William M. Weaver

A Critical Role for Dnmt1 and DNA Methylation in T Cell Development, Function, and Survival

The role of DNA methylation and of the maintenance DNA methyltransferase Dnmt1 in the epigenetic regulation of developmental stage- and cell lineage-specific gene expression in vivo is uncertain. This is addressed here through the generation of mice in which Dnmt1 was inactivated by Cre/loxP-mediated deletion at sequential stages of T cell development. Deletion of Dnmt1 in early double-negative thymocytes led to impaired survival of TCRalphabeta(+) cells and the generation of atypical CD8(+)TCRgammadelta(+) cells. Deletion of Dnmt1 in double-positive thymocytes impaired activation-induced proliferation but differentially enhanced cytokine mRNA expression by naive peripheral T cells. We conclude that Dnmt1 and DNA methylation are required for the proper expression of certain genes that define fate and determine function in T cells.

The Proximal Regulatory Element of the Interferon-γ Promoter Mediates Selective Expression in T Cells

Interferon-γ (IFN-γ) is produced by natural killer cells and certain subsets of T cells, but the basis for its selective expression is unknown. Within the region between −108 and −40 base pairs of the IFN-γ promoter are two conserved and essential regulatory elements, which confer activation-specific expression in T cells. This report describes studies indicating that the most proximal of these two regulatory elements is an important determinant of its restricted expression. The proximal element is a composite site that binds members of the CREB/ATF, AP-1, and octamer families of transcription factors. Jun is essential for activation-induced transcription and binds preferably as a heterodimer with ATF-2. In contrast, CREB appears to dampen transcription from this element. The CpG dinucleotide in this element is selectively methylated in Th2 T cells and other cells that do not express IFN-γ, and methylation markedly reduces transcription factor binding. As a target for DNA methylation and for binding of transcription factors that mediate or impede transcription, this element appears to play a central role in controlling IFN-γ expression.

Ontogeny of T Lymphocyte Function in the Neonate

ABSTRACT: T cell precursors are first detected in the thymus at eight weeks of gestation. By 15 to 20 weeks of gestation, T‐cell precursors expressing αβ and γδ T‐cell receptors are present in the thymus in numbers relatively similar to those found in postnatal life. However, recent data suggest that T‐cell receptor diversity is more limited during fetal and neonatal life than in adults. Additionally, the functional capacity of T cells in the fetus and neonate is immature, in that neonatal T cells express a limited repertoire of lymphokines in response to activation. Specifically, the production of the lymphokines, interferon‐γ and interleukin‐4, which participate in the maturation of cytotoxic cells, activation of macrophages, and the maturation and modulation of B cell function and isotype expression, is reduced more than tenfold compared to cells from adults. This appears to result primarily from the lack of memory T cells in the fetus and neonate, reflecting their antigenic naivete. The difference in lymphokine expression is due to diminished transcription of these genes in neonatal T cells in response to activation. Preliminary data indicate that differences in essential promoter elements regulating transcription of these lymphokine genes plays a role in their differential expression in T cells.

Isolation, purification and characteristics of mononuclear phagocytes from human placentas

Methods for the isolation of mononuclear phagocytes from human placentas by digestion with collagenase or with trypsin are described. Mononuclear phagocytes were approximately 35% of the cells obtained. Preparations were enriched for mononuclear phagocytes by sequential density gradient centrifugation over Ficoll-Hypaque and Percoll, adherence to plastic and removal of contaminating trophoblastic cells with brief trypsin exposure. Monolayers obtained by these methods were greater than 85% mononuclear phagocytes. These cells were predominantly of fetal origin; most were mature macrophages but up to 20% appeared to be recently derived from blood monocytes as determined by ultrastructural peroxidase cytochemistry.

RESISTANCE OF GROUP B STREPTOCOCCI (GBS) TO KILLING BY OXYGEN METABOLITES

Known risk factors for early onset GBS sepsis include lack of specific antibody, density of maternal colonization and prematurity. An additional proposed risk factor, is decreased production of microbicidal oxygen metabolites by phagocytes of susceptible newborns(NB). However, GBS lack catalase, produce and release H2O2, and thus should be readily killed by phagocytes with a diminished respiratory burst. Surprisingly, CBS III were equally or more resistant than Staph aureus (SA) to reagent H2O2(60 min assay, n=5-7): log10 cfu/ml at none, 10−3M, 5×10−3M, 10−2M H2O2 for GBS=7.1,7.3,5.9,3.1* and for SA=7.2,6.6,4.5*,4.4*-(*p < 0.001 vs none). Results with other strains of GBS were similar; Group A strep were more susceptible. Neither GBS nor SA were killed in 60 min by a xanthine oxidase-acetaldehyde - Fe++ - EDTA system by a flux of 0.7 nmoles O2/ml/min; both were killed by 3.5 nmoles O2/ml/min. Killing of both was inhibited by omission of Fe++ or EDTA and by addition of mannitol, catalase or superoxide dismutase, suggesting that hydroxyl radical (OH·) played a major role in microbicidal activity and was active at concentrations &Tilde; 2 log10 less than H2O2. Although GBS lacked catalase, GBS compared to SA contained more glutathione(G) (68.2 vs 0.2 nmoles/mg) and G reductase (44.2 vs 1.3 nmoles/mg/min); SOD and G peroxidase were comparable. G may protect GBS from H2O2 and OH: Defective phagocyte production of oxygen radicals, particularly OH· as reported by Ambruso(Ped 64:722,1979), may contribute to the NBs susceptibility to GBS.

978 MURAMYL DIPEPTIDE (MDP) ENHANCES SUPEROXIDE (O 2 − ) PRODUCTION BUT NOT ANTIMICROBIAL ACTIVITY OF HUMAN MACROPHAGES (Mø)

MDP, a subunit of bacterial peptidoglycan, enhances resistance of animals to certain infections; MDP treatment enhances animal Mø phorbolmyristate acetate (PMA) stimulated O2− production and antimicrobial activity (indices of Mø activation). We examined in vitro effects of MDP on human monocyte-derived Mø O2− production and antimicrobial activity against T. gondii (T), an intracellular pathogen, and S. aureus (S), an extracellular pathogen. PMA stimulated O2− generation by MDP treated Mø (MDP-Mø) was always increased (55% ± 12%) compared to control Mø (NMø) (n=7; p <.02); without PMA no O2− was generated. In contrast, O2− generation by MDP-Mø and by NMø phagocytosing T,S or zymosan was similar. Survival of T within NMø (53% ± 11%) and within MDP-Mø (43% ± 12%) did not differ significantly; T replicated equally well in either Mø. Phagocytosis of [14C] -labelled S, determined after removal of extracellular S with lysostaphin, by NMø and by MDP Mø was equal (log10 5.4 ± .1); killing of intracellular S by NMø (63% ± 11%) and by MDP Mø (61% ± 8%) was comparable. By enhancing Mø release of inflammatory mediators such as O2− in response to non-specific stimuli, while failing to enhance Mø antimicrobial activity, MDP might adversely affect human response to infection. Chemical modification of MDP alters its effects in animals; modifications may yield drugs that enhance human host defenses without enhancing inflammation.