Table 1.
Summary of differences between LLO56 and LLO118 T cells.
Fig 1.
LLO56 naïve helper T cells have higher calcium mobilization in vitro.
Naïve T cells from LLO56 and LLO118 TCR transgenic mice were obtained from the spleen using negative selection and calcium levels were measured using live cell microscopy. T cells were added to antigen presenting cells (bone marrow derived macrophages) that were loaded overnight with the L. monocytogenes peptide (LLO190-205). A. Average curves of intracellular Ca2+ mobilization from LLO56 and LLO118 naïve T cells (340/380 ratios) (n = 40+). Error bars show the SEM at the influx peak and every 5 minutes after the peak (n = 40+). B. Statistical analysis of peak calcium influx of stimulated LLO56 and LLO118 naïve T helper cells (n = 40+). C. Statistical analysis of the sustained intracellular Ca2+ levels (Average 340/380 values between minutes 5 and 20) after initial stimulation response (n = 40+). D. Standard deviation was determined by linear regression analysis and shows variability in the calcium signal for each group (n = 40+). (* = p < .05; NS = not significant).
Fig 2.
Naïve LLO56 T helper cells have higher levels of CD5.
Flow cytometry analysis of CD5 expression of LLO56 and LLO118 T helper cells was done using LLO118 and LLO56 splenocytes at different time points after stimulation with the LLO190-205 peptide from L. monocytogenes. A. Gating strategy for measuring CD4 and CD5 mean fluorescent intensity on LLO118 and LLO56 T cells. B. CD5 levels of naïve T cells, T cell stimulated for 3 days, and T cells stimulated for 8 days. T helper cells from LLO56 (red line) overlaid with the CD5 levels from LLO118 (shaded blue). Unstained cells are also included (black dots). C-D. Comparison of mean fluorescence intensity (MFI) profiles for LLO56 and LLO118 expression levels of CD5 at different time points were determined by flow cytometry quantitative analysis. (* = p < .05; ** = p < .01; *** = p < .001; NS = not significant).
Fig 3.
LLO118 helper T cells have higher calcium signaling on day 3 post stimulation.
LLO56 and LLO118 splenocytes were isolated and cultured with the LLO190-205 peptide from L. monocytogenes for 72 hours in vitro. 24 hours before live imaging, a second set of splenocytes were isolated and cultured in an 8-chamber slide loaded with LLO190-205 peptide of L. monocytogenes for use as antigen presenting cells. T cells were stained with Fura-2AM, added to the antigen presenting cells and Ca2+ influx was measured. A. Average curves of intracellular Ca2+ mobilization from stimulated LLO56 and LLO118 splenocytes (340/380 ratios; n = 30) on day 3 post stimulation. Error bars show the SEM at the influx peak. B. Statistical analysis of peak calcium influx of stimulated LLO56 and LLO118 naïve T helper cells (n = 30+). C. Statistical analysis of the sustained intracellular Ca2+ levels (Average 340/380 values between minutes 5 and 20) after initial stimulation response (n = 30+). D. Standard deviation was determined by linear regression analysis and shows variability in the calcium signal for each group (n = 30+). (** = p < .01; *** = p < .001; NS = not significant).
Fig 4.
No calcium differences between LLO56 and LLO118 on day 8 post stimulation.
LLO56 and LLO118 transgenic splenocytes were isolated and cultured with the LLO190-205 peptide for a week. 24 hours before live imaging, a second set of splenocytes were isolated and cultured in an 8-chamber slide loaded with LLO190-205 peptide of L. monocytogenes for use as antigen presenting cells. 8 days stimulated T cells were stained with Fura-2AM and Ca2+ influx was measured using live imaging microscopy. A. Average curves of intracellular Ca2+ mobilization from stimulated LLO56 and LLO118 splenocytes (340/380 ratios; n = 30) on day 8 post stimulation. Error bars show the SEM at the influx peak. B. Statistical analysis of peak calcium influx of stimulated LLO56 and LLO118 naïve T helper cells (n = 30+). C. Statistical analysis of the sustained intracellular Ca2+ levels (Average 340/380 values between minutes 5 and 20) after initial stimulation response (n = 30+). D. Standard deviation was determined by linear regression analysis and shows variability in the calcium signal for each group (n = 30+). (NS = not significant).
Fig 5.
Flow cytometry calcium analysis confirms improved calcium mobilization for naïve LLO56 T cells and higher calcium mobilization for LLO118 T cells at day 3 post-stimulation.
LLO56 and LLO118 splenocytes were isolated and cultured at different time points with the LLO190-205 peptide. Calcium levels were quantified using the FlowJo kinetics tool to determine the area under the curve (AUC) for each sample. Calcium mobilization levels for LLO118 and LLO56 are quantified (mean ± SEM of the area under the curve). A. Statistical analysis of naïve LLO118 and LLO56 T helper cell calcium mobilization after activation with PMA and Ionomycin. B. Statistical analysis of day 3 post stimulated LLO118 and LLO56 calcium mobilization and C. Statistical analysis of day 8 post stimulated LLO118 and LLO56 calcium mobilization. (NS = not significant).
Table 2.
Summary of CD5 and calcium findings for LLO56 and LLO118.
Fig 6.
CD5 expression in naïve LLO56 T helper cells is correlated with higher Ca2+ mobilization.
Flow cytometry analysis was performed to determine Ca2+ mobilization levels in LLO56, LLO118, LLO56-CD5 knockout and LLO118-CD5 knockout T cells stimulated with the L. monocytogenes peptide (naïve, day 3, and day 8 time points). Calcium levels were quantified using the FlowJo kinetics tool to determine the area under the curve (AUC) for each sample (mean ± SEM). A-C. Statistical analysis of calcium mobilization of LLO118 and LLO118-CD5 knockout T cells stimulated with PMA/Ionomycin. Data is shown for naive (A), day 3 post stimulation (B) and day 8 post stimulation (C). D-F. Statistical analysis of calcium mobilization of naïve LLO56 and LLO56-CD5 knockout T cells stimulated with PMA/Ionomycin. Data is shown for naive (D), day 3 post stimulation (E) and day 8 post stimulation (F). (* = p < .05; NS = not significant).