Double Negative (CD3+4−8−) TCRαβ Splenic Cells from Young NOD Mice Provide Long-Lasting Protection against Type 1 Diabetes
Figure 2
NOD DNCD3 protection against diabetes transfer in NOD/Scid mice.
Panel A, diabetogenic splenocytes (5×105 cells) from a pool of 4–5 month-old diabetic (hyperglycemic) NOD mice were injected i.p. into 4 week-old NOD/Scid female mice (n = 6, control diabetes group) and glycemia was measured on a weekly-basis. Dotted horizontal line at 200 mg/dL indicates the upper limit of euglycemia as previously established in a cohort of 12 non-treated, 4 week-old NOD/Scid females. Colored symbols refer to individual mice. Arrow on the X-axis indicates the time of cell infusion. Panel B, FACS-sorted DNCD3 splenic cells (5×105 cells) from a pool of 14 day-old NOD (dark symbols) and NON.NOD females (light symbols) (n = 20 donors per group) were infused i.p into 4 week-old NOD/Scid females (n = 6 recipients per group) and glycemia was monitored on a weekly-basis. Shown are the glycemia values (mean ± SD) in each group of NOD/Scid recipients. Arrow on the X-axis indicates the time of cell infusion. Panel C, glucose tolerance test carried out in NOD/Scid mice 2 months after infusion of DNCD3 splenocytes from 14 day-old NOD mice. Shown are two representative NOD/Scid mice infused with diabetogenic splenic cells from a hyperglycemic NOD mouse and tested 2 weeks post-cell infusion (diabetes control, red symbols); a representative NOD/Scid mouse infused with DNCD3 splenocytes from 14 day-old NOD mice (grey symbols), and a representative NOD/Scid mouse infused with DNCD3 splenocytes from 14 day-old NON.NOD mice (white symbols). Arrow on the X axis indicates the time of glucose i.p. injection (60 mg/mouse). Panel D, FACS-sorted DNCD3 splenic cells (5×105 cells) from 14 day-old NOD or NON.NOD females (pool of 20 mice per group) were co-infused i.p. in the same day with diabetogenic splenocytes (5×105 cells) from diabetic NOD females into 4 week-old NOD/Scid females (n = 6 recipients per group). Shown are the glycemia values in individual NOD/Scid recipients of DNCD3 splenocytes from NOD mice (dark symbols) or NON.NOD control mice (blue symbols). Arrows (dark and red) indicate the same time of cell co-infusion. Panel E, FACS-sorted DNCD3 splenic cells (5×105 cells) from 14 day-old NOD females (pool of 20 mice) were infused i.p. and 1 month later co-infused i.p. with diabetogenic splenocytes (5×105 cells) from diabetic NOD females into 4 week-old NOD/Scid females (n = 6 recipients/group). Shown are the glycemia values in individual NOD/Scid recipients. The time-points of cell infusion with DNCD3 splenocytes (dark arrow) and co-infusion of diabetogenic cells (red arrow) are indicated. A single NOD/Scid recipient became hyperglycemic 10 weeks after co-transfer, whereas the other 5 NOD/Scid mice remained euglycemic (p = 0.002 for the group of protected NOD/Scid mice by NOD DNCD3 splenocytes as compared with non-protected NOD/Scid mice by NON.NOD DNCD3 splenocytes). Panel F, hematoxylin-eosin staining of paraffin-embedded sections of the pancreas for a representative euglycemic, non-injected NOD/Scid mouse (row 1, left panel); an euglycemic NOD/Scid mouse 13 weeks after co-transfer of NOD DNCD3 splenocytes with diabetogenic splenocytes as in panel E (row 2, left panel); and the only hyperglycemic NOD/Scid mouse 13 weeks after cell co-transfer of NOD DNCD3 splenocytes with diabetogenic splenocytes as in panel E (row 3, left panel). Shown is the staining of adjacent pancreatic cross-sections from the same mice with a rabbit anti-mouse insulin Ab-HRP conjugate (rows 1–3, right panels). Panel G, FACS-sorted DNCD3 splenic cells (5×105 cells) from 14 day-old NON.NOD females (pool of 20 mice) were infused i.p. into 4 week-old NOD/Scid females (n = 6 recipients per group) and 1 month later co-infused i.p. with diabetogenic splenocytes (5×105 cells) from diabetic NOD females. Shown are the glycemia values in individual NOD/Scid recipients. The time-points of cell infusion with DNCD3 splenocytes (dark arrow) followed 1 month later by co-infusion of diabetogenic cells (red arrow) are indicated. All NOD/Scid recipients became hyperglycemic within 3 to 4 weeks as the diabetes control group did (see panel A) (p≤0.001 for two combining experiments). Each panel represents one of two representative experiments for which the significance of diabetes incidence was p ≤0.005.