Ligand-dependent Inhibition of CD1d-restricted NKT Cell Development in Mice Transgenic for the Activating Receptor Ly49D

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM–ITAM signaling is likely to play an important role in the developmental program of NKT cells

Intracellular and Extracellular Leukemia Inhibitory Factor Proteins Have Different Cellular Activities That Are Mediated by Distinct Protein Motifs

Although many growth factors and cytokines have been shown to be localized within the cell and nucleus, the mechanism by which these molecules elicit a biological response is not well understood. The cytokine leukemia inhibitory factor (LIF) provides a tractable experimental system to investigate this problem, because translation of alternatively spliced transcripts results in the production of differentially localized LIF proteins, one secreted from the cell and acting via cell surface receptors and the other localized within the cell. We have used overexpression analysis to demonstrate that extracellular and intracellular LIF proteins can have distinct cellular activities. Intracellular LIF protein is localized to both nucleus and cytoplasm and when overexpressed induces apoptosis that is inhibited by CrmA but not Bcl-2 expression. Mutational analysis revealed that the intracellular activity was independent of receptor interaction and activation and reliant on a conserved leucine-rich motif that was not required for activation of cell surface receptors by extracellular protein. This provides the first report of alternate intracellular and extracellular cytokine activities that result from differential cellular localization of the protein and are mediated by spatially distinct motifs

Ly49D-mediated ITAM signaling in immature thymocytes impairs development by bypassing the pre-TCR checkpoint.

Activating and inhibitory NK receptors regulate the development and effector functions of NK cells via their ITAM and ITIM motifs, which recruit protein tyrosine kinases and phosphatases, respectively. In the T cell lineage, inhibitory Ly49 receptors are expressed by a subset of activated T cells and by CD1d-restricted NKT cells, but virtually no expression of activating Ly49 receptors is observed. Using mice transgenic for the activating receptor Ly49D and its associated ITAM signaling DAP12 chain, we show in this article that Ly49D-mediated ITAM signaling in immature thymocytes impairs development due to a block in maturation from the double negative (DN) to double positive (DP) stages. A large proportion of Ly49D/DAP12 transgenic thymocytes were able to bypass the pre-TCR checkpoint at the DN3 stage, leading to the appearance of unusual populations of DN4 and DP cells that lacked expression of intracellular (ic) TCRβ-chain. High levels of CD5 were expressed on ic TCRβ(-) DN and DP thymocytes from Ly49D/DAP12 transgenic mice, further suggesting that Ly49D-mediated ITAM signaling mimics physiological ITAM signaling via the pre-TCR. We also observed unusual ic TCRβ(-) single positive thymocytes with an immature CD24(high) phenotype that were not found in the periphery. Importantly, thymocyte development was completely rescued by expression of an Ly49A transgene in Ly49D/DAP12 transgenic mice, indicating that Ly49A-mediated ITIM signaling can fully counteract ITAM signaling via Ly49D/DAP12. Collectively, our data indicate that inappropriate ITAM signaling by activating NK receptors on immature thymocytes can subvert T cell development by bypassing the pre-TCR checkpoint

Regulated expression of alternate transcripts from the mouse oncostatin M gene: Implications for interleukin-6 family cytokines

Oncostatin M (OSM) is a member of the IL-6 family of polyfunctional cytokines. The characterized murine OSM transcript consists of three exons and encodes a secreted protein. Investigations of mOSM expression using the ribonuclease protection assay demonstrated novel sites of expression in undifferentiated but not differentiated pluripotent cells, and revealed the existence of alternatively spliced mOSM transcripts. cDNAs representing a novel mOSM transcript (mOSM 13) containing exon 1 spliced directly to exon 3 were isolated from bone marrow using Rapid Amplification of cDNA Ends (RACE) PCR and RT-PCR approaches. Expression of the mOSM 13 transcript was regulated in a tissue-specific manner and independently of mOSM transcript production, suggesting that its production is biologically significant. Splicing of exon 1 directly to exon 3 disrupts the OSM open reading frame of mOSM 13. Initiation of translation at sites within exon 3 of mOSM 13 would yield N-terminally truncated OSM proteins that are localized within the cell. The omission of exon 2 by alternate splicing and the production of intracellular proteins with alternate biological activities are conserved among several IL-6 family cytokines and are one manifestation of a more general phenomenon; the production of alternate cytokine transcripts encoding intracellular and extracellular proteins.http://www.sciencedirect.com/science/journal/1043466