A Novel, LAT/Lck Double Deficient T Cell Subline J.CaM1.7 for Combined Analysis of Early TCR Signaling.

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex.

Measurements of heterotypic associations between cluster of differentiation CD74 and CD44 in human breast cancer-derived cells.

Interactions between pairs of membrane-bound receptors can enhance tumour development with implications for targeted therapies for cancer. Here we demonstrate clear heterotypic interaction between CD74 and CD44, which might act in synergy and hence contribute to breast cancer progression. CD74, a type II transmembrane glycoprotein, is a chaperone for MHC class II biosynthesis and a receptor for the MIF. CD44 is the receptor for hyaluronic acid and is a Type I transmembrane protein. Interactions between CD74, MIF and the intra-cytoplasmic domain of CD44 result in activation of ERK1/2 pathway, leading to increased cell proliferation and decreased apoptosis. The level of CD44 in the breast tumor cell lines CAMA-1, MDA-MB-231, MDA-MB-435 and the immortalized normal luminal cell line 226LDM was higher than that of CD74. It was also observed that CD74 and CD44 exhibit significant variation in expression levels across the cells. CD74 and CD44 were observed to accumulate in cytoplasmic compartments, suggesting they associate with each other to facilitate tumour growth and metastasis. Use of a novel and validated colocalisation and image processing approach, coupled with co-immunoprecipitation, confirmed that CD74 and CD44 physically interact, suggesting a possible role in breast tumour growth. This is the first time that CD74 and CD44 colocalization has been quantified in breast cancer cells using a non-invasive and validated bioimaging procedure. Measuring the co-expression levels of CD74 and CD44 could potentially be used as a 'biomarker signature' to monitor different stages of breast cancer.

Interaction of HLA-DR and CD74 at the cell surface of antigen-presenting cells by single particle image analysis.

Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.

Changes in CDKN2D , TP53, and miR125a expression: potential role in the evaluation of human amniotic fluid-derived mesenchymal stromal cell fitness.

Human amniotic fluid-derived mesenchymal stromal cells (hAMSC) have become one of the main cell populations used in regenerative medicine and for the study of various clinical disorders. These cells have a great capacity for proliferation and differentiation and do not form teratomas when transplanted into animal models, and their stemness seems to be between embryonic cells and adult mesenchymal cells. Before their use in cell therapy, they must be cultured and expanded in vitro, but the effect this process has on their fitness, a determining factor for the success or failure of cell therapy, is unknown. We undertook a follow-up of gene and microRNAs (miRNAs) expression using microarray of hAMSC for the first 15 passages. Significant changes were noted in the expression of various mRNAs and miRNAs, particularly down-regulation of TP53, increased expression of hsa-miR-125a and up-regulation of CDKN2D . The variations in TP53 and hsa-miR-125a may act as an indicator of the stemness of the hAMSC, whereas CDKN2D may indicate the begging of early senescence process in a p53-independent mechanism. The genes described in this study will help evaluate the fitness of hAMSC, thus guaranteeing their biological quality for use in regenerative medicine.

Key cellular components and interactive histocompatibility molecules regulating tolerance to the fetal allograft.

Implantation is a major landmark in life. It involves the correct apposition of the embryo in the maternal endometrium. The cellular environment influences placenta development, and direct contact of the fetus with maternal tissues is achieved through decidual cells. At the decidua, and at systemic level, the correct balance of cells potentially acting as antigen-presenting cells and histocompatibility products play a pivotal role in achieving feto-maternal tolerance. Here, we review some of the current issues associated with the interplay between cells and molecules needed for pregnancy development.