Although not as well studied as other similar lymphoid tissues, it is clear that the NALT plays an important role in the immune response to some respiratory pathogens, such as reoviruses [11]. However others have shown that removal of the NALT has no effect on influenza or pneumococcal infection [14] and [15] although depletion of CD4 or CD8 T-cells in vivo does increase influenza virus titres in the nose after challenge [16]. These data suggest that the NALT may not be essential for induction of immune responses to respiratory pathogens but nevertheless antigen-specific cells located in the URT may play a role in containment of respiratory infections. As the NALT

would be the first structure to encounter M.tb during aerosol infection we analysed whether it contributes to protection against M.tb following intra-nasal immunisation with a vaccine candidate, Ad85A. By comparing an immunisation regime that preferentially targets the NALT Sotrastaurin mw VE-821 clinical trial to one targeting the whole respiratory tract, we show that only regimes

that induce strong deep lung immune responses protect against aerosol M.tb challenge. All experiments were performed with 6–8-week-old female BALB/c mice (Harlan Orlac, Blackthorn, UK), were approved by the animal use ethical committee of Oxford University and fully complied with the relevant Home Office guidelines. Human adenovirus serotype 5 expressing antigen 85A was produced as described previously [9]. Mice were anaesthetised with Ketamine/Domitor intra-peritoneally and immunised i.n. with 2 × 109 v.p. of Ad85A suspended in different volumes from 5 to 50 μl. The mice were allowed

to slowly inhale the virus suspension, half of which was dropped into each nostril. BCG (SSI, kindly provided by Dr. Amy Yang, CBER/FDA, MD, USA) was administered subcutaneously in the left hind footpad at a dose of 2 × 105 crotamiton colony forming units (CFU) in 30 μl volume. For i.n. boosting, Ad85A was given 10–12 weeks post-BCG. Mice were challenged by aerosol with M.tb (kindly provided by Dr. Amy Yang, Erdman strain, CBER/FDA), using a modified Henderson apparatus [17] 4 weeks post-Ad85A or 4 months post-BCG immunisation. Deposition in the lung was measured 24 h post-challenge as ∼200CFU of M.tb per mouse. Mice were culled 4–6 weeks post-challenge, lungs and spleen homogenized and 10-fold dilutions plated on Middlebrook 7H11 agar plates (E & O Laboratories Ltd., Bonnybridge, UK). Colonies were counted after 3–4 weeks of incubation at 37 °C in 5% CO2. The organized NALT (O-NALT) was extracted by removing the head from the body, dissecting away the lower jaw, tongue and connective tissue to expose the soft palette of the upper jaw. The front incisors were then cut away to reveal the anterior end of the soft palette. The palette was then peeled back from the anterior end, including the paired NALT structures at the posterior of the hard palette. The diffuse NALT (D-NALT) was not removed.