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 2
Intravenous administration of CG results in strong Th1 T cell responses in the spleen of TC-1 tumour bearing mice. Group of 6-7 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 CG spores, (B) 3x108 CFU/kg amount of CG spores, or (C) PBS or received (D) Nil treatment. 11 days after CG spore injection, mice were sacrificed and spleens were isolated and single cell suspension was made by physical disruption of spleen described in Materials and Methods. A total of 1x106 of splenic cells were stimulated with PMA and ionomycin in the presence of protein transport inhibitor for 10 hours, and then surface was stained with anti-CD3, anti-CD4, anti-CD8a, followed by intracellular staining with anti-IFNγ, anti-IL-5, anti-IL-9, or anti-IL-10, respectively. Live and CD45+ T cells were gated. (a) CD3+IFNγ+ cells; (b) CD3+IL-5+; (c) CD3+IL-10+ T cells; (d) CD3+IL-9+; (e) or 1 × 106 of splenic cells were stimulated with different amount of MHC I restricted HPV16E7 or unrelated MHC I restricted peptide overnight or left unstimulated. Each sample was added to the ELISPOT plate in triplicate. Antigen specific CD8+IFNγ+ T cell responses were measured by ELISPOT assay as described in the section of Materials and Methods. P<0.05 is considered statistically significant. Results shown represent one of two independent experiments.