Transcriptional and Clonal Characterization of Cytotoxic T Cells in Crescentic Glomerulonephritis.

SIGNIFICANCE STATEMENT: T-cell infiltration is a hallmark of crescentic GN (cGN), often caused by ANCA-associated vasculitis. Pathogenic T-cell subsets, their clonality, and downstream effector mechanisms leading to kidney injury remain to be fully elucidated. Single-cell RNA sequencing and T-cell receptor sequencing revealed activated, clonally expanded cytotoxic CD4 + and CD8 + T cells in kidneys from patients with ANCA-associated cGN. In experimental cGN, kidney-infiltrating CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), which induced apoptosis in renal tissue cells by activation of procaspase-3, and aggravated disease pathology. These findings describe a pathogenic function of (clonally expanded) cytotoxic T cells in cGN and identify GzmB as a mediator and potential therapeutic target in immune-mediated kidney disease. BACKGROUND: Crescentic GN (cGN) is an aggressive form of immune-mediated kidney disease that is an important cause of end stage renal failure. Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis is a common cause. T cells infiltrate the kidney in cGN, but their precise role in autoimmunity is not known. METHODS: Combined single-cell RNA sequencing and single-cell T-cell receptor sequencing were conducted on CD3 + T cells isolated from renal biopsies and blood of patients with ANCA-associated cGN and from kidneys of mice with experimental cGN. Functional and histopathological analyses were performed with Cd8a-/- and GzmB-/- mice. RESULTS: Single-cell analyses identified activated, clonally expanded CD8 + and CD4 + T cells with a cytotoxic gene expression profile in the kidneys of patients with ANCA-associated cGN. Clonally expanded CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), in the mouse model of cGN. Deficiency of CD8 + T cells or GzmB ameliorated the course of cGN. CD8 + T cells promoted macrophage infiltration and GzmB activated procaspase-3 in renal tissue cells, thereby increasing kidney injury. CONCLUSIONS: Clonally expanded cytotoxic T cells have a pathogenic function in immune-mediated kidney disease.

Antigen Cross-Presentation by Murine Proximal Tubular Epithelial Cells Induces Cytotoxic and Inflammatory CD8+ T Cells.

Immune-mediated glomerular diseases are characterized by infiltration of T cells, which accumulate in the periglomerular space and tubulointerstitium in close contact to proximal and distal tubuli. Recent studies described proximal tubular epithelial cells (PTECs) as renal non-professional antigen-presenting cells that stimulate CD4+ T-cell activation. Whether PTECs have the potential to induce activation of CD8+ T cells is less clear. In this study, we aimed to investigate the capacity of PTECs for antigen cross-presentation thereby modulating CD8+ T-cell responses. We showed that PTECs expressed proteins associated with cross-presentation, internalized soluble antigen via mannose receptor-mediated endocytosis, and generated antigenic peptides by proteasomal degradation. PTECs induced an antigen-dependent CD8+ T-cell activation in the presence of soluble antigen in vitro. PTEC-activated CD8+ T cells expressed granzyme B, and exerted a cytotoxic function by killing target cells. In murine lupus nephritis, CD8+ T cells localized in close contact to proximal tubuli. We determined enhanced apoptosis in tubular cells and particularly PTECs up-regulated expression of cleaved caspase-3. Interestingly, induction of apoptosis in the inflamed kidney was reduced in the absence of CD8+ T cells. Thus, PTECs have the capacity for antigen cross-presentation thereby inducing cytotoxic CD8+ T cells in vitro, which may contribute to the pathology of immune-mediated glomerulonephritis.

IL-10 Receptor Signaling Empowers Regulatory T Cells to Control Th17 Responses and Protect from GN.

Background Th17 cells are central pathogenic mediators of autoimmune disease, including many forms of GN. IL-10 receptor signaling (IL-10R) in regulatory T cells (Tregs) has been implicated in the downregulation of Th17 cells, but the underlying molecular mechanisms and functional relevance of this process remain unclear.Methods We generated mice with Treg-specific IL-10Ra deficiency and subjected these mice to nephrotoxic serum-induced nephritis as a model of crescentic GN. Immune responses and Treg phenotypes were extensively analyzed.Results Compared with controls, mice with IL-10Ra-/- Tregs showed a spontaneously overshooting Th17 immune response. This hyper-Th17 phenotype was further boosted during GN and associated with aggravated renal injury. Notably, abrogation of IL-10Ra signaling in Tregs increased dendritic cell activation and production of Th17-inducing cytokines. In contrast, Treg trafficking and expression of chemokine receptor CCR6 remained unaffected, indicating mechanisms of Th17 control, differing from those of previously identified CCR6+ Treg17 cells. Indeed, the capacity for direct in vitro suppression of Th17 responses by IL-10Ra-/- Tregs was significantly impaired. As underlying pathology, analyses conducted in vitro and in vivo using double-fluorescent reporter mice revealed strikingly decreased IL-10 production by IL-10Ra-/- Tregs. To assess, whether reduced IL-10 could explain the hyper Th17 phenotype, competitive cotransfer experiments were performed. Supporting our concept, IL-10Ra-/- T cells differentiated into Th17 cells at much higher frequencies than wild type T cells did during GN.Conclusions IL-10R engagement optimizes Treg-mediated suppression of Th17 immunity. We hypothesize a feed-forward loop, in which IL-10Ra signaling reinforces IL-10 secretion by Tregs which potently controls Th17 development via direct and indirect mechanisms. IL-10R thus may be a promising therapeutic target for the treatment of GN.

Carcinoembryonic antigen-related cell adhesion molecule 1 controls IL-2-dependent regulatory T-cell induction in immune-mediated hepatitis in mice.

A dysbalance between effector T cells (Tconv) and regulatory T cells (Tregs) and impaired Treg function can cause autoimmune liver disease. Therefore, it is important to identify molecular mechanisms that control Treg homeostasis. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a) is an immune coreceptor with dichotomous roles in T-cell regulation: its short isoform (CEACAM1S) can activate T cells and induce Tregs, whereas its long isoform (CEACAM1L), containing two intracellular immune receptor tyrosine-based inhibitory motifs, can inhibit activated T-cell function. In the liver, CEACAM1 has antifibrotic effects in models of nonalcoholic steatohepatitis. However, its role in immune-mediated hepatitis is unknown. In the mouse model of concanavalin A-induced CD4+ T-cell-dependent liver injury, liver damage was aggravated and persisted in Ceacam1-/- mice. Concomitantly, we observed hyperexpansion of Tconv, but reduction of interleukin (IL)-2 production and hepatic forkhead box protein P3+ (Foxp3+ )CD4+ Treg numbers. CEACAM1-/- CD4+ T cells showed impaired IL-2-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation, which correlated with a failure of naïve CEACAM1-/- CD4+ T cells to convert into Tregs in vitro. Furthermore, CEACAM1-/- Tregs expressed reduced levels of Foxp3, CD25, and B-cell lymphoma 2. Adoptive transfer experiments demonstrated that hepatic Treg expansion and suppressive activity required CEACAM1 expression on both CD4+ T cells and Tregs. We identified predominant CEACAM1S expression on hepatic CD4+ T cells and Tregs from mice with acute liver injury and expression of both isoforms in liver-derived CD4+ T-cell clones from patients with liver injury. CONCLUSION: Our data suggest that CEACAM1S expression in CD4+ T cells augments IL-2 production and STAT5 phosphorylation leading to enhanced Treg induction and stability, which, ultimately, confers protection from T-cell-mediated liver injury. (Hepatology 2018;68:200-214).

A Protective Function of IL-22BP in Ischemia Reperfusion and Acetaminophen-Induced Liver Injury.

Acute liver injury can be secondary to a variety of causes, including infections, intoxication, and ischemia. All of these insults induce hepatocyte death and subsequent inflammation, which can make acute liver injury a life-threatening event. IL-22 is a dual natured cytokine which has context-dependent protective and pathogenic properties during tissue damage. Accordingly, IL-22 was shown to promote liver regeneration upon acute liver damage. However, other studies suggest pathogenic properties of IL-22 during chronic liver injury. IL-22 binding protein (IL-22BP, IL-22Ra2) is a soluble inhibitor of IL-22 that regulates IL-22 activity. However, the significance of endogenous IL-22BP in acute liver injury is unknown. We hypothesized that IL-22BP may play a role in acute liver injury. To test this hypothesis, we used Il22bp-deficient mice and murine models of acute liver damage induced by ischemia reperfusion and N-acetyl-p-aminophenol (acetaminophen) administration. We found that Il22bp-deficient mice were more susceptible to acute liver damage in both models. We used Il22 × Il22bp double-deficient mice to show that this effect is indeed due to uncontrolled IL-22 activity. We could demonstrate mechanistically increased expression of Cxcl10 by hepatocytes, and consequently increased infiltration of inflammatory CD11b+Ly6C+ monocytes into the liver in Il22bp-deficient mice upon liver damage. Accordingly, neutralization of CXCL10 reversed the increased disease susceptibility of Il22bp-deficient mice. In conclusion, our data indicate that IL-22BP plays a protective role in acute liver damage, via controlling IL-22-induced Cxcl10 expression.

A Proinflammatory Role of Type 2 Innate Lymphoid Cells in Murine Immune-Mediated Hepatitis.

Type 2 innate lymphoid cells (ILC2) mediate inflammatory immune responses in the context of diseases triggered by the alarmin IL-33. In recent years, IL-33 has been implicated in the pathogenesis of immune-mediated liver diseases. However, the immunoregulatory function of ILC2s in the inflamed liver remains elusive. Using the murine model of Con A-induced immune-mediated hepatitis, we showed that selective expansion of ILC2s in the liver was associated with highly elevated hepatic IL-33 expression, severe liver inflammation, and infiltration of eosinophils. CD4+ T cell-mediated tissue damage and subsequent IL-33 release were responsible for the activation of hepatic ILC2s that produced the type 2 cytokines IL-5 and IL-13 during liver inflammation. Interestingly, ILC2 depletion correlated with less severe hepatitis and reduced accumulation of eosinophils in the liver, whereas adoptive transfer of hepatic ILC2s aggravated liver inflammation and tissue damage. We further showed that, despite expansion of hepatic ILC2s, 3-d IL-33 treatment before Con A challenge potently suppressed development of immune-mediated hepatitis. We found that IL-33 not only activated hepatic ILC2s but also expanded CD4+ Foxp3+ regulatory T cells (Treg) expressing the IL-33 receptor ST2 in the liver. This Treg subset also accumulated in the liver during resolution of immune-mediated hepatitis. In summary, hepatic ILC2s are poised to respond to the release of IL-33 upon liver tissue damage through expression of type 2 cytokines thereby participating in the pathogenesis of immune-mediated hepatitis. Inflammatory activity of ILC2s might be regulated by IL-33-elicited ST2+ Tregs that also arise in immune-mediated hepatitis.

T-Bet Enhances Regulatory T Cell Fitness and Directs Control of Th1 Responses in Crescentic GN.

Th1 cells are central pathogenic mediators of crescentic GN (cGN). Mechanisms responsible for Th1 cell downregulation, however, remain widely unknown. Recently, it was proposed that activation of the Th1-characteristic transcription factor T-bet optimizes Foxp3+ regulatory T (Treg) cells to counteract Th1-type inflammation. Because very little is known about the role of T-bet+ Treg1 cells in inflammatory diseases, we studied the function of these cells in the nephrotoxic nephritis (NTN) model of cGN. The percentage of Treg1 cells progressively increased in kidneys of nephritic wild-type mice during the course of NTN, indicating their functional importance. Notably, naïve Foxp3CrexT-betfl/fl mice, lacking Treg1 cells, showed spontaneous skewing toward Th1 immunity. Furthermore, absence of Treg1 cells resulted in aggravated NTN with selectively dysregulated renal and systemic Th1 responses. Detailed analyses of Treg cells from Foxp3CrexT-betfl/fl mice revealed unaltered cytokine production and suppressive capacity. However, in competitive cotransfer experiments, wild-type Treg cells outcompeted T-bet-deficient Treg cells in terms of population expansion and expression levels of Foxp3, indicating that T-bet expression is crucial for general Treg fitness. Additionally, T-bet-deficient Treg cells lacked expression of the Th1-characteristic trafficking receptor CXCR3, which correlated with significant impairment of renal Treg infiltration. In summary, our data indicate a new subtype of Treg cells in cGN. These Treg1 cells are characterized by activation of the transcription factor T-bet, which enhances the overall fitness of these cells and optimizes their capacity to downregulate Th1 responses by inducing chemokine receptor CXCR3 expression.

The Limonoids TS3 and Rubescin E Induce Apoptosis in Human Hepatoma Cell Lines and Interfere with NF-κB Signaling.

Hepatocellular carcinoma (HCC) is extremely resistant towards pharmacological therapy. To date, the multi-kinase inhibitor Sorafenib is the only available therapeutic agent with the potential to prolong patient survival. Using the human hepatoma cell lines HepG2 and Huh7, we analyzed anti-cancer activities of 6 purified havanensin type limonoids isolated from the traditional African medicinal plant Trichilia rubescens Oliv. Our results show that two of the compounds, TR4 (TS3) and TR9 (Rubescin E) reduced hepatoma cell viability, but not primary hepatocyte viability, at TC50s of 5 to 10 µM. These were significantly lower than the TC50s for Sorafenib, the histone deacetylase inhibitor SAHA or 5-Fluoruracil. In comparison, TR3 (Rubescin D), a limonoid isolated in parallel and structurally highly similar to TR4 and TR9, did not interfere with hepatoma cell viability. Both, TR4 and TR9, but not TR3, induced apoptosis in hepatoma cells and interfered with NF-κB activation. TR4 as well as TR9 significantly supported anti-cancer activities of Sorafenib. In summary, the limonoids TR4 and TR9 exhibit anti-cancer activities and support Sorafenib effects in vitro, having the potential to support future HCC therapy.

CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN.

Chemokines and chemokine receptors are implicated in regulatory T cell (Treg) trafficking to sites of inflammation and suppression of excessive immune responses in inflammatory and autoimmune diseases; however, the specific requirements for Treg migration into the inflamed organs and the positioning of these cells within the tissue are incompletely understood. Here, we report that Tregs expressing the TH1-associated chemokine receptor CXCR3 are enriched in the kidneys of patients with ANCA-associated crescentic GN and colocalize with CXCR3(+) effector T cells. To investigate the functional role of CXCR3(+) Tregs, we generated mice that lack CXCR3 in Tregs specifically (Foxp3(eGFP-Cre) × Cxcr3(fl/fl)) and induced experimental crescentic GN. Treg-specific deletion of CXCR3 resulted in reduced Treg recruitment to the kidney and an overwhelming TH1 immune response, with an aggravated course of the nephritis that was reversible on anti-IFNγ treatment. Together, these findings show that a subset of Tregs expresses CXCR3 and thereby, acquires trafficking properties of pathogenic CXCR3(+) TH1 cells, allowing Treg localization and control of excessive TH1 responses at sites of inflammation.

Treg17 cells are programmed by Stat3 to suppress Th17 responses in systemic lupus.

Systemic lupus erythematosus (SLE) is a complex and potentially fatal autoimmune disorder. Although Th17 cells are thought to be central mediators of SLE, mechanisms underlying their counter regulation remain largely unknown. To help define this, we studied the function of the newly defined Stat3-dependent Th17-specific regulatory T cells (Treg17). Treg-specific deletion of Stat3 was achieved by generating Foxp3(Cre) × Stat3(fl/fl) mice and SLE was induced by intraperitoneal injection of pristane. Lack of Treg17 cells in these mice caused selectively enhanced peritoneal Th17 inflammation. Importantly, Treg17 deficiency also resulted in aggravated pulmonary vasculitis with increased percentages of Th17 cells and significantly higher mortality. Similarly, 4 and 9 months after pristane injection, analysis of renal and systemic immunity showed overshooting Th17 responses in the absence of Treg17 cells, associated with the aggravation of lupus nephritis. Expression of the Th17 characteristic trafficking receptor CCR6 was strikingly reduced on Tregs of Foxp3(Cre) × Stat3(fl/fl) mice, resulting in impaired renal Treg infiltration. Thus, Stat3-induced Treg17 cells are novel antiinflammatory mediators of SLE. One mechanism enabling Treg17 cells to target pathogenic Th17 responses is shared expression of the chemokine receptor CCR6.

RORγt(+)Foxp3(+) Cells are an Independent Bifunctional Regulatory T Cell Lineage and Mediate Crescentic GN.

Cells expressing both the regulatory T cell (Treg)-inducing transcription factor Foxp3 and the Th17 transcription factor RORγt have been identified (biTregs). It is unclear whether RORγt(+)Foxp3(+) biTregs belong to the Th17-specific Treg17 cells, represent intermediates during Treg/Th17 transdifferentiation, or constitute a distinct cell lineage. Because the role of biTregs in inflammatory renal disease is also unknown, we studied these cells in the nephrotoxic nephritis (NTN) model of acute crescentic GN. Induction of NTN resulted in rapid renal and systemic expansion of biTregs. Notably, analyses of the biTreg expression profile revealed production of both anti-inflammatory (IL-10, IL-35) and proinflammatory (IL-17) cytokines. Additionally, biTregs expressed a signature of surface molecules and transcription factors distinct from those of Th17 cells and conventional Tregs (cTregs), and biTregs were identified in Treg17-deficient mice. Finally, fate reporter and cell transfer studies confirmed that biTregs are not Treg/Th17 transdifferentiating cells. Therapeutic transfer of biTregs suppressed the development of nephritis to an extent similar to that observed with transferred cTregs, but in vitro studies indicated different mechanisms of immunosuppression for biTregs and cTregs. Intriguingely, as predicted from their cytokine profile, endogenous biTregs displayed additional proinflammatory functions in NTN that were abrogated by cell-specific deletion of RORγt. In summary, we provide evidence that RORγt(+)Foxp3(+) biTregs are a novel and independent bifunctional regulatory T cell lineage distinct from cTregs, Treg17 cells, and Th17 cells. Furthermore, biTregs appear to contribute to crescentic GN and hence may be novel therapeutic targets.

Testosterone suppresses hepatic inflammation by the downregulation of IL-17, CXCL-9, and CXCL-10 in a mouse model of experimental acute cholangitis.

Autoimmune liver diseases predominantly affect women. In this study, we aimed to elucidate how sex affects autoimmune hepatic inflammation. Acute experimental cholangitis was induced by adoptive transfer of OVA-specific CD8(+) T cells into mice, which express the cognate Ag on cholangiocytes. In contrast to previous mouse models of cholangitis, this model displayed a strong sexual dimorphism: female mice developed marked cholangitis, whereas male mice were resistant to cholangitis induction. The recruitment of endogenous CD4(+) T cells, but not transferred CD8(+) T cells into female livers was strongly increased. These cells expressed higher amounts of the proinflammatory cytokine IL-17, which was at least in part responsible for the liver inflammation observed. The recruitment of endogenous CD4(+) T cells was associated with increased expression of the chemokines CXCL-9 and CXCL-10 in female livers. The sex-specific factor responsible for the observed differences was found to be testosterone: male mice could be rendered susceptible to liver inflammation by castration, and testosterone treatment was sufficient to completely suppress liver inflammation in female mice. Accordingly, testosterone treatment of female mice significantly reduced the expression of IL-17A, CXCL-9, and CXCL-10 within the liver. Serum testosterone levels of untreated mice negatively correlated with the IL-17, CXCL-9, and CXCL-10 expression in the liver, further supporting a role for testosterone in hepatic immune homeostasis. In conclusion, testosterone was found to be the major determinant of the observed sexual dimorphism. Further study into the role of testosterone for liver inflammation could lead to novel treatment targets in human autoimmune liver diseases.

CEACAM1 confers resistance toward oxygen-induced vessel damage in a mouse model of retinopathy of prematurity.

PURPOSE: To determine a functional role for the carcinoembryonic antigen-related cell-adhesion molecule 1 (CEACAM1) in retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). METHODS: In a 21/75/21% OIR mouse model, retinal neovascularization was compared in wild-type and CEACAM1-deficient mice. Animals were housed under normoxic conditions until postnatal day 7, followed by exposure to 75% oxygen for 5 days, and further housing under normoxic conditions. Retinal vascular anatomy, vaso-obliteration, neovascularization, and tuft formation were characterized and quantified in retinal flat-mounts from untreated mice and from experimental mice during and at different time points after exposure to high oxygen levels. The vascular network was stained with fluorescently labeled isolectin B4. RESULTS: Mice deficient in CEACAM1 did not present any apparent abnormalities in their postnatal retinal vascular development under normoxic housing conditions. However, after hyperoxia and under relative hypoxic conditions, retinal neovascularization and tuft formation were aggravated in the mutant. Congruently, revascularization and vessel maturation were delayed in CEACAM1-deficient mice whereas in wild-type mice, tuft regression and vascular remodeling occurred efficiently after exposure to high oxygen levels. CONCLUSIONS: Our report describes a functional role for CEACAM1 in retinal neovascularization in a mouse model of OIR. This is the first study demonstrating that CEACAM1 enhances vascular remodeling and tuft regression by increasing endothelial resistance to alterations in oxygen tension, thus accelerating vascular recovery after systemic hypoxia.

Stat3 programs Th17-specific regulatory T cells to control GN.

A pathogenic role for Th17 cells in inflammatory renal disease is well established. The mechanisms underlying their counter-regulation are, however, largely unknown. Recently, Th17 lineage-specific regulatory T cells (Treg17) that depend on activation of the transcription factor Stat3 were identified. We studied the function of Treg17 in the nephrotoxic nephritis (NTN) model of crescentic GN. The absence of Treg17 cells in Foxp3(Cre)×Stat3(fl/fl) mice resulted in the aggravation of NTN and skewing of renal and systemic immune responses toward Th17. Detailed analysis of Stat3-deficient Tregs revealed that the survival, activation, proliferation, and suppressive function of these cells remained intact. However, Tregs from Foxp3(Cre)×Stat3(fl/fl) mice lacked surface expression of the chemokine receptor CCR6, which resulted in impaired renal trafficking. Furthermore, aggravation of NTN was reversible in the absence of Th17 responses, as shown in CD4(Cre)×Stat3(fl/fl) mice lacking both Treg17 and Th17 cells, suggesting that Th17 cells are indeed the major target of Treg17 cells. Notably, immunohistochemistry revealed CCR6-bearing Treg17 cells in kidney biopsy specimens of patients with GN. CCR6 expression on human Treg17 cells also appears dependent on STAT3, as shown by analysis of Tregs from patients with dominant-negative STAT3 mutations. Our data indicate the presence and involvement of Stat3/STAT3-dependent Treg17 cells that specifically target Th17 cells in murine and human crescentic GN, and suggest the kidney-specific action of these Treg17 cells is regulated by CCR6-directed migration into areas of Th17 inflammation.

Regulatory T cell-derived IL-10 ameliorates crescentic GN.

Regulatory T cells (Tregs) exert their immunosuppressive activity through several immunoregulatory mechanisms, including the production of anti-inflammatory cytokines such as IL-10. Although several studies suggest a role for Tregs in modulating crescentic GN, the underlying mechanisms are not well understood. Here, using IL-10 reporter mice, we detected IL-10-producing Foxp3(+) T cells in the kidney, blood, and secondary lymphoid tissue in a mouse model of crescentic GN. Specific inactivation of Il10 in Foxp3(+) Tregs eliminated the ability of these cells to suppress renal and systemic production of IFNγ and IL-17; these IL-10-deficient Tregs lost their capacity to attenuate renal tissue injury. These data highlight the suppressive functions of Tregs in crescentic GN and suggest the importance of Treg-derived IL-10 in ameliorating disease severity and in modulating both the Th1 and most notably Th17 immune response.

Immature renal dendritic cells recruit regulatory CXCR6(+) invariant natural killer T cells to attenuate crescentic GN.

Immature renal dendritic cells (DCs) are protective early in murine crescentic GN, but the mechanisms underlying this protection are unknown. Here, depletion of DCs reduced the recruitment of invariant natural killer T (iNKT) cells, which attenuate GN, into the kidney in the early stage of experimental crescentic GN. More than 90% of renal iNKT cells expressed the chemokine receptor CXCR6, and renal DCs produced high amounts of the cognate ligand CXCL16 early after induction of nephritis, suggesting that renal DC-derived CXCL16 might attract protective CXCR6(+) iNKT cells. Consistent with this finding, CXCR6-deficient mice exhibited less iNKT cell recruitment and developed nephritis that was more severe, similar to the aggravated nephritis observed in mice depleted of immature DCs. Finally, adoptive transfer of CXCR6-competent NKT cells ameliorated nephritis. Taken together, these results suggest an immunoprotective mechanism involving immature DCs, CXCL16, CXCR6, and regulatory iNKT cells, which might stimulate the development of new therapeutic strategies for GN.

A novel BAT3 sequence generated by alternative RNA splicing of exon 11B displays cell type-specific expression and impacts on subcellular localization.

BACKGROUND: The human lymphocyte antigen (HLA) encoded BAT3/BAG6 recently attracted interest as a regulator of protein targeting and degradation, a function that could be exerted in the cytosol and in the nucleus. The BAT3 gene was described to consist of 25 exons. Diversity of transcripts can be generated by alternative RNA splicing, which may control subcellular distribution of BAT3. METHODOLOGY/PRINCIPAL FINDINGS: By cDNA sequencing we identified a novel alternatively spliced sequence of the BAT3 gene located between exons 11 and 12, which was designated as exon 11B. Using PCR and colony hybridization we identified six cDNA variants, which were produced by RNA splicing of BAT3 exons 5, 11B and 24. In four examined cell types the content of BAT3 splice variants was examined. Most of the cDNA clones from monocyte-derived dendritic cells contain exon 11B, whereas this sequence was almost absent in the B lymphoma Raji. Exon 5 was detected in most and exon 24 in approximately half of the cDNA clones. The subcellular distribution of endogenous BAT3 largely correlates with a cell type specific splicing pattern. In cells transfected with BAT3 variants, full-length and Δ24 BAT3 displayed nearly exclusive nuclear staining, whereas variants deleted of exon 11B showed substantial cytosolic expression. We show here that BAT3 is mainly expressed in the cytosol of Raji cells, while other cell types displayed both cytosolic and nuclear staining. Export of BAT3 from the nucleus to the cytosol is inhibited by treatment with leptomycin B, indicating that the Crm1 pathway is involved. Nuclear expression of BAT3 containing exon 11B suggests that this sequence plays a role for nuclear retention of the protein. CONCLUSIONS/SIGNIFICANCE: Cell type-specific subcellular expression of BAT3 suggests distinct functions in the cytosol and in the nucleus. Differential expression of BAT3 variants may reconcile the multiple roles described for BAT3.

CXCR3 deficiency exacerbates liver disease and abrogates tolerance in a mouse model of immune-mediated hepatitis.

The chemokine receptor CXCR3 is preferentially expressed by Th1 cells and critically involved in their recruitment to inflamed tissue. In a mouse model of immune-mediated liver injury inducible by Con A, we investigated the role of CXCR3 in acute IFN-γ-mediated hepatitis as well as in tolerance induction, which has been shown to depend on IL-10-producing CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Induction of Con A hepatitis resulted in increased intrahepatic expression of the CXCR3 ligands CXCL9, CXCL10, and CXCL11. CXCR3(-/-) mice developed a more severe liver injury with higher plasma transaminase activities and a more pronounced Th1/Th17 response compared with wild-type (wt) animals upon Con A injection. Moreover, CXCR3(-/-) mice did not establish tolerance upon Con A restimulation, although Tregs from CXCR3(-/-) mice were still suppressive in an in vitro suppression assay. Instead, Tregs failed to accumulate in livers of CXCR3(-/-) mice upon Con A restimulation in contrast to those from wt animals. Con A-tolerant wt mice harbored significantly increased numbers of intrahepatic CXCR3(+)T-bet(+) Tregs that produced IL-10 compared with nontolerant animals. IFN-γ deficiency or anti-IFN-γ Ab treatment demonstrated that conversion to CXCR3(+)T-bet(+) Tregs depended on a Th1 response. Accordingly, in an immunotherapeutic approach, CD4(+)CD25(+)Foxp3(+) Tregs from Con A-pretreated CXCR3-deficient mice failed to protect against Con A-induced hepatitis, whereas Tregs from Con A-tolerant wt mice allowed CXCR3-deficient mice to recover from Con A hepatitis. In summary, CXCR3(+)T-bet(+)IL-10(+) Tregs are generated in the liver in dependence of IFN-γ, then disseminated into the organism and specifically migrate into the liver, where they limit immune-mediated liver damage.

CCR6 recruits regulatory T cells and Th17 cells to the kidney in glomerulonephritis.

T cells recruited to the kidney contribute to tissue damage in crescentic and proliferative glomerulonephritides. Chemokines and their receptors regulate T cell trafficking, but the expression profile and functional importance of chemokine receptors for renal CD4+ T cell subsets are incompletely understood. In this study, we observed that renal FoxP3+CD4+ regulatory T cells (Tregs) and IL-17-producing CD4+ T (Th17) cells express the chemokine receptor CCR6, whereas IFNgamma-producing Th1 cells are CCR6-. Induction of experimental glomerulonephritis (nephrotoxic nephritis) in mice resulted in upregulation of the only CCR6 ligand, CCL20, followed by T cell recruitment, renal tissue injury, albuminuria, and loss of renal function. CCR6 deficiency aggravated renal injury and increased mortality (from uremia) among nephritic mice. Compared with wild-type (WT) mice, CCR6 deficiency reduced infiltration of Tregs and Th17 cells but did not affect recruitment of Th1 cells in the setting of glomerulonephritis. Adoptive transfer of WT but not CCR6-deficient Tregs attenuated morphologic and functional renal injury in nephritic mice. Furthermore, reconstitution with WT Tregs protected CCR6-/- mice from aggravated nephritis. Taken together, these data suggest that CCR6 mediates renal recruitment of both Tregs and Th17 cells and that the reduction of anti-inflammatory Tregs in the presence of a fully functional Th1 response aggravates experimental glomerulonephritis.