The Role of IL-17 and Related Cytokines in Inflammatory Autoimmune Diseases.

Interleukin-17 (IL-17) induces the production of granulocyte colony-stimulating factor (G-CSF) and chemokines such as CXCL1 and CXCL2 and is a cytokine that acts as an inflammation mediator. During infection, IL-17 is needed to eliminate extracellular bacteria and fungi, by inducing antimicrobial peptides such as defensin. This cytokine also plays an important role in chronic inflammation that occurs during the pathogenesis of autoimmune diseases and allergies such as human rheumatoid arthritis (RA) for which a mouse model of collagen-induced arthritis (CIA) is available. In autoimmune diseases such as RA and multiple sclerosis (MS), IL-17 is produced by helper T (Th) cells that are stimulated by IL-1ß and IL-6 derived from phagocytes such as macrophages and from tissue cells. IL-17 contributes to various lesions that are produced by Th17 cells, one subset of helper T cells, and by γδ T cells and innate lymphoid cells. It strongly contributes to autoimmune diseases that are accompanied by chronic inflammation. Thus, a functional understanding of Th17 cells is extremely important. In this review, we highlight the roles of cytokines that promote the development and maintenance of pathogenic Th17 cells in autoimmune diseases.

Uterine natural killer cells severely decrease in number at gestation day 6 in mice.

Uterine natural killer (uNK) cells remarkably increase in number after implantation. NK cells or their precursors migrate from the blood stream and contribute to the increase. However, the contribution of uNK cells present in the virgin uterus has been unclear. To elucidate this issue, we examined uterine leukocyte subsets during pregnancy in BALB/c mice. The most dramatic change was the massive decrease in CD11b⁻ or Gr-1⁻ cells at gestation day (gd) 6. Uterine NK cells at gd 0 were CD11b⁻, and severely decreased at gd 6. The decrease was selective, and the proportion of other cells examined did not decrease. Uterine NK cells almost recovered at gd 12. These cells at gd 12 were more mature and/or activated in terms of expression of CD11b, CD27, CD127, or B220 than at gd 0. CXCL12 expression was observed on uterine cells at gd 0 or 6, but not at gd 12, whereas CXCR4 was detected on uNK cells at gds 0 and 12. A much higher expression of IL-15 in uterine cells or interferon-gamma expression in uNK cells was observed at gd 12 than at gd 0. IL-15 receptor alpha chain was detected on uNK cells at gd 12, but not at gd 0. Taken together, these findings were consistent with our interpretation that uNK cells present at gd 0 do not contribute to the increase of uNK cell number after implantation, and NK cells or their precursors migrate into the uterus, mature, and produce interferon-gamma to support pregnancy.

CCR7 ligands up-regulate IL-23 through PI3-kinase and NF-κ B pathway in dendritic cells.

We reported previously that the production of IL-23 is impaired in DCs from mice that lack expression of the chemokines CCL19 and CCL21, which share the receptor CCR7, suggesting that these chemokines are required for IL-23 expression. However, the molecular mechanism of CCR7-mediated IL-23 production in DCs is unknown. We found that CCL19 and CCL21 stimulated DCs through CCR7 and induced transcription of IL-23p19 mRNA and IL-23 production in splenic and BMDC. Stimulation of DCs with CCR7 ligands induced phosphorylation of MAPK family members and of Akt, but only a specific PI3K inhibitor, LY294002, not inhibitors of ERK, JNK, or p38, decreased IL-23p19 transcription and IL-23 production. In DCs stimulated with CCL19 or CCL21, I κ B α was degraded, and NF-κ B was translocated into the nucleus. Prevention of NF-κ B activation blocked chemokine-mediated IL-23p19 transcription. A PI3K inhibitor abolished NF-κ B activation and IL-23 production. Based on these findings, we concluded that PI3K and NF-κ B signaling pathways play a critical role in CCR7-mediated IL-23 production in murine DCs. As IL-23 contributes to Th17 cell generation, and Th17 cells are pathogenic in autoimmune diseases, precise elucidation of these mechanisms would contribute to the development of strategies to control autoimmune diseases.

[Mechanism of alcohol consumption-mediated Th2-polarized immune response].

Alcohol consumption impairs Th1-mediated cellular immune responses and enhances serum IgE levels. It has been reported that the elevated IgE levels are associated with a Th2 polarization response, but the mechanisms for enhancing Th2 polarization by the ethanol treatment remain to be elucidated. The aim of this review is to present and discuss the mechanism of Th2 polarization response by alcohol. IL-12 production by APCs such as monocytes, macrophages, and dendritic cells (DCs) preferentially leads to Th1 polarization. Acute ethanol consumption results in a significant decrease in IL-12 production in LPS-stimulated DCs and a CD40/CD40L interaction between CD40 on the DCs and CD40 ligand expressed on activated T cells. This suggests that Th2 polarization by ethanol is caused by impaired IL-12 production from APCs. In contrast, the induction of IL-10 by LPS is enhanced by ethanol treatment, suggesting that elevated IL-10 may play a role in ethanol-induced suppression of IL-12. However, ethanol inhibited IL-12 production in LPS-stimulated DCs devoid of IL-10 (IL-10/DC), suggesting that down-regulation of IL-12 by ethanol is independent of the IL-10 levels. Furthermore, several studies report that PGE2, cAMP and linolic acid, and endogenous lipid mediators released in inflammatory conditions, also inhibit IL-12 production. These inhibitory effects are similar to the IL-12 inhibition by ethanol. In addition, increase in the levels of these lipid mediators is induced by ethanol treatment. Alternatively, cytokine signaling studies indicate that IL-12 production by DCs is negatively regulated by PI3K and GSK-3, but positively regulated by p38 MAPK, mTOR, and NF-kappa B. Thus, it seems possible that ethanol may interact on the upstream of IL-12 producing a signal pathway. In fact, ethanol alters the stability of cell membrane, and suppresses clustering of TLR4 and recruitment of signaling molecules into lipid rafts, where it associates with the Ser/Thr kinase and the adaptor proteins, and forms a signaling complex. Down-regulation of lipid raft signaling is results in the impaired IL-12 production leading to the Th1 polarization, and causes CD4+ T cells to differentiation toward the Th2 lineage.

Novel ribbon-type nuclear factor of activated T cells decoy oligodeoxynucleotides preclude airways hyperreactivity and Th2 cytokine expression in experimental asthma.

BACKGROUND: Nuclear factor of activated T cells (NFAT) is required for the differentiation of Th2 responses, so we examined its role in mouse experimental asthma and tested the hypothesis that an NFAT blockade with a decoy against NFAT can prevent asthma progression. OBJECTIVE: To determine the effects of the NFAT decoy oligodeoxynucleotides (ODNs) on the development of airway inflammation, we designed a novel ribbon-type ODN containing two binding sites for NFAT in a single decoy molecule without an open end, which is more stable than a conventional decoy, and largely preserved its structural integrity in the presence of nucleases. METHODS: Ribbon-type NFAT decoy ODNs were transfected into ovalbumin (OVA)-sensitized CD3+ T cells in vitro. OVA-immunized mice received these cells by intraperitoneal injection. Airway hyperreactivity (AHR) was measured and the transfected CD3+ T cells' responses to the airways were characterized. RESULTS: Development of AHR after OVA challenge was effectively abolished after adoptive transfer of ribbon-type NFAT decoy ODN transfected CD3+ T cells. Transfer of ribbon-type decoy significantly reduced the number of inflammatory cells and the concentrations of IL-4, IL-5 and IL-13, but not IFN-γ, in the bronchoalveolar lavage of the recipient mice. CONCLUSION: These results suggest the inhibitory effect of ribbon-type decoy ODNs against NFAT on the induction of bronchial asthma. Adoptively transferred CD3+ T cells, which are transfected with NFAT decoy, may be an effective strategy for the treatment of asthma.

Liposome-coupled antigens are internalized by antigen-presenting cells via pinocytosis and cross-presented to CD8 T cells.

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen-presenting cells (APCs) to CD8+ T cells, and that this process resulted in the induction of antigen-specific cytotoxic T lymphocytes. In the present study, the mechanism by which the liposome-coupled antigens were cross-presented to CD8+ T cells by APCs was investigated. Confocal laser scanning microscopic analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-based liposomes received processing at both MHC class I and class II compartments, while most of the antigens coupled to the surface of saturated-fatty-acid-based liposomes received processing at the class II compartment. In addition, flow cytometric analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-liposomes were taken up by APCs even in a 4°C environment; this was not true of saturated-fatty-acid-liposomes. When two kinds of inhibitors, dimethylamiloride (DMA) and cytochalasin B, which inhibit pinocytosis and phagocytosis by APCs, respectively, were added to the culture of APCs prior to the antigen pulse, DMA but not cytochalasin B significantly reduced uptake of liposome-coupled antigens. Further analysis of intracellular trafficking of liposomal antigens using confocal laser scanning microscopy revealed that a portion of liposome-coupled antigens taken up by APCs were delivered to the lysosome compartment. In agreement with the reduction of antigen uptake by APCs, antigen presentation by APCs was significantly inhibited by DMA, and resulted in the reduction of IFN-γ production by antigen-specific CD8+ T cells. These results suggest that antigens coupled to the surface of liposomes consisting of unsaturated fatty acids might be pinocytosed by APCs, loaded onto the class I MHC processing pathway, and presented to CD8+ T cells. Thus, these liposome-coupled antigens are expected to be applicable for the development of vaccines that induce cellular immunity.

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.

Liposome-coupled peptides induce long-lived memory CD8 T cells without CD4 T cells.

CD8(+) T cells provide broad immunity to viruses, because they are able to recognize all types of viral proteins. Therefore, the development of vaccines capable of inducing long-lived memory CD8(+) T cells is desired to prevent diseases, especially those for which no vaccines currently exist. However, in designing CD8(+) T cell vaccines, the role of CD4(+) T cells in the induction and maintenance of memory CD8(+) T cells remains uncertain. In the present study, the necessity or not of CD4(+) T cells in the induction and maintenance of memory CD8(+) T cells was investigated in mice immunized with liposome-coupled CTL epitope peptides. When OVA-derived CTL epitope peptides were chemically coupled to the surfaces of liposomes and inoculated into mice, both primary and secondary CTL responses were successfully induced. The results were further confirmed in CD4(+) T cell-eliminated mice, suggesting that CD4(+) T cells were not required for the generation of memory CD8(+) T cells in the case of immunization with liposome-coupled peptides. Thus, surface-linked liposomal antigens, capable of inducing long-lived memory CD8(+) T cells without the contribution of CD4(+) T cells, might be applicable for the development of vaccines to prevent viral infection, especially for those viruses that evade humoral immunity by varying their surface proteins, such as influenza viruses, HIV, HCV, SARS coronaviruses, and Ebola viruses.

CCR7 ligands are required for development of experimental autoimmune encephalomyelitis through generating IL-23-dependent Th17 cells.

CCL19 and CCL21 are thought to be critical for experimental autoimmune encephalomyelitis (EAE) induction, but their precise role is unknown. We examined the role of these chemokines in inducing EAE. C57BL/6 mice lacking expression of these chemokines (plt/plt mice) or their receptor CCR7 were resistant to EAE induced with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55)) and pertussis toxin. However, passive transfer of pathogenic T cells from wild-type mice induced EAE in plt/plt mice, suggesting a defect independent of the role of CCR7 ligands in the migration of immune cells. Examination of draining lymph node (DLN) cells from MOG(35-55)-immunized plt/plt mice found decreased IL-23 and IL-12 production by plt/plt dendritic cells (DCs) and a concomitant defect in Th17 cell and Th1 cell generation. In contrast, production of the Th17 lineage commitment factors IL-6 and TGF-beta were unaffected by loss of CCR7 ligands. The adoptive transfer of in vitro-generated Th17 cells from DLN cells of MOG(35-55)-immunized plt/plt mice developed EAE in wild-type recipient mice, whereas that of Th1 cells did not. Pathogenic Th17 cell generation was restored in plt/plt DLNs with the addition of exogenous IL-23 or CCL19/CCL21 and could be reversed by inclusion of anti-IL-23 mAb in cultures. Exogenous CCL19/CCL21 induced IL-23p19 expression and IL-23 production by plt/plt or wild-type DCs. Therefore, CCR7 ligands have a novel function in stimulating DCs to produce IL-23 and are important in the IL-23-dependent generation of pathogenic Th17 cells in EAE induction.

Blood-derived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1 immune responses.

T helper type 1 (T(H)1)-polarized immune responses, which confer protection against intracellular pathogens, are thought to be initiated by dendritic cells (DCs) that enter lymph nodes from peripheral tissues. Here we found after viral infection or immunization, inflammatory monocytes were recruited into lymph nodes directly from the blood to become CD11c(+)CD11b(hi)Gr-1(+) inflammatory DCs, which produced abundant interleukin 12p70 and potently stimulated T(H)1 responses. This monocyte extravasation required the chemokine receptor CCR2 but not the chemokine CCL2 or receptor CCR7. Thus, the accumulation of inflammatory DCs and T(H)1 responses were much lower in Ccr2(-/-) mice, were preserved in Ccl2(-/-) mice and were relatively higher in CCL19-CCL21-Ser-deficient plt mutant mice, in which all other lymph node DC types were fewer in number. We conclude that blood-derived inflammatory DCs are important in the development of T(H)1 immune responses.

Adiponectin stimulates IL-8 production by rheumatoid synovial fibroblasts.

The adipokines are linked not only to metabolic regulation, but also to immune responses. Adiponectin, but not leptin or resistin induced interleukin-8 production from rheumatoid synovial fibroblasts (RSF). The culture supernatant of RSF treated with adiponectin induced chemotaxis, although adiponectin itself had no such effect. Addition of antibody against adiponectin, and inhibition of adiponectin receptor gene decreased adiponectin-induced IL-8 production. Nuclear translocation of nuclear factor-kappa B was increased by adiponectin. The induction of interleukin-8 was inhibited by mitogen-activated protein kinase inhibitors. These findings suggest that adiponectin contributes to the pathogenesis of rheumatoid arthritis.

Experimental scrapie in 'plt' mice: an assessment of the role of dendritic-cell migration in the pathogenesis of prion diseases.

Peripherally acquired transmissible spongiform encephalopathies display strikingly long incubation periods, during which increasing amounts of prions can be detected in lymphoid tissues. While precise sites of peripheral accumulation have been described, the mechanisms of prion transport from mucosa and skin to lymphoid and nervous tissues remain unknown. Because of unique functional abilities, dendritic cells (DCs) have been suspected to participate in prion pathogenesis. In mice inoculated subcutaneously with scrapie-infected DCs, the incubation was shorter when cells were alive as compared with killed cells, suggesting that DC functions may facilitate prion neuroinvasion. However, early propagation in lymphoid tissues seemed not importantly affected by DC vitality. Mutant (plt) mice that have deficient CCL19/CCL21 expression and DC migration displayed similar infection of secondary lymphoid organs as normal mice, regardless of the route of inoculation and scrapie strain. Under certain conditions of transcutaneous inoculation, the incubation and duration of disease were moderately prolonged in plt mice. This was not related to a milder neuropathogenesis, since plt and normal mice were equally susceptible to intracerebral prion challenge. We conclude that peripheral spreading of prions appears poorly dependent on cell migration through the chemokine/receptor system CCL19/CCL21/CCR7, although DCs might be able to help prions reach sites of neuroinvasion.

Enhancement of IFN-gamma production for Th1-cell therapy using negatively charged liposomes containing phosphatidylserine.

We have developed an efficient method of interferon-gamma (IFN-gamma) induction for Th1-cell therapy. OVA (ovalbumin)-specific Th1 clone 42-6A cells cocultured with antigen presenting cells (APCs) from spleen resulted in high levels of OVA-specific IFN-gamma production by the treatment of phosphatidylserine (PS), but not phosphatidic acid (PA), liposomes-encapsulated OVA (OVA-liposomes). The IFN-gamma production was increased in a manner dependent on the PS content of the liposomes and inhibited by the addition of annexin V, a PS binding protein. Furthermore, coadministration of Th1 cells plus OVA-liposomes in mice strikingly enhanced IFN-gamma levels in serum and in spleen cells compared with that of Th1 cells plus OVA. In addition, serum levels of IL-12 p70 increased and ongoing OVA-specific IgE immune response was dramatically attenuated. These results first suggest that antigen delivery using negatively charged liposomes containing PS is very useful for the enhancement of IFN-gamma production in Th1-cell therapy.

Chemokines CCL19 and CCL21 promote activation-induced cell death of antigen-responding T cells.

Secondary lymphoid organs (SLOs) provide a niche for the initiation and regulation of T-cell responses, but the mechanisms have been poorly understood. We investigated the influence of chemokines CCL19 and CCL21 constitutively expressed in SLOs on activation-induced cell death (AICD) of CD4+ T cells. When paucity of lymph node T cells (plt) mutant mice lacking expression of CCL19/CCL21 were primed with OVA/CFA, both expansion of OVA-responding CD4+ T cells in the draining lymph nodes and an in vitro recall response were prolonged as compared with responses in wild-type (WT) mice. The apoptotic cell frequency among OVA-responding CD4+ T cells was similarly low in plt/plt and WT mice during the clonal expansion phase. However, during the clonal contraction phase, the frequency never increased in plt/plt mice, whereas in WT mice it continuously increased to a peak 18 days after immunization. The presence of CCL19/CCL21 during the in vitro stimulation of CD4+ T cells with anti-CD3 plus anti-CD28 significantly enhanced in vitro AICD induction of the restimulated T cells, partially through enhancing expression of Fas ligand. Our results suggest that CCL19/CCL21 produced by stromal cells and antigen-presenting cells regulate CD4+ T-cell immune responses in SLOs by promoting AICD.

Inhibition of MHC class II expression and immune responses by c-MIR.

We previously reported a novel E3 ubiquitin ligase (E3), designated as c-MIR, which targets B7-2 to lysosomal degradation and down-regulates the B7-2 surface expression through ubiquitination of its cytoplasmic tail. B7-2 is well known as a costimulatory molecule for Ag presentation, suggesting that the manipulation of c-MIR expression modulates immune responses in vivo. To examine this hypothesis, we generated genetically modified mice in which c-MIR was expressed under an invariant chain (Ii) promoter. Dendritic cells derived from genetically engineered mice showed low ability to present Ags. In addition, these mice showed resistance to the onset of experimental autoimmune encephalomyelitis and an impaired development of CD4 T cells in the thymus and the periphery. These findings led us to conclude that MHC class II (MHC II) is an additional target for c-MIR. Indeed, forced expression of c-MIR in several B cell lines down-regulated the surface expression of MHC II, and down-regulation was found to depend on the presence of a single lysine residue in the cytoplasmic tail of the I-A beta-chain. In a reconstitution system using 293T cells, we found that the lysine residue at position 225 in the I-A beta-chain was ubiquitinated by c-MIR. To our knowledge, c-MIR is the first example of an E3 that is capable of inhibiting MHC II expression. Our findings suggest that c-MIR might potently regulate immune responses in vivo.

Role of CCL21 and CCL19 in allergic inflammation in the ovalbumin-specific murine asthmatic model.

BACKGROUND: Dendritic cells are the most powerful of the antigen-presenting cells and are known to play important roles in sensitization and inflammation in allergen-specific asthma. Various cytokines and chemokines are involved in the maturation and activation of dendritic cells. Among them is CC chemokine ligand (CCL)21, a key chemokine in the entry of naive T cells and antigen-stimulated dendritic cells into the T-cell zones of secondary lymphoid organs, which is a critical process in antigen-specific T-cell activation. OBJECTIVE: We studied the role of CCL21 in airway inflammation in asthma by using BALB/c-plt/plt (plt) mice, which possess genetic defects in expression of both CCL21 and CCL19. METHODS: Plt and control BALB/c mice were immunized with ovalbumin and alum 4 times and thereafter were subjected to a 2-week regimen of ovalbumin inhalation. RESULTS: In plt mice, ovalbumin-specific IgE response was delayed compared with control BALB/c mice, but they had the same level of response after final immunization. Although airway inflammation and response to acetylcholine were significantly reduced compared with BALB/c mice, significant eosinophilic inflammation and hyperresponsiveness were also observed in plt mice after 2 weeks of inhalation. Four weeks after cessation of inhalation, airway inflammation and hyperresponsiveness in plt mice were greater than in BALB/c mice. At the time of resolution of airway inflammation, IL-10 production was enhanced in BALB/c mice but not in plt mice. CONCLUSION: The chemokines CCL21 and CCL19 were critical for resolution of airway inflammation. CLINICAL IMPLICATIONS: The findings about the chemokines for induction and resolution of inflammation are key to establishing a new strategy for asthma immunotherapy.

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.

Negatively charged phospholipids suppress IFN-gamma production in T cells.

The effect of phospholipids on IFN-gamma production in mouse T cells was investigated. Phosphatidylserine (PS), which has a negatively charged head group, completely inhibited IFN-gamma production in splenic naïve T cells and antigen-dependent IFN-gamma production in Th1 clone 42-6A cells, whereas other phospholipids, which have neutrally charged head group, had no effect. The structural requirements for IFN-gamma inhibitory effects by PS were investigated, and dimyristoyl-PS (C14: 0) and dipalmitoyl-PS (C16: 0) had no effect on IFN-gamma production, and interestingly, distearoyl-PS (18: 0) increased IFN-gamma production. Dioleoyl-PS (C18: 1), dilinoleoyl-PS (C18: 2), and oleoyl-lyso-PS (C18: 1) completely inhibited IFN-gamma production. To clarify this mechanism, we focused on the stability of IFN-gamma mRNA, and the treatment of splenic naïve T cells with PS brought about 40% reductions in IFN-gamma mRNA expression in the presence of actinomycin D. Collectively, IFN-gamma inhibitory effects by PS are highly dependent on the molecular structure of PS and involve the decreasing of the stability of IFN-gamma mRNA.

CXCL9 antagonism further extends prolonged cardiac allograft survival in CCL19/CCL21-deficient mice.

CCL19/MIP-3beta and CCL21/SLC are essential for chemotactic recruitment of mature dendritic cells (DC) to T-cell areas of secondary lymphoid tissue. Paucity of lymph node T-cells (plt/plt) mice lack CCL21-serine (ser) and CCL19 expression. We tested plt/plt and wild type (wt) BALB/c (H2d) mice as recipients of heart or skin allografts from C57BL/10J (H2b) donors. Donor DC trafficking to secondary lymphoid tissue was markedly reduced in plt heart but not skin allograft recipients. Heart, but not skin grafts survived significantly longer in plt recipients. Accordingly, T cells from plt heart transplant recipients demonstrated poor anti-donor responses in ex vivo MLR, compared to wt heart or wt and plt skin recipients. Moreover, donor-reactive T cells from plt heart recipients exhibited Th2-skewing in comparison to T cells from wt heart or skin graft recipients. Anti-CXCL9/Mig was administered for 2 weeks post-transplant to determine whether impairment of activated T-cell migration could further prolong cardiac allograft survival in plt recipients. CXCL9-antagonism extended graft survival significantly only in plt mice, likely due, in part, to retention of alloactivated T cells in secondary lymphoid tissue/reduction of graft-infiltrating T cells. Thus, targeting DC and activated T-cell migration concomitantly has additive effects in prolonging heart graft survival with potential for therapeutic application.

The role of CCL21 in recruitment of T-precursor cells to fetal thymi.

During embryonic development, T-lymphoid precursor cells colonize the thymus. Chemoattraction by the fetal thymus is thought to mediate T-precursor cell colonization. However, the molecules that attract T-precursor cells to the thymus remain unclear. By devising time-lapse visualization in culture, the present results show that alymphoid fetal thymus lobes attract T-precursor cells from fetal liver or fetal blood. CD4(-)CD8(-)CD25(-)CD44+ fetal thymocytes retained the activity to specifically re-enter the thymus. The attraction was predominantly due to I-A-expressing thymic epithelial cells and was mediated by pertussis toxin-sensitive G-protein signals. Among the chemokines produced by the fetal thymus, CCL21, CCL25, and CXCL12 could attract CD4(-)CD8(-)CD25(-)CD44+ fetal thymocytes. However, fetal thymus colonization was markedly diminished by neutralizing antibodies specific for CCL21 and CCL25, but not affected by anti-CXCL12 antibody. Fetal thymus colonization was partially defective in CCL21-deficient plt/plt mice and was further diminished by anti-CCL25 antibody. These results indicate that CCL21 is involved in the recruitment of T-cell precursors to the fetal thymus and suggest that the combination of CCL21 and CCL25 plays a major role in fetal thymus colonization.