Anderson, Elinor Julie Rae
The role of the CCX-CKR chemokine receptor in immunity and tolerance.
PhD thesis, University of Glasgow.
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CCX-CKR is an atypical chemokine receptor for the homeostatic chemokines CCL19, CCL21 and CCL25. CCL19 and CCL21 are also ligands for CCR7 and are crucial for the induction of antigen specific immunity and tolerance, whereas CCL25 is the sole ligand for CCR9 and is involved in the recruitment of immune effector cells to the small intestine. CCX-CKR does not signal after binding its ligands, as determined by a failure to induce the rapid increase in intracellular calcium that is typical of G-protein mediated signalling. CCX-CKR also does not become desensitised to chemokine binding and therefore is proposed to act as a scavenger receptor that can regulate the activity of CCR7 and CCR9 by affecting the availability of their ligands in vivo. At the time of starting my project, there were no published reports describing the biological function of CCX-CKR in vivo and the principal aim of my thesis was to characterise the immune system of the recently generated CCX-CKR KO mouse with particular focus on the intestinal immune compartment where all three of the chemokine ligands are expressed.
Firstly, as described in Chapter 3, I analysed the cellular composition of the secondary lymphoid organs of CCX-CKR KO mice. These studies revealed normal proportions and absolute numbers of lymphocytes, CD11c+ dendritic cells (DC), macrophages and natural killer (NK) cells in the absence of CCX-CKR. The proliferative responses of lymphocytes to mitogenic or TCR stimulation in vitro were also normal, although there was a decreased production of IFNγ by CD4+ T cells from CCX-CKR KO mice. Although most of the phenotypic subsets of conventional DC were present in comparable numbers in the mesenteric lymph nodes (MLN) of CCX-CKR KO and WT mice, there was a consistent and dramatic reduction in the numbers of CD11cloPDCA-1+ plasmacytoid DC (pDC) in CCX-CKR KO MLN. In parallel, fewer CD11cloB220+ cells from CCX-CKR KO MLN than WT expressed CCR9, despite this marker being expressed normally by lymphocytes in these mice. The proportions of pDC in CCX-CKR KO inguinal lymph nodes (ILN) were also significantly reduced compared to WT and pDC from CCX-CKR KO MLN, ILN and spleen all appeared to express higher levels of class II MHC than WT pDC. These data suggest that CCX-CKR may play an important role in the recruitment and/or survival of pDC in the LN and that in its absence, pDC in secondary lymphoid organs may have a more mature phenotype.
In Chapter 4, I examined the cellularity of the intestinal immune compartment in resting CCX-CKR KO mice, as well as the effects of Flt3L administration in vivo. Although CCX-CKR KO mice displayed no histological abnormalities in their small intestinal architecture and had normal numbers of T cells in the lamina propria, they did have significantly reduced numbers of intra-epithelial lymphocytes (IEL) as well as decreased proportions of CD19+ B cells and increased proportions of CD11c+ cells in the lamina propria compared with WT mice. The proportions and absolute numbers of CD103+ DC were normal in the lamina propria and MLN of CCX-CKR KO mice, suggesting that CCX-CKR has little to no role in regulating DC migration from the lamina propria to the MLN, a process that is critically dependent on CCR7. Although the proportions and absolute numbers of B cells and CD11c+ cells were normal in CCX-CKR KO Peyer’s patches (PP), there were significantly decreased proportions of pDC compared with WT PP. In vivo treatment of CCX-CKR KO mice with the DC differentiation factor Flt3L, expanded CD11c+ DC numbers dramatically in both CCX-CKR KO and WT small intestinal lamina propria, ILN, spleen, MLN and PP. Although Flt3L abolished the apparent defects in pDC populations in the ILN and PP, this was not the case for CCX-CKR KO MLN, which remained significantly deficient in pDC compared to WT MLN. Work in Chapter 6 examined parallel effects in the blood and bone marrow.
As the CCX-CKR ligands CCL19 and CCL21 are involved in the development of all adaptive immune responses, and together with the other CCX-CKR ligand, CCL25, orchestrate immune responses to antigen encountered in the gut, I next investigated the development of antigen specific immunity and tolerance in CCX-CKR KO mice. There were no significant differences in systemic immune responses to subcutaneous immunisation with ovalbumin (OVA) emulsified in complete Freunds adjuvant (CFA) between CCX-CKR KO and WT mice when assessed in vivo or in vitro. However, the development of oral tolerance in CCX-CKR KO mice was impaired, with no suppression of OVA specific delayed type hypersensitivity (DTH) responses or of serum OVA specific IgG2a as was seen in WT mice. In parallel with defective systemic tolerance after feeding OVA, CCX-CKR KO mice appeared to be more susceptible to priming of systemic and local antibody responses after feeding OVA with cholera toxin (CT) as a mucosal adjuvant. In addition, there was some evidence of priming of OVA specific antibody responses in CCX-CKR KO mice fed OVA alone, which was not seen in WT mice. Despite this evidence of abnormal mucosal immunity, CCX-CKR KO mice developed DSS colitis normally, with all indices of disease being identical in CCX-CKR KO and WT animals. Together, these data suggest that there are selective defects in the regulation of antigen specific mucosal immune responses in the small intestine of CCX-CKR KO mice that may predispose these animals towards exaggerated active immune responses.
Finally, I performed some preliminary experiments to try and relate DC function to the immune dysregulation I observed in CCX-CKR KO mice and to explore the basis of the pDC defect in the MLN. Bone marrow derived and splenic DC from CCX-CKR KO mice showed a reduced ability to process and present intact protein antigens although endocytic activity was normal. MLN DC from normal and Flt3L treated CCX-CKR KO mice responded similarly to in vitro stimulation with the synthetic TLR7 agonist R848, showing an expansion in the numbers of CD11chiPDCA-1+ cells that nearly all expressed CD40 and CD86. In addition, in vivo administration of R848 triggered identical migration of CD11chiclassIIMHChiCD103+ DC into the MLN of CCX-CKR KO and WT mice, suggesting that lamina propria pDC can effectively mobilise local DC to the MLN in the absence of CCX-CKR. There were no differences in the proportions or absolute numbers of pDC in the liver of CCX-CKR KO and WT mice despite the fact that liver pDC have been implicated in other studies of oral tolerance. Although the proportions of pDC were normal in the bone marrow of resting and Flt3L treated CCX-CKR KO mice, there did appear to be a defective recruitment of Flt3L expanded pDC into the blood of these animals. I used an adoptive transfer approach to study the in vivo localisation of pDC into lymphoid organs and although these experiments were not entirely conclusive, they indicated that WT pDC could enter the MLN and other lymphoid tissues of CCX-CKR KO mice normally and that pDC from CCX-CKR KO mice may have a defect in their ability to enter WT MLN.
Taken together, my data suggest that CCX-CKR is involved in the entry and/or survival of pDC in secondary lymphoid organs, a process that normally involves migration across high endothelial venules (HEV). This was associated with impaired oral tolerance induction and heightened immune responses to antigen delivered orally, indicating that CCX-CKR contributes to the regulation of mucosal immunity and tolerance by an as yet unclear mechanism. Further study of these animals will hopefully better define the relationship between pDC and the regulation of mucosal immune responses.
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