The B-subunits of LT-1 and LT-IIa increased maintenance of DO.11.10 T cells A20 WT B cells were cultured with OVA in presence of LT-IB, LT-IIaB or LT-IB + LTIIaB (Mix), for 4-6 hrs. OVA-specific DO.11.10 T cells were CFSE labelled, added to the culture and incubated for 48 hrs. Cells were then analyzed by Flow cytometry. Dead cells were gated out based on their low forward and side scatter (A). Other cells were analyzed for the proportion of T cells in culture (B). The data is representative of 3 similar independent experiments.

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... cells were pre-incubated with LT-IB, LT-IIaB and LT-IB+LT-IIaB in the presence of OVA. CFSE-labeled DO.11.10 T cells were subsequently added. Flow cytometry analysis revealed that the proportion of CFSE + T cells was significantly higher in the presence of LT-IB +OVA (approx. 70%) and LT-IIaB+OVA (approx. 46%) compared to OVA alone (approx. 32%) (Fig. 4B). As DO.11.10 is a continuously dividing cell line, the increase in the proportion of CFSE + cells could reflect retention of CFSE in these cells (see below). Further, in wells containing each of the enterotoxins B-subunits, the proportion of some CFSE + T and unlabeled cells with higher FSC (a readout of cell size) increased compared ...

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... dividing cell line, the increase in the proportion of CFSE + cells could reflect retention of CFSE in these cells (see below). Further, in wells containing each of the enterotoxins B-subunits, the proportion of some CFSE + T and unlabeled cells with higher FSC (a readout of cell size) increased compared to OVA alone and untreated control (Fig. 4B). This increase in cell size is also evident in the selected gate in the cultures incubated with LT-IB (Fig. 4A) where the spread of the cells from center to forward position in the FSC channel is noticeable. The increase in FSC of these cells suggests an increase in cell size due to their activation. In support, examination of these ...

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... cells (see below). Further, in wells containing each of the enterotoxins B-subunits, the proportion of some CFSE + T and unlabeled cells with higher FSC (a readout of cell size) increased compared to OVA alone and untreated control (Fig. 4B). This increase in cell size is also evident in the selected gate in the cultures incubated with LT-IB (Fig. 4A) where the spread of the cells from center to forward position in the FSC channel is noticeable. The increase in FSC of these cells suggests an increase in cell size due to their activation. In support, examination of these by light microscopy revealed some single large cells compared to untreated control and OVA-treated controls (not ...

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... was significantly higher (approx. 61%) compared to OVA (approx. 32%). However, it is noted that the total number of B and T cells in the live gate was lower in this cell culture compared to the others. This effect can be seen by the increase in the density of the Low FSC/high SSC cells, most likely dead cells, compared to other treatments (Fig. 4A). The reason for the cell death is unclear; particularly that analysis of cells undergoing apoptosis after 24 hrs incubation with the B-subunits of LTs did not reveal a difference from other treatments including the untreated control culture (Fig. 3B). However, this response may reflect an increase in activation of these cells. After 48 ...

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... of cells to advanced generations. There was also a slight increase in the total number of CFSE + T cells. Incubation with LT-IB +OVA or LT-IIaB+OVA increased T cell division when compared to OVA alone and was associated with an increase in the total number of CFSE + -low T cells, which is consistent with the results from the dot plot analysis (Fig. 4B). T cell division in wells containing LT-IB+OVA was slower compared to those containing LT-IIaB+OVA since most of the cells remained in g 0 and g 2 . Further, comparison between the T cell division profiles of LT-IIaB with and without OVA showed higher division in LT-IIaB+OVA. This effect is revealed by a shift of the cells from g 0 to ...

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... or OVA-treated controls, suggesting increased survival of T cells in these cultures. Stimulation by the mix +OVA slightly increased T cell division, shown by a shift from g 0 to g 1 . However, the total number of T cells did not noticeably increase over that of untreated or OVA-treated control, suggesting a loss of cells following activation (Fig. 4). It should be noted that T cell division in the presence of LT-IB+OVA, mix, and mix+OVA was slower compared to LT-IIaB +OVA or the other treatments, in that most of the cells did not advance further in their division. Collectively, these results show that stimulation by LT-IIaB+OVA results in higher division of OVA-responsive T cells ...