(A) The expression of CD127 was measured on CD2⁺ cells in the mesenteric lymph nodes (MLNs) in mice treated with hIg or teplizumab in the presence or absence of antibiotics (Abx). In hIg- and antibiotic-treated mice, CD127 expression was reduced compared with mice that had not been treated with antibiotics (^†P = 0.016). The MFI was significantly reduced after teplizumab in mice that had not received antibiotics (*P = 0.02) but not in the mice treated with antibiotics (n = 3–4 mice/group). (B) There was an interactive effect of antibiotic treatment on the frequency of CD8CM in the mesenteric lymph nodes after teplizumab treatment (*P = 0.035). The frequency increased in mice that received teplizumab without antibiotics (^†P = 0.049) but not in mice that received antibiotics (n = 3–4 mice/group), and was higher than in mice treated with teplizumab and antibiotics (P = 0.035). (C and D) The frequency of IL-2–producing CD4⁺ and CD8⁺ T cells in the spleen was analyzed by flow cytometry following stimulation with PMA/ionomycin for 6 hours, 18 hours following treatment with teplizumab or hIg (n = 10 per group). There was a significant effect of antibiotic treatment on the frequency of IL-2⁺ CD4⁺ and CD8⁺ T cells. *P = 0.03, **P = 0.006, 2-way ANOVA. (E) p-STAT5 was increased in the CD8⁺ splenocytes from mice treated with antibiotics and anti-CD3 mAb compared with mice that received anti-CD3 mAb without antibiotics (*P = 0.02, 2-way ANOVA; each symbol represents 1 mouse). (F) Representative histograms from a mouse treated with teplizumab alone or teplizumab plus antibiotics are shown (red = isotype control, blue = p-STAT5 staining without IL-2, green = p-STAT5 staining with IL-2). Statistical significance in all panels was determined by multiple comparison from 2-way ANOVA.