underlying lymphocyte entry and organization in the thyroid remain unknown. We tested the hypothesis that CCL21, a chemokine that regulates homeostatic lymphocyte trafficking, and whose expression has been detected in AITD, is involved in the migration of lymphocytes to the thyroid. We show that transgenic mice expressing CCL21 from the thyroglobulin promoter (TGCCL21 mice) have significant lymphocytic infiltrates, which are topologically segregated into B and T cell areas. Although high endothelial venules expressing peripheral lymph node addressin were frequently observed in the thyroid tissue, lymphocyte recruitment was independent of L-selectin or lymphotoxin-alpha but required CCR7 expression. Taken together, these results indicate that CCL21 is sufficient to drive lymphocyte recruitment to the thyroid, suggest that CCL21 is involved in AITD pathogenesis, and establish TGCCL21 transgenic mice as a novel model to study the formation and function of lymphoid follicles in the thyroid.     

Involvement of CCL18 in allergic asthma

Allergic asthma is associated with a pulmonary recruitment of Th type 2 cells, basophils, and eosinophils, mainly linked to chemokine production. CCL18 is a chemokine preferentially expressed in the lung, secreted by APCs, induced by Th2-type cytokines, and only present in humans. Therefore, CCL18 may be involved in allergic asthma. PBMC from asthmatics allergic to house dust mite cultured in the presence of Dermatophagoides pteronyssinus 1 (Der p 1) allergen secreted CCL18, 48 and 72 h after stimulation, whereas those from healthy donors did not. Part of CCL18 was directly derived from Der p 1-stimulated plasmacytoid dendritic cells, whereas the other part was linked to monocyte activation by IL-4 and IL-13 produced by Der p 1-stimulated T cells. In bronchoalveolar lavages from untreated asthmatic allergic patients, CCL18 was highly increased compared with controls. Functionally, CCL18 preferentially attracted in vitro-polarized Th2 cells and basophils, but not eosinophils and Th1 cells, and induced basophil histamine and intracellular calcium release. These data show a new function for CCL18, i.e., the recruitment of Th2 cells and basophils, and suggest that CCL18 may play a predominant role in allergic asthma.     

Dopamine selectively induces migration and homing of naive CD8+ T cells via dopamine receptor D3

The nervous systems affect immune functions by releasing neurohormones and neurotransmitters. A neurotransmitter dopamine signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. The secondary lymphoid tissues are highly innervated by sympathetic nerve fibers that store dopamine at high contents. Lymphocytes also produce dopamine. In this study, we examined expression and function of dopamine receptors in lymphocytes. We found that D3 was the predominant subtype of dopamine receptors in the secondary lymphoid tissues and selectively expressed by naive CD8+ T cells of both humans and mice. Dopamine induced calcium flux and chemotaxis in mouse L1.2 cells stably expressing human D3. These responses were almost completely inhibited by pertussis toxin, indicating that D3 was coupled with the Galphai class of G proteins. Consistently, dopamine selectively induced chemotactic responses in naive CD8+ T cells of both humans and mice in a manner sensitive to pertussis toxin and D3 antagonists. Dopamine was highly synergistic with CCL19, CCL21, and CXCL12 in induction of chemotaxis in naive CD8+ T cells. Dopamine selectively induced adhesion of naive CD8+ T cells to fibronectin and ICAM-1 through activation of integrins. Intraperitoneal injection of mice with dopamine selectively attracted naive CD8+ T cells into the peritoneal cavity. Treatment of mice with a D3 antagonist U-99194A selectively reduced homing of naive CD8+ T cells into lymph nodes. Collectively, naive CD8+ T cells selectively express D3 in both humans and mice, and dopamine plays a significant role in migration and homing of naive CD8+ T cells via D3.     

Novel CCL21-vault nanocapsule intratumoral delivery inhibits lung cancer growth

Background

Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer.

Principal Findings

Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3⁺T, CCR7⁺T, IFNγ⁺T lymphocytes, DEC205⁺ DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells.

Significance

This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an “off the shelf” approach to deliver antitumor cytokines to treat a broad range of malignancies.     

Low CXCL13 expression, splenic lymphoid tissue atrophy and germinal center disruption in severe canine visceral leishmaniasis

Visceral leishmaniasis is associated with atrophy and histological disorganization of splenic compartments. In this paper, we compared organized and disorganized splenic lymphoid tissue from dogs naturally infected with Leishmania infantum assessing the size of the white pulp compartments, the distribution of T, B and S100⁺ dendritic cells, using immunohistochemistry and morphometry and the expression of CCR7 and the cytokines, CXCL13, lymphotoxin (LT)-α, LT-β, CCL19, CCL21, TNF-α, IL-10, IFN-γ and TGF-β, using by real time RT-PCR. The lymphoid follicles and marginal zones were smaller (3.2 and 1.9 times, respectively; Mann-Whitney, P<0.02) in animals with disorganized splenic tissue in comparison to those with organized splenic lymphoid tissue. In spleens with disorganized lymphoid tissue, the numbers of T cells and S100⁺ dendritic cells were decreased in the follicles, and the numbers of B cells were reduced in both the follicles and marginal zones. CXCL13 mRNA expression was lower in animals with disorganized lymphoid tissue (0.5±0.4) compared to those with organized lymphoid tissue (2.7±2.9, both relative to 18S expression, P = 0.01). These changes in the spleen were associated with higher frequency of severe disease (7/12) in the animals with disorganized than in animals with organized (2/13, Chi-square, P = 0.01) splenic lymphoid tissue. The data presented herein suggest that natural infection with Leishmania infantum is associated with the impairment of follicular dendritic cells, CXCL13 expression, B cell migration and germinal center formation and associates these changes with severe clinical forms of visceral leishmaniasis. Furthermore the fact that this work uses dogs naturally infected with Leishmania infantum emphasizes the relevance of the data presented herein for the knowledge on the canine and human visceral leishmaniasis.     

Permanent survival of fully MHC-mismatched islet allografts by targeting a single chemokine receptor pathway

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 approximately 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.