Leslie B. King

MMTV Env encodes an ITAM responsible for transformation of mammary epithelial cells in three-dimensional culture

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

Rapid B Cell Receptor-induced Unfolded Protein Response in Nonsecretory B Cells Correlates with Pro- Versus Antiapoptotic Cell Fate

The adaptive unfolded protein response (UPR) is essential for the development of antibody-secreting plasma cells. B cells induced by lipopolysaccharide (LPS) to differentiate into plasma cells exhibit a nonclassical UPR reported to anticipate endoplasmic reticulum stress prior to immunoglobulin production. Here we demonstrate that activation of a physiologic UPR is not limited to cells undergoing secretory cell differentiation. We identify B cell receptor (BCR) signaling as an unexpected physiologic UPR trigger and demonstrate that in mature B cells, BCR stimulation induces a short lived UPR similar to the LPS-triggered nonclassical UPR. However, unlike LPS, BCR stimulation does not induce plasma cell differentiation. Furthermore, the BCR-induced UPR is not limited to cells in which BCR induces activation, since a UPR is also induced in transitional immature B cells that respond to BCR stimulation with a rapid apoptotic fate. This response involves sustained up-regulation of Chop mRNA indicative of a terminal UPR. Whereas sustained Chop expression correlates with the ultimate fate of the BCR-triggered B cell and not its developmental stage, Chop–/– B cells undergo apoptosis, indicating that CHOP is not required for this process. These studies establish a system whereby a terminal or adaptive UPR can be alternatively triggered by physiologic stimuli.

Positive and negative selection during B lymphocyte development.

Our laboratory is interested in a variety of issues related to lymphocyte development. More specifically, we have focused on the processes that regulate the decision to commit to the B lymphocyte (B cell) lineage, then the subsequent signals that are involved in maintaining this commitment to the B cell lineage. These signals result in the positive selection of those B cells that properly execute the complex genetic changes associated with B cell development, then trigger the elimination of B cells that are responsive to self-antigens and, therefore, possess the potential to mediate autoimmune disease. Our general experimental approach has been to address these issues from the perspective of signal transduction. Our goal is to define the biochemical and genetic processes that are integrated in order to accomplish these selection processes. To do so, we employ in vivo animal models as well as more defined in vitro studies, using both primary and transformed cell lines. For the past several years, we have been primarily interested in the precise mechanisms by which the B cell antigen receptor (BCR), and intermediate forms of this receptor, regulate these complex developmental processes. We have used the ongoing studies described below as two representative examples of how we are approaching these issues and some of the insights that we have made. To place both of these studies in context, we will begin with a brief introduction into B cell development.

Distinct calcium channels regulate responses of primary B lymphocytes to B cell receptor engagement and mechanical stimuli

Intracellular Ca2+ plays a central role in controlling lymphocyte function. Nonetheless, critical gaps remain in our understanding of the mechanisms that regulate its concentration. Although Ca2+-release-activated calcium (CRAC) channels are the primary Ca2+ entry pathways in T cells, additional pathways appear to be operative in B cells. Our efforts to delineate these pathways in primary murine B cells reveal that Ca2+-permeant nonselective cation channels (NSCCs) operate in a cooperative fashion with CRAC. Interestingly, these non-CRAC channels are selectively activated by mechanical stress, although the mechanism overlaps with BCR-activated pathways, suggesting that they may operate in concert to produce functionally diverse Ca2+ signals. NSCCs also regulate the membrane potential, which activates integrin-dependent binding of B cells to extracellular matrix elements involved in their trafficking and localization within secondary lymphoid organs. Thus, CRAC and distinct Ca2+ permeant NSCCs are differentially activated by the BCR and mechanical stimuli and regulate distinct aspects of B cell physiology.

B‐lymphocyte calcium inFlux

Summary:  Dynamic changes in cytoplasmic calcium concentration dictate the immunological fate and functions of lymphocytes. During the past few years, important details have been revealed about the mechanism of store‐operated calcium entry in lymphocytes, including the molecular identity of calcium release‐activated calcium (CRAC) channels and the endoplasmic reticulum (ER) calcium sensor (STIM1) responsible for CRAC channel activation following calcium depletion of stores. However, details of the potential fine regulation of CRAC channel activation that may be imposed on lymphocytes following physiologic stimulation within an inflammatory environment have not been fully addressed. In this review, we discuss several underexplored aspects of store‐operated (CRAC‐mediated) and store‐independent calcium signaling in B lymphocytes. First, we discuss results suggesting that coupling between stores and CRAC channels may be regulated, allowing for fine tuning of CRAC channel activation following depletion of ER stores. Second, we discuss mechanisms that sustain the duration of calcium entry via CRAC channels. Finally, we discuss distinct calcium permeant non‐selective cation channels (NSCCs) that are activated by innate stimuli in B cells, the potential means by which these innate calcium signaling pathways and CRAC channels crossregulate one another, and the mechanistic basis and physiologic consequences of innate calcium signaling.

Igα/Igβ Complexes Generate Signals for B Cell Development Independent of Selective Plasma Membrane Compartmentalization

Ligand-induced BCR association with detergent-resistant plasma membrane compartments (lipid rafts) has been argued to be essential for initiating and/or sustaining Igα/Igβ-dependent BCR signaling. Because a fraction of the BCR and an even larger fraction of the preBCR associates with lipid rafts in the apparent absence of ligand stimulation, it has been proposed that raft-associated receptor complexes mediate the ligand-independent basal signaling events observed in resting B lineage cells. However, there is no direct evidence that localization of Igα/Igβ-containing complexes to detergent-resistant membrane compartments is absolutely required for the signaling events that drive B cell development. To address these issues we have designed surrogate preBCR/Igα/Igβ complexes that are incapable of ligand-induced aggregation and that are preferentially targeted to either raft or nonraft compartments. An analysis of their ability to promote the preBCR-dependent proB→preB cell transition of murine B cell progenitors revealed that expression of these surrogate receptor complexes at levels that approximate that of the conventional preBCR can drive B cell development in a manner independent of both aggregation and lipid raft localization.

Molecular Mechanisms Regulating Negative Selection in Immature-Stage B-Cells

Although the tremendous clonal diversity expressed in the B-cell receptor (BCR) repertoire is beneficial for mounting immune responses to foreign antigens, it is simultaneously detrimental in that it may include a multitude of BCRs capable of recognizing self antigens. Therefore, mechanisms must exist to functionally silence these self-reactive B-cells. Negative selection of autoreactive cells has been proposed to occur by clonal elimination or abortion (deletion), clonal silencing (anergy), and clonal alteration (receptor editing). Although there has been a significant advance in our understanding of these various modes of tolerance in recent years, the biochemical mechanisms regulating tolerance remain largely unknown. Rather than present a comprehensive review of the findings in this area, the authors will concentrate on the role of clonal deletion in the tolerization of immature B-cells. In particular, the following questions will be addressed: 1. Which target cell populations are sensitive to deletion? 2. What are the molecular mechanisms responsible for rendering these target populations tolerance sensitive? and 3. Is clonal deletion necessary for the maintenance of B-cell tolerance?