Lymphocyte repertoire selection and intracellular self/non-self-discrimination: historical overview.

Immunological self/non-self-discrimination is conventionally seen as an extracellular event, involving interactions been receptors on T cells pre-educated to discriminate and peptides bound to major histocompatibility complex proteins (pMHCs). Mechanisms by which non-self peptides might first be sorted intracellularly to distinguish them from the vast excess of self-peptides have long been called for. Recent demonstrations of endogenous peptide-specific clustering of pMHCs on membrane rafts are indicative of intracellular enrichment before surface display. The clustering could follow the specific aggregation of a foreign protein that exceeded its solubility limit in the crowded intracellular environment. Predominantly entropy-driven, this homoaggregation would colocalize identical peptides, thus facilitating their collective presentation. Concentrations of self-proteins are fine-tuned over evolutionary time to avoid this. Disparate observations, such as pyrexia and female susceptibility to autoimmune disease, can be explained in terms of the need to cosegregate cognate pMHC complexes internally before extracellular display.

PIP3 regulation as promising targeted therapy of mast-cell-mediated diseases.

It is well established that mast cells play a key regulatory role in allergy and inflammation involving engagement of antigen with IgE bound to high-affinity IgE receptors (FcεRI). The most aggressive efforts in regulating mast cell function have focused on selectively inhibiting cell activation and subsequent mediator synthesis and release, or alternatively, blocking the action of proinflammatory mediators in order to prevent or reduce disease severity. More recently, the goal for rationally designed pharmacotherapy has shifted focus to targeting and disrupting signaling pathways leading to inhibition of specific cell function(s). In this context, the PI-3K/PIP3/Akt pathway represents a potent target for pharmacologic intervention in mast cell-mediated inflammatory disorders. A pivotal component of this cascade is the activation of phosphatidylinositol-3-kinase (PI-3K) leading to a rise in intracellular levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 has broad effects on mast cell signaling and function as well as on proliferation and survival. We propose that PIP3 represents a potent target for developing therapeutic approaches to down regulate mast cell function and, in turn, reduce the severity of mast cell dependent disease. In this article we review approaches that have been taken to regulate the PI-3K pathway in mast cells. Moreover, we review a novel approach to target the signaling lipid, PIP3, and deplete intracellular levels of this phosphoinositol using a chimeric toxin composed of the FcεRI binding region of IgE and the active subunit of the cytolethal distending toxin, CdtB, which we have recently demonstrated to function as a PIP3 phosphatase.

An Fcγ receptor-dependent mechanism drives antibody-mediated target-receptor signaling in cancer cells.

Antibodies to cell-surface antigens trigger activatory Fcγ receptor (FcγR)-mediated retrograde signals in leukocytes to control immune effector functions. Here, we uncover an FcγR mechanism that drives antibody-dependent forward signaling in target cells. Agonistic antibodies to death receptor 5 (DR5) induce cancer-cell apoptosis and are in clinical trials; however, their mechanism of action in vivo is not fully defined. Interaction of the DR5-agonistic antibody drozitumab with leukocyte FcγRs promoted DR5-mediated tumor-cell apoptosis. Whereas the anti-CD20 antibody rituximab required activatory FcγRs for tumoricidal function, drozitumab was effective in the context of either activatory or inhibitory FcγRs. A CD40-agonistic antibody required similar FcγR interactions to stimulate nuclear factor-κB activity in B cells. Thus, FcγRs can drive antibody-mediated receptor signaling in target cells.

Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 exvivo expansion.

Peripheral blood natural killer (NK) cell therapy for acute myeloid leukemia has shown promise in clinical trials after allogeneic stem cell transplantation. Cord blood (CB) is another potentially rich source of NK cells for adoptive immune therapy after stem cell transplantation. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. However, despite expressing normal surface activating and inhibitory NK receptors, CB-derived NK cells have poor cytolytic activity. In this study, we investigate the cellular mechanism and demonstrate that unmanipulated CB-NK cells exhibit an impaired ability to form F-actin immunologic synapses with target leukemia cells compared with peripheral blood-derived NK cells. In addition, there was reduced recruitment of the activating receptor CD2, integrin leukocyte function-associated antigen-1, and the cytolytic molecule perforin to the CB-NK synapse site. Exvivo interleukin (IL)-2 expansion of CB-NK cells enhanced lytic synapse formation including CD2 and leukocyte function-associated antigen-1 polarization and activity. Furthermore, the acquired antileukemic function of IL-2-expanded CB-NK cells was validated using a nonobese diabetic severe combined immunodeficient IL-2 receptor common γ-chain null mouse model. We believe our results provide important mechanistic insights for the potential use of IL-2-expanded CB-derived NK cells for adoptive immune therapy in leukemia.

The influence of IgG density and macrophage Fc (gamma) receptor cross-linking on phagocytosis and IL-10 production.

We have previously demonstrated that the addition of immune complexes (IC) to stimulated macrophages could profoundly influence cytokine production. In the present work we sought to determine the density of IgG on immune complexes necessary to mediate phagocytosis, inhibit IL-12 production and induce IL-10 production from stimulated macrophages. We developed immune complexes with predictable average densities of surface-bound immunoglobulin. We show that a threshold amount of IgG was necessary to mediate attachment of IC to macrophages. At progressively higher densities of IgG, Fc receptor-mediated phagocytosis resulted in an inhibition of IL-12 production and then an induction of IL-10. The reciprocal alterations in these two cytokines occurred when as little as one optimally opsonized SRBC was bound per macrophage. Macrophage IL-10 induction by immune complexes was associated with the activation of the MAP kinase, ERK, which was progressively increased as a function of IgG density. We conclude that signal transduction through the macrophage Fcγ receptors vary as a function of signal strength. At moderate IgG densities, especially in the presence of complement, efficient phagocytosis occurs in the absence of cytokine alterations. At slightly higher IgG densities IL-12 production is shut off and eventually IL-10 induction occurs. Thus, the myriad events emanating from FcγR ligation depends on the density of immune complexes, allowing the Fc receptors to fine-tune cellular responses depending on the extent of receptor cross-linking.

An immunoglobulin-like receptor, Allergin-1, inhibits immunoglobulin E-mediated immediate hypersensitivity reactions.

Anaphylaxis is a life-threatening immediate hypersensitivity reaction triggered by antigen capture by immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcvarepsilonRI) on mast cells. However, the regulatory mechanism of mast cell activation is not completely understood. Here we identify an immunoglobulin-like receptor, Allergin-1, that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain, and show it was preferentially expressed on mast cells. Mouse Allergin-1 recruited the tyrosine phosphatases SHP-1 and SHP-2 and the inositol phosphatase SHIP. Coligation of Allergin-1 and FcvarepsilonRI suppressed IgE-mediated degranulation of bone marrow-derived cultured mast cells. Moreover, mice deficient in Allergin-1 developed enhanced passive systemic and cutaneous anaphylaxis. Thus, Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice.

Antigen-induced oligomerization of the B cell receptor is an early target of Fc gamma RIIB inhibition.

The FcgammaRIIB is a potent inhibitory coreceptor that blocks BCR signaling in response to immune complexes and, as such, plays a decisive role in regulating Ab responses. The recent application of high-resolution live cell imaging to B cell studies is providing new molecular details of the earliest events in the initiation BCR signaling that follow within seconds of Ag binding. In this study, we report that when colligated to the BCR through immune complexes, the FcgammaRIIB colocalizes with the BCR in microscopic clusters and blocks the earliest events that initiate BCR signaling, including the oligomerization of the BCR within these clusters, the active recruitment of BCRs to these clusters, and the resulting spreading and contraction response. Fluorescence resonance energy transfer analyses indicate that blocking these early events may not require molecular proximity of the cytoplasmic domains of the BCR and FcgammaRIIB, but relies on the rapid and sustained association of FcgammaRIIB with raft lipids in the membrane. These results may provide novel early targets for therapies aimed at regulating the FcgammaRIIB to control Ab responses in autoimmune disease.

Low-affinity IgE receptor (FcepsilonRII)-mediated activation of human monocytes by both monomeric IgE and IgE/anti-IgE immune complex.

Monocytes and macrophages of individuals with allergic diseases express increased levels of the low-affinity IgE receptors (FcepsilonRII or CD23) on their surfaces. The cross-linking of CD23-bound IgE antibody by allergen activates the cells to release inflammatory mediators. In mast cells, the binding of IgE to the high-affinity IgE receptors (FcepsilonRI) has recently been shown to activate these cells independent of allergen. It has not been determined if such is true of the binding of IgE to the low-affinity receptors. The purpose of this study was, therefore, to determine whether monomeric IgE alone can activate CD23-bearing human monocytes and how this may relate to the activation by IgE/anti-IgE immune complex. Purified monocytes, cultured for 48 h with IL-4 to up-regulate CD23 were sensitized with human myeloma IgE and further cultured for 24 h with or without anti-human IgE antibody. The release of cytokines TNF-alpha and MIP-1alpha (as an index of activation) was determined by enzyme immunoassay. Results showed that in IL-4-treated/CD23-bearing monocytes, sensitization with IgE alone caused a release of TNF-alpha and MIP-1alpha. The addition of anti-IgE antibody to cross-link the bound IgE resulted in the enhancement of the response. Such activation by monomeric IgE and IgE/anti-IgE immune complex was blocked with an anti-CD23 antibody, confirming the specific involvement of CD23 molecules. Neither of the activation modalities elevated intracellular cAMP, contrary to previous report. These results show for the first time, that in CD23-bearing monocytes, IgE sensitization alone can activate monocytes, and that ligation of such IgE by anti-IgE antibody only enhances the response. These observations have implications for the understanding of the pathophysiology of IgE-dependent inflammation accompanying many allergic diseases.

Regulation of hematopoietic cell function by inhibitory immunoglobulin G receptors and their inositol lipid phosphatase effectors.

Numerous autoimmune and inflammatory disorders stem from the dysregulation of hematopoietic cell activation. The activity of inositol lipid and protein tyrosine phosphatases, and the receptors that recruit them, is critical for prevention of these disorders. Balanced signaling by inhibitory and activating receptors is now recognized to be an important factor in tuning cell function and inflammatory potential. In this review, we provide an overview of current knowledge of membrane proximal events in signaling by inhibitory/regulatory receptors focusing on structural and functional characteristics of receptors and their effectors Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 and SH2 domain-containing inositol 5-phosphatase-1. We review use of new strategies to identify novel regulatory receptors and effectors. Finally, we discuss complementary actions of paired inhibitory and activating receptors, using Fc gammaRIIA and Fc gammaRIIB regulation human basophil activation as a prototype.

Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21.

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 +/- 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 +/- 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2(-/-)/arrestin 3(-/-) murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.

C3a-derived peptide binds to the type I FcepsilonR and inhibits proximal-coupling signal processes and cytokine secretion by mast cells.

A peptide with the natural sequence derived from the complement component C3a, designated C3a7, and C3a9, having a modified sequence of that, was previously shown to inhibit the high-affinity IgER (FcepsilonRI)-induced secretory response of both mucosal and serosal-type mast cells. In addition, several processes that couple the FcepsilonRI stimulus to the cellular response were all suppressed in the presence of these peptides. Here, we show that peptide C3a9 binds to the FcepsilonRI on the surface of unperturbed mast cells (rat mucosal-type RBL-2H3 cell line) and remains bound even after FcepsilonRI-IgE aggregation by antigen as assessed by confocal microscopy. Moreover, that peptide interferes the initial steps of FcepsilonRI-coupling network. Namely, peptide binding to the FcepsilonRI beta-chain interrupts this chain's association with both src family protein tyrosine kinases Lyn and Fyn and enhances the internalization of the receptor. C3a9 was further found to inhibit the phosphorylation of two members of the mitogen-activated protein kinase family, extracellular signal-regulated kinase (ERK) and p38. Although ERK is usually activated via the ras-raf-mitogen-activated protein kinase/ERK kinase (MEK) pathway, our results show that C3a9 has no effect on the c-raf phosphorylation, suggesting that this complement-derived peptide inhibits ERK activation via an alternative route. C3a9 also inhibits the late-phase response to FcepsilonRI stimulus of bone marrow-derived mast cells, reducing secretion of the inflammatory cytokines IL-6 and tumor necrosis factor-alpha. Taken together, the consequence of its interference with the earliest steps of FcepsilonRI stimulus-response coupling and the C3a-derived peptide inhibits both the immediate and the late-phase responses of mast cells.

Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells.

Interleukin 2 and interleukin 15 (IL2 and IL15, respectively) provide quite distinct contributions to T-cell-mediated immunity, despite having similar receptor composition and signaling machinery. As most of the proposed mechanisms underlying this apparent paradox attribute key significance to the individual alpha-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2Ralpha and IL15Ralpha at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2Ralpha and IL15Ralpha at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2Ralpha molecules reside outside the clustered domains, whereas approximately 30% of IL15Ralpha molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2Ralpha and IL15Ralpha domains remained constant, suggesting a 'building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two alpha-chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.

Fc gamma RI-mediated activation of human mast cells promotes survival and induction of the pro-survival gene Bfl-1.

Activation of mast cells through either FcepsilonRI or FcgammaRI leads to release of mediators contributing to the inflammatory response. One of the biologic characteristics of mast cells in allergic pathology is that these cells have the capacity to recover and regranulate after aggregation of FcepsilonRI. We have previously demonstrated that the pro-survival protein A1/Bfl-1 is required for mast cells to survive IgE-mediated activation. In the present study, we have investigated whether human mast cells show similar induction of bfl-1 and activation-induced survival after aggregation of FcgammaRI. Human cord blood-derived mast cells were activated by aggregation of either FcepsilonRI or FcgammaRI, and activation-induced survival and induction of bfl-1 was measured. We found that aggregation of FcgammaRI-induced expression of Bfl-1 and caused a comparable activation-induced mast cell survival as FcepsilonRI does. These data suggests that activation through Fc-receptors contribute to mast cell survival during antibody-dependent mast cell mediated inflammatory responses.

[Characterization and analysis of the aggregation of a single-chain chimeric anti-ErbB2 antibody].

We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.

IL-6 contributes to the expression of RAGs in human mature B cells.

Mature B cells acquire the capacity to revise rearranged Ig V region genes in secondary lymphoid organs. In previous studies, we demonstrated that cross-linking the BCR and the CD40 induces the expression of the RAG1 and RAG2 enzymes and, thereby, secondary rearrangements. We examine herein the mechanism that underpins RAG1 and RAG2 expression in peripheral and tonsil B cells. Coordinated engagement of the BCR and CD40 promoted the synthesis of IL-6 and, thereby, up-regulation of its receptor on activated B lymphocytes. Furthermore, we provide evidence that IL-6 initiates the expression of RAGs in circulating B cells, and extends those in tonsil B cells. Thus, neutralization of IL-6 or blocking of its receptor inhibits RAG expression. Moreover, we demonstrate that IL-6 impedes BCR-mediated termination of RAG gene expression in both population of B cells. The recovered inhibition of RAG gene transcription by IL-6 receptor blockade supports the notion that once recombination is launched, its termination is also regulated by IL-6. Taken together, these studies provide new insight into the dual role of IL-6 in inducing and terminating expression of the recombinase machinery for secondary rearrangements in mature human B cells.

Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis.

Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands.

UNLABELLED: B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. CONCLUSION: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.

CD43 cross-linking increases the Fas-induced apoptosis through induction of Fas aggregation in Jurkat T-cells.

CD43 (sialophorin, leukosialin) is a heavily sialylated surface protein expressed on most leukocytes and platelets including T cells. Although CD43 antigen is known to have multiple and complex structure, exact function of CD43 in each cell type is not completely understood. Here we evaluated the role of CD43 in Fas (CD95)-induced cell death in human T lymphoblastoid cell line, Jurkat. Crosslinking CD43 antigen by K06 mAb increased the Fas-mediated Jurkat cell apoptosis and the augmentation was inhibited by treatment with caspase inhibitors. Further, CD43 signaling of Jurkat cells induced Fas oligomerization on the cell surfaces implying that CD43 ligation have effects on early stage of Fas-induced T cell death. These also suggest that CD43 might play an important role in contraction of the immune response by promotion of Fas-induced apoptosis in human T cells.

Clustering of pre-B cell integrins induces galectin-1-dependent pre-B cell receptor relocalization and activation.

Interactions between B cell progenitors and bone marrow stromal cells are essential for normal B cell differentiation. We have previously shown that an immune developmental synapse is formed between human pre-B and stromal cells in vitro, leading to the initiation of signal transduction from the pre-BCR. This process relies on the direct interaction between the pre-BCR and the stromal cell-derived galectin-1 (GAL1) and is dependent on GAL1 anchoring to cell surface glycosylated counterreceptors, present on stromal and pre-B cells. In this study, we identify alpha(4)beta(1) (VLA-4), alpha(5)beta(1) (VLA-5), and alpha(4)beta(7) integrins as major GAL1-glycosylated counterreceptors involved in synapse formation. Pre-B cell integrins and their stromal cell ligands (ADAM15/fibronectin), together with the pre-BCR and GAL1, form a homogeneous lattice at the contact area between pre-B and stromal cells. Moreover, integrin and pre-BCR relocalizations into the synapse are synchronized and require actin polymerization. Finally, cross-linking of pre-B cell integrins in the presence of GAL1 is sufficient for driving pre-BCR recruitment into the synapse, leading to the initiation of pre-BCR signaling. These results suggest that during pre-B/stromal cell synapse formation, relocalization of pre-B cell integrins mediated by their stromal cell ligands drives pre-BCR clustering and activation, in a GAL1-dependent manner.

Specific patterns of Cdc42 activity are related to distinct elements of T cell polarization.

T cell polarization toward and within the cellular interface with an APC is critical for effective T cell activation. The Rho family GTPase Cdc42 is a central regulator of cellular polarization. Using live-cell imaging, we characterized the spatiotemporal patterns of Cdc42 activity and their physiological regulation. Using three independent means of experimental manipulation of Cdc42 activity, we established that Cdc42 is a critical regulator of T cell actin dynamics, TCR clustering, and cell cycle entry. Using quantification of three-dimensional data, we could relate distinct spatiotemporal patterns of Cdc42 activity to specific elements of T cell activation. This result suggests that Cdc42 activity in specific locations at specific times is most critical for its function in T cell activation.