The Dosage of the Derivative of Clostridium Ghonii (DCG) Spores Dictates Whether an IFNγ/IL-9 or a Strong IFNγ Response Is Elicited in TC-1 Tumour Bearing Mice
Figure 4
Intravenous administration of DCG leads to strong inflammation responses within tumor of TC-1 tumour bearing mice. Group of 5-7 six-week-old female C57/BL6 mice were inoculated with 2x106 of TC-1 tumour cells subcutaneously. When tumour grew to 300 mm3 in size, tumour bearing mice were injected intravenously with either (A) 1x108 CFU/kg amount of DCG, (B) 3x108 CFU/kg amount of DCG, or (C) PBS or received (D) Nil treatment. 11 days after DCG injection, mice were sacrificed and tumours were isolated and single cells were made by digest with Collagenase D. Cells were subject to Ficoll separation to enrich mononuclear cells in tumour cells, were stimulated with PMA and ionomycin in the presence of protein transport inhibitor overnight, and were surface stained for CD45, CD3, followed by intracellular stain with anti-IFNγ (a), anti-IL-9 (b), and anti-IL-10 (c), respectively. Cells were gated on CD45+ cells. Or cells were stimulated with PMA and ionomycin overnight, and supernatants from stimulated mononuclear cells of the tumour were measured by multicytokine ELISA for cytokines secreted by tumour infiltrating immune cells. The multicytokine ELISA can detect IL1α, IL1β, IL4, IL5, IL6, IL10, IL13, IL17α, IL23, IFNγ, TNFα, and GM-CSF (d). The result represents one of two independent experiments.