Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents

Source: Smith SG, Lalor MK, Gorak-Stolinska P, Blitz R, Beveridge NE, Worth A, McShane H, Dockrell HM. Mycobacterium tuberculosis PPD-induced immune biomarkers
measurable in vitro following BCG vaccination of UK adolescents by multiplex bead
array and intracellular cytokine staining. BMC Immunol. 2010 Jul 7;11:35.

Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents by multiplex bead array and intracellular cytokine staining

Clinical trails of BCG vaccination have previously shown variable efficacy against pulmonary tuberculosis in adults (0-80%) and high efficacy in children. BCG vaccination has demonstrated a protective efficacy of 77% against pulmonary tuberculosis when administered to UK adolescents. In this paper, the authors have used this setting to investigate the nature of immunity induced by BCG vaccination in a cohort of school children (12-15 years) in UK.
The Th-1-type immune response is most effective against TB. IFN-gamma which is an important part of Th-1-type immune response plays important role in protection against TB, as mice and human who are deficient in IFN-gamma signaling pathways are highly susceptible to TB. However, it has also been observed that patients with a latent TB infection and positive IFN-gamma release status can progress to active infection and IFN-gamma can also be detected in samples from patients with active disease. Thus other components of type-1 response e.g. TNF-alpha, IL-2 and IL-12 may also play important roles.
A critical role of Th-17 response involving IL-17 has also been suggested by some studies. CD4 cells play important role in TB immunity. Other cells that are also important include CD8 cells, natural killer T cells and gamma delta T cells. A protective biomarker profile thus defines the presence of all these cytokines and cells that secrete these cytokines and absence of certain biomarkers (e.g., IL-4 and IL-10) which may subvert the response to TB and allow bacterial infection to proceed.

Results:
Biomarkers detected by diluted whole blood assay and multiplex bead array analysis: Blood sample from 12 adolescents were taken before and after BCG vaccination. These blood samples were diluted with RPMI 1640 and cultured for six days in the presence or absence of Mtb purified protein derivative (PPD). Assay supernatants were collected and frozen and analyzed for biomarker content by multiplex bead array. Of the 20 biomarkers that were measured in this study, 10 were found to be raised in samples form adolescents taken after one month of vaccination. These included Th1 type cytokines, IFN-gamma and IL-2, proinflammatory cytokines and chemokines, TNF-alpha, IL-17, IL-1alpha, MIP1alpha, IL-6 and IP-10. GM-CSF and anti-inflammatory cytokine, IL-10 were also elevated. No significant increase was observed in G-CSF, IL-4 or IL-8. Some cytokines were undetectable in both pre and post vaccination samples. These were IL-12p70, IL-7, IL-15, IL-5, IL-1β and eotaxin. The authors further wanted to know which biomarkers were associated with IFN-gamma responses in these assays. They found that there was a correlation between the magnitude of IFN-gamma responses and those of other Th-1 or proinflammatory cytokines such as TNF-alpha, MIP-1alpha, IL-2, and IL-17. The authors also observed a strong correlation between IFN-gamma and GM-CSF and between IFN-gamma and IL-10. The authors then analyzed biomarker profile in children after 12 months of vaccination. It was observed that out of 10 biomarkers that were significantly increased in post 1 month vaccinated samples, 8 were also increased after 12 months of vaccination. IL-6 and IL-10 returned to levels comparable with pre-vaccinated samples.
Thus, the authors concluded that BCG vaccination results in a broad biomarker response measurable at one month in peripheral blood and detected following stimulation with Mtb PPD. The authors also observed that this biomarker response is maintained up to 12 months of vaccination.

Flow-cytometric analysis of cellular cytokine production
The authors isolated and preserved PBMCs from blood samples of 12 months post BCG vaccinated children. The recovered cells were stimulated for 24 hrs in the presence or absence of Mtb PPD and then stained with a panel of antibodies that recognize phenotype markers of lymphocytes and cytokines. First the authors applied gating on singlet and live cells and then excluded CD14 or CD19 expressing cells. After this, the single cytokine expression was examined on CD4 (CD3+ and CD4+), CD8 (CD3+ and CD8+) and NK (CD3- and CD56+) cells. The authors found that IFN-gamma was detected in all three set of cells. TNF-alpha was detected in CD4 cells and NK and it was very low in CD8 cells. Next the authors studied the expression of both IFN-gamma and TNF-alpha in these samples. They found that majority of cytokine producing cells were either producing IFN-gamma or TNF-alpha and very few were producing both.
Thus, from these studies the authors concluded that cytokine responses are less readily detected by flow cytometry compared to multiplex bead array analysis involving a six day culture.

Conclusions:
In the present study, the authors demonstrated Mtb PPD specific response after 1 month and 12 month of BCG vaccination. This response was quite broad in terms of cytokines released. These cytokines included Th-1 responses and pro-inflammatory cytokines and chemokines. When the PBMCs from 12 month vaccinated samples were analyzed by flow cytometry, CD4 and CD8 cells that can release more than one cytokines were not detected. The majority of these cells and also NK cells were single cytokine producers.

Future aspects:
IL-6, IL-8 and IP-10 were elevated in post vaccine samples on stimulation with Mtb PPD, however, they were also high in pre vaccine samples on stimulation with Mtb PPD. The reason for this is unknown. The authors hypothesized that there could be some antigen non- specific stimulation of macrophages and monocytes via the TLRs in response to undefined components of Mtb PPD protein cocktail.

IL-17 was found to be highly increased in samples from post vaccinated children. However, the precise nature of IL-17 detected remains unknown. Protein antigens in Mtb PPD that are processed and presented by MHC II molecules can lead to CD4+ Th17 response. The other possible source could be gamma delta T cells.