FIGURE 6.

Removal of dam Tregs from splenocytes of foster pups enhanced lactationally transferred immunity in FVB/NJ mice. FVB/NJ (A) or BALB/cJ (B) pups were foster nursed by B6.Foxp3^EGFP dams exposed to M. tuberculosis and challenged with PPD 4 wk before the onset of lactation. At 5 wk of age, pup splenocytes were exposed to PPD for 16–18 h, and the percentage of responsive CD8⁺ splenocytes was determined. All are nonimmunized pups fostered by immunized dam: no PPD negative control (bar 1) (n = 8 for FVB/NJ; n = 11 for BALB/cJ from two litters each); with PPD (bar 2) (n = 7 for FVB/NJ, n = 6 for BALB/cJ from two litters each); with dam Tregs removed by flow sorting (bar 3) (n = 7 for FVB/NJ, n = 6 for BALB/cJ on two occasions). After the removal of dam Tregs, Abs to H-2K^b, H-2K^d, and H-2K^q were used to determine whether the responding cell was of dam or pup origin (right panel, n = 3 for FVB/NJ from one litter and n = 6 for BALB/cJ from two litters). The ∼90% illustrated are those positively identified as pup derived on the basis of positive staining for H-2K^d or H-2K^q and negative for dam H-2K^b, using isotype controls for gating (right panel). All analyses were done using flow cytometry. All results are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.