B cell ligand discrimination through a spreading and contraction response.

B cells recognize foreign antigens by virtue of cell surface immunoglobulin receptors and are most effectively activated by membrane-bound ligands. Here, we show that in the early stages of this process, B cells exhibit a two-phase response in which they first spread over the antigen-bearing membrane and then contract, thereby collecting bound antigen into a central aggregate. The extent of this response, which is both signaling- and actin-dependent, determines the quantity of antigen accumulated and hence the degree of B cell activation. Brownian dynamic simulations reproduce essential features of the antigen collection process and suggest a possible basis for affinity discrimination. We propose that dynamic spreading is an important step of the immune response.

A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector.

We have investigated the minimal time required for efficient transduction of human hematopoietic repopulating cells using a surrogate nonobese diabetic (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay. Cord blood CD34+ cells were transduced to high levels over 24-48 hr in the presence of Flt-3 ligand, stem cell factor, interleukin 3, and interleukin 6. Under these conditions, high levels of NOD/SCID repopulating activity were preserved, but the levels of gene marking in engrafting cell populations measured by expression of a reporter transgene were low. Extension of the transduction period by 24 hr (total culture period, 72 hr) under the same cytokine conditions resulted in high levels of gene marking, but on closer analysis expression was limited predominantly to the myeloid population. Efficient transduction of both lymphoid and myeloid lineages could be achieved only if the transduction protocol was extended by a further 24 hr (total culture period, 96 hr), suggesting that myeloid lineage-committed precursors are capable of repopulation, and that over shorter time periods transduction is largely restricted to this population. This adds to the emerging evidence of heterogeneity within the SRC compartment, and has important implications for the interpretation of this assay in stem cell transplantation and gene transfer studies.

Presence of inulin and oligofructose in the diets of Americans.

The U.S. Department of Agriculture 1994-1996 Continuing Survey of Food Intakes by Individuals was used to estimate the intake of naturally occurring inulin and oligofructose by the U.S. population. Two nonconsecutive 24-h dietary recalls from >15,000 Americans of all ages were conducted, and a special database of inulin and oligofructose was developed specifically for the analyses. American diets provided on average 2.6 g of inulin and 2.5 g of oligofructose. Intakes varied by gender and age, ranging from 1.3 g for young children to 3.5 g for teenage boys and adult males. When standardized for amount of food consumed, the intakes showed little difference across gender and age. Significant differences in intake of these components were seen between categories within region of the country, season, income, and race and origin; however, the actual differences were relatively small. Major food sources of naturally occurring inulin and oligofructose in American diets were wheat, which provided about 70% of these components, and onions, which provided about 25% of these components. The estimation of the presence of inulin and oligofructose in the diets of Americans has not been published to date.

Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor gamma chain.

Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human haemopoiesis and lymphoid cell function. However, the utility of existing mouse strains can be limited by shortened life-spans, spontaneous production of functional lymphocytes with ageing, and residual innate immunity leading to variable levels of engraftment. Mice with a deletion of the common cytokine receptor gamma chain (gamma c) gene have reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2)-deficient strain produced mice doubly homozygous for the gamma c and RAG2 null alleles (gamma c-/RAG2-). These mice have a stable phenotype characterized by the absence of all T lymphocyte. B lymphocyte and NK cell function. Injection of human B-lymphoblastoid cells resulted in earlier fatal metastatic lymphoproliferative disease than in NOD/LtSz-scid controls. This was particularly evident in animals injected intravenously, possibly because of residual NK activity in NOD/LtSz-scid mice. Levels of engraftment with peripheral-blood-derived human lymphocytes were also increased and associated with higher CD4/CD8 ratios. These findings demonstrate that this new strain of immunodeficient mice has significant advantages over existing strains for engraftment of human cells, and may be useful for study of adoptive immunotherapy and novel therapies for GvHD and HIV infection.

The role of c-Myb or a related factor in regulating the T cell receptor gamma gene enhancer.

An enhancer has been localized 3 kb downstream of the C gamma 1 gene segment of the murine TCR-gamma locus. One element, the gamma 3 site, has been shown to be critical for its functional activity. Here we have determined that Myb-related transcription factors bind to the gamma 3 site and appear to be critical for the full activity of the TCR-gamma enhancer. c-myb products can transactivate the gamma 3 site in cell lines that do not ordinarily support enhancer activity of the gamma 3 site. Mutations in the myb site or an adjacent site for core-binding factor(s) prevent transactivation. c-myb expression in various cell lines is consistent with their capacity to activate the gamma 3 enhancer element using transient transfection assays. Therefore, c-Myb or a related factor appears to play an important role in regulating the murine TCR-gamma enhancer.

Ordered rearrangement of variable region genes of the T cell receptor gamma locus correlates with transcription of the unrearranged genes.

T cell receptor V gamma genes rearrange to the J gamma 1 gene segment in a highly ordered fashion during development. We demonstrate a striking correlation between the pattern of expression of unrearranged V gamma genes and the timing of their rearrangement. Thus, the increases in V gamma 2 rearrangements, and decreases in V gamma 3 and V gamma 4 rearrangements observed during development are paralleled by increasing or decreasing levels of the corresponding unrearranged V gene transcript. We also provide evidence that both the V gamma 3 and V gamma 4 genes are accessible in mature V gamma 3+ cells, but that the V gamma 4 gene may be inaccessible in the progenitors of V gamma 3 cells. The results suggest that regulated local accessibility of the chromatin surrounding V gamma genes is responsible for ordered V gamma gene rearrangement during development.

Identification of a T-cell-specific transcriptional enhancer located 3' of C gamma 1 in the murine T-cell receptor gamma locus.

A transcriptional enhancer element has been localized 3 kilobases 3' of the murine T-cell receptor C gamma 1 locus using a chloramphenicol acetyltransferase reporter gene construct. As a monomer the enhancer functions only in PEER gamma delta cells and Jurkat alpha beta cells of the T-cell lines tested. However, a tetramer of the enhancer functions in virtually all T-cell lines tested, including alpha beta T-cell lines, but not in other cell types. These results suggest that elements other than the enhancer are responsible for the failure of rearranged C gamma 1 genes to be expressed in alpha beta T cells. The enhancer has been localized to a 200-base-pair Rsa I restriction fragment, which contains sequence motifs similar to those found in the other T-cell receptor enhancers but not in the immunoglobulin enhancers.