B cell antigen receptor-induced activation of Akt promotes B cell survival and is dependent on Syk kinase.

Signaling through the B cell Ag receptor (BCR) is a key determinant in the regulation of B cell physiology. Depending on additional factors, such as microenvironment and developmental stage, ligation of the BCR can trigger B lymphocyte activation, proliferation, or apoptosis. The regulatory mechanisms determining B cell apoptosis and survival are not known. Using the chicken B lymphoma cell line DT40 as a model system, we investigated the role of the serine/threonine kinase Akt in B cell activation. While parental DT40 cells undergo apoptosis in response to BCR cross-linking, cells overexpressing Akt show a greatly diminished apoptotic response. By contrast, limiting the activation of Akt, either by inhibiting phosphatidylinositol 3-kinase or by ectopic expression of the phospholipid phosphatase MMAC1, results in a significant increase in the percentage of apoptotic cells after BCR cross-linking. Using various DT40 knockout cell lines, we further demonstrate that the tyrosine kinase Syk is required for Akt activation and that Lyn tyrosine kinase inhibits Akt activation. Taken together, the data demonstrate that Akt plays an important role in B cell survival and that Akt is activated in a Syk-dependent pathway.

Gene dose-dependent maturation and receptor editing of B cells expressing immunoglobulin (Ig)G1 or IgM/IgG1 tail antigen receptors.

Conserved differences between the transmembrane and cytoplasmic domains of membrane immunoglobulin (Ig)M and IgG may alter the function of antigen receptors on naive versus memory B cells. Here, we compare the ability of these domains to signal B cell allelic exclusion and maturation in transgenic mice. A lysozyme-binding antibody was expressed in parallel sets of mice as IgM, IgG1, or a chimeric receptor with IgM extracellular domains and transmembrane/cytoplasmic domains of IgG1. Like IgM, the IgG1 or chimeric IgM/G receptors triggered heavy chain allelic exclusion and supported development of mature CD21(+) B cells. Many of the IgG or IgM/G B cells became CD21(high) and downregulated their IgG and IgM/G receptors spontaneously, resembling memory B cells and B cells with mutations that exaggerate B cell antigen receptor signaling. Unlike IgM-transgenic mice, "edited" B cells that carry non-hen egg lysozyme binding receptors preferentially accumulated in IgG and IgM/G mice. This was most extreme in lines with the highest transgene copy number and diminished in variant offspring with fewer copies. The sensitivity of B cell maturation to transgene copy number conferred by the IgG transmembrane and cytoplasmic domains may explain the diverse phenotypes found in other IgG-transgenic mouse strains and may reflect exaggerated signaling.

Ig heavy chain extracellular spacer confers unique glycosylation of the Mb-1 component of the B cell antigen receptor complex.

A unique functional role for the B cell Ag receptor, IgD, has not yet been identified. A number of properties of IgD, such as distinct intron-exon organization and a conserved pattern of developmental expression, suggest a selective evolutionary advantage for this receptor isotype. To explore structural features of IgD that may confer unique functions, chimeric Ag receptors were generated in which small segments of the IgD heavy chain membrane proximal regions were substituted for corresponding segments of the IgM heavy chain. Polypeptides that associate with the Ig receptors were analyzed. Mb-1/Ig-alpha, a signal transduction molecule, is known to be glycosylated in a distinct manner when associated with IgD compared to when associated with other isotypes. We report that this differential glycosylation results because one N-linked carbohydrate on Mb-1 fails to be processed into an Endo-H-resistant form when associated with IgM or IgG, whereas both N-linked carbohydrates are processed on Mb-1 associated with IgD. By preparing chimeric IgM-IgD heavy chains, substitution of the IgD extracellular spacer segment alone was found to be necessary and sufficient to confer an IgD-type glycosylation pattern on the Mb-1 molecule. This altered glycosylation pattern, however, did not confer a detectable difference in calcium mobilization or in protein tyrosine phosphorylation upon receptor stimulation. Interestingly, a similar altered glycosylation pattern has been reported for the T cell Ag receptor complex in which carbohydrates on the CD3 delta-chain are fully processed in gamma delta-T cell receptors but only partially processed in alpha beta-T cell receptors.

Inhibition of tumor cell growth by interferon-gamma is mediated by two distinct mechanisms dependent upon oxygen tension: induction of tryptophan degradation and depletion of intracellular nicotinamide adenine dinucleotide.

Growth of a variety of human tumor cell lines is inhibited by interferon-gamma (IFN-gamma) in vitro. This mechanism is not well understood. The present experiments identify two separate mechanisms which account for the growth inhibitory activity of IFN-gamma. Cell lines most sensitive to IFN-gamma (inhibited by 10-30 U/ml IFN-gamma in 3 d) were stimulated by IFN-gamma to oxidize tryptophan in media to kynurenine and completely eliminated tryptophan from the culture media after 48-72 h. Addition of L-tryptophan, but not other aromatic amino acids, other essential amino acids, or D-tryptophan, prevented inhibition of cell growth by IFN-gamma. The amount of IFN-gamma required to yield 50% inhibition of cell growth was directly related to the concentration of L-tryptophan in culture media and increased from approximately 3 to 600 U/ml as the concentration of tryptophan in the media was increased from 25 to 1,000 microM. By contrast, inhibition of growth of the cell lines, BT20 and HT29, was not prevented by addition of tryptophan. Inhibition by IFN-gamma (100-300 U/ml after 5-6 d) was, however, completely prevented by addition of two inhibitors of adenosine diphosphate-ribosyl transferase (ADP-RT), 3-aminobenzamide or nicotinamide. Activity of ADP-RT was increased in these cell lines after addition of IFN-gamma. ADP-RT catalyzes the incorporation of the ADP moiety of nicotinamide adenine dinucleotide (NAD) into proteins and causes depletion of intracellular NAD. All tumor cell lines tested had reduced levels of intracellular NAD after treatment with IFN-gamma and loss of NAD preceded inhibition of cell growth by 12-24 h. Inhibitors of IFN-gamma-mediated inhibition of cell growth prevented loss of levels of intracellular NAD. Generation of reactive oxygen species lead to DNA strand breaks which result in activation of ADP-RT. Increased DNA strand breaks were induced in BT20 and HT29 cells but not ME180 and A549 cells after culture with IFN-gamma. The two enzymes known to catalyze the decyclization of tryptophan to kynurenine require superoxide anion for activity. Increased amounts of superoxide anion were released from ME180 and A549 cells after culture with IFN-gamma. Reduced oxygen concentration decreased the ability of IFN-gamma to inhibit tumor cell growth in vitro. Intracellular glutathione has been shown to protect cells against oxidative damage by various agents. Elevation or reduction of intracellular glutathione concentrations lowered or raised sensitivity of cell lines to IFN-gamma, respectively. These data indicate that at least two distinct mechanisms can account for IFN-gamma-madiated inhibition of tumor cell growth. Both mechanisms appear to be sensitive to oxygen tension and to changes in intracellular glutathione concentrations, and both mechanisms lead to loss of intracellular NAD.

Generation and characterization of continuous lines of CD8+ suppressor T lymphocytes.

The ability to grow normal T lymphocytes in long term culture has advanced our understanding of T cell biology. The growth of CD4+ cell lines allowed a further evaluation and appreciation of functional subtypes within this group. Cytotoxic CD8+ T cells have been characterized as well. The routine and continuous culture of Ag-nonspecific CD8+ Ts cells has been difficult to achieve. We have found that CD8+ T cells that suppress T cell proliferation and lack cytotoxic activity against T cells can be routinely obtained from PWM or PHA-stimulated PBMC. Continuous culture of T cell blasts from PWM or PHA-stimulated PBMC resulted in the growth of CD4+ and CD8+ T cells. These lines developed suppressor cell activity within 7 days after stimulation with PWM and 3 to 4 wk after stimulation with PHA. Concomitant with the development of suppressor activity was the loss of CD4+ T cells resulting in homogeneous lines of CD8+ suppressor cells. These cell lines have been maintained in continuous culture for greater than 6 mo by addition of rIL-2 twice weekly and restimulation with feeder cells and PHA every 2 wk. Activity of these cell lines was relatively resistant to irradiation or treatment with mitomycin C. Both cell lines suppressed proliferation of autologous or heterologous CD4+ T cells stimulated with PWM, OKT3, or tetanus toxoid but failed to suppress proliferation of CD4+ T cells in a mixed lymphocyte reaction. CD4+ T cells stimulated with PWM produced equivalent amounts of IL-2 in the presence or absence of Ts cells but failed to express the IL-2R (TAC) on their surface in the presence of Ts cells. By contrast, CD4+ T cell lines or cytotoxic CD8+ T cell lines failed to suppress proliferation of CD4+ T cells. With these results we describe methods for the generation and continuous culture of Ag-nonspecific CD8+ Ts cells and define some of their properties. These cells lines should be helpful in further elucidating the functional and phenotypic repertoire of CD8+ Ts cells.