P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish

The recruitment and migration of macrophages and neutrophils is an important process during the early stages of the innate immune system in response to acute injury. Transgenic pu.1:EGFP zebrafish permit the acquisition of leukocyte migration trajectories during inflammation. Currently, these high-quality live-imaging data are mainly analysed using general statistics, for example, cell velocity. Here, we present a spatio-temporal analysis of the cell dynamics using transition matrices, which provide information of the type of cell migration. We find evidence that leukocytes exhibit types of migratory behaviour, which differ from previously described random walk processes. Dimethyl sulfoxide treatment decreased the level of persistence at early time points after wounding and ablated temporal dependencies observed in untreated embryos. We then use pharmacological inhibition of p38 and c-Jun N-terminal kinase mitogen-activated protein kinases to determine their effects on in vivo leukocyte migration patterns and discuss how they modify the characteristics of the cell migration process. In particular, we find that their respective inhibition leads to decreased and increased levels of persistent motion in leukocytes following wounding. This example shows the high level of information content, which can be gained from live-imaging data if appropriate statistical tools are used

Nanometre-scale organisation of the inhibitory human natural killer cell receptor KIR3DL1 and its HLA class I ligands

Natural killer (NK) cells express an array of activating and inhibitory receptors, which enable the detection of stress-induced markers and ‘self’ human leukocyte antigen (HLA) class I molecules on target cells. The distribution of these receptors at the cell surface is thought to be important for signal integration at the immune synapse. Killer-cell immunoglobulin-like receptors (KIR) and HLA class I proteins are highly polymorphic. In particular, allelic variation affecting the expression and function of the inhibitory receptor KIR3DL1 and its HLA class I ligands, can influence HIV infection outcome. It is not known, however, if genetic variation can affect the organisation of KIR and HLA proteins at the cell surface and at the NK-cell immune synapse. Here, single-molecule localisation microscopy was used to investigate the spatial distribution of KIR3DL1 and HLA class I proteins within the plasma membrane. HLA class I proteins were relatively evenly distributed at the nanometre-scale, with fewer than 5% of molecules forming clusters in unstimulated primary B cells. In contrast, over half of KIR3DL1 receptors were constitutively arranged in clusters sized 4064 ± 384 nm², at the surface of resting NK cells. Receptor ligation induced the reorganisation of KIR3DL1, resulting in the formation of larger and denser clusters. Interestingly, the extent of these changes varied for different alleles of KIR3DL1, suggesting that genetic variability may affect KIR3DL1 recruitment at the NK-cell synapse. Allelic variation did not influence the constitutive organisation of KIR and HLA proteins, when expressed at the same level. However, their nanoscale distribution was influenced by cell surface levels; at higher expression levels, HLA class I clustering decreased, whereas the number of KIR3DL1 clusters increased when expressed at higher levels. These results demonstrate the distinct nanoscale organisation of KIR3DL1 and HLA class I molecules. Moreover, factors that influence their expression, including genetic variation, may have an important impact on the distribution of KIR3DL1 and HLA class I proteins at the cell surface.Open Acces

HLA-B and HLA-C differ in their nanoscale organization at cell surfaces

The particular HLA class I variants an individual carries influences their resistance and susceptibility to a multitude of diseases. Expression level and variation in the peptide binding region correlates with, for example, a person's progression to AIDS after HIV infection. One factor which has not yet been addressed is whether or not different HLA class I proteins organize differently in the cell membrane on a nanoscale. Here, we examined the organization of three HLA-B allotypes (B*2705, B*5301, and B*5701) and two HLA-C allotypes (C*0602 and C*0702) in the membrane of 721.221 cells which otherwise lack expression of HLA-B or HLA-C. All these allotypes are ligands for the T cell receptor and leukocyte immunoglobulin-like receptors, but additionally, the HLA-B allotypes are ligands for the killer-cell immunoglobulin-like receptor family member KIR3DL1, HLA-C*0602 is a ligand for KIR2DL1, and HLA-C*0702 is a ligand for KIR2DL2/3. Using super-resolution microscopy, we found that both HLA-B and HLA-C formed more clusters and a greater proportion of HLA contributed to clusters, when expressed at lower levels. Thus, HLA class I organization is a covariate in genetic association studies of HLA class I expression level with disease progression. Surprisingly, we also found that HLA-C was more clustered than HLA-B when expression level was controlled. HLA-C consistently formed larger and more numerous clusters than HLA-B and a greater proportion of HLA-C contributed to clusters than for HLA-B. We also found that the organization of HLA class I proteins varied with cell type. T cells exhibited a particularly clustered organization of HLA class I while B cells expressed a more uniform distribution. In summary, HLA class I variants are organized differently in the cell surface membrane which may impact their functions