Regulation of B cell function by plasmacytoid dendritic cells

Abstract

Dendritic cells (DCs) are early sentinels of pathogen exposure and central in the initiation and orchestration of adaptive immune responses. Apart from the prominent role of DCs in the activation of T cells, DCs have been shown to influence humoral B cell mediated responses. DCs are therefore important cells for regulating immune responses to vaccines. Many of the vaccines under development today are against pathogens such as Mycobacterium tuberculosis and HIV-1 that likely require induction of both cellular and humoral responses to cause protection. This generates a need for new safe and effective vaccine adjuvants that can stimulate such responses. One class of adjuvants that has attracted a lot of interest over the last years is TLR (toll-like receptor)-ligands. Selected TLR-ligands can specifically activate subsets of DCs and B cells according to their cognate receptor expression and therefore represent promising candidates to shape vaccine-induced responses.

The first aim of this thesis was to investigate the responsiveness of B cells to TLR stimulation to proliferate and differentiate into antibody (Ab) producing cells. Furthermore, the aims were to study, whether the distinctly different myeloid (MDCs) and plasmacytoid (PDCs) can support these responses in a T helper cell-independent or dependent manner. In addition, differences in the responses of B cells and DCs from humans versus non-human primates (NHP) were addressed.

In paper I, we established and refined isolation protocols for subsets of primary human PDCs, MDCs and B cells from blood and methods to examine their functions. We found that total B cells responded strongly to engagement of TLR9, less to TLR7/8 and not to engagement of TLR3. Furthermore, PDCs but not MDCs markedly enhanced B cell proliferation and differentiation into Ab producing cells in response to TLR7/8-ligand stimulation and to a lesser extent to TLR9-ligands (CpG ODN classes A, B, and C). PDCs strongly enhanced TLR7/8-ligand-induced proliferation of both memory and naive B cells but were only able to support memory cells to differentiate to CD27high plasmablasts. Type I IFN produced to high levels by PDCs was the principal mediator of the enhanced responses upon TLR7/8 stimulation. This effect may at least in part be explained by the reported upregulation of TLR7 and MyD88 by IFNα. Although MDCs expressed high levels of the known B cell growth factors IL-6, IL-10, and B cell-activating factor (BAFF) in response to TLR7/8 stimulation, they were unable to enhance B cell responses in this system.

In paper II, we found that PDCs also had the ability to augment naïve B cell responses induced by BCR engagement and T cell help. The presence of supernatants from TLR-stimulated PDCs increased B cell proliferation, the frequency of B cells that differentiated to CD27high CD38high cells, and secretion of IgM. IFNα produced by PDCs was again instrumental in these processes and increased cell viability or proliferation were not main reasons for the improved B cell function. We found that PDC supernatants or IFNα induced upregulation of the co-stimulatory molecule CD86 on B cells. Further, these B cells showed improved ability to interact with and activate T cells. Thus, increased B cell responsiveness to T cell contact, mediated by PDCs via their production of IFNα, may facilitate B cell proliferation and differentiation into Ab producing cells.

In order to further explore the influence of IFNα and PDCs on B cell functions in vivo in humans, models such as NHPs that more closely resemble humans than rodents need to be utilized. NHPs have the advantage that they to a large extent exhibit similar subpopulations of DC and B cell subsets as well as similar TLR expression as humans. However, similarities and potential disparities between the species need to be carefully investigated to facilitate the translation of NHP studies into clinical trials. In paper III, we therefore examined whether the effect of activated PDCs or IFNα enhanced B cell functions was comparable in human and NHP rhesus macaque cells in response to TLR ligands. We found similar responses in human and rhesus cultures to the selected TLR ligands in terms of B cell proliferation. B cell proliferation to the TLR7/8-L and CpG class C showed a significant and comparable increase in presence of IFNα. However, upon stimulation only human B cells acquired high expression of CD27, associated with plasmablast formation, although both human and rhesus B cells produced increased levels of IgM. Instead, rhesus B cell differentiation was associated with a more prominent downregulation of CD20. This validates that rhesus macaques are relevant and appropriate in vivo models to study TLR induced B cell responses although the choice of B cell differentiation markers to measure must be considered.

In conclusion, the studies included in this thesis highlight the potential of PDCs and IFNα to shape B cell differentiation to Ab secreting cells. These studies add to the understanding on the role of DCs in modulation of B cell responses, which is crucial information for the design of novel vaccines, adjuvants and immuno-modulatory treatment formulations.