Characterization of a novel murine T cell-activating factor

Purified resting peripheral lymph node T cells can be activated to produce interleukin 2 (IL 2) and to proliferate in the presence of Concanavalin A (Con A) and an apparently novel lymphokine that we call T cell activating factor (TAF). TAF is biochemically distinct from IL 1, IL 2, IL 3, and other colony stimulating factors, IL 4 (BSF-1) and interferons. Furthermore, of the recombinant and natural cytokines tested, only IL 2 and TAF are active in the TAF assay. In the presence of Con A, TAF stimulates an increase in the steady-state level of IL 2 mRNA in T cells, the secretion of active IL 2 into the culture medium, and the proliferation of the T cells. We propose that TAF is a previously undescribed molecule the function of which is to stimulate IL 2 production by T cells that have encountered antigen, and we propose that TAF has an important role in primary T cell immune responses.     

IL-6 and IL-1 synergize to stimulate IL-2 production and proliferation of peripheral T cells

Purified T cells can be induced to proliferate and to produce the autocrine growth factor IL-2 with mAb to the TCR and costimulatory cytokines. In a previous report we demonstrated that human IL-6 stimulates IL-2 production and proliferation of purified T cells, in conjunction with the insolubilized anti-TCR V beta 8 mAb, F23.1. Here we show that when CD4+ T cells are rigorously purified to greater than 99% CD4+CD8-, they respond only weakly to F23.1 and IL-6. Instead, there is an additional requirement for IL-1, which dramatically synergizes with IL-6 to induce prolonged (greater than 7 days) proliferative responses and IL-2 production. Similar results were observed when the highly mitogenic anti-CD3 mAb 145-2C11 was substituted for F23.1. The proliferation induced by F23.1, IL-1, and IL-6 was substantially (greater than 80%) inhibited by a mAb to mouse IL-2, and was not inhibited by an anti-IL-4-mAb. In accordance with this finding, medium conditioned by the activated CD4+ cells contained large amounts of IL-2, which increased over a 7-day culture period. These results demonstrate that IL-6 and IL-1 stimulate T cell proliferation by inducing production of the autocrine growth factor IL-2. In addition, the two lymphokines must be present simultaneously for activation to occur. The possible roles of IL-6 and IL-1 in IL-2 gene regulation and in Ag-induced T cell activation are discussed.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Infection-induced regulation of natural killer cells by macrophages and collagen at the lymph node subcapsular sinus

Infection leads to heightened activation of natural killer (NK) cells, a process that likely involves direct cell-to-cell contact, but how this occurs in vivo is poorly understood. We have used two-photon laser-scanning microscopy in conjunction with Toxoplasma gondii mouse infection models to address this question. We found that after infection, NK cells accumulated in the subcapsular region of the lymph node, where they formed low-motility contacts with collagen fibers and CD169(+) macrophages. We provide evidence that interactions with collagen regulate NK cell migration, whereas CD169(+) macrophages increase the activation state of NK cells. Interestingly, a subset of CD169(+) macrophages that coexpress the inflammatory monocyte marker Ly6C had the most potent ability to activate NK cells. Our data reveal pathways through which NK cell migration and function are regulated after infection and identify an important accessory cell population for activation of NK cell responses in lymph nodes.     

Diversity, rearrangement, and expression of murine T cell gamma genes

Although the T cell gamma genes are similar in many respects to T cell receptor alpha and beta genes, earlier studies suggested that only a single gamma variable (V gamma) gene is expressed in mature T cells. We report the isolation and characterization of three new rearranged V gamma genes from murine fetal thymocytes. Although each of the new V gamma gene rearrangements is present in fetal thymocytes, two of them are undetectable in mature T cells. The levels of mRNA corresponding to each type of V gamma gene rearrangement in mature T cells are dramatically diminished compared with those in fetal thymocytes, although the abundance of two of the rearranged genes is increased in mature T cells. Our results demonstrate that there is significant expressed variability of gamma genes in immature T cells. Furthermore, the dynamics of gamma gene rearrangement and expression support the idea that gamma genes function in immature T cells.