Abstract
Tuberculosis (TB) is a lung disease caused by Mycobacterium tuberculosis (Mtb), which was responsible for 1.6 million deaths in 2017. The only vaccine against TB; bacille Calmette Guérin (BCG), protects against miliary and meningeal TB in neonates and offers some protection in animal models. However, BCG provides unreliable protection against the predominant form of TB; adult pulmonary TB. Consequently, a new more efficacious vaccine against adult pulmonary TB is urgently required.
A major challenge preventing the development of a new vaccine against adult pulmonary TB is that very little is known about which cell types and memory responses contribute to protection against mycobacterial infections following BCG vaccination. Recent evidence has suggested that innate lymphoid cells (ILCs), a subset of immune cells, may contribute to the generation of a protective immune response against Mtb since ILC1 and ILC3 subsets have been found to upregulate the production of the cytokine; interferon gamma and accumulate in the lungs of BCG-inoculated mice compared to naïve mice. Consequently, the aim of this project was to identify whether ILC1s and ILC3s isolated from the lungs of BCG-inoculated mice could inhibit the growth of mycobacteria in vitro, which would indicate directly for the first time whether ILCs can protect against mycobacterial infections.
Flow cytometry was used to identify ILC subsets based on the expression of surface markers to enable sorting of live ILCs. However, poor fidelity between surface marker expression and the canonical ILC subset transcription factor expression, which is the gold-standard method for categorising ILCs, meant that ILC subsets could not be confidently categorised. Additionally, insufficient numbers of ILCs in the lungs of BCG-inoculated mice meant that it was not feasible to sort live ILC subsets. Instead, total ILCs isolated from the lungs of BCG-inoculated mice were used to establish an in vitro mycobacterial growth inhibition assay, whereby total ILCs sorted by flow cytometry were cultured with BCG-infected splenocytes. The ability of ILCs to enhance mycobacterial killing was measured by enumerating BCG colony forming units when the cultures were plated onto agar. Although sorting and cultures were successful, there was high variability in BCG colony forming units per mL detected between technical replicates and no enhancement in splenocyte killing of mycobacteria was detected when ILCs were cultured with BCG-infected splenocytes compared to when BCG-infected splenocytes were cultured alone. These findings indicate that further optimisation of the in vitro mycobacterial growth inhibition assay is required to reduce the variability and that alternative approaches such as adoptive transfer studies could be used to assess ILC enhancement of mycobacterial killing.