PMC

M. tuberculosis Ag85B FF15 tetramer⁺ Tregs can be detected in milk from C57BL/6 dams. Dot plot (A) and data plot (B) (n = 6) showing FF15-specific (SP) tetramer⁺CD4⁺Foxp3⁺ Tregs in stomach milk samples from midlactation neonates nursed by an M. tuberculosis–immunized and then challenged dam. Irrelevant tetramer PA15 (NS) was used as a negative control. Gating was first for lymphoid cells, then for CD4⁺, then for Foxp3⁺, and then for tetramer⁺. (B) Data are the percentage of CD4⁺Foxp3⁺ cells also positive for the specific tetramer. All results are presented as mean ± SD. *p < 0.05.

Lactational transfer of maternal Tregs to the spleen and thymi of foster pups. C57BL/6J dams were used as foster dams for BALB/cJ (n = 5) and FVB/NJ (n = 6) pups from equivalently timed matings on two occasions. Tissues were analyzed in 3-wk-old weanlings. Cells were characterized as lymphoid, then as CD4⁺ or CD8⁺, then as of maternal origin (H-2K^b positivity), and then as Foxp3⁺ by Ab staining using flow cytometry. Data are shown as the percentages of CD4⁺ or CD8⁺ cells also positive for maternal MHC and Foxp3 (A) and as numbers per organ (B). All results are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

PPD-specific IL-10–producing Tregs in milk of M. tuberculosis–immunized C57BL/6 dams. Twelve-day-old pups (n = 7) nursed by M. tuberculosis–immunized and then challenged dams were used as donors of whey clots from their stomachs. Cells from the whey clots were exposed ex vivo to PPD for 16–18 h, and the functionality of CD4⁺Foxp3⁺ (A) and CD8⁺Foxp3⁺ (B) Tregs was judged by their ability to produce IL-10. Lymphoid cells were first gated for CD4⁺ or CD8⁺, then for Foxp3⁺, and then for IL-10⁺. Example of CD4⁺Foxp3⁺ cells (C), and from those, the percentage producing IL-10 in the absence (D) or presence (E) of PPD stimulation. Data in the bar graphs are presented as a percentage of CD4⁺ or CD8⁺ and Foxp3⁺ cells also positive for IL-10. Data are expressed as mean ± SD. *p < 0.05, ****p < 0.0001.

C57BL/6J dam cells are lactationally transferred to the spleens and thymi of BALB/cJ and FVB/NJ foster recipients. C57BL/6J (H-2K^b) dams were used as foster dams for BALB/cJ (H-2K^d) (n = 5) and FVB/NJ (H-2K^q) (n = 6) pups from equivalently timed matings on two occasions. Lymphoid cell populations were first gated on the basis of forward versus side scatter from all flow acquired splenocytes and/or thymocytes. Subsequent gating was for CD4⁺ cells and/or CD8⁺ single-positive cells, followed by gating for maternal MHC (H-2K^b). Results are presented as percentages of total CD4⁺ or CD8⁺ single-positive cells (A) and numbers per organ (B). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ******p < 0.000001.

Lactational transfer of immunity from Th1 dam to Th2 pups. FVB/NJ (A) or BALB/cJ (B) pups were directly immunized and then challenged, or nonimmunized pups were foster nursed by C57BL/6J dams immunized and then challenged 4 wk before the onset of lactation. Splenocytes were exposed to PPD for 16–18 h and then analyzed by flow cytometry, gating first on lymphoid cells, then CD8⁺ cells, and then CD8⁺ cells also positive for IFN-γ. The resulting percentages of Ag-responsive CD8⁺ splenocytes are shown: no PPD negative control (bar 1) (n = 30 for FVB/NJ from six litters, n = 15 for BALB/cJ from three litters); directly immunized pups (bar 2) (n = 8 for FVB/NJ, n = 10 for BALB/cJ from two litters each); nonimmunized and fostered by immunized dam (bar 3) (n = 22 for FVB/NJ from four litters, n = 7 for BALB/cJ from two litters); and nonimmunized and fostered by immunized dam with all dam cells removed (bar 4 and right panel) (n = 12 for FVB/NJ from two litters, n = 6 for BALB/cJ from two litters). Pup splenocytes were analyzed at 5 wk and 12–16 wk [right panels, (B)] wk of age. All analyses were done by flow cytometry. All results are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, *****p < 0.00001, ******p < 0.000001.

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.