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Dendritic cells (DC) play a key role in driving the adaptive immune response to Mycobacterium tuberculosis (MTB), the causative pathogen of tuberculosis (TB). However, studying these important yet very sparse immune cells in the context of MTB pathogenesis is severely restricted by the lack of suitable cell lines and the complexity of culturing of DC progenitors, usually obtained from the bone marrow. However, significant advances have been made towards generating long-term DC cultures from various lymphoid tissues. Here, we report the evidence for generating a long-term, self-renewing DC culture from the Balb/c mouse spleen. We demonstrate that these cells, termed IDC-3, have a myeloid DC origin, i.e. they are CD11c(+) CD11b(++) CD8-α(-) F4/80(+/-) and that they also display a phenotype MHC-II(+) CD16/32(++) CD80(+/-) CD86(+) , indicating that they are immature DC. Following incubation with Mycobacterium bovis BCG (Bacillus Calmette Guerin), the IDC-3 efficiently took up bacteria and acquired the morphology of mature DC. Importantly though, when IDC-3 were pre-stimulated with a mycobacterial antigen in vitro, they were able to induce proliferation of T lymphocytes from mice immunized with the same antigen. The T-cell stimulatory potential of IDC-3 was further enhanced when the cells were co-stimulated with an anti-CD40 mAb. We therefore suggest that the IDC-3 culture system could be a useful tool for studying the interaction of DC with mycobacteria., (© 2010 The Authors. Journal Compilation © 2010 APMIS.)

Background: In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed.Methods: We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen.Results: Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay.Conclusions: We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms. [ABSTRACT FROM AUTHOR]