We grafted fetal thymi from wild-type mice into immunodeficient RAG-2-/- or class II-/-RAG-2-/- (class II MHC-) recipients and followed the fate of naive CD4+ T cells derived from the grafts. In both types of recipients, newly generated CD4+ T cells proliferated to the same extent in the periphery and rapidly filled the empty T cell compartment. However, CD4+ T cells in class II- recipients gradually decreased in number over 6 months. These results show that interactions between the TCR and class II molecules are not required for newly generated CD4+ T cells to survive and proliferate, but are necessary to maintain the size of the peripheral T cell pool for extended periods.

Human DNA polymerase N (POLN or pol ) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiationinduced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.The human genome contains 15 distinct known DNA polymerase genes, and these are classified into four families A, B, X, and Y based on their amino acid sequences (1). Human DNA polymerase N (POLN), 2 the most recently discovered nuclear DNA polymerase, is an A-family enzyme with unknown function. The gene on chromosome 4p16.2 encodes a protein of 900 amino acid residues with a molecular mass of 100 kDa (2). The prototypical A-family DNA polymerase, Escherichia coli pol I is a high fidelity DNA polymerase that contributes to the maturation of Okazaki fragments during DNA replication and in gap-filling during base excision repair (BER), nucleotide excision repair (NER), and repair of DNA interstrand cross-links.Human POLQ, another A-family DNA polymerase, is similar to the Drosophila nuclear DNA polymerase Mus308 (3) in that it encodes both a DNA/RNA helicase domain and an A-family DNA polymerase domain (4, 5). By contrast, POLN has only the DNA polymerase domain. The POLN gene is encoded only in vertebrate genomes, but not in invertebrates or any lower eukaryotes. Possibly POLN has a role related to organ systems that are especially developed in vertebrates, such as the adaptive immune system or the brain. Expression studies of POLN are limited, but expression of the gene has been detected by Northern blotting in testes, heart and skeletal muscle tissue (2) and by expression sequence tagging in prostate, muscle, brain, and other organs. In cells from a human neuroblastoma patient, a chromosome fusion (1, 4) disrupting the DNA polymerase domain coding sequence of POLN was observed at diagnosis and at relapse. A (4, 17) fusion was detected at relapse only (6). It is possible that POLN might serve as a tumor suppressor in some cell types and that loss...

Genes were isolated using the suppression subtractive hybridization method by stimulation of pro/pre B cells with anti-CD40 and interleukin (IL)-4 to mature Sμ-Sε–switched cells. One of the strongly upregulated genes encodes a novel murine CC chemokine we have named ABCD-1. The ABCD-1 gene has three exons separated by 1.2- and 2.7-kb introns. It gives rise to a 2.2-kb transcript containing an open reading frame of 276 nucleotides. Two polyadenylation sites are used, giving rise to cDNAs with either 1550 or 1850 bp of 3′ untranslated regions. The open reading frame encodes a 24 amino acid–long leader peptide and a 68 amino acid–long mature protein with a predicted molecular mass of 7.8 kD. ABCD-1 mRNA is found in highest quantities in activated splenic B lymphocytes and dendritic cells. Little chemokine mRNA is present in lung, in unstimulated splenic cells, in thymocytes, and in lymph node cells. No ABCD-1 mRNA is detected in bone marrow, liver, kidney, or brain, in peritoneal exudate cells as well as in the majority of all unstimulated B lineage cells tested. It is also undetectable in Concanavalin A–activated/IL-2–restimulated splenic T cells, and in bone marrow–derived IL-2–induced natural killer cells and IL-3–activated macrophages. Recombinant ABCD-1 revealed a concentration-dependent and specific migration of activated splenic T lymphoblasts in chemotaxis assays. FACS® analyses of migrated cells showed no preferential difference in migration of CD4+ versus CD8+ T cell blasts. Murine as well as human T cells responded to ABCD-1. Freshly isolated cells from bone marrow, thymus, spleen, and lymph node, IL-2–activated NK cells, and LPS-stimulated splenic cells, all did not show any chemotactic response. Thus, ABCD-1 is the first chemokine produced in large amounts by activated B cells and acting selectively on activated T lymphocytes. Therefore, ABCD-1 is expected to play an important role in the collaboration of dendritic cells and B lymphocytes with T cells in immune responses.

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides ( In innate immune systems, toll-like receptors (TLRs) 2 are implicated in recognition and signaling of pathogen-associated molecular patterns (1). Stimulation of different TLRs induces distinct patterns of gene expression, which leads to the activation of innate immunity and instructs the development of antigen-specific acquired immunity (2). Among the TLR family, TLR4 plays a critical role in recognition and signaling of bacterial lipopolysaccharide (LPS) (3). TLR4 requires accessory protein MD-2 for an efficient response to LPS (4). We have recently demonstrated the direct interaction between MD-2 and extracellular TLR4 domain (5, 6). MD-2 binds LPS (7), but LPS has been demonstrated to be cross-linked with TLR4 and MD-2 only when coexpressed with CD14 (8), suggesting that LPS is in close proximity to the receptor complex.The lung is constantly challenged by inhaled pathogens, pollutants, and particles that are present in the environment. Pulmonary surfactant, a mixture of lipids and proteins that serves to reduce the surface tension of the alveoli, is involved in the innate immune system of the lung. Recent studies demonstrate that the most abundant component of surfactant protein, surfactant protein A (SP-A), plays important roles in pathogen clearance and inflammatory responses (9 -12). SP-A belongs to the collectin subgroup of the C-type lectin superfamily along with surfactant protein D (SP-D) and mannose-binding lectin. The primary structure of SP-A subunits are composed of a short amino-terminal segment, a collagen-like sequence characterized by Gly-X-Y repeats with an interruption near the midpoint of the domain, a neck domain, and a carbohydrate recognition domain (CRD) (13). Trimeric association occurs by the folding of collagenous domains into triple helices (14) and coiled-coil bundling of ␣-helices in the neck (15). Fully assembled SP-A is a bouquet-like octadecamer consisting of six trimeric subunits that are stabilized by the amino-terminal sequences and disulfide bonds (16).Recent studies from this and other laboratories have demonstrated that SP-A modulates inflammation by interacting with cell surface receptors including CD14 (17), TLR2 (18, 19), signal-inhibitory regulatory protein ␣, and calreticulin/CD91 (20). Although it has been suggested that SP-A activates cellular responses dependent on TLR4 (21), the interactions of SP-A * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan and by Akiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.

During B-cell development the surrogate light (SL) chain is selectively expressed in progenitor and precursor B cells during the developmental stages of D(H) to J(H) and V(H) to D(H)J(H) rearrangements. Approximately half of all muH chains produced by these rearrangements cannot pair with SL chains and cannot form a pre-B-cell receptor (pre-BCR). A spectrum of affinities between VpreB and individual V(H) domains generates preB cells with pre-BCR of different fitness which, in turn, determines the extent of the pre-B II-cell proliferation and the fidelity of allelic exclusion of the H chain locus. Once pre-BCR is expressed, SL chain expression is turned off. As pre-B II cells proliferate, SL is diluted out, thus limiting pre-BCR formation. As a consequence, pre-B II cells stop proliferating, become small and resting and begin to rearrange the L chain loci. Multiple rearrangements of the kappaL chain alleles are often detected in wild-type small pre-B II cells. Around 20% of the muH chain-expressing small pre-B II cells also express L chains but do not display the Ig on the surface. Hence, it is likely that not all L chains originally generated in resting pre-B II cells can pair with the muH chain previously present in that cell. The best fitting ones are selected preferentially to generate sIg+ B cells. Furthermore, the transition of immature B cells from the bone marrow to spleen and their development to mature cells appear as two separate steps controlled by different genes.

At the precursor B cell stage during bone marrow B cell development, Ig μH chain associates with surrogate L (SL) chain, which is encoded by the three genes VpreB1, VpreB2, and λ5, to form the pre-B cell receptor (pre-BCR). Surface expression of the pre-BCR is believed to signal both proliferation and allelic exclusion of the IgH locus. Mice which lack either VpreB1/VpreB2 or λ5 show a lack of precursor B cell expansion but normal IgH allelic exclusion. This would suggest that one of either λ5 or VpreB can make a pre-BCR-like complex which is still able to signal allelic exclusion but not proliferation. To investigate this, we established mice lacking all components of the SL chain. These mice showed severely impaired B cell development which was similar to that previously found in mice lacking either λ5 or VpreB1/VpreB2. Surprisingly, the IgH locus was still allelically excluded and thus the SL chain appears not to be involved in allelic exclusion.

The pre-B cell receptor consists of immunoglobulin (Ig) μ heavy chains and surrogate light chain, i.e., the VpreB and λ5 proteins. To analyze the role of the two VpreB proteins, mice lacking the VpreB1 and VpreB2 genes were generated. VpreB1 − /−VpreB2 − /− mice were impaired in their B cell development at the transition from pre-BI to large pre-BII cells. Pre-BII cells did not expand by proliferation, consequently 40-fold less small pre-BII and immature B cells were found in bone marrow, and the generation of immature and mature conventional B cells in spleen appeared reduced. In addition, only low numbers of B-1a cells were detected in the peritoneum. Surprisingly, Ig heavy chain allelic exclusion was still active, apparently ruling out a signaling role of a VpreB1/VpreB2–containing receptor in this process.