Lymphocyte Interaction

In the Lymphocyte Interaction Laboratory our chief concern is the characterisation of the molecular and cellular interactions underlying B cell activation. B cells comprise a critical component of adaptive immune responses to potentially pathogenic invaders by mediating the production of specific antibodies and contributing to the induction of immunological memory. Indeed realisation of the therapeutic potential of antibodies for the diagnosis and treatment of cancers has added further clinical significance to the drive for a more comprehensive understanding of the B cell activation process.

Antigen-induced B cell activation

B cell activation is initiated in response to the recognition of specific antigen through the surface B cell receptor (BCR). The BCR is a complex consisting of a heterotetrameric membrane immunoglobulin (mIg), together with a membrane embedded Igα/β sheath. Antigen engagement of the BCR triggers the phosphorylation of tyrosine residues within the Igα/β leading to the recruitment and activation of a plethora of intracellular signalling molecules and adaptors. These molecules are extensively organised such that they can co-ordinate cellular responses such as cytoskeleton rearrangements and induction of gene expression. One established consequence of B cell activation in response to membrane-bound antigen is the reorganisation of proteins within the plasma membrane. This reorganisation allows BCRs to concentrate in a central cluster and act as a platform for antigen internalisation, allowing the subsequent recruitment of T cell help and maximal activation of B cells.

Microclusters of BCRs organise intracellular signalling and orchestrate B cell spreading

In previous work we showed that during activation, B cells rapidly spread across antigen-containing surfaces, prior to a more prolonged contraction phase (Fleire et al., Science, 2006). This two-phase cellular response has important consequences as it allows the B cell to accumulate greater amounts of antigen, and thus shapes the outcome of B cell activation. In order to elucidate the molecular mechanism underlying the B cell spreading response, we have examined an extensive panel of knock-outs generated in the DT40 B cell line (Weber et al., J Exp Med. 2008).

This screen, verified in primary cells, established an important role for the kinases Lyn and Syk, and for intracellular effectors and adaptors such as Vav, PLCγ2 and Blnk. Subsequently high-resolution TIRFM was used to visualise the spatiotemporal dynamics of these effectors within the B cell. In response to membrane containing antigen, we observed the rapid formation of BCR microclusters throughout the contact area, in agreement withour previous observations (Depoil et al., Nat Immunol. 2008). As these microclusters are the sites for the sequential recruitment of Lyn and Syk, and coordinate the subsequent assembly of numerous 'microsignalosomes', we suggested that they might be the common signalling units in lymphocytes. Furthermore we detected cooperation between PLC&gammal2 stickies and Vav, components of the 'microsignalosome', such that each enhances the recruitment and retention of the other (Weber et al., J Exp Med. 2008). Interestingly this offers a molecular explanation for the essential role of CD19 in mediating B-cell spreading and activation in response to membrane-bound antigen (Depoil et al., Nat Immunol. 2008). As the cytoplasmic domain of CD19 contains binding sites for numerous intracellular signalling molecules, CD19 through transient association with BCR-microclusters can mediate the recruitment of additional molecules of Vav and PI3K. Thus CD19 can enhance signalling through the BCR in response to membrane-bound antigen, propagating spreading and thus facilitating B cell activation.

iNKT cells and TLR9 stimulation can mediate the formation of extrafollicular plasma B cells

Following antigen-induced activation, B cells can differentiate along two alternative pathways. The first of these is important in early immune responses and results in the formation of extrafollicular plasma cells capable of the rapid production of low-affinity antibodies. In contrast, the second involves entry to the germinal centre for affinity maturation, and yields plasma cells able to secrete extremely high affinity antibodies and memory cells that confer long-lived protection. However at this stage, the factors responsible for determining the differentiation pathway and thus shaping B cell fate remain poorly characterised. To shed light on aspects of this complex process, we have designed and employed particulates directly conjugated with both antigen and other immunostimulants as an investigative tool. As these particulates cannot be taken up by B cells through phagocytosis, they provide a method for selectively targeting the activity of the immunostimulant to specific B cells. We observed that immunisation with particulates containing antigen and αGalCer, an iNKT cell stimulatory ligand, resulted in the enhanced formation of antigen-specific extrafollicular plasma cells (Barral et al., PNAS, 2008). A dissection of the mechanism underlying the observed enhancement revealed that BCR-mediated particulate internalisation was required such that αGalCer could be loaded onto endosomal CD1d. This internalisation was dependent on the avidity of the BCR-antigen interaction surpassing a tightly regulated threshold. The subsequent presentation of αGalCer-CD1d on the B cell surface allowed for the recruitment of iNKT cell help, resulting in the formation of extrafollicular plasma cells capable of class switched antibodies. In a similar manner, we have observed that immunization with particulates containing antigen together with a TLR9 ligand generated enhanced formation of antigen-specific extrafollicular plasma cells (Eckl-Dorna and Batista, in press). Thus these particulates have aided the identification of factors intrinsic and extrinsic to the B cells that can influence the outcome of B cell differentiation.

Future directions

Taken together, research in the Lymphocyte Interaction Laboratory within the past year has made a considerable contribution to the understanding of the processes underlying B cell activation. In spite of our significant progress a number of challenges remain, namely the characterisation of the dynamics and regulation of cytoskeleton reorganisations during B cell activation and the determination of factors responsible for shaping B cell fate in vivo. We are seeking to address these, and other, issues through the development and application of innovative high-resolution imaging techniques including single-particle tracking and multi-photon microscopy.

For a list of refereed research papers, see Publications (in navigation on left).