The Role of the CC-¬¨‚ↂÄövÑv™chemokine receptor 6, CCR6, in B cell differentiation during the humoral immune response

The T cell-dependent humoral immune response consists of a series of discrete stages that culminate in the differentiation of effector B cells, capable of secreting antibodies. These cellular events occur in specific microenvironments within secondary lymphoid organs. The positioning of B cells during the humoral response allows them to receive the appropriate cellular and genetic cues necessary for their subsequent differentiation. Consequently, the precise positioning of B cells is essential for the generation of an effective humoral response. The chemokine receptors, a family of G-protein coupled receptors, are responsible for directing the migration and organisation of lymphocytes in the immune system. It has been established that the chemokine receptors, CXCR5, CCR7, CXCR4 and EBI2, work together to co-ordinate the movement of B cells in the humoral response, by integrating stimuli from various chemokine ligands in the surrounding environment. Whilst a number of chemokine receptors on B cells have been functionally defined, others are not well understood. One such chemokine receptor, CCR6, has an expression pattern consistent with a role in B cell differentiation, however, the contribution of CCR6 to the humoral response remains undefined. This thesis focused on addressing the role of CCR6 in the humoral response. I hypothesised that CCR6 expression is necessary for efficient B cell differentiation during T cell-dependent humoral immune responses. Specifically, this thesis has examined 1) the expression level of CCR6 during B cell activation, 2) the consequences of B cell differentiation in the absence of CCR6, 3) the potential mechanisms that underlie the contribution of CCR6 to B cell differentiation and finally, 4) the relevance of CCR6 in B cell-mediated autoimmunity. Initially, CCR6 expression was assessed on B cells after activation with T cell-dependent antigens. Flow cytometry analysis demonstrated that antigen-specific B cells significantly upregulate CCR6 expression upon activation in vitro. This finding was also confirmed in vivo. Furthermore, cell sorting and immunofluorescence revealed that CCR6 is expressed during T-B cell interactions in vivo. Consequently, this work established that CCR6 is upregulated on activated B cells. Considering the distinct expression of CCR6 during B cell differentiation, particularly activation, the impact of CCR6 on the humoral response was evaluated. B cell differentiation was quantified in WT and CCR6-/- mice that were challenged with the immunogen, NP-KLH, over a 6-week period. Flow cytometry and immunofluorescence analysis revealed that the CCR6-/- mice generated more germinal centre B cells at 3 and 5 days after antigen challenge, compared to WT mice. This correlated with a significant reduction in the frequency of na‚àövòve B cells at days 3, 5 and 14, as well as a significant increase in the memory B cell population at days 3, 5, and 10, after challenge in the CCR6-/- mice compared to the WT mice. In addition, when extrafollicular B cells were analysed in MD4 and MD4.CCR6-/- mice immunised with HEL-SRBC, the MD4.CCR6-/- mice were found to have significantly fewer extrafollicular B cells 6 days after challenge, compared to the MD4 mice. Even though B cell differentiation was found to be dysregulated in the absence of CCR6, this dysregulation was not permanent as it only occurred early in the response, indicating a role for CCR6 in the initiation of the humoral response. Investigation into the potential mechanisms underlying the B cell dysregulation observed in the CCR6-/- mice resulted in several findings. Immunofluorescence of splenic germinal centre follicles showed no gross structural abnormalities in the CCR6-/- mice. In addition, qRT-PCR indicated that somatic hypermutation and differentiation in CCR6-/- germinal centre B cells was not affected. However, CCR6-/- germinal centre B cells contained significantly more Bcl-6 mRNA than WT, potentially accounting for the increased germinal centre response. Furthermore, this increase in Bcl- 6 mRNA was attributed to a significant increase in T follicular helper cell-secreted IL-21. Also, flow cytometry analysis demonstrated that the T follicular helper cell and follicular dendritic cell populations of CCR6-/- mice were reduced compared to WT mice, indicating that germinal centre affinity selection is impaired in the absence of CCR6. Despite the increased germinal centre response observed in the CCR6-/- mice, antibody quantification by ELISA demonstrated that the production of antigen-specific IgM and IgG in CCR6-/- mice was equivalent to that in WT mice. Additionally, an adoptive transfer model demonstrated that the loss of CCR6 on leukocytes other than B cells is responsible for the B cell dysregulation identified in the CCR6-/- mice. Taken together, the reduced extrafollicular response and increased germinal centre response likely counterbalance each other in the CCR6-/- mice. Hence, the loss of CCR6 does not have an overall detrimental effect on the humoral response. Finally, the clinical relevance of CCR6 was examined in a mouse model of systemic autoimmunity and in the human B cell-mediated systemic autoimmune disease, Systemic Lupus Erythematosus (SLE). Flow cytometry analysis showed that the FasLgld mice, which develop a spontaneous generalised systemic autoimmune disease, have a significantly lower frequency of CCR6+ B cells than WT mice. However, CCR6 expression on B cells was significantly higher in the FasLgld mice compared to the WT mice. This finding was examined in a preliminary study of SLE. Participants diagnosed with SLE had a significantly higher expression of CCR6 on B cells, compared to healthy controls. The increased CCR6 expression observed in autoimmunity highlights the potential for chemokine receptors to be used as biomarkers and therapeutic targets of disease. Overall, this thesis presents several novel findings. It is the first to demonstrate the upregulation of CCR6 on activated B cells, as well as the first to define the T cell-dependent humoral response in CCR6-/- mice and to examine the potential of chemokine receptors to be used in clinical medicine. As a result of this work, I propose that CCR6 aids the organisation of activated B cells during the T cell-dependent humoral response. This thesis provides a significant contribution to our understanding of the development of efficient humoral responses and insight into how responses can be manipulated in disease.