Pakistan has more than 90% of Bacillus Calmette-Guerin (BCG) coverage at birth through Extended Program on Immunization (EPI). Though, BCG does not provide protection against pulmonary tuberculosis, it protects children under five from severe form of tuberculosis. BCG is known to have immuno-modulatory effects, as being live attenuated vaccine, which activates multiple cells of innate immunity such as Macrophages, Dendritic Cells and Neutrophils. One of the immune modulatory mechanisms is the instruction of specialized T cells population. In this study, we identified novel immune modulatory genes (Butyrophyllin, Granzyme) identified from Micro-array data of long survival of melanoma skin cancer patients treated with intra-lesional BCG therapy. We compared the expression of Butyrophillin (BTN3A2), Granzyme A (GZMA) and TNF genes in BCG vaccinated and non-vaccinated healthy volunteers. In addition, we compared the cytokine profiles (Th1, Th2 &Th17) between vaccinated and non-vaccinated groups to determine if prior vaccination status has any effect on cytokine secretion, and whether stimulation of BCG using whole blood (WB) and PBMCs has differential response in cytokine secretion.
Venous blood (16–20ml) was collected from vaccinated and non-vaccinated adults (n=20), aged between 14 to 40 years in heparinized tube (20IU/ml). 4-5ml of blood was used for whole blood assay (WBA) and rest for isolation of PBMCs. After counting the cells, whole blood and PBMCs were stimulated with BCG vaccine (MOI 1.2x106) at 12 hours. Supernatant were tested for cytokine Th1[IL-2, IFN and TNFα], Th2 [IL-4, IL-10], Th17[IL-17A,] using Bio-Plex Pro Human Cytokine Group I Assay kit (BioRad labs, Hercules CA, USA) on Luminex 200.
There was no difference in gene expression profiling of above genes with the exception of GZMA (BCG “yes”: 0.51±0.851; “no”: 8.92±16.69; p=0.045) in WB assay. Although, there was a trend of >2 fold higher responses in non-vaccinated controls. No difference was observed in the secretion of cytokines in vaccinated and non-vaccinated groups. In terms of comparison between stimulation of WBA and PBMCs, BCG stimulation in PBMCs showed a higher median response in IL10 (p =0.013), IL17 (p =0.003) but not in IL2 (p =0.011) and IFN (p =0.004). Of all the six cytokines, TNFα levels were similar in the both groups (p =0.112), and showed a highest stimulation index for all six cytokines.
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Faculty of Health Sciences Research Committee