Responsiveness of B cells is regulated by the hinge region of IgD.

Mature B cells express immunoglobulin M (IgM)- and IgD-isotype B cell antigen receptors, but the importance of IgD for B cell function has been unclear. By using a cellular in vitro system and corresponding mouse models, we found that antigens with low valence activated IgM receptors but failed to trigger IgD signaling, whereas polyvalent antigens activated both receptor types. Investigations of the molecular mechanism showed that deletion of the IgD-specific hinge region rendered IgD responsive to monovalent antigen, whereas transferring the hinge to IgM resulted in responsiveness only to polyvalent antigen. Our data suggest that the increased IgD/IgM ratio on conventional B-2 cells is important for preferential immune responses to antigens in immune complexes, and that the increased IgM expression on B-1 cells is essential for B-1 cell homeostasis and function.

N-linked glycosylation selectively regulates autonomous precursor BCR function.

Developing B cells express distinct classes of B cell antigen receptors (BCRs) that differ in their heavy chain (HC). Although only muHC is expressed in early stages, deltaHC-containing BCRs dominate on the surface of mature B cells. The reason for the tightly regulated expression of these receptors is poorly understood. Here we show that muHC was specifically required for precursor BCR (pre-BCR) function and that deltaHC was unable to form a functional pre-BCR. A conserved asparagine (N)-linked glycosylation site at position 46 (N46) in the first conserved domain of muHC was absolutely required for pre-BCR function, and swapping that domain with deltaHC resulted in a functional deltaHC-containing pre-BCR. When tested in the context of the BCR, muHC with a mutant N46 showed normal function, which indicated that N46-glycosylation is specifically required for pre-BCR function. Our results suggest an unexpected mode of pre-BCR function, in which binding of the surrogate light chain to N46 mediates autonomous crosslinking and, concomitantly, receptor formation.

Differences in Self-Recognition between Secreted Antibody and Membrane-Bound B Cell Antigen Receptor.

The random gene segment rearrangement during B cell development ensures Ab repertoire diversity. Because this process might generate autoreactive specificities, it has been proposed that stringent selection mechanisms prevent the development of autoreactive B cells. However, conventional assays to identify autoreactive B cells usually employ in vitro-generated Abs, which differ from membrane-bound BCRs. In this study, we used a cell-based assay to investigate the autoreactivity of membrane-bound BCRs derived from different B cell developmental stages of human peripheral blood. Contrasted to soluble Ab counterparts, only a few of the tested BCRs were autoreactive, although the cell-based assay sensitively detects feeble Ag recognition of a germline-reverted murine BCR that was selected after OVA immunization of mice, whereas conventional assays failed to do so. Together, these data suggest that proper identification of autoreactive B cells requires the membrane-bound BCR, as the soluble Ab may largely differ from its BCR counterpart in Ag binding.

Idelalisib for optimized CD19-specific chimeric antigen receptor T cells in chronic lymphocytic leukemia patients.

Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less-differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long-term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib-induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib-based CART cell generation resulted in an enrichment of less-differentiated naïve-like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD-1 and Tim-3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib-treated CART cells was validated in a xenograft mouse model. Intracellular TNF-α and IFN-γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib-based CART cell generation protocols are warranted.

Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling.

B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.

Assembly and Function of the Precursor B-Cell Receptor.

During early stages of development, precursor B lymphocytes express a characteristic type of antigen receptor known as the pre-B-cell receptor (pre-BCR). This receptor differs from conventional BCRs in that it possesses a germ line-encoded surrogate light chain (SLC), which is associated with the signal transduction machinery via heavy chain (HC) proteins that have been generated by productive rearrangement of the immunoglobulin HC genes. The pre-BCR marks a key step of B-cell commitment, as it activates the B-cell-specific signaling cascade and mediates the selection, expansion, and differentiation of cells expressing a productively rearranged HC protein. Another difference between the pre-BCR and conventional BCR might be the initial event that triggers receptor activation, as the pre-BCR is activated in the absence of external ligands, while conventional BCRs require antigen for activation. Nonetheless, the pre-BCR downstream signaling cascade is largely similar to that of the BCR suggesting that the characteristic LC of the pre-BCR mediates important receptor interactions thereby providing distinctive, germ line-encoded features to the pre-BCR. In fact, the SLC enables the pre-BCR to act as a surrogate autoreactive receptor. Here, we outline the structure and function of the pre-BCR and how the autonomous signaling capacity might be a direct consequence of pre-BCR assembly. In addition to its role in early B-cell development, we discuss how the ordered activation of downstream signaling cascades enables the pre-BCR to activate seemingly opposing cellular programs such as proliferation and differentiation.

Autoreactivity and the positive selection of B cells.

The diversity among B-cell antigen receptor (BCR) specificities is generated by random rearrangement of gene segments during early B-cell development. While such stochastic recombination of gene segments is important for diversity, it introduces the risk of producing self-reactive BCRs which might lead to the development of autoimmune diseases. Therefore, it has been proposed that negative selection of autoreactive BCR specificities during early B-cell development are required to establish tolerance towards self. In fact, transgenic mouse models have identified a number of "tolerance mechanisms" such as receptor editing, clonal deletion and anergy, all of which prevent the development of autoreactive B cells. Recent data, however, reveal that self-recognition is crucial for the generation of B cells, and that the precursor-BCR (pre-BCR), which is essential for early B-cell development, basically plays the role of an autoreactive BCR. Moreover, although it has become clear that autoreactive B cells are present in the periphery of healthy individuals, the role of autoantigen in their development, persistence and regulation is unclear. This review outlines the important role of autoreactivity in early B-cell development and presents potential models for the regulation of the activation of peripheral B cells by different forms of self or foreign antigens.

Insulin-like growth factor-1 receptor (IGF1R) as a novel target in chronic lymphocytic leukemia.

The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is implicated in various tumor entities including chronic lymphocytic leukemia (CLL), but its functional significance in this disease remains poorly characterized. Here, we show that the IGF1R protein is overexpressed in various CLL subsets, suggesting a contribution to CLL pathology. Indeed, we show that IGF1R knockdown in primary human CLL cells compromised their viability. Likewise, IGF1R inhibition with 3 structurally distinct compounds induced apoptosis, even in the presence of protective stroma components. Furthermore, IGF1R inhibition effectively limited CLL development in Eµ-TCL1 transgenic mice and of primary human CLL xenografts. In agreement with its prosurvival function, IGF1R inhibition affected the phosphorylation and/or expression of multiple signaling proteins. The multikinase inhibitor sorafenib yielded similar effects on these signaling elements as IGF1R inhibitors. Indeed, IGF1R appears to be a direct sorafenib target because sorafenib decreased IGF1R expression and phosphorylation, counteracted insulin-like growth factor-1 (IGF-1) binding to CLL cells, and lowered the in vitro kinase activity of recombinant, purified IGF1R. Thus, we demonstrate that blockade of IGF1R-mediated signaling represents a novel mechanism of action for sorafenib in CLL. Importantly, IGF1R inhibitors compromise CLL viability in their microenvironment context, implicating this RTK as a promising therapeutic target.

Carbohydrate-dependent B cell activation by fucose-binding bacterial lectins.

Bacterial lectins are typically multivalent and bind noncovalently to specific carbohydrates on host tissues to facilitate bacterial adhesion. Here, we analyzed the effects of two fucose-binding lectins, BambL from Burkholderia ambifaria and LecB from Pseudomonas aeruginosa, on specific signaling pathways in B cells. We found that these bacterial lectins induced B cell activation, which, in vitro, was dependent on the cell surface expression of the B cell antigen receptor (BCR) and its co-receptor CD19, as well as on spleen tyrosine kinase (Syk) activity. The resulting release of intracellular Ca2+ was followed by an increase in the cell surface abundance of the activation marker CD86, augmented cytokine secretion, and subsequent cell death, replicating all of the events that are observed in vitro upon canonical and antigen-mediated B cell activation. Moreover, injection of BambL in mice resulted in a substantial, BCR-independent loss of B cells in the bone marrow with simultaneous, transient enlargement of the spleen (splenomegaly), as well as an increase in the numbers of splenic B cells and myeloid cells. Together, these data suggest that bacterial lectins can initiate polyclonal activation of B cells through their sole capacity to bind to fucose.

Pathogen-specific B-cell receptors drive chronic lymphocytic leukemia by light-chain-dependent cross-reaction with autoantigens.

Several lines of evidence indirectly suggest that antigenic stimulation through the B-cell receptor (BCR) supports chronic lymphocytic leukemia (CLL) development. In addition to self-antigens, a number of microbial antigens have been proposed to contribute to the selection of the immunoglobulins expressed in CLL. How pathogen-specific BCRs drive CLL development remains, however, largely unexplored. Here, we utilized mouse models of CLL pathogenesis to equip B cells with virus-specific BCRs and study the effect of antigen recognition on leukemia growth. Our results show that BCR engagement is absolutely required for CLL development. Unexpectedly, however, neither acute nor chronic exposure to virus-derived antigens influenced leukemia progression. Rather, CLL clones preferentially selected light chains that, when paired with virus-specific heavy chains, conferred B cells the ability to recognize a broad range of autoantigens. Taken together, our results suggest that pathogens may drive CLL pathogenesis by selecting and expanding pathogen-specific B cells that cross-react with one or more self-antigens.

Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia.

Cell-autonomous B-cell receptor (BcR)-mediated signalling is a hallmark feature of the neoplastic B lymphocytes in chronic lymphocytic leukaemia (CLL). Here we elucidate the structural basis of autonomous activation of CLL B cells, showing that BcR immunoglobulins initiate intracellular signalling through homotypic interactions between epitopes that are specific for each subgroup of patients with homogeneous clinicobiological profiles. The molecular details of the BcR-BcR interactions apparently dictate the clinical course of disease, with stronger affinities and longer half-lives in indolent cases, and weaker, short-lived contacts mediating the aggressive ones. The diversity of homotypic BcR contacts leading to cell-autonomous signalling reconciles the existence of a shared pathogenic mechanism with the biological and clinical heterogeneity of CLL and offers opportunities for innovative treatment strategies.