Supraphysiological FOXP3 expression in human CAR-Tregs results in improved stability, efficacy, and safety of CAR-Treg products for clinical application.

The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Endophilin A2 regulates B-cell endocytosis and is required for germinal center and humoral responses.

Antigen-specific B-cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B-cell endosomal trafficking pathways and their specific roles in B-cell responses have not been systematically investigated. Here, we report high-throughput identification of genes regulating B-cell receptor (BCR)-mediated antigen internalization using genome-wide functional screens. We show that antigen internalization depends both on constitutive, clathrin-mediated endocytosis and on antigen-induced, clathrin-independent endocytosis mediated by endophilin A2. Although endophilin A2-mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B-cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2-deficient mice show defects in GC B-cell responses and production of high-affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin-independent intracellular trafficking in GC B-cell clonal expansion and antibody responses.

Physiological and Pathophysiological Roles of IgM Fc Receptor (FcµR) Isoforms.

IgM is the first antibody to emerge during phylogeny, ontogeny, and immune responses and serves as a first line of defense. Effector proteins interacting with the Fc portion of IgM, such as complement and its receptors, have been extensively studied for their functions. IgM Fc receptor (FcµR), identified in 2009, is the newest member of the FcR family and is intriguingly expressed by lymphocytes only, suggesting the existence of distinct functions as compared to the FcRs for switched Ig isotypes, which are expressed by various immune and non-hematopoietic cells as central mediators of antibody-triggered responses by coupling the adaptive and innate immune responses. Results from FcµR-deficient mice suggest a regulatory function of FcµR in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes. In this article, we discuss conflicting views about the cellular distribution and potential functions of FcµR. The signaling function of the Ig-tail tyrosine-like motif in the FcµR cytoplasmic domain is now formally shown by substitutional experiments with the IgG2 B cell receptor. The potential adaptor protein associating with FcµR and the potential cleavage of its C-terminal cytoplasmic tail after IgM binding are still enigmatic. Critical amino acid residues in the Ig-like domain of FcµR for interacting with the IgM Cµ4 domain and the mode of interaction are now defined by crystallographic and cryo-electron microscopic analyses. Some discrepancies on these interactions are discussed. Finally, elevated levels of a soluble FcµR isoform in serum samples are described as the consequence of persistent B cell receptor stimulation, as seen in chronic lymphocytic leukemia and probably in antibody-mediated autoimmune disorders.

Memory control by the B cell antigen receptor.

The generation of memory B cells (MBCs) that have undergone immunoglobulin class switching from IgM, which dominates primary antibody responses, to other immunoglobulin isoforms is a hallmark of immune memory. Hence, humoral immunological memory is characterized by the presence of serum immunoglobulins of IgG subtypes known as the γ-globulin fraction of blood plasma proteins. These antibodies reflect the antigen experience of B lymphocytes and their repeated triggering. In fact, efficient protection against a previously encountered pathogen is critically linked to the production of pathogen-specific IgG molecules even in those cases where the primary immune response required cellular immunity, for example, T cell-mediated clearance of intracellular pathogens such as viruses. Besides IgG, also IgA and IgE can provide humoral immunity depending on the microbe's nature and infection route. The molecular mechanisms underlying the preponderance of switched immunoglobulin isotypes during memory antibody responses are a matter of active and controversial debate. Here, we summarize the phenotypic characteristics of distinct MBC subpopulations and discuss the decisive roles of different B cell antigen receptor isotypes for the functional traits of class-switched B cell populations.

Vav family proteins constitute disparate branching points for distinct BCR signaling pathways.

Antigen recognition by B-cell antigen receptors (BCRs) activates distinct intracellular signaling pathways that control the differentiation fate of activated B lymphocytes. BCR-proximal signaling enzymes comprise protein tyrosine kinases, phosphatases, and plasma membrane lipid-modifying enzymes, whose function is furthermore coordinated by catalytically inert adaptor proteins. Here, we show that an additional class of enzymatic activity provided by guanine-nucleotide exchange factors (GEFs) of the Vav family controls BCR-proximal Ca2+ mobilization, cytoskeletal actin reorganization, and activation of the PI3 kinase/Akt pathway. Whereas Vav1 and Vav3 supported all of those signaling processes to different extents in a human B-cell model system, Vav2 facilitated Actin remodeling, and activation of Akt but did not promote Ca2+ signaling. On BCR activation, Vav1 was directly recruited to the phosphorylated BCR and to the central adaptor protein SLP65 via its Src homology 2 domain. Pharmacological inhibition or genetic inactivation of the substrates of Vav GEFs, small G proteins of the Rho/Rac family, impaired BCR-induced Ca2+ mobilization, probably because phospholipase Cγ2 requires activated Rac proteins for optimal activity. Our findings show that Vav family members are key relays of the BCR signalosome that differentially control distinct signaling pathways both in a catalysis-dependent and -independent manner.

Recruitment of the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B cells.

The improved antibody responses of class-switched memory B cells depend on enhanced signaling from their B cell antigen receptors (BCRs). However, BCRs on both naive and antigen-experienced B cells use the canonical immunoglobulin-associated alpha and beta-protein signaling subunits. Here we identified a BCR isotype-specific signal-amplification mechanism. Whereas immunoglobulin M (IgM)-containing BCRs initiated intracellular signals exclusively through immunoglobulin-associated alpha- and beta-proteins, IgG- and IgE-containing BCRs also used a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. When phosphorylated, this tyrosine recruited the adaptor Grb2, resulting in sustained protein kinase activation and prolonged generation of second messengers, which together culminated in enhanced B cell proliferation. Hence, membrane-bound IgG and IgE exert antigen recognition as well as costimulatory functions, thereby rendering memory B cells less dependent on T cell help.

A fluorescent probe for STED microscopy to study NIP-specific B cells.

We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the functional principles of B cell antigen receptors (BCRs). We successfully used them in flow-cytometry, confocal and super-resolution microscopy techniques.

The B-cell antigen receptor of IgE-switched plasma cells regulates memory IgE responses.

BACKGROUND: Allergic inflammation is driven by IgE-producing plasma cells (PCs), which are required for IgE-mediated activation of mast cells and basophils. Repeated antigen encounter elicits a memory IgE response with elevated serum IgE titers and accumulation of IgE-producing PCs. However, the cellular compartment and molecular signals that underlie the immunologic memory of IgE responses remain unclear. OBJECTIVE: With this study we aimed at clarifying whether inactivation of the cytoplasmic immunoglobulin tail tyrosine (ITT) motif in transmembrane IgE (mIgE) impairs the memory IgE response in mice. METHODS: We generated mice with an inactivated mIgE-ITT motif and analyzed serum IgE levels as well as the generation of IgE-producing germinal center B cells and PCs subsequent to primary and secondary infection with helminths. In vitro cultures were used to study the mIgE-ITT-controlled expression of mIgE on the surface of PCs. Systemic mast cell activation was determined by serum Mcpt1 ELISA in response to ovalbumin challenge. RESULTS: mIgE-ITT-mutant mice showed an impaired memory IgE response subsequent to helminth infection. Furthermore, sensitization and challenge of mIgE-ITT-mutant mice with ovalbumin resulted in diminished serum IgE titers and reduced mast cell activation. The mIgE-ITT motif was required for optimal cell surface expression of mIgE B-cell antigen receptors but not for intracellular IgE expression in PCs. CONCLUSION: These results indicate that the mIgE B-cell antigen receptor plays a critical role in establishing or maintaining the population of IgE-producing PCs during memory IgE responses.

The extracellular membrane-proximal domain of membrane-bound IgE restricts B cell activation by limiting B cell antigen receptor surface expression.

Immunoglobulin E (IgE) antibodies are key mediators of allergic reactions. Due to their potentially harmful anaphylactic properties, their production is tightly regulated. The membrane-bound isoform of IgE (mIgE), which is an integral component of the B cell antigen receptor, has been shown to be critical for the regulation of IgE responses in mice. In primate species including humans, mIgE can be expressed in two isoforms that are produced by alternative splicing of the primary ε Ig heavy chain transcript, and differ in the absence or presence of an extracellular membrane-proximal domain (EMPD) consisting of 52 amino acids. However, the function of the EMPD remains unclear. Here, we demonstrate that the EMPD restricts surface expression of mIgE-containing BCRs in human and murine B cells. The EMPD does not interfere with BCR assembly but acts as an autonomous endoplasmic reticulum retention domain. Limited surface expression of EMPD-containing mIgE-BCRs caused impaired activation of intracellular signaling cascades and hence represents a regulatory mechanism that may control the production of potentially anaphylactic IgE antibodies in primate species.

The Memory Function of the B Cell Antigen Receptor.

Activated B lymphocytes preserve their antigen experience by differentiating into long-lived pools of antibody-secreting plasma cells or various types of memory B cells (MBCs). The former population constantly produces serum immunoglobulins with sufficient specificity and affinity to thwart infections with recurrent pathogens. By contrast, memory B cell populations retain their antigen receptors on the cell surface and hence need pathogen-induced differentiation steps before they can actively contribute to host defense. The terminal differentiation of MBCs into antibody-secreting plasma cells is hallmarked by the absence of the lag phase characteristic for primary antibody responses. Moreover, secondary antibody responses are predominantly driven by MBCs that bear an antigen receptor of the IgG class on their surface although IgM-positive memory populations exist as well. These fundamental principles of B cell memory were enigmatic for decades. Only recently, we have begun to understand the underlying mechanisms. This review summarizes our current understanding of how different subpopulations of MBCs are generated during primary immune responses and how their functional heterogeneity on antigen recall is controlled by different signaling capabilities of B cell antigen receptor (BCR) isotypes and by the nature of the antigen.

The non-Ig parts of the VpreB and λ5 proteins of the surrogate light chain play opposite roles in the surface representation of the precursor B cell receptor.

The VpreB and λ5 proteins, together with Igµ-H chains, form precursor BCRs (preBCRs). We established λ5(-/-)/VpreB1(-/-)/VpreB2(-/-) Abelson virus-transformed cell lines and reconstituted these cells with λ5 and VpreB in wild-type form or with a deleted non-Ig part. Whenever preBCRs had the non-Ig part of λ5 deleted, surface deposition was increased, whereas deletion of VpreB non-Ig part decreased it. The levels of phosphorylation of Syk, SLP65, or PLC-γ2, and of Ca(2+) mobilization from intracellular stores, stimulated by µH chain crosslinking Ab were dependent on the levels of surface-bound preBCRs. It appears that VpreB probes the fitness of newly generated VH domains of IgH chains for later pairing with IgL chains, and its non-Ig part fixes the preBCRs on the surface. By contrast, the non-Ig part of λ5 crosslinks preBCRs for downregulation and stimulation.

Epstein-Barr virus LMP2A signaling in statu nascendi mimics a B cell antigen receptor-like activation signal.

BACKGROUND: The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is expressed during different latency stages of EBV-infected B cells in which it triggers activation of cytoplasmic protein tyrosine kinases. Early studies revealed that an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic N-terminus of LMP2A can trigger a transient increase of the cytosolic Ca2+ concentration similar to that observed in antigen-activated B cells when expressed as a chimeric transmembrane receptor. Even so, LMP2A was subsequently ascribed an inhibitory rather than an activating function because its expression seemed to partially inhibit B cell antigen receptor (BCR) signaling in EBV-transformed B cell lines. However, the analysis of LMP2A signaling has been hampered by the lack of cellular model systems in which LMP2A can be studied without the influence of other EBV-encoded factors. RESULTS: We have reanalyzed LMP2A signaling using B cells in which LMP2A is expressed in an inducible manner in the absence of any other EBV signaling protein. This allowed us for the first time to monitor LMP2A signaling in statu nascendi as it occurs during the EBV life cycle in vivo. We show that mere expression of LMP2A not only stimulated protein tyrosine kinases but also induced phospholipase C-γ2-mediated Ca2+ oscillations followed by activation of the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase pathway and induction of the lytic EBV gene bzlf1. Furthermore, expression of the constitutively phosphorylated LMP2A ITAM modulated rather than inhibited BCR-induced Ca2+ mobilization. CONCLUSION: Our data establish that LMP2A expression has a function beyond the putative inhibition of the BCR by generating a ligand-independent cellular activation signal that may provide a molecular switch for different EBV life cycle stages and most probably contributes to EBV-associated lymphoproliferative disorders.

The Small GTPase RHOA Links SLP65 Activation to PTEN Function in Pre B Cells and Is Essential for the Generation and Survival of Normal and Malignant B Cells.

The generation, differentiation, survival and activation of B cells are coordinated by signals emerging from the B cell antigen receptor (BCR) or its precursor, the pre-BCR. The adaptor protein SLP65 (also known as BLNK) is an important signaling factor that controls pre-B cell differentiation by down-regulation of PI3K signaling. Here, we investigated the mechanism by which SLP65 interferes with PI3K signaling. We found that SLP65 induces the activity of the small GTPase RHOA, which activates PTEN, a negative regulator of PI3K signaling, by enabling its translocation to the plasma membrane. The essential role of RHOA is confirmed by the complete block in early B cell development in conditional RhoA-deficient mice. The RhoA-deficient progenitor B cells showed defects in activation of immunoglobulin gene rearrangement and fail to survive both in vitro and in vivo. Reconstituting the RhoA-deficient cells with RhoA or Foxo1, a transcription factor repressed by PI3K signaling and activated by PTEN, completely restores the survival defect. However, the defect in differentiation can only be restored by RhoA suggesting a unique role for RHOA in B cell generation and selection. In full agreement, conditional RhoA-deficient mice develop increased amounts of autoreactive antibodies with age. RHOA function is also required at later stage, as inactivation of RhoA in peripheral B cells or in a transformed mature B cell line resulted in cell loss. Together, these data show that RHOA is the key signaling factor for B cell development and function by providing a crucial SLP65-activated link between BCR signaling and activation of PTEN. Moreover, the identified essential role of RHOA for the survival of transformed B cells offers the opportunity for targeting B cell malignancies by blocking RHOA function.

NOTCH Activation via gp130/STAT3 Signaling Confers Resistance to Chemoradiotherapy.

Resistance of tumor cells to chemoradiotherapy represents a fundamental problem in clinical oncology. The underlying mechanisms are actively debated. Here we show that blocking inflammatory cytokine receptor signaling via STAT3 re-sensitized treatment-refractory cancer cells and abolished tumor growth in a xenograft mouse model when applied together with chemoradiotherapy. STAT3 executed treatment resistance by triggering the expression of RBPJ, the key transcriptional regulator of the NOTCH pathway. The mandatory RBPJ interaction partner, NOTCH intracellular domain, was provided by tumor cell-intrinsic expression of NOTCH ligands that caused tonic NOTCH proteolysis. In fact, NOTCH inhibition phenocopied the effect of blocking STAT3 signaling. Moreover, genetic profiling of rectal cancer patients revealed the importance of the STAT3/NOTCH axis as NOTCH expression correlated with clinical outcome. Our data uncovered an unprecedented signal alliance between inflammation and cellular development that orchestrated resistance to chemoradiotherapy. Clinically, our findings allow for biomarker-driven patient stratification and offer novel treatment options.

Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling.

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.

Conformational plasticity and navigation of signaling proteins in antigen-activated B lymphocytes.

Over the past two decades our view of the B cell antigen receptor (BCR) has fundamentally changed. Being initially regarded as a mute antibody orphan of the B cell surface, the BCR turned out to be a complex multimolecular machine monitoring almost all stages of B cell development, selection, and activation through a plethora of ubiquitously and cell-type-specific effector proteins. A comprehensive understanding of the many BCR signaling facets is still out but a few common biochemical principles outlined in this review operate at the level of receptor activation and orchestrate specific wiring of intracellular transducer cascades. First, initiation and processing of antigen-induced signal transduction relies on transient conformational changes in the signaling proteins to trigger their physical interaction with downstream elements. Second, this dynamic assembly of signalosomes occurs at distinct subcellular locations, most prominently the plasma membrane, which requires dynamic relocalization of one or more of the engaged molecules. For both, precise complex formation and efficient subcellular targeting, B cell signaling components are equipped with a variety of protein interaction domains. Here we provide an overview on how these simple rules are applied by a limited number of transmembrane and cytosolic proteins to convert BCR ligation into Ca(2+) mobilization and Ras activation in an adjustable manner.

Differential organization of tonic and chronic B cell antigen receptors in the plasma membrane.

Stimulation of the B cell antigen receptor (BCR) triggers signaling pathways that promote the differentiation of B cells into plasma cells. Despite the pivotal function of BCR in B cell activation, the organization of the BCR on the surface of resting and antigen-activated B cells remains unclear. Here we show, using STED super-resolution microscopy, that IgM-containing BCRs exist predominantly as monomers and dimers in the plasma membrane of resting B cells, but form higher oligomeric clusters upon stimulation. By contrast, a chronic lymphocytic leukemia-derived BCR forms dimers and oligomers in the absence of a stimulus, but a single amino acid exchange reverts its organization to monomers in unstimulated B cells. Our super-resolution microscopy approach for quantitatively analyzing cell surface proteins may thus help reveal the nanoscale organization of immunoreceptors in various cell types.

The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications.

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG®- or myc-tag. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

Grb2 and GRAP connect the B cell antigen receptor to Erk MAP kinase activation in human B cells.

The B cell antigen receptor (BCR) employs enzymatically inactive adaptor proteins to facilitate activation of intracellular signaling pathways. In animal model systems, adaptor proteins of the growth factor receptor-bound 2 (Grb2) family have been shown to serve critical functions in lymphocytes. However, the roles of Grb2 and the Grb2-related adaptor protein (GRAP) in human B lymphocytes remain unclear. Using TALEN-mediated gene targeting, we show that in human B cells Grb2 and GRAP amplify signaling by the immunoglobulin tail tyrosine (ITT) motif of mIgE-containing BCRs and furthermore connect immunoreceptor tyrosine-based activation motif (ITAM) signaling to activation of the Ras-controlled Erk MAP kinase pathway. In contrast to mouse B cells, BCR-induced activation of Erk in human B cells is largely independent of phospholipase C-É£ activity and diacylglycerol-responsive members of Ras guanine nucleotide releasing proteins. Together, our results demonstrate that Grb2 family adaptors are critical regulators of ITAM and ITT signaling in naïve and IgE-switched human B cells.