Frank Leithäuser

Commensal Gut Flora Drives the Expansion of Proinflammatory CD4 T Cells in the Colonic Lamina Propria under Normal and Inflammatory Conditions

We tested in B6 mice whether the local expansion of CD4 T cells producing proinflammatory cytokines including IL-17 (Th17 cells) in the colonic lamina propria (cLP) depends on the commensal microflora. High numbers of CD4 Th17 cells were found in the lamina propria of the ileum and colon but not the duodenum, jejunum, mesenteric lymph nodes, spleen, or liver of specific pathogen-free (SPF) mice. The microflora is required for the accumulation of cytokine (IL-17, IFN-γ, TNF-α, IL-10)-producing CD4 T cells in the cLP because only low numbers of cytokine-producing cLP CD4 T cells were found in syngeneic (age- and sex-matched) germfree mice. The fraction of cLP Th17 cells was higher in (type I and type II) IFN- but not IL-4- or IL-12p40-deficient SPF congenics. cLP CD4 Th17 cells produce IL-17 but not IFN-γ, TNF-α, IL-4, or IL-10. cLP CD4 Th17 cells accumulate locally in colitis induced by adoptive transfer of IFN-γ+/+ or IFN-γ−/− CD4 T cells into congenic SPF (but not germfree) RAG−/− hosts. In this colitis model, cLP CD4 T cells that “spontaneously” produce IL-17 progressively increase in number in the inflamed cLP, and increasing serum IL-17 levels appear as the disease progresses. Commensal bacteria-driven, local expansion of cLP CD4 Th17 cells may contribute to the pathogenesis of this inflammatory bowel disease.

Colonic lamina propria dendritic cells in mice with CD4+ T cell-induced colitis.

CD11c+ (F4/80– CD68–) dendritic cells (DC) in the colonic lamina propria (cLP) of normal and immunodeficient (RAG1–/–) C57BL/6 (B6) mice show high surface expression of MHC class I/II molecules and CD1d, and low surface expression of CD40, CD80, CD86 costimulator molecules. CD4+ α β T cells from normal or MHC class II‐deficient B6 micetransferred into congenic RAG1–/– hosts induce a progressive, lethal colitis. Concomitant with colitis development, DC in the inflamed cLP increase in number and up‐regulate surface expression of CD1d, MHC class II molecules and CD40, CD80, CD86 costimulator molecules. cLP DC from non‐transplanted (healthy) and transplanted (diseased) mice produce similar amounts of IL‐12 p70 and IL‐10 in response to CD40 signaling, but the inducible IL‐12 p40 release is 5–15‐fold higher in mice with colitis than in non‐transplanted mice. Binding of IL‐12 p40 to p19 generates IL‐23. Freshly isolated cLP lymphocytes (cLPL) from transplanted, diseased mice express 3–10‐fold more p19 transcripts than cLPL from non‐transplanted, healthy mice. p19 expression by cLPL is further up‐regulated in response to CD40 ligation. Freshly isolated cLP DC from transplanted mice with colitis (but not from non‐transplanted controls) stimulate IFN‐γ (but not IL‐4 or IL‐13) release by co‐cultured NKT cells. Incolitis, DC accumulate in the cLP, show an activated surface phenotype, up‐regulate IL‐12 p40 and p19 expression, and ‘spontaneously’ stimulate NKT‐like cells. cLP DC may be interesting targets for novel therapeutic approaches to modulate mucosal T cell responses in situ.

Activating immunity in the liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-gamma release by liver NKT cells.

A prominent subset of the hepatic innate immune system is α-galactosylceramide (αGalCer)-reactive, (CD4+ and CD4−CD8−) CD1d-restricted NKT cells. We investigated in C57BL/6 (B6) mice which hepatic cell type stimulates hepatic NKT cell activation. Surface expression of CD1d but not CD40, CD80, or CD86 costimulator molecules was detected in hepatocytes. Pulsed in vitro or in vivo with αGalCer, hepatocytes triggered IL-4 release by liver NKT cells but required exogenous IL-12 to trigger IFN-γ release by NKT cells. Liver dendritic cells (DC) isolated from nontreated mice showed low surface expression of MHC, CD1d, and CD40, CD80, or CD86 costimulator molecules that were strikingly up-regulated after αGalCer injection. Although liver CD11c+ DC displayed lower CD1d surface expression than hepatocytes, they were potent stimulators of IFN-γ and IL-4 release by liver NKT when pulsed with αGalCer in vitro or in vivo. Liver DC are thus potent stimulators of proinflammatory cytokine release by NKT cells, are activated themselves in the process of NKT cell activation, and express an activated phenotype after the NKT cell population is eliminated following αGalCer stimulation.

Mediastinal (thymic) large B-cell lymphoma: where do we stand?

Mediastinal (thymic) B-cell lymphoma (MBL) is a locally highly aggressive tumour that was first definitively described in the early 1980s. The incidence of MBL is low, which made disease characterisation difficult initially. However, MBL has several peculiar clinical, morphological, immunological, and genetic features. Collectively, these characteristics distinguish it from other diffuse, large B-cell lymphomas. Consequently, MBL has become a defined subtype of diffuse large B-cell lymphoma with its own code (9679/3) in the International Classification of Diseases. New insights into the biological and clinical aspects of MBL have been gained from the study of large numbers of cases. Nevertheless, the histogenesis of the disease is not yet fully understood. We review the available data on MBL with special emphasis on its morphological, immunological, and genetic properties. Also discussed are recent data on molecular genetics, biology, and treatment.

FOXO1 is a tumor suppressor in classical Hodgkin lymphoma

The FOXO transcription factors control proliferation and apoptosis in different cell types. Their activity is regulated by posttranslational modifications, mainly by the PI3K-PKB pathway, which controls nuclear export and degradation. We show that FOXO1 is highly expressed in normal germinal center B cells as well as in non-Hodgkin lymphomas, including follicular lymphoma, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, and mantle cell lymphoma. In contrast, in 31 of 32 classical Hodgkin lymphoma (cHL) cases, Hodgkin and Reed-Sternberg cells were FOXO1 negative. Neoplastic cells of nodular lymphocyte-predominant Hodgkin lymphoma were negative in 14 of 20 cases. FOXO1 was down-regulated in cHL cell lines, whereas it was expressed in non-Hodgkin lymphoma cell lines at levels comparable with normal B cells. Ectopic expression of a constitutively active FOXO1 induced apoptosis in cHL cell lines and blocked proliferation, accompanied with cell-cycle arrest in the G(0)/G(1) phase. We found that, in cHL cell lines, FOXO1 is inactivated by multiple mechanisms, including constitutive activation of AKT/PKB and MAPK/ERK kinases and up-regulation of microRNAs miR-96, miR-182, and miR-183. These results suggest that FOXO1 repression contributes to cHL lymphomagenesis.

Hepatic dendritic cell subsets in the mouse

The CD11c+ cell population in the non‐parenchymal cell population of the mouse liver contains dendritic cells (DC), NK cells, B cells and T cells. In the hepatic CD11c+ DC population from immunocompetent or immunodeficient [recombinase‐activating gene‐1 (RAG1)–/–] C57BL/6 mice (rigorously depleted of T cells, B cells and NK cells), we identified a B220+ CD11cint subset of ‘plasmacytoid’ DC, and a B220– CD11c+ DC subset. The latter DC population could be subdivided into a major, immature (CD40lo CD80lo CD86lo MHC class IIlo) CD11cint subset, and a minor, mature (CD40hi CD80hi CD86hi MHC class IIhi) CD11chi subset. Stimulated B220+ but not B220– DC produced type I interferon. NKT cell activation in vivo increased the number of liver B220– DC three‐ to fourfold within 18 h post‐injection, and up‐regulated their surface expression of activation marker, while it contracted the B220+ DC population. Early in virus infection, the hepatic B220+ DC subset expanded, and both, the B220+ as well as B220– DC populations in the liver matured. In vitro, B220– but not B220+ DC primed CD4+ or CD8+T cells. Expression of distinct marker profiles and functions, and distinct early reaction to activation signals hence identify two distinct B220+ and B220– subsets in CD11c+ DC populations freshly isolated from the mouse liver.

KLF4 is a tumor suppressor in B-cell non-Hodgkin lymphoma and in classic Hodgkin lymphoma

The transcription factor KLF4 may act both as an oncogene and a tumor suppressor in a tissue-depending manner. In T- and pre-B-cell lymphoma, KLF4 was found to act as tumor suppressor. We found the KLF4 promoter methylated in B-cell lymphoma cell lines and in primary cases of B-cell lymphomas, namely, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, and in classic Hodgkin lymphoma (cHL) cases. Promoter hypermethylation was associated with silencing of KLF4 expression. Conditional overexpression of KLF4 in Burkitt lymphoma cell lines moderately retarded proliferation, via cell-cycle arrest in G(0)/G(1). In the cHL cell lines, KLF4 induced massive cell death that could partially be inhibited with Z-VAD.fmk. A quantitative reverse-transcribed polymerase chain reaction array revealed KLF4 target genes, including the proapoptotic gene BAK1. Using an shRNA-mediated knock-down approach, we found that BAK1 is largely responsible for KLF4-induced apoptosis. In addition, we found that KLF4 negatively regulates CXCL10, CD86, and MSC/ABF-1 genes. These genes are specifically up-regulated in HRS cells of cHL and known to be involved in establishing the cHL phenotype. We conclude that epigenetic silencing of KLF4 in B-cell lymphomas and particularly in cHL may favor lymphoma survival by loosening cell-cycle control and protecting from apoptosis.

Molecular-cytogenetic comparison of mucosa-associated marginal zone B-cell lymphoma and large B-cell lymphoma arising in the gastro-intestinal tract

Extranodal B‐cell lymphoma of mucosa‐associated lymphoid tissue (MALT) type may represent a model of lymphoma progression, because a small cell component frequently occurs in the large cell variants. We studied 52 extranodal B‐cell lymphomas: 18 extranodal marginal zone B‐cell lymphomas of MALT type (MZBL,MT), 7 MZBL,MT of the gastro‐intestinal tract with a diffuse large B‐cell component (giMZBLplusLBCL), and 27 diffuse large B‐cell lymphomas of the gastro‐intestinal tract without small cell component (giLBCL). Analytical techniques were comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The translocation t(11;18) was found as the sole aberration in two MZBL,MT only. In contrast to this, t(11;18)‐negative MZBL,MT were characterized by frequent gains on chromosome 3 and DNA amplifications on 2p13–p15. Furthermore, we found a clonal lymphoma progression from the small to the large cell component with accumulation of gains and losses of chromosomal material in the large cell component in giMZBLplusLBCL. Aberrations overlapping with MZBL,MT and giMZBLplusLBCL included losses on chromosome 13, amplifications of the REL proto‐oncogene, or gains on chromosome 12. In addition, the large cell component revealed gains on 8q24, including amplifications of the MYC proto‐oncogene, and losses on 2q. The giLBCL had frequent gains on chromosomes 12 and 9, as well as on 11q, and losses on 6q. We conclude that, based on the distinctive and partly overlapping patterns of genetic aberrations, MALT lymphomas can be divided into different genetic subgroups.

Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo

Background: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. Aims: We analysed the expression levels of CK1 delta and epsilon (CK1δ/∊) in pancreatic tumour cells in order to validate the effects of CK1 inhibition by 3-[2,4,6-(trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) on their proliferation and sensitivity to anti-CD95 and gemcitabine. Methods: CK1δ/∊ expression levels were investigated by using western blotting and immunohistochemistry. Cell death was analysed by FACS analysis. Gene expression was assessed by real-time PCR and western blotting. The putative anti-tumoral effects of IC261 were tested in vivo in a subcutaneous mouse xenotransplantation model for pancreatic cancer. Results: We found that CK1δ/∊ are highly expressed in pancreatic tumour cell lines and in higher graded PDACs. Inhibition of CK1δ/∊ by IC261 reduced pancreatic tumour cell growth in vitro and in vivo. Moreover, IC261 decreased the expression levels of several anti-apoptotic proteins and sensitised cells to CD95-mediated apoptosis. However, IC261 did not enhance gemcitabine-mediated cell death either in vitro or in vivo. Conclusions: Targeting CK1 isoforms by IC261 influences both pancreatic tumour cell growth and apoptosis sensitivity in vitro and the growth of induced tumours in vivo, thus providing a promising new strategy for the treatment of pancreatic tumours.

Clustering of Colonic Lamina Propria CD4 + T Cells to Subepithelial Dendritic Cell Aggregates Precedes the Development of Colitis in a Murine Adoptive Transfer Model

Initial lesions in inflammatory bowel disease induced during the repopulation of immunodeficient RAG1−/− mice with immunocompetent CD4+ T cells have not been previously described. In this transfer colitis model, we followed CD4+ T cell repopulation in the host by injecting autofluorescent CD4+ T cells from congenic, enhanced green fluorescent protein (eGFP)-transgenic mice. This allowed the direct, sensitive, and unambiguous histological detection of the repopulation of the intestinal tract, mesenteric lymph nodes, and spleen of the host with donor eGFP+ CD4+ T cells. We identified in RAG1−/− mice intestinal dendritic cell (DC) aggregates under the basal crypt epithelium at the mucosa/submucosa junction from which F4/80+ macrophages were excluded. At Days 8 to 11 posttransfer (before colitis was manifest), CD4+ T cells clustered and proliferated in CD11c+ DC aggregates. T cell clustering was most pronounced in the cecum where histologically overt colitis became manifest 5 to 10 days later. Junctional DC aggregates were thus prevalent in the triggering phase of the disease. The data suggest that pathogenic T cell responses inducing inflammatory bowel disease are primed or restimulated in situ in junctional CD4+ T cell/DC aggregates.