(A) Schematic diagram of the microbiome-depleting antibiotic treatment regimen (Abx). Three days before adoptive CD4⁺ T cell transfer (blue arrow), mice were initiated on Abx daily (yellow triangles) and harvested 15 days after adoptive T cell transfer (red arrow). (B) Weight of Rag1^KOTslpr^KO (n = 6) and Rag1^KO (n = 7) treated with Abx and receiving adoptive CD4⁺ T cell transfer of 2 million purified naive WT CD4⁺CD25^– T cells. Unpaired, 2-tailed t test. (C) Colitis scoring of Rag1^KOTslpr^KO (n = 5) and Rag1^KO (n = 5) mice treated with Abx on day 15 after T cell transfer. Fisher’s exact test. (D) Representative colon H&E staining of Rag1^KOTslpr^KO and Rag1^KO mice treated with Abx on day 15 after T cell transfer. Insets show the high magnification of the mucosal inflammatory environment in the test and control groups. Note the mucosal hyperplasia in the Rag1^KOTslpr^KO sample, but the retention of goblet cells in both samples. Scale bars: 50 μm. (E) Representative CD3/CD4 staining of colons from Rag1^KOTslpr^KO and Rag1^KO mice treated with Abx on day 15 after T cell transfer. Scale bar: 50 μm. (F) CD4⁺ T cell counts in Rag1^KOTslpr^KO (n = 5) and Rag1^KO (n = 5) colons treated with Abx. CD4⁺CD3⁺ T cells in 10 randomly selected HPFs per colon were counted. Each dot represents 1 HPF. Experimental data were verified in a second independent experiment. Bar graph shows mean + SD. Unpaired, 2-tailed t test. NS, not significant.