E. Charles Snow

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.

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.

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.

Defective CD19-dependent signaling in B-1a and B-1b B lymphocyte subpopulations

Peritoneal and pleural cavities in mice and humans contain a unique population of B-lymphocytes called B-1 cells that are defective in B cell antigen receptor (BCR) signaling but have an increased propensity to produce autoantibodies. Several molecules such as Btk, Vav, and CD19 known to be important for BCR signaling have been shown to be critical for the development of B-1 cells from undefined precursors. Here we demonstrate that B-1 cell unresponsiveness to BCR cross-linking is in part due to defective signaling through CD19, a molecule known to modulate signaling thresholds in B cells. The defective CD19 signaling is manifested in reduced synergy between mIgM and CD19 to stimulate calcium mobilization in B-1 cells. BCR induced tyrosine phosphorylation of CD19 was transient in B-1 cells while it was prolonged in splenic B-2 cells. In both B-1 and B-2 cells BCR cross-linking induced a modest increase of CD19 associated Lyn, a Src family protein tyrosine kinase (PTK) thought to be important for CD19 phosphorylation. However, the tyrosine phosphorylated CD19 in B-1 cells binds less phosphatidylinositol 3-kinase (PI3-K) compared to B-2 cells. Most interestingly, we find that Vav-1 and Vav-2, proteins thought to be critical for CD19 signal transduction, are severely reduced in B-1 cells resulting in a complete absence of any CD19 associated Vav. Also we showed that both B-1a and B-1b B cells failed to proliferate in response to BCR cross-linking which in part appears to be due to defects in CD19 mediated amplification of BCR induced calcium mobilization.

Characterization and function of autoreactive T-lymphocyte clones isolated from normal, unprimed mice

Self-Ia-reactive cloned T-cell lines, designated PK, were established by long-term culture of T cells from normal DBA/2 mice with irradiated syngeneic splenic adherent cells (SAC), rich in macrophages and dendritic cells. The cell lines were Thy 1+, Lyt 1+, Lyt 2-, produced IL-2 following stimulation with syngeneic spleen cells, and did not exhibit alloreactivity when screened against six different H-2 haplotypes. Of the five cloned PK cell lines tested, four were I-Ed restricted while one was I-Ad restricted as determined by genetic mapping and blocking studies carried out with monoclonal anti-Ia sera. Extensive specificity studies suggested that the PK cells reacted to syngeneic Ia molecules alone and not to foreign antigens such as fetal calf serum (FCS) used in the culture medium, in association with self-Ia. SAC pulsed with FCS or other protein antigens such as turkey gamma-globulin (TGG) were tested for their ability to induce proliferation of autoreactive T cells and other antigen-specific T cells using culture conditions consisting of serumless medium and interleukin 2 (IL-2). The data showed that the autoreactive T cells proliferated better in response to antigen-unpulsed SAC, while FCS-specific and TGG-specific cell lines, developed independently, proliferated only in response to FCS- or TGG-pulsed SAC, respectively, but not to antigen-unpulsed SAC. These results clearly distinguished the autoreactive T-cell clones from the antigen-specific T-cell clones. Preliminary studies carried out to investigate the functions of autoreactive T cells suggested that these cells helped in the in vitro differentiation of alloantigen-specific cytotoxic T lymphocytes (CTL) from CTL precursors obtained from the thymus and augmented syngeneic, allogeneic, and antigen-specific immune responses in vitro. The autoreactive T cells were also capable of inducing both proliferation and differentiation of antigen-specific populations of B cells in the absence of antigen. The present investigation suggests that autoreactive, non-antigen-reactive T cells can be cloned from normal, unimmunized mice and that such cell lines may provide a powerful tool for analyzing the role of the syngeneic mixed lymphocyte reaction in induction and maintenance of both T-and B-cell immune responses.

A Nonimmunoglobulin Transgene and the Endogenous Immunoglobulin μ Gene Are Coordinately Regulated by Alternative RNA Processing during B-Cell Maturation

ABSTRACT 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 phosphatidylinositol response is an early event in the physiologically relevant activation of antigen-specific B lymphocytes

Receptor ligand-induced turnover of plasma membrane phosphatidylinositol (PI) has been implicated as part of a membrane receptor signal transduction system in a number of mammalian cell types. Signaling through B-lymphocyte surface immunoglobulin (sIg2) has been explored polyclonally through the use of anti-Ig reagents, with the assumption that anti-Ig mimics the process of antigen binding to the antigen-specific cell. We have utilized a method of obtaining trinitrophenyl (TNP)-specific populations of B lymphocytes in order to determine if antigen binding to these antigen-specific cells initiates PI turnover. This method has allowed us to explore the membrane phospholipid events following antigen binding directly, rather than with inference from the anti-Ig system. We have found that both thymus-dependent and thymus-independent antigens (with the exception of TNP-lipopolysaccharide) produced an increase in PI turnover comparable to that generated by anti-IgM stimulation. The lack of increased PI turnover following TNP-LPS stimulation may be attributable to the action of LPS on the biochemical events of the PI cycle. In a B-cell subpopulation depleted of antigen-specific cells, only anti-IgM produced a PI effect. These results represent the first demonstration of PI turnover as an early activation event in a physiologically relevant lymphocyte system.

Thymus-dependent antigenic stimulation of hapten-specific B lymphocytes

ThyraUS-dependent antigenic stimulation of B-cells requires the delivery of sequential signals which drive resting B cells into the cell cycle and allow some of the cells to differentiate into anti hod y-secre ting cells. This review will consider intervals during the activation process at which potentially important signals can be delivered to the resting B cells. The salient events involved with triggering of B cells which will be considered are I) the binding of antigen to the specific surface receptor for antigen [membrane immunoglobulin (mig)], 2) the interaction of T cells with B cells via the T-cell antigen receptor recognition of B-cell surface la in association with processed antigen, and 3) the binding of nonspecific Tcell-derived lymphokines (B-cell growth and difTerentiation factors) to specific receptors on the B-cell surface membrane. For this purpose we have analyzed the response of hapten-specific B-cells which have received physiologically relevant stimuli.

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.

Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii

ABSTRACT The Toxoplasma genome encodes the capacity for distinct architectures underlying cell cycle progression in a life cycle stage-dependent manner. Replication in intermediate hosts occurs by endodyogeny, whereas a hybrid of schizogony and endopolygeny occurs in the gut of the definitive feline host. Here, we characterize the consequence of the loss of a cell cycle-regulated ovarian tumor (OTU family) deubiquitinase, OTUD3A of Toxoplasma gondii (TgOTUD3A; TGGT1_258780), in T. gondii tachyzoites. Rather than the mutation being detrimental, mutant parasites exhibited a fitness advantage, outcompeting the wild type. This phenotype was due to roughly one-third of TgOTUD3A-knockout (TgOTUD3A-KO) tachyzoites exhibiting deviations from endodyogeny by employing replication strategies that produced 3, 4, or 5 viable progeny within a gravid mother instead of the usual 2. We established the mechanistic basis underlying these altered replication strategies to be a dysregulation of centrosome duplication, causing a transient loss of stoichiometry between the inner and outer cores that resulted in a failure to terminate S phase at the attainment of 2N ploidy and/or the decoupling of mitosis and cytokinesis. The resulting dysregulation manifested as deviations in the normal transitions from S phase to mitosis (S/M) (endopolygeny-like) or M phase to cytokinesis (M/C) (schizogony-like). Notably, these imbalances are corrected prior to cytokinesis, resulting in the generation of normal progeny. Our findings suggest that decisions regarding the utilization of specific cell cycle architectures are controlled by a ubiquitin-mediated mechanism that is dependent on the absolute threshold levels of an as-yet-unknown target(s). Analysis of the TgOTUD3A-KO mutant provides new insights into mechanisms underlying the plasticity of apicomplexan cell cycle architecture. IMPORTANCE Replication by Toxoplasma gondii can occur by 3 distinct cell cycle architectures. Endodyogeny is used by asexual stages, while a hybrid of schizogony and endopolygeny is used by merozoites in the definitive feline host. Here, we establish that the disruption of an ovarian-tumor (OTU) family deubiquitinase, TgOTUD3A, in tachyzoites results in dysregulation of the mechanism controlling the selection of replication strategy in a subset of parasites. The mechanistic basis for these altered cell cycles lies in the unique biology of the bipartite centrosome that is associated with the transient loss of stoichiometry between the inner and outer centrosome cores in the TgOTUD3A-KO mutant. This highlights the importance of ubiquitin-mediated regulation in the transition from the nuclear to the budding phases of the cell cycle and provides new mechanistic insights into the regulation of the organization of the apicomplexan cell cycle. IMPORTANCE Replication by Toxoplasma gondii can occur by 3 distinct cell cycle architectures. Endodyogeny is used by asexual stages, while a hybrid of schizogony and endopolygeny is used by merozoites in the definitive feline host. Here, we establish that the disruption of an ovarian-tumor (OTU) family deubiquitinase, TgOTUD3A, in tachyzoites results in dysregulation of the mechanism controlling the selection of replication strategy in a subset of parasites. The mechanistic basis for these altered cell cycles lies in the unique biology of the bipartite centrosome that is associated with the transient loss of stoichiometry between the inner and outer centrosome cores in the TgOTUD3A-KO mutant. This highlights the importance of ubiquitin-mediated regulation in the transition from the nuclear to the budding phases of the cell cycle and provides new mechanistic insights into the regulation of the organization of the apicomplexan cell cycle.