Rat invariant TCR α-chains and NKT cells were investigated to clarify whether CD1d-mediated recognition by NKT cells is conserved further in evolution. Rats had multiple-copies of TRAV14 genes, which can be categorized into two types according to the diversity accumulated in the CDR2 region. Rats retained invariant TCRα forms with the homogeneous junctional region similar to mouse invariant TRAV14-J281. The proportion of invariant TCR among Vα14+ clones was 12.9% in the thymus and increased in the periphery, 31% in the spleen and 95% in hepatic sinusoidal cells. The invariant TRAV14-J281 was expressed by liver sinusoidal and splenic NKT cells with CD8, CD44high, and TCR Vβ8. Type 1 invariant TCRα was expressed more frequently in hepatic lymphocytes, while type 2 invariant TCRα was expressed predominantly in the spleen. Both types of cells cytolyzed to and were stimulated to proliferate by CD1d-expressing cells in a CD1d-restricted manner. These results suggested that rat NKT cells bearing distinct Vα14 chains are distributed in a tissue-specific pattern. NKT cell populations in rats were more variable than those in mice, indicating that they play novel roles in nature. The implication of the molecular interaction between the structurally diverse invariant TCRα and CD1d/ligand complex in different organs is discussed.
A novel idea is emerging that a large molecular repertoire is common to the nervous and immune systems, which might reflect the existence of novel neuronal functions for immune molecules in the brain. Here, we show that the transmembrane adaptor signaling protein CD3, first described in the immune system, has a previously uncharacterized role in regulating neuronal development. Biochemical and immunohistochemical analyses of the rat brain and cultured neurons showed that CD3 is mainly expressed in neurons. Distribution of CD3 in developing cultured hippocampal neurons, as determined by immunofluorescence, indicates that CD3 is preferentially associated with the somatodendritic compartment as soon as the dendrites initiate their differentiation. At this stage, CD3 was selectively concentrated at dendritic filopodia and growth cones, actin-rich structures involved in neurite growth and patterning. siRNA-mediated knockdown of CD3 in cultured neurons or overexpression of a loss-of-function CD3 mutant lacking the tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAMs) increased dendritic arborization. Conversely, activation of endogenous CD3 by a CD3 antibody reduced the size of the dendritic arbor. Altogether, our findings reveal a novel role for CD3 in the nervous system, suggesting its contribution to dendrite development through ITAM-based mechanisms.
We previously showed that the cell surface-expressed Mr 70,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3+, CD4-, CD8-, T-cell receptor (TCR)alphabeta-, natural killer recetor-P1- T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the Vdelta6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR gammadelta chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR Vdelta6 family are likely to contribute to host resistance to tumor cells.
The antigen recognition system of NKT cells acts via an invariant T-cell receptor (TR) which recognizes CD1d and is highly conserved in mice, rats and humans. NKT cells expressing an invariant mouse TR composed of TRAV11-TRAJ18 (formerly Valpha14-Jalpha281) are positively selected by CD1d, and recognize an antigen in context with CD1d. Here we show ten distinct TRAV11 genes (previously designated by us as TRAV14) on rat Chromosome 15 (BN/SsNHsd/MCW strain). In the rat TRAV11 genes, the splicing sites, the recombination signal sequences, and the possible promoter regions were well conserved, indicating that they were functional. Predicted protein sequences of rat TRAV11 genes were analyzed, including the three loops (CDR1-3) which connect the beta-strands of the domain encoded by the TRA V-REGION and is hypervariable in sequence. The CDR1-IMGT sequence (from 27 to 32; VTPFNN) was conserved among most rat TRAV11 genes. The CDR2-IMGT sequences (from 56 to 61) were grouped into two types: type 1 [L(T/K)NKEE], and type 2 [LAYKKE]. The mRNAs of both types have a different tissue distribution. The CDR3 sequences were short and invariant, the rat TRAV11 genes being preferentially rearranged with rat TRAJ18 (Jalpha281), with the joint consisting of a single amino acid (A or G). Thus, rats had multiple TRAV11 chains with diversified CDR2-IMGT and homogenous CDR1-IMGT and CDR3-IMGT.
Copper (Cu) is an indispensable metal for normal development and function of humans, especially in central nervous system (CNS). However, its redox activity requires accurate Cu transport system. ATP7A, a main Cu2+ transporting-ATPase, is necessary to efflux Cu across the plasma membrane and synthesize cuproenzymes. Menkes disease (MD) is caused by mutations in ATP7A gene. Clinically, MD is Cu deficiency syndrome and is treated with Cu-histidine injections soon after definite diagnosis. But outcome of the most remains poor. To estimate the standard therapy, Cu distribution in the treated classic MD patients is analyzed by synchrotron-generated X-ray fluorescence technique (SR-XRF), which identifies and quantifies an individual atom up to at subcellular level of resolution with wide detection area. SR-XRF analysis newly reveals that Cu exists in spinal cord parenchyma and flows out via venous and lymph systems. By systemic analysis, excess Cu is detected in the proximal tubular cells of the kidney, the mucosal epithelial cells of the intestine, and the lymph and venous systems. The current study suggests that the standard therapy supply almost enough Cu for patient tissues. But given Cu passes through the tissues to venous and lymph systems, or accumulate in the cells responsible for Cu absorption.
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