The role of langerin+ CD8+ dendritic cells during systemic bacterial infection

Abstract

Splenic langerin⁺ CD8⁺ dendritic cells (DCs) are a recently identified DC subset found to be superior to langerin⁻ DCs in their ability to cross-prime cytotoxic CD8⁺ T cell responses in mice. Due to their location in the marginal zone of the spleen, langerin⁺ CD8⁺ DCs are ideally positioned to scan the blood for foreign pathogens. We hypothesised that langerin⁺ CD8⁺ DCs were a protective DC subset during systemic bacterial infection, due to their location and cross-presenting ability. Mycobacterium bovis bacille Calmette Guerin (BCG) was used to model a systemic infection. Initiation of CD8⁺ T cell responses during mycobacterial infection is incompletely understood, and it was hypothesised that langerin⁺ CD8⁺ DCs are important for the activation of CD8⁺ T cell immunity during systemic BCG infection. To examine the role of langerin⁺ CD8⁺ DCs during systemic BCG infection, diphtheria toxin was administered to langerin-diphtheria toxin receptor (DTR) mice, which express the DTR on all langerin⁺ DCs. Due to the rapid turnover of CD8⁺ DCs in the spleen, langerin⁺ CD8⁺ DC depletion lasted approximately three days. Therefore, to assess their role during chronic BCG infection, a protocol was developed for the prolonged depletion of langerin⁺ CD8⁺ DCs. Using this protocol, langerin⁺ CD8⁺ DCs were depleted in langerin-DTR mice receiving intravenous infection with BCG. It was found that langerin⁺ CD8⁺ DC depleted mice had a significantly increased bacterial burden in the spleen one week post infection, suggesting a beneficial role for langerin⁺ CD8⁺ DCs during systemic BCG infection.

To determine how the adaptive immune response was affected by the loss of langerin⁺ CD8⁺ DCs, an adoptive transfer model was established, using a recombinant strain of BCG expressing ovalbumin (OVA), and OVA specific transgenic CD8⁺ T cells (OT-I cells). Depletion of langerin⁺ CD8⁺ DCs resulted in delayed activation of CD8⁺ T cells. It was also noted that IL-12p40 levels in the serum of both naïve and BCG infected mice was significantly reduced in the absence of langerin⁺ CD8⁺ DCs. To determine if the protective effects of langerin⁺ CD8⁺ DCs could be attributed to their ability to initiate CD8⁺ T cell responses or produce IL-12p40, monoclonal antibodies to deplete CD8⁺ T cells or neutralise IL-12 were administered to mice before BCG infection. It was found that neither CD8⁺ T cells nor IL-12 was required for control of the bacterial burden during the first week of infection.

It was subsequently discovered that depletion of langerin⁺ CD8⁺ DCs resulted in an increased bacterial burden in the spleen as early as 30 minutes post infection. Therefore, the innate immune response was assessed. Using fluorescent BCG, it was found that langerin⁺ CD8⁺ DCs were not associated with BCG during the first 24 hours of infection. A large proportion of the infected cells were neutrophils and macrophages/monocytes. An in vitro model of DC and BCG co-culture was developed, and it was found that langerin⁺ CD8⁺ DCs did not need to be in contact with infected cells to induce early bacterial killing, and that a soluble molecule produced by langerin⁺ CD8⁺ DCs mediated early bacterial killing.

The work in this thesis revealed that langerin⁺ CD8⁺ DCs stimulate both innate and adaptive immunity during systemic bacterial infection, and that their presence early after infection is beneficial for bacterial protection.