Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway.

Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-1 (TCF-1) (gene name Tcf7). To identify additional regulators of T cell specification, a cDNA library from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expression of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin(-)Sca-1(+)Kit(+)CD27(-)) and multipotent progenitors (Lin(-)Sca-1(+)Kit(+)CD27(+)), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bcl11b, TCF-1 (Tcf7), and HEBalt, Notch target Deltex1, Deltex3L, Fkbp5, Eva1, and Tmem131. Like GATA3 and Deltex1, Bcl11b, Fkbp5, and Eva1 were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only Bcl11b and HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative 1 and double negative 2) corresponding to T lineage specification. Bcl11b was uniquely T lineage restricted and induced by Notch/Delta signaling specifically upon entry into the T lineage differentiation pathway.

Bovine peptidoglycan recognition protein-S: antimicrobial activity, localization, secretion, and binding properties.

Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern recognition molecules of innate immunity that are conserved from insects to humans. Various PGRPs are reported to have diverse functions: they bind bacterial molecules, digest PGN, and are essential to the Toll pathway in Drosophila. One family member, bovine PGN recognition protein-S (bPGRP-S), has been found to bind and kill microorganisms in a PGN-independent manner, raising questions about the identity of the bPGRP-S ligand. Addressing this, we have determined the binding and microbicidal properties of bPGRP-S in a range of solutions approximating physiologic conditions. In this study we show that bPGRP-S interacts with other bacterial components, including LPS and lipoteichoic acid, with higher affinities than for PCP, as determined by their abilities to inhibit bPGRP-S-mediated killing of bacteria. Where and how PGRPs act in vivo is not yet clear. Using Immunogold electron microscopy, PGRP-S was localized to the dense/large granules of naive neutrophils, which contain the oxygen-independent bactericidal proteins of these cells, and to the neutrophil phagolysosome. In addition, Immunogold staining and secretion studies demonstrate that neutrophils secrete PGRP-S when exposed to bacteria. Bovine PGRP-S can mediate direct lysis of heat-killed bacteria; however, PGRP-S-mediated killing of bacteria is independent of this activity. Evidence that bPGRP-S has multiple activities and affinity to several bacterial molecules challenges the assumption that the PGRP family of proteins recapitulates the evolution of TLRs. Mammalian PGRPs do not have a single antimicrobial activity against a narrow range of target organisms; rather, they are generalists in their affinity and activity.

Isolation, characterization, and antimicrobial properties of bovine oligosaccharide-binding protein. A microbicidal granule protein of eosinophils and neutrophils.

Peptidoglycan recognition proteins (PGRPs) constitute a recently characterized family of pattern-recognition molecules that are conserved from insects to humans and are implicated in mammalian innate immunity. Here we report the isolation, characterization, cDNA cloning, and antimicrobial activities of a bovine PGRP ortholog termed bovine oligosaccharide-binding protein (bOBP). Milligram quantities of bOBP were purified from peripheral leukocytes, thus allowing for the characterization of the disulfide array and for determining the in vitro antimicrobial activities of the native protein. Of the tissues analyzed, bOBP mRNA was detected only in bone marrow where the protein is synthesized as a 190 amino acid precursor. The mature 169 amino acid protein is stored in the cytoplasmic granules of neutrophils and eosinophils but is absent from lymphocytes, monocytes, and platelets. bOBP was microbicidal for Gram-positive and Gram-negative bacteria and yeast at low micromolar concentrations. The finding that bOBP was microbicidal for organisms in which peptidoglycan is absent (Cryptococcus neoformans) or buried (Salmonella typhimurium) indicates that previous conclusions about the specificity of peptidoglycan recognition proteins must be reevaluated and suggests that other envelope components may mediate the antimicrobial action of PGRP family members.