Number of Peyer’s patches in mice of various immunodeficient genotypes. Peyer’s patches were counted using stereo microscopical transillumination (magnification, ×16) of the small intestine after filling with PBS. The box and whiskers plot shows range and quartiles. The box extends from the 25th to the 75th percentile, with a line at the median (the 50th percentile). The whiskers extend above and below the box to show the highest and lowest values.

Morphological appearance of Peyer’s patches and spleens in β7^−/−, RAG-1^−/−, μMT, TNFα^−/−× LTα^−/− and wild-type mice. The atrophic Peyer’s patches found in β7^−/− mice contained an almost complete absence of B cells (B220) compared to wild-type mice and a reduced FDC network within Peyer’s patches. Instead, there was a normal composition of B cell follicles and FDC networks in spleens of β7^−/− mice. The microarchitecture of spleen and Peyer’s patches in RAG-1^−/− and μMT mice was severely impaired. However, B220 staining revealed single positive cells in RAG-1^−/− and μMT tissues, these most likely being NK cells (see Results). Surprisingly, positive staining with the FDC marker M1 was observed in the Peyer’s patches (but not spleens) of these mice. In the case of TNFα^−/− × LTα^−/− mice a portion of the small intestine was stained. Original magnifications, ×100 and ×200 for B220 staining and FDC-M1, respectively.

Presence and distribution of M cells in Peyer’s patches in several B cell-deficient mice. A–D: Enterocytes with high alkaline phosphatase (AP) activity of wild-type (A), β7^−/− (B), μMT (C), and RAG-1^−/− (D). Peyer’s patches were stained red, and mature M cells lacking AP activity appeared white. Goblet cells that also lack AP activity were selectively stained in blue using Alcian blue. Some pink cells observed with this technique were considered to represent cells with a phenotype intermediate between mature enterocytes and M cells. Original magnification of all large figures, ×100. Insets represent a high-power view (×400) of part of the domes. E: Quantification of intestinal M cells on whole-mount preparation Peyer’s patches stained for AP. Whole mounts were stained as in A–D and AP-negative and AP-positive cells were counted. The abundance of M cells (% of the total epithelial cells within the FAE) was enhanced in B cell-reduced/deficient mice. Values are presented as a box and whiskers plot as in Figure 1 .

Western blot analysis of brains and spleens. Presence or absence of PrP^Sc was determined by western blots of spleen or brain material electrophoresed natively (-), or after digestion with PK (+). Molecular weights of the proteins were indicated in kilodaltons on the left. Large amounts of PrP^Sc were detected in the brains and spleens of either β7^−/− or wild-type mice that had developed scrapie (terminal sick) after the time points indicated. PrP^Sc was detected only in mice which were challenged orally with high-dose (8 log LD50) RML or i.p. with 6 log LD₅₀ but not in tissues of clinically healthy mice, thereby excluding subclinical disease.

Morphological changes in the brains of mice orally exposed to prions. Pronounced astrogliosis (GFAP) in the hippocampus of scrapie-sick (terminal disease) β7^−/− and wild-type mice was obvious, whereas no gliosis and vacuolization (H&E) was observed in clinically healthy RAG-1^−/−, μMT, and TNFα^−/− × LTα^−/− mice. Original magnification, ×200.

Determination of prion infectivity titers in spleens, Peyer’s patches, or small intestines of scrapie-challenged mice with different alterations in the GALT. Titers were determined in spleens (blue symbols) and Peyer’s patches (red symbols) of wild-type (crosses), β7^−/−(circles), RAG-1^−/ (diamonds), μMT (squares) and TNFα^−/− × LTα^−/− (triangles) mice. Since TNFα^−/− × LTα^−/− mice lacked PPs, segments of the small intestine were processed for transmission studies to indicator mice. Mice were challenged orally with 8 log LD₅₀ or i.p. with 6 log LD₅₀ of scrapie prions as indicated. Standard deviations within groups are presented only when they exceed ± 0.75 log LD₅₀. Symbols on the abscissa and below the threshold of the prion titer 1.5 log LD₅₀ indicate prion titers below detection limit (none of the four indicator mice developed scrapie). If one or more indicator mice survived >180 dpi, or if the mean incubation time was more than 120 days, titer was assumed to be close to the detection threshold of the bioassay at prion titer of 1.5 log LD₅₀. For these samples (labeled with small letters) the numbers of animals succumbing to scrapie out of four inoculated tga20 mice and incubation time (in days) to terminal scrapie were as follows: a, 2/4(87,117); b, 2/4(109,111); c, 1/4(92); e, 2/4(117,134); f, 1/4(109), g, 3/4(124,134,151). d: In this transmission experiment one of the four tga20 mice died 24 hours after inoculation, most likely as a result of intracerebral bleeding following the injection procedure.