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The Role of the CC-­‐chemokine receptor 6, CCR6, in B cell differentiation during the humoral immune response

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Bannan, J (2014) The Role of the CC-­‐chemokine receptor 6, CCR6, in B cell differentiation during the humoral immune response. PhD thesis, University of Tasmania.

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Abstract

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 naïve 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.

Item Type: Thesis (PhD)
Keywords: CCR6, B cells, humoral immune response
Copyright Holders: The Author
Copyright Information:

Copyright 2014 the author

Date Deposited: 02 Jun 2016 02:51
Last Modified: 12 Apr 2017 17:00
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