Reinhard Andreesen

Elevation of serum interleukin-6 concentration precedes acute-phase response and reflects severity in acute pancreatitis

Experimental studies have shown that interleukin-6 induces all major acute-phase proteins in the liver, including C-reactive protein. In 50 patients with acute pancreatitis, the serum concentrations of interleukin-6 and C-reactive protein were determined daily during the first week of hospitalization. Patients were divided into three groups according to clinical criteria: mild pancreatitis (less than or equal to 1 complication; n = 25), severe pancreatitis (greater than or equal to 2 complications; n = 15), and lethal outcome (n = 10). Patients with mild disease showed initially slightly elevated levels of interleukin-6 (22.0 +/- 9.8 U/mL) that decreased to low levels within 4 days (5.0 +/- 1.0 U/mL). In patients with severe pancreatitis, serum concentrations of interleukin-6 were initially clearly elevated (35.0 +/- 7.5 U/mL) and remained slightly elevated until day 7 (13.0 +/- 2.0 U/mL). Patients with lethal outcome had markedly elevated initial interleukin-6 concentrations (61.0 +/- 15.0 U/mL) that decreased but were still elevated at day 7 (26.0 +/- 2.5 U/mL). In all three groups, C-reactive protein concentrations followed the course of interleukin-6 concentrations by 1 day. There was a positive correlation between maximal interleukin 6 concentrations and maximal increases in the serum concentrations of C-reactive protein (r = 0.66). At days 1 and 2, increased (greater than 15 U/mL) interleukin-6 concentrations (positive predictive value, 91%; negative predictive value, 82%) predicted a severe or lethal course of the disease more accurately than elevated [greater than 0.10 g/L (greater than 10 mg/dL)] C-reactive protein concentrations (positive predictive value, 67%; negative predictive value, 79%). In conclusion, elevated serum concentrations of interleukin-6 followed by increased levels of C-reactive protein reflect the severity of acute pancreatitis.

Phase I study of adoptive T-cell therapy using antigen-specific CD8+ T cells for the treatment of patients with metastatic melanoma.

PURPOSE The adoptive transfer of in vitro generated tumor antigen-specific cytotoxic T lymphocytes (CTL) provides a promising approach to the immunotherapy of cancer. A phase I study was conducted to test the feasibility, safety, and survival of adoptively transferred Melan-A-specific CTL lines in melanoma patients. PATIENTS AND METHODS Eleven HLA-A2+ patients with metastatic melanoma received at least three intravenous infusions of Melan-A-specific CTL at 2-week intervals. CTL were generated by four rounds of in vitro stimulation of purified CD8+ peripheral blood lymphocytes with autologous dendritic cells pulsed with an HLA-A2 binding Melan-A peptide. Each T-cell infusion was accompanied by a 6-day course of low-dose interleukin-2. RESULTS A total of 52 T-cell infusions were administered, averaging 2.1 x 10(8) Melan-A-specific CTL per infusion. Clinical adverse effects were mild and consisted of chills and low-grade fever in seven of 11 patients. Clinical and immunologic responses revealed an antitumor response in three of 11 patients (one complete regression, one partial regression, one mixed response), an elevated frequency of circulating Melan-A tetramer+ T cells up to 2 weeks in all the patients with a maximal frequency of 2% of total CD8+ T cells, an increase in eosinophils to up to 50% in seven of 11 patients, and a selective loss of Melan-A expression in lymph node metastases in two evaluated patients after T-cell transfer. CONCLUSION Our data indicate that the adoptive transfer of antigen-specific T cells in melanoma patients can induce clinical tumor-specific immune responses without major adverse effects.

Blockade of PD-L1 (B7-H1) augments human tumor-specific T cell responses in vitro.

Human tumors frequently escape immune destruction, despite the presence of cyototoxic T cells (CTL) recognizing tumor‐associated antigens (TAA). We have previously shown that programmed death ligand‐1 (PD‐L1), a recently identified ligand of the B7 superfamily, is expressed on murine tumors and can inhibit antitumor immune responses. To evaluate the clinical relevance of our animal model findings, we examined human tumors and tumor‐specific T cells. We found PD‐L1 to be constitutively expressed on human renal cell carcinoma (RCC) cell lines and upregulated on human melanoma cell lines upon exposure to interferon‐gamma. Similarly, we found binding of anti‐PD‐L1 monoclonal antibody (mAb) on frozen sections from RCC and melanomas, but not on normal tissues. The corresponding inhibitory receptor of PD‐L1, PD‐1, revealed a higher expression on tumor‐infiltrating lymphocytes than on peripheral blood lymphocytes (PBL) from melanoma patients upon specific antigen stimulation. Stimulation of PBL from healthy donors with peptide‐loaded dendritic cells in the presence of anti‐PD‐L1 mAb altered neither the total T cell numbers after expansion, nor the percentage of peptide‐specific CTL, when providing a T cell help by addition of cytokines. However, when stimulating TAA‐specific CTL and T helper cells with Ag‐pulsed dendritic cells in the absence of exogenous cytokines, PD‐L1 blockade increased the cytokine production. Similar to the data achieved in the murine system, the blockade of PD‐L1 on human tumors resulted in enhanced cytolytic activity of TAA‐specific CTLs and cytokine production of TAA‐specific T helper cells when interacting directly with the tumor. In summary, our data suggest that PD‐L1/PD‐1 interactions negatively regulate T cell effector functions predominately in the absence of exogenous cytokine support, indicating an important role for this pathway in tumor evasion.

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation.

The adoptive transfer of CD4+CD25+ natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high‐grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell‐specific surface markers. Depletion of CD127+ cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3+ cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4+CD25+CD127low Treg, correlating with loss of FOXP3 expression and emergence of pro‐inflammatory cytokines. Further analysis identified CD45RA−FOXP3+ memory‐type Treg as the main source of converting cells, whereas CD45RA+FOXP3+ Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell‐type‐specific characteristics after repetitive TCR stimulation.

Surface Phenotype Analysis of Human Monocyte to Macrophage Maturation

Cells of the mononuclear phagocyte system arise from circulating blood monocytes. Upon emigration from the vasculature, monocytes differentiate into macrophages, a process that monocytes similarly undergo in vitro. We have established primary cultures from elutriated or adherence‐purified blood monocytes and analyzed the antigenic modulation during monocyte to macrophage transformation, which could be followed by the expression of specific antigens and which required as yet unknown inducer signals present in the serum. It is shown that in the absence of serum monocytes only survive in vitro when cultured adherent to plastic but rapidly die in suspension culture. Starting at 0.5%, serum induced maturation dose‐dependently, with the optimal concentration being 2 to 5%. Of those antigens not present on monocyte, the low‐affinity Fc receptor (CD16), the α‐chain of the vitronectin receptor (CD51), gp65‐MAX.1, and gp68‐MAX.3 were expressed only upon serum‐induced macrophage differentiation, whereas the transferrin receptor (CD71), MAX.26, and to some degree also gp65‐MAX.11 appeared to be independent of maturation and were also found on primary cultures of adherent monocytes under serum‐free conditions. In addition, the rapid induction of HLA class II antigens (within 24 hr) was similar with and without serum, as was the continued high‐density expression in long‐term culture. The monocyte‐specific CD14 antigen was down‐regulated in the absence of serum but kept its level of expression on differentiated macrophages. In comparison, alveolar and peritoneal macrophages, respectively, differed in their antigenic phenotype: Alveolar macrophages expressed high HLA class II antigens but low CD14, whereas for peritoneal macrophages the opposite was found. Both interferon‐γ and ‐α suppressed macrophage maturation in vitro but had contrary effects on HLA class II and CD16 expression: Interferon‐γ up‐regulated the two types of antigens, which, in contrast, were down‐regulated by interferon‐α.

Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity

DNA methylation participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. With few exceptions, research thus far has focused on gene promoters, and little is known about the extent, functional relevance, and regulation of cell type-specific DNA methylation at promoter-distal sites. Here, we present a comprehensive analysis of differential DNA methylation in human conventional CD4(+) T cells (Tconv) and CD4(+)CD25(+) regulatory T cells (Treg), cell types whose differentiation and function are known to be controlled by epigenetic mechanisms. Using a novel approach that is based on the separation of a genome into methylated and unmethylated fractions, we examined the extent of lineage-specific DNA methylation across whole gene loci. More than 100 differentially methylated regions (DMRs) were identified that are present mainly in cell type-specific genes (e.g., FOXP3, IL2RA, CTLA4, CD40LG, and IFNG) and show differential patterns of histone H3 lysine 4 methylation. Interestingly, the majority of DMRs were located at promoter-distal sites, and many of these areas harbor DNA methylation-dependent enhancer activity in reporter gene assays. Thus, our study provides a comprehensive, locus-wide analysis of lineage-specific methylation patterns in Treg and Tconv cells, links cell type-specific DNA methylation with histone methylation and regulatory function, and identifies a number of cell type-specific, CpG methylation-sensitive enhancers in immunologically relevant genes.

Interleukin-8 and neutrophil activation in acute pancreatitis

Abstract. It has been suggested that leucocytes play an important role in the pathogenesis of complicated pancreatitis. Indeed, increased plasma concentrations of neutrophil elastase as a marker of neutrophil activation could be detected in patients with a severe course of the disease. Recently, interleukin‐8 (IL‐8) has been described as a novel neutrophil activating peptide. To determine the role of IL‐8 in acute pancreatitis we measured its serum concentrations by a specific enzyme‐linked immunosorbent assay in 10 patients with acute pancreatitis daily during the first week of hospitalization. IL‐8 levels were compared with plasma concentrations of neutrophil elastase and the clinical course of the disease. Three of the patients had uncomplicated pancreatitis, while seven showed various extrapancreatic complications. Patients with complicated pancreatitis had statistically significant (P<0.05) higher mean values of IL‐8 (121 ±41 pg ml‐1 vs. 13 ± 6 pg ml‐1, mean ± SEM) and neutrophil elastase (547 ± 35 ng ml‐1 vs. 250±20 ng ml‐1) than patients with uncomplicated disease. There was a positive correlation (r= 0.52, P < 0.0001) between IL‐8 and neutrophil elastase in the lower concentration range of IL‐8 (< 100 pg ml‐1). At IL‐8 levels > 100 pg ml‐1 neutrophil elastase was always greatly elevated; however, under these conditions the relationship between IL‐8 and elastase was no longer linear. No measurable IL‐8 concentrations were found when plasma elastase was < 200 ng ml‐1. During follow‐up, initially elevated IL‐8 concentrations decreased in correlation with clinical improvement. In conclusion, the results suggest that IL‐8 contributes to initial neutrophil activation during acute pancreatitis. IL‐8 seems thus to be a factor involved in the pathogenesis of complicated pancreatitis.

Reverse Signaling Through Transmembrane TNF Confers Resistance to Lipopolysaccharide in Human Monocytes and Macrophages

We have previously reported that the CD14+ monocytic subpopulation of human PBMC induces programmed cell death (apoptosis) in cocultured endothelial cells (EC) when stimulated by bacterial endotoxin (LPS). Apoptosis is mediated by two routes, first via transmembrane TNF-α (mTNF) expressed on PBMC and, in addition, by TNF-independent soluble factors that trigger apoptosis in EC. Neutralizing anti-TNF mAb completely blocked coculture-mediated apoptosis, despite the fact that there should have been additional soluble cell death factors. This led to the hypothesis that a reverse signal is transmitted from the TNF receptor on EC to monocytes (MO) via mTNF that prevents the production of soluble apoptotic factors. Here we have tested this hypothesis. The results support the idea of a bidirectional cross-talk between MO and EC. Peripheral blood MO, MO-derived macrophages (MΦ), or the monocytic cell line Mono Mac 6 were preincubated with human microvascular EC that constitutively express TNF receptor type I (TNF-R1) and subsequently stimulated with LPS. Cell-free supernatants of these preparations no longer induced EC apoptosis. The preincubation of MO/MΦ with TNF-reactive agents, such as mAb and soluble receptors, also blocked the production of death factors, providing further evidence for reverse signaling via mTNF. Finally, we show that reverse signaling through mTNF mediated LPS resistance in MO/MΦ as indicated by the down-regulation of LPS-induced soluble TNF and IL-6 as well as IL-1 and IL-10.

Genome-Wide Profiling of CpG Methylation Identifies Novel Targets of Aberrant Hypermethylation in Myeloid Leukemia

The methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. Because aberrant hypermethylation may be used as a marker for disease, a sensitive method for the global detection of DNA methylation events is of particular importance. We describe a novel and robust technique, called methyl-CpG immunoprecipitation, which allows the unbiased genome-wide profiling of CpG methylation in limited DNA samples. The approach is based on a recombinant, antibody-like protein that efficiently binds native CpG-methylated DNA. In combination with CpG island microarrays, the technique was used to identify >100 genes with aberrantly methylated CpG islands in three myeloid leukemia cell lines. Interestingly, within all hypermethylation targets, genes involved in transcriptional regulation were significantly overrepresented. More than half of the identified genes were absent in microarray expression studies in either leukemia or normal monocytes, indicating that hypermethylation in cancer may be largely independent of the transcriptional status of the affected gene. Most individually tested genes were also hypermethylated in primary blast cells from acute myeloid leukemia patients, suggesting that our approach can identify novel potential disease markers. The technique may prove useful for genome-wide comparative methylation analysis not only in malignancies.

Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 μmol/L to 11.8 ± 2.8 μmol/L in all post-transplant samples and to 3.5 ± 3 μmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.