Ikaros DNA-Binding Proteins as Integral Components of B Cell Developmental-Stage-Specific Regulatory Circuits

Ikaros DNA-binding proteins are critical for the development of lymphocytes and other hematopoietic lineages, but it remains unclear how they cooperate with other regulators of signaling and transcription to achieve ordered gene expression during development. Here, we show that Ikaros proteins regulate the pre-BCR component λ5 in a stage-specific manner. In pre-BI cells, Ikaros modulated λ5 expression in competition with the transcriptional activator EBF. This required Ikaros binding to the Igll1 (λ5) promoter and was abolished either by mutation of the Ikaros DNA-binding domain or by deletion of a single Ikaros site from the Igll1 promoter. At the transition from the pre-BI to pre-BII stage, the expression of the Ikaros family member Aiolos was upregulated and required for the efficient silencing of Igll1. Aiolos expression was controlled by pre-BCR signals via the adaptor protein SLP-65. Thus, pre-BCR signaling regulates Aiolos and the silencing of Igll1 via a developmental-stage-specific feedback loop

The Ig-α ITAM is required for efficient differentiation but not proliferation of pre-B cells

Signals from the pre-B cell receptor (pre-BCR) mediated by the cytoplasmic tails of Ig-α/Ig-β are essential for developing B cells. To analyze the role of Ig-α ITAM and non-ITAM tyrosines in pre-BCR signaling, we reconstituted individual tyrosine mutants of Ig-α in src homology 2 domain-containing leukocyte protein of 65 kDa (SLP-65)/Ig-α double-deficient pre-B cells. We show that the Ig-α mutants led to comparable pre-BCR expression on the cell surface, while the pre-BCR-induced tyrosine phosphorylation was different. We further show that the reconstitution of Ig-α and the resulting pre-BCR expression led to enrichment of the pre-BCR-expressing cells in vitro irrespective of the introduced Ig-α mutation. We show that, even though the enrichment rate increased by lowering the IL-7 concentration, residual amounts of IL-7 were required for optimal enrichment. Our results indicate that surface IL-7 receptor expression is modulated by the pre-BCR, thereby increasing the IL-7 sensitivity of the respective cells. In contrast to the comparable pre-B cell proliferation, however, the Ig-α mutants differed in their capacity to induce calcium flux and activate efficient pre-B cell differentiation. Together, our data suggest that ITAM tyrosines and Y204 are required for efficient pre-B cell differentiation but not proliferation

Mice with human immune system components as in vivo models for infections with human pathogens

Many pathogens relevant to human disease do not infect other animal species. Therefore, animal models that reconstitute or harbor human tissues are explored as hosts for these. In this review, we will summarize recent advances to utilize mice with human immune system components, reconstituted from hematopoietic progenitor cells in vivo. Such mice can be used to study human pathogens that replicate in leukocytes. In addition to studying the replication of these pathogens, the reconstituted human immune system components can also be analyzed for initiating immune responses and control against these infections. Moreover, these new animal models of human infectious disease should replicate the reactivity of the human immune system to vaccine candidates and, especially, the adjuvants contained in them, more faithfully

Conventional Light Chains Inhibit the Autonomous Signaling Capacity of the B Cell Receptor

Signals from the B cell antigen receptor (BCR), consisting of μ heavy chain (μHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of μHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca2+ flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of μHC to induce Ca2+ flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous μHC ability and resulted in antigen-dependent Ca2+ flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation

SLP-65 regulates immunoglobulin light chain gene recombination through the PI(3)K-PKB-Foxo pathway

Although the essential role of the adaptor protein SLP-65 in pre-B cell differentiation is established, the molecular mechanism underlying its function is poorly understood. In this study, we uncover a link between SLP-65-dependent signaling and the phosphoinositide-3-OH kinase (PI(3)K)-protein kinase B (PKB)-Foxo pathway. We show that the forkhead box transcription factor Foxo3a promotes light chain rearrangement in pre-B cells. Our data suggest that PKB suppresses light chain recombination by phosphorylating Foxo proteins, whereas reconstitution of SLP-65 function counteracts PKB activation and promotes Foxo3a and Foxo1 activity in pre-B cells. Together, these data illuminate a molecular function of SLP-65 and identify a key role for Foxo proteins in the regulation of light chain recombination, receptor editing and B cell selection

Autoreactive B Cell Receptors Mimic Autonomous Pre-B Cell Receptor Signaling and Induce Proliferation of Early B Cells

The majority of early immature B cells express autoreactive B cell receptors (BCRs) that are, according to the current view, negatively selected to avoid the production of self-reactive antibodies. Here, we show that polyreactive BCRs, which recognize multiple self-antigens, induced autonomous signaling and selective expansion of B cell precursors in a manner comparable to the pre-BCR. We found that the pre-BCR was capable of recognizing multiple self-antigens and that a signaling-deficient pre-BCR lacking the non-Ig region of the surrogate-light-chain component λ5 was rescued by the complementarity-determining region 3 derived from heavy chains of polyreactive receptors. Importantly, bone marrow B cells from mice carrying Ig transgenes for an autoreactive BCR showed increased cell-cycle activity, which could not be detected in cells lacking the transgenic BCR. Together, the pre-BCR has evolved to ensure self-recognition because autoreactivity is required for positive selection of B cell precursors

Human NK cells of mice with reconstituted human immune system components require pre-activation to acquire functional competence

To investigate human natural killer (NK)– cell reactivity in vivo we have reconstituted human immune system components by transplantation of human hematopoietic progenitor cells into NODscid IL2Rnull mice. We demonstrate here that this model allows the development of all NK-cell subsets that are also found in human adult peripheral and cord blood, including NKp46CD56 NK cells. Similar to human cord blood, NK cells from these reconstituted mice require preactivation by interleukin-15 to reach the functional competence of human adult NK cells. Mainly the terminally differentiated CD16 NK cells demonstrate lower reactivity without this stimulation. After preactivation, both CD16 and CD16 NK cells efficiently produce interferon- and degranulate in response to stimulation with NK cell–susceptible targets, including K562 erythroleukemia cells. NK-cell lines, established from reconstituted mice, demonstrate cytotoxicity against this tumor cell line. Importantly, preactivation can as well be achieved by bystander cell maturation via poly I:C stimulation in vitro and injection of this maturation stimulus in vivo. Preactivation in vivo enhances killing of human leukocyte antigen class I negative tumor cells after their adoptive transfer. These data suggest that a functional, but resting, NK-cell compartment can be established in immune-compromised mice after human hematopoietic progenitor cell transfer

Robust T cell stimulation by Epstein-Barr virus-transformed B cells after antigen targeting to DEC-205

DEC-205 is a type I transmembrane multilectin receptor that is predominantly expressed on dendritic cells (DCs). Therefore, previous studies primarily focused on processing of DEC-205–targeted antigens by this potent antigen presenting cell type. Here we show that Epstein-Barr virus (EBV) transformed lymphoblastoid B-cell lines (LCLs) not only express DEC-205 at similar levels to DCs, but also efficiently present targeted EBV nuclear antigen 1 (EBNA1) and EBV-latent membrane protein 1 (LMP1) to EBNA1- and LMP1-specific CD4+ and CD8+ T-cell clones in vitro. Targeting of antigens to DEC-205 on B cells led to more efficient MHC class II than I loading, and stimulated T cells more efficiently than targeting to DEC-205 on DCs. Although LCLs internalized DEC-205–targeted antigens less efficiently than DCs, they retained them for longer time periods and delivered them to endosomal compartments that receive also B-cell receptor targeted proteins. This could facilitate prolonged T-cell stimulation and efficient MHC class II loading, and, indeed, CD4+ T-cell expansion by DEC-205–targeted vaccination was significantly compromised in B-cell deficient mice. These studies suggest that B cells, activated by virus transformation or other means, can contribute to T-cell stimulation after DEC-205 targeting of antigens during vaccination