Stromal mesenteric lymph node cells are essential for the generation of gut-homing T cells in vivo.

T cells primed in the gut-draining mesenteric lymph nodes (mLN) are imprinted to express alpha4beta7-integrin and chemokine receptor CCR9, thereby enabling lymphocytes to migrate to the small intestine. In vitro activation by intestinal dendritic cells (DC) or addition of retinoic acid (RA) is sufficient to instruct expression of these gut-homing molecules. We report that in vivo stroma cells, but not DC, allow the mLN to induce the generation of gut tropism. Peripheral LN (pLN) transplanted into the gut mesenteries fail to support the generation of gut-homing T cells, even though gut-derived DC enter the transplants and prime T cells. DC that fail to induce alpha4beta7-integrin and CCR9 in vitro readily induce these factors in vivo upon injection into mLN afferent lymphatics. Moreover, uniquely mesenteric but not pLN stroma cells express high levels of RA-producing enzymes and support induction of CCR9 on activated T cells in vitro. These results demonstrate a hitherto unrecognized contribution of stromal cell delivered signals, including RA, on the imprinting of tissue tropism in vivo.

Stromal cells confer lymph node-specific properties by shaping a unique microenvironment influencing local immune responses.

Lymph nodes (LN) consist not only of highly motile immune cells coming from the draining area or from the systemic circulation, but also of resident stromal cells building the backbone of the LN. These two cell types form a unique microenvironment which is important for initiating an optimal immune response. The present study asked how the unique microenvironment of the mesenteric lymph node (mLN) is influenced by highly motile cells and/or by the stromal cells. A transplantation model in rats and mice was established. After resecting the mLN, fragments of peripheral lymph node (pLN) or mLN were inserted into the mesentery. The pLN and mLN have LN-specific properties, resulting in differences of, for example, the CD103(+) dendritic cell subset, the adhesion molecule mucosal addressin cell adhesion molecule 1, the chemokine receptor CCR9, the cytokine IL-4, and the enzyme retinal dehydrogenase 2. This new model clearly showed that during regeneration stromal cells survived and immune cells were replaced. Surviving high endothelial venules retained their site-specific expression (mucosal addressin cell adhesion molecule 1). In addition, the low expression of retinal dehydrogenase 2 and CCR9 persisted in the transplanted pLN, suggesting that stromal cells influence the lymph node-specific properties. To examine the functional relevance of this different expression pattern in transplanted animals, an immune response against orally applied cholera toxin was initiated. The data showed that the IgA response against cholera toxin is significantly diminished in animals transplanted with pLN. This model documents that stromal cells of the LN are active players in shaping a unique microenvironment and influencing immune responses in the drained area.

Dendritic cell subsets in lymph nodes are characterized by the specific draining area and influence the phenotype and fate of primed T cells.

Dendritic cells (DC) are important in differential T-cell priming. Little is known about the local priming by DC in the microenvironment of different lymph nodes and about the fate of the imprinted T cells. Therefore, freshly isolated rat DC from mesenteric lymph nodes (mLN) and axillary lymph nodes (axLN) were phenotyped and cultured with blood T cells in the presence of the superantigen Mycoplasma arthritidis mitogen (MAM). The phenotype, proliferation and apoptosis of the primed T cells were analysed. Our data show that a common DC population exists in both mLN and axLN. In addition, region-specific DC with an organotypical marker expression imprinted by the drained area were found. Coculture of T cells with DC from mLN or axLN resulted in a distinct shift in the CD4 and CD8 expression of T cells and their phenotype. Furthermore, when these differentially primed mLN and axLN T cells were injected into recipients, mLN-primed T cells survived longer in other lymphoid organs. The results show that the region-specific DC have a unique phenotype and an impact on the ratio of CD4 : CD8 T cells during an immune response in vivo.

Estimation of glomerular filtration rates after orthotopic liver transplantation: Evaluation of cystatin C-based equations.

Early detection of renal dysfunction in patients after orthotopic liver transplantation is important. Creatinine-based equations to estimate glomerular filtration rate (GFR) were found to be less accurate in liver transplant recipients than in their original populations. Since cystatin C (CysC) is independent from muscle mass and hepatic biosynthesis, we evaluated the diagnostic accuracy of 3 CysC-based equations (Larson, Hoek, and Filler formulae) that are based on the same CysC method as that of our center in comparison to the abbreviated creatinine-based modification of diet in renal disease (MDRD) formula in 59 liver transplant recipients. "True GFR" was measured by 99mTc-diethylene triamine pentaacetic acid ((99m)Tc-DTPA) clearance. Neither correlation with the GFR (correlation coefficients: 0.594-0.640) nor precision (root mean square error: 15.7-18.17 mL/min/1.73 m(2)) differed significantly between the tested formulae. The biases of the Hoek and Larsson formulae were significantly smaller than those of the MDRD and Filler equations (-0.1 and -2.3 vs. 10.1 and 7.9 mL/min/1.73 m(2), respectively; P

Comparative study of NK cell-mediated cytotoxicity using radioactive and flow cytometric cytotoxicity assays.

Cell-mediated cytotoxicity is a major effector pathway of the immune system. Thus far, radioactive assays have been widely used, but have significant disadvantages. Meanwhile, flow cytometric assays have been established but have not all been assessed simultaneously relative to the radioactive assays. Here, we have evaluated flow cytometric enumeration of surviving target cells, annexin-V binding and detection of activated caspase-3 and caspase-6 in direct comparison to the 51chromium (51Cr) release assay, and the JAM test. For assay evaluation NKL effector cells Fas-resistant K562 and Fas-sensitive Jurkat target cells were studied. Percent specific lysis measured for each E:T ratio was fitted to a sigmoid dose response curve. Both the flow cytometric and radioactive cytotoxicity assays showed equivalent background lysis (1-13%) but differed considerably with respect to maximum cytotoxicity (11-82% in K562 and 49-75% in Jurkat cells). Half maximum lysis ranged from 4:1 to 28:1 E:T ratios in K562 cells and from 1:3 to 24:1 in Jurkat cells, respectively. Flow cytometric enumeration of surviving target cells was the only assay which permitted detection of cytotoxicity at considerable lower E:T ratios (in K562 cells 1:4 to 2:1 and in Jurkat cells 1:4 to 1:1) than the conventional assays. Prolonged incubation over 24 h did not improve the sensitivity for flow cytometric enumeration of surviving target cells or the JAM test. The observed differences in the lysis of target cells are likely to reflect different sensitivity of cell death-associated changes which are measured by each assay. Thus, the particular choice of a cytotoxicity assay must be carefully adapted to the experimental situation under study.