FIGURE 7 (SUPPLEMENTARY INFORMATION)
A. Splenic T-cells from AS-MSP-treated diabetes-free NOD mice proliferate when co-cultured with allogeneic splenocytes. T-cells from diabetes-free NOD mice treated with AS-MSP were obtained over enrichment columns and co-cultured with -irradiated splenocytes from Balb/c, C57BL6 or syngeneic diabetes-free NOD mice (10 weeks of age). Proliferation was measured four days later using the CyQuant reagent. Spl refers to allogeneic irradiated splenocytes; AS-MSP Tc refers to splenic T-cells obtained from diabetes-free NOD mice treated with AS-MSP (randomly selected from the AS-MSP cohorts in FIGURE 1D). The bars show the mean of relative fluorescence units, indicating T-cell proliferation in vitro and the error bars the SEM of one of three mice, representative of the other two (assay in triplicate).
B. Pooled lymph node-derived T-cells from AS-MSP-treated diabetes-free NOD mice proliferate when co-cultured with allogeneic splenocytes.
C. Splenic T-cells from AS-MSP-treated, diabetes-free NOD mice exhibit suppressed proliferation in the presence of syngeneic islet lysate in vitro. T-cells were enriched from the spleen or the pooled lymph nodes of AS-MSP-treated diabetes-free mice selected at random from the AS-MSP diabetes-free cohort shown in FIGURE 1D. -irradiated NOD splenocytes (from diabetes-free 10 week-old NOD mice) were used as antigen-presenting cells and parallel cultures were pulsed with NIT-1 lysate (1 g/well)or PBS vehicle. As positive control, cocultures of irradiated splenocytes and T-cells from new-onset diabetic NOD mice were pulsed with NIT-1 lysate (NOD Tc). The data shown derive from T-cells of one of the three randomly selected AS-MSP-treated diabetes-free mice, where the cocultures were in triplicate and are representative of the differences in the other two mice. Bars indicate the relative fluorescence unit mean and the error bars the SEM.
D. Pooled lymph node-derived T-cells from AS-MSP-treated, diabetes-free NOD mice exhibit suppressed proliferation in the presence of syngeneic islet lysate in vitro.
E.Splenic and pooled lymph node-derived T-cells from AS-MSP-treated, diabetes-free NOD mice proliferate in the presence of syngeneic irradiated splenocytes and ovalbumin in vitro. T-cells were enriched from the spleen or the pooled lymph nodes of AS-MSP-treated diabetes-free mice selected at random from the AS-MSP diabetes-free cohort shown in FIGURE 1D. -irradiated NOD splenocytes (from diabetes-free 10 week-old NOD mice) were used as antigen-presenting cells and parallel cultures were pulsed with intact ovalbumin (5 g/well)or PBS vehicle. The data shown derive from T-cells of one of the three randomly selected AS-MSP-treated diabetes-free mice, where the cocultures were in triplicate and are representative of the differences in the other two mice. Bars indicate the relative fluorescence unit mean and the error bars the SEM.
F. Cytokine production by splenic T-cells from AS-MSP-treated, diabetes-free NOD mice in the presence or absence of NIT-1 lysate in vitro. The supernatants from the co-cultures indicated in Figure 6C above were probed for cytokines in a Luminex multi-analyte system. The bars show the fluorescence unit means and the error bars the SEM.