TMTregs inhibit responder cell expansion and effector function but not Th1 differentiation. CD4⁺ T cells purified from 6.5 Tg mice (Thy1.1⁺/Thy1.2⁺) were labeled with CFSE and transferred as responder cells, either alone or together with an equal number of sorted TMTregs or effector cells (Thy1.1^+/+), into BALB/c recipients. All mice received vacHA immunization the next day. (A) Cells from tail blood collected at the indicated time points were stained with anti-Thy1.1–PE and anti-Thy1.2–APC mAbs. Percentages of the gated populations are indicated. (B) Spleen cells were harvested, counted, and the absolute numbers of Thy1.1⁺/Thy1.2⁺ and Thy1.1^+/+ cells were determined 5 days after vaccination. *P < .05; **P < .01. (C) The divided fractions of the Thy1.1⁺/Thy1.2⁺ and Thy1.1^+/+ populations were sorted by FACS either separately or together and analyzed for proliferation and IFN-γ production in the presence of varied amounts of peptide. (D) In vitro suppression assay. Responder cells (Rag2^–/– 6.5CD4⁺) were mixed with sorted cells at the indicated ratios in the presence of HA peptide and irradiated BALB/c splenocytes. The dotted line represents the proliferation of responders cultured alone with peptide and splenocytes; ▪, sorted responder cells when transferred alone; ▴, cotransferred responders and TMTregs sorted together; ×, separated responders when cotransferred with TMTregs; and □, separated TMTregs when cotransferred with responders. Results were shown as mean ± SE of triplicate cultures. The data shown are representative of 2 separate experiments. Similar results were obtained when TMTregs were transferred in excess of naive responder cells by a 2:1 ratio.