Cutting edge: recruitment of the CD19/CD21 coreceptor to B cell antigen receptor is required for antigen-mediated expression of Bcl-2 by resting and cycling hen egg lysozyme transgenic B cells.

Recruitment of the CD19/CD21 coreceptor is thought to lower the threshold for effective signaling through the B cell Ag receptor. We provide evidence supporting a second role for coreceptor recruitment, and that is to enhance the survival/proliferative potential of the responding B cells. We show that B cell Ag receptor signaling in the absence of coreceptor recruitment induces cellular accumulation of the anti-apoptotic protein Bcl-xL, whereas CD19-mediated signals are required for Bcl-2 accumulation. The expression of both anti-apoptotic proteins correlates with the enhanced responsiveness of both resting and cycling B cells to growth-promoting signals delivered through CD40. These results provide further evidence for the necessity of coreceptor recruitment during Ag-dependent B cell activation and indicate that Ags derived from inflammatory sites function as better thymus-dependent Ags than their counterparts not coated with complement fragments.

CD40-mediated induction of p21 accumulation in resting and cycling B cells.

The accumulation of G1 cell cycle-related proteins by resting or cycling B cells stimulated with B cell antigen receptor (BCR)- and T helper (Th) cell-derived signals is documented. Resting B cells constitutively express cyclin dependent kinase (cdk)4, cdk2 and the cyclin dependent kinase inhibitor (CKI), p27. The initiation of optimal proliferation with F(ab')2 anti-mu plus paraformaldehyde-fixed CD40 ligand-baculovirus-infected Sf9 cells (CD40L/Sf9 cells) increases accumulation of both cdk4 and cdk2 while decreasing p27 levels. B cells express cyclin D2 early during cycle progression, while cyclin D3 and E are not expressed until 18 h poststimulation and cyclin A by 24 h poststimulation. Cycling B cells express heightened levels of all these cyclins and cdks. Although neither BCR- nor CD40-mediated signals appreciably alter cycling B cell accumulation of cyclins D2, cdk4 and cdk2, the absence of BCR-derived signals results in a decreased accumulation of cyclins D3 and E. Finally, CD40-mediated signals induce resting B cells to accumulate the CKI, p21, while cycling B cells require both BCR- and CD40-mediated signals to maintain increased expression of p21. Thus, a Th cell-derived signal may impact upon both resting and cycling B cell cycle progression, at least in part, by regulating the accumulation of p21. The functional consequences of p21 accumulation as cells enter and move through the cell cycle are discussed.

An in vitro approach for the characterization of the cycling B cell response.

Because isolation of sufficient numbers of cycling, germinal center B cells from mice for biochemical characterization of BCR-derived signals can be problematic, we have designed an experimental approach for generating large numbers of cycling B cells for further study. In the experiments reported here, small, resting B cells were polyclonally stimulated with lipopolysaccharide (LPS), and cycling B cells isolated as two bands on three-step Percoll gradients. Cycling B cells isolated at Days 2, 4, or 6 of preactivation showed an increased expression of Fas receptor and peanut agglutinin binding, with a concomitant decrease in sIgD positivity. These cells phenotypically resembled extrafollicular or early germinal center B cells. These cycling B cells were used to study the functional consequences of differential signaling through the BCR. Strong cross-linking of BCR, by restimulation of cycling normal B cells with either immobilized or soluble F(ab')2 anti-mu and cycling hen egg lysozyme (HEL) transgenic B cells with either soluble or immobilized HEL, extended cellular proliferation by 2-3 d. In contrast, cycling B cells either restimulated with soluble, whole anti-mu (to mimic binding of soluble immune complexes) or cultured in the absence of restimulation (to mimic cycling B cells not competitive for antigen) resulted in the rapid exit of the cells from cycle. This system will enable the molecular and biochemical characterization of signal delivery to cycling B cells.

Strength of signal through BCR determines the fate of cycling B cells by regulating the expression of the Bcl-2 family of survival proteins.

Cycling, splenic B cells were recultured with: (1) no stimulant to reflect poorly competitive clones; (2) soluble, whole anti-mu to reflect clones that bind soluble immune complexes; (3) soluble F(ab')2 anti-mu to reflect clones that bind soluble antigen; and (4) immobilized anti-mu to reflect clones that bind antigen presented by FDC. All four groups displayed similar levels of the death proteins Bax and Bcl-xS. In contrast, cycling B cells restimulated with either soluble F(ab')2 or immobilized anti-mu expressed heightened levels of the survival protein Bcl-xL, and only cells restimulated with immobilized anti-mu expressed the survival protein Bcl-2. Cycling B cells restimulated with either soluble F(ab')2 or immobilized anti-mu displayed a selective survival advantage over cycling B cells receiving no stimulus or soluble, whole anti-mu by both enhancing their responsiveness to CD40 ligand, a Th-cell-derived signal, and increasing the period that the cycling B cells remained responsive to this Th-cell-derived signal. The Th-cell-derived signal did not appreciably alter cycling B cell expression of Bcl-2 family members.

A nonimmunoglobulin transgene and the endogenous immunoglobulin mu gene are coordinately regulated by alternative RNA processing during B-cell maturation.

The immunoglobulin (Ig) genes have been extensively studied as model systems for developmentally regulated alternative RNA processing. Transcripts from these genes are alternatively processed at their 3' ends to yield a transcript that is either cleaved and polyadenylated at a site within an intron or spliced to remove the poly(A) site and subsequently cleaved and polyadenylated at a downstream site. Results obtained from expressing modified genes in established tissue culture cell lines that represent different stages of B-lymphocyte maturation have suggested that the only requirement for regulation is that a pre-mRNA contain competing cleavage-polyadenylation and splice reactions whose efficiencies are balanced. Since several non-Ig genes modified to have an Ig gene-like structure are regulated in cell lines, Ig-specific sequences are not essential for this control. This strongly implies that changes in the amounts or activities of general RNA processing components mediate the processing regulation. Despite numerous studies in cell lines, this model of Ig gene regulation has never been tested in vivo during normal lymphocyte maturation. We have now introduced a non-Ig gene with an Ig gene-like structure into the mouse germ line and demonstrate that RNA from the transgene is alternatively processed and regulated in murine splenic B cells. This establishes that the balance and arrangement of competing cleavage-polyadenylation reactions are sufficient for RNA processing regulation during normal B-lymphocyte development. These experiments also validate the use of tissue culture cell lines for studies of Ig processing regulation. This is the first transgenic mouse produced to test a specific model for regulated mRNA processing.

The regulated expression of cell cycle-related proteins as B-lymphocytes enter and progress through the G1 cell cycle stage following delivery of complete versus partial activation stimuli.

Resting B-cells lack both cyclins D and E while constitutively expressing low levels of cdk4 and cdk2. B-cells receiving a complete growth stimulus express cyclin D2 by 10hr and cyclin E by 10-24hr poststimulation while increasing their protein levels of cdk4 and cdk2. B-cells receiving partial growth stimuli move into G1 without passing the G1 restriction point and transiently increase cyclin D2 mRNA levels without accumulating cyclin D2 protein. In the absence of cyclin D2 accumulation, cdk4 is not activated, and cyclin E is not expressed. These results suggest that signals responsible for moving B-cells through the G1 restriction point impact at the level of cyclin D2 protein accumulation. The possible implications of these results are discussed.

Combined Hoechst 33342 and merocyanine 540 staining to examine murine B cell cycle stage, viability and apoptosis.

A procedure is described for the dual staining of lymphocytes with Hoechst 33342 (Ho342) to examine cell cycle position, and merocyanine 540 (MC540) that allows for the analysis of cells entering the early stages of apoptosis. Ho342 is a DNA specific dye and MC540 detects membrane phospholipid domain changes, some of which are associated with apoptotic cells. Flow analysis of B cells dually stained with Ho342 and MC540 allows for the discrimination of five distinct subpopulations. Two of these subpopulations represent viable, MC540 negative/dull cells with either 2n or 4n DNA. As 2n and 4n DNA B cells become MC540 bright they move into two distinct subpopulations representing cells entering and progressing through the early stages of apoptosis. As the apoptotic, MC540 bright cells move into the latter stages of apoptosis, they localize into a fifth subpopulation displaying reduced staining with Ho342 indicative of late stage apoptotic cells in the process of fragmenting their DNA. This experimental approach enables the characterization of lymphocyte populations for percentages of viable, early apoptotic, and late apoptotic cells. The cells are not fixed during this procedure, and since both dyes are viable dyes there is an additional opportunity to obtain sorted cells from any of the defined subpopulations for reculturing and functional analysis.

T helper cell regulation of normal and neoplastic B cell growth.

Following challenge with a thymus-dependent antigen T helper cells regulate B cell growth and differentiation in several ways. Initially, the T cells physically associate with antigen presenting B cells. While in conjugate, the two cells communicate with each other through the actions of cell surface receptors whose ligands are integral membrane proteins expressed on the surface of the apposed cell. The ensuing biochemical pathways regulate the expression of genes required for B cell cycle progression. As a consequence of this interaction, the T cells are induced to synthesize and secrete soluble mediators that also affect B cell proliferation, as well as determining the ultimate fate of the activated B cell. We also suggest a role for normal, Th cells during the development, and continued expansion of certain types of B cell lymphomas.

Appearance of CAD activity, the rate-limiting enzyme for pyrimidine biosynthesis, as B cells progress into and through the G1 stage of the cell cycle.

CAD is a multifunctional protein which mediates the first three enzymatic steps of pyrimidine biosynthesis. Previous studies have implicated CAD as a cell cycle regulated protein. In the present paper CAD activity is studied as polyclonally stimulated, murine B cells progress through the early stages of the cell cycle. CAD activity is seen to increase in a biphasic manner. The initial increase in activity occurs prior to or as the cells increase CAD mRNA suggesting that post-translational modification of preformed enzyme may account for at least a portion of this initial enhancement. Increases in CAD mRNA occur by 12 hr poststimulation and precede the second, more dramatic increase in B cell CAD activity. Preliminary experiments failed to provide support of a role for IL-4 in regulating the expression of CAD as B cells progress into G1. CAD enzymatic activity does represent, however, a marker for early B cell cycle progression.

Expression of polyamine transporter activity during B lymphocyte cell cycle progression.

The movement of resting B cells into the early stages of the cell cycle is accompanied by elevations in cellular polyamine levels. Our previous results indicate that part of this increase is a consequence of enhanced levels of ornithine decarboxylase activity in activated B cells. However, the levels of ornithine decarboxylase wane within 30 h post-stimulation, suggesting that B cells may acquire polyamines from an alternative source. Results in the present communication indicate that B cells initiate the transport of extracellular polyamines as they enter the G1 stage of the cell cycle. This induction of polyamine transport activity is seen after the stimulation of B cell cycle progression by both PMA and ionomycin, as well as cognate help from activated Th cells. The transport activities are energy and temperature sensitive, and can be competitively inhibited by unlabeled ligands. The results of the competition studies are compatible with the presence of at least two types of transporters, a low affinity transporter for both putrescine and spermidine, and a higher affinity transporter selective for spermidine. The results are discussed in terms of biochemical events associated with Th cell-mediated, B cell activation.

T helper cells.

B-cell proliferation and differentiation is controlled by T helper cells. Recent studies have determined that the expression of a novel, 39 kD, T-cell membrane protein is responsible for inducing T-cell-dependent B-cell activation. The receptor for this protein on the resting B cell is CD40. Once activated, B cells are induced to grow and differentiate by the elaboration of interleukin-4 and interleukin-5 from activated T cells. Together, T cell-B cell contact and soluble factors provide all the signals required for B-cell growth and differentiation.

Induction and regulation of casein kinase II during B lymphocyte activation.

This study evaluates the regulation of casein kinase II (CK II) activity in resting B cells induced to enter the cell cycle. The induction of B cell cycle progression PMA and ionomycin results in an oscillatory expression of CK II. This kinase activity is also elicited after direct physical interaction between B cells and activated, fixed Th cells, indicating that the increase seen in CK II activity is probably associated with the delivery of the competence-inducing signal to resting B cells. The selective inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis, during PMA and ionomycin-induction of B cell cycle progression, inhibits the expression of CK II activity. The addition of polyamines to cytosolic preparations recovered from cells in which ODC is inhibited results in the appearance of CK II activity, showing that the ODC inhibitor does not directly inhibit the kinase. The treatment of B cells with cycloheximide results in the appearance of CK II activity within 15 min, and this induction is partially explainable by a cycloheximide-elicited increase in cellular levels of polyamines. The artificial elevation of cellular levels of cAMP simultaneous with the addition of PMA and ionomycin results in a 150 to 200% increase in detectable CK II levels, suggesting that the cAMP-dependent signaling cascade may participate during the early regulation of CK II. In contrast, the inhibition of protein kinase C does not adversely influence the early expression of CK II, while actually enhancing kinase activity by 18 h poststimulation.

T helper cell-dependent B cell activation.

Small, resting B lymphocytes are driven into the cell cycle as a consequence of receiving multiple signals from elements found within their local environment. The first of these signals results from the binding of specific antigen to membrane immunoglobulin (mIg) receptors on the B cells. Pursuant to antigen binding, signals are transduced and the B cell commences to endocytose and degrade the antigen. Fragments of the antigen are expressed on the B cell surface in noncovalent association with class II major histocompatibility complex (MHC) molecules. The antigen-class II MHC complex serves as a recognition complex for CD4+ helper T cells (Th). As a consequence of recognition, Th form stable physical conjugates with the B cells. Over an extended period of time the Th and B cells bilaterally signal one another. This interchange of signals results in the growth and differentiation of both cells. This review will discuss the sequence of events that culminate in the growth and differentiation of B lymphocytes to antibody-producing cells.

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.

The proteolysis of membrane-associated protein kinase C as a possible component of the signalling pathway leading to c-myc induction in B lymphocytes.

Occupancy of surface immunoglobulin (sIg) receptor for antigen expressed on resting B cells initiates increased turnover of membrane-associated phosphatidylinositol (PI), which ultimately leads to the enhanced expression of c-myc mRNA. The mechanism which links these initial membrane biochemical changes to subsequent alterations in c-myc transcription is unclear. The present study examines the possible involvement of PKC and its calpain-generated proteolytic fragment, protein kinase M (PKM), in conveying the membrane-associated signal to the nucleus. Utilizing an in vitro phosphorylation assay, we have shown that a calcium-dependent protease, similar to calpain, is involved in the downregulation of membrane-associated PKC induced by anti-immunoglobulin or phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation of resting B cells. In addition, we have confirmed previous studies showing that PMA and ionomycin are both required for optimal expression of c-myc mRNA. The enhanced expression of c-myc mRNA is sensitive to inhibitors of PKC, such as H-7 and sangavimycin, providing evidence for a prominent role of PKC and/or PKM in the receptor-mediated up-regulation of c-myc message expression. Finally, a calpain inhibitor interferes with the transmission of the membrane-associated signal which induces the increased expression of c-myc mRNA. Our results are consistent with the hypothesis that the calpain-mediated proteolysis of membrane-associated PKC is involved in the sIg-mediated signal transduction pathway.

Cognate interactions between helper T cells and B cells.

The mechanism by which mammals produce an antibody response after exposure to antigen has intrigued biologists for over a hundred years. Here, Randolph Noelle and Charles Snow review some of the experimental findings since the early 1970s that have advanced understanding of the mechanisms operating during B-cell activation by thymus-dependent (TD) antigens. They also propose a model for B-cell activation that emphasizes the critical role played by direct cellular interactions between B cells and helper T(TH) cells and seek to place into perspective the role played by the membrane immunoglobulin (mlg) receptor in cognate responses.

Insulin modulates the interleukin 2 responsiveness of T lymphocytes.

Insulin has been shown to enhance the proliferation and differentiation of T cells stimulated by both polyclonal stimulants and specific antigen. This study describes an experimental system designed to understand the mechanism(s) by which occupancy of the insulin receptor mediates the enhancement seen in T cell expansion. These experiments demonstrate that the ability of insulin to influence T cell expansion resides in an insulin-mediated maintenance of the interleukin-2 (IL-2) responsiveness of the activated cells. This was analyzed by following the decay pattern of T cell IL-2 responsiveness by placing the activated T cells into serum-free cultures in the presence or absence of insulin. This maintenance of responsiveness was not mediated by insulin regulating the expression of the IL-2 receptor alpha chain. We feel that this experimental approach will prove useful for dissecting the biochemical and molecular changes mediated by insulin which interface with the ability of activated T cells to effectively respond to IL-2.