Incidence of diabetes in two independently derived NOD.MyD88KO and heterozygous NOD.MyD88KO/+ stocks (J-Jackson Laboratory and Y-Yale) (12 back-crosses to NOD). F, females; M, males. n, number of animals per group.

Diabetes incidence in SPF NOD.MyD88KO (J) females and control heterozygous NOD.MyD88KO/⁺ littermates treated with a broad-spectrum antibiotic from birth.Diabetes incidence in GF NOD.MyD88KO and control MyD88KO/⁺ mice. Incidence is shown for male mice; 100% of female NOD.MyD88KO and NOD.MyD88KO/⁺ female GF mice became diabetic (Supplemental Fig.1).Diabetes incidence in gnotobiotic male NOD.MyD88KO and control NOD.MyD88KO/⁺ mice colonized with a consortium of 6 bacterial strains (ASF 361,519,356,492,502, and 500; see Supplemental results for descriptions). The incidence of diabetes in gnotobiotic NOD.MyD88KO mice was significantly different from the incidence in GF NOD.MyD88KO animals (p<0.001), and in gnotobiotic NOD.MyD88KO/+ mice (p<0.05) (Kaplan Meier test).Histological scores of islet destruction in SPF, GF and ASF-colonized NOD.MyD88KO/⁺ and NOD.MyD88KO mice. Mice in all groups were males, except for the SPF NOD.MyD88KO group, which included both genders.

The overall reactivity of T cells from secondary lymphoid organs of MyD88-sufficient (solid bars) and MyD88-negative (open bars) NOD mice was calculated as a mean of the percentages of mice in the given group reacting to individual diabetes-associated peptides (see Supplemental Fig. 2c).The frequency of CD8⁺ T cells producing IFN-γ in response to peptide recognized by diabetogenic clone 8.3 was determined in the spleens, MLN and PLN of MyD88-sufficient (solid bars) and MyD88-negative (open bars) NOD mice. Error bars – s.e.m.Proliferation of CFSE-labeled BDC2.5 CD4⁺ T cells, depleted of CD25⁺ T cells, in the PLNs of SPF NOD and NOD.MyD88KO mice assayed on day 3 after i.v. injection. Representative CFSE dilution profiles are shown for BDC2.5⁺-gated cells in PLN (red) and control skin-draining lymph nodes (blue).
p values in all experiments were determined using unpaired Student's t test. Error bars – s.e.m. n, number of animals per group.

Ratio of Firmicutes to Bacteroidetes in the cecal microbiota of NOD.MyD88KO/+ and NOD.MyD88KO mice who were or were not treated with Sulfatrim. Mean values per mouse ± s.e.m. Untreated NOD.MyD88KO mice had, on average, a significantly lower F/B ratio than all other mice combined (one-tailed t-test, t=-2.31, p=0.013). When comparing post-hoc the effect of sulfatrim in MyD88KO mice only, no significant difference in F/B ratios was observed (t= 0.85, p=0.20).Abundance of members of three different bacterial families in the cecal contents of NOD.MyD88KO/+ and NOD.MyD88KO mice (not treated with Sulfatrim). Mean values ± s.e.m. Each of the three families is enriched in NOD.MyD88KO mice (Lactobacilliaceae t=-1.54, p=0.07, Porphyromonadaceae t=2.1, p=0.03, Rikenellaceae t=2.74, p=0.007; one-tailed t-test).Clustering of mouse cecal bacterial communities using the unweighted UniFrac metric n=7,223 sequences. Diversity, not abundance of bacteria was taken into consideration. Top panel: Sulfatrim-treated litters. Bottom panel: Untreated litters. Line colors indicate families. Each label is a mouse: red labels are NOD.MyD88KO/+ (H); black labels are NOD.Myd88KO (K). The number is the common mother, S and N designate exposure to Sulfatrim or no Sulfatrim, respectively.Histological examination of the pancreata of 8 wk old males from SPF NOD, GF NOD, as well as GF NOD and GF NOD.MyD88KO exposed from birth to microbiota of an SPF NOD.MyD88KO female. The percentages (mean ±s.e.m.) of affected islets with no infiltration (open bars), periinsulitis (blue bars) and true infiltration (combined grades II and III) are shown. p values were obtained using unpaired Student's t test. n, number of animals per group.