Figure 1

OT-II regulatory T (T reg) cells suppress antigen-dependent expansion of naïve OT-I T cells. (A) OVA_257-264 concentration (1 fM-1 nM) dependence of OT-I splenocyte proliferation assessed after 5 days of stimulation using flow cytometry after prelabeling with Cell Proliferation Dye eFluor 670. (B) CD4⁺ CD25⁺ T cells from wild-type and OT-II mice show robust forkhead box P3 (FoxP3) expression following polyclonal T cell receptor (TCR) stimulation (CD3) together with costimulation (CD28). Representative flow cytometry analysis of FoxP3 expression levels (dark gray) compared to isotype controls (light gray) in activated CD4⁺ CD25⁺ T reg cells (left) from wild-type (middle) and OT-II (right) mice. (C) Representative flow cytometry analysis of the reduction of cell proliferation as revealed by prelabeling with Cell Proliferation Dye eFluor 670 upon stimulation of naïve OT-I splenocytes with OVA_257-264 (0.1 pM) for 5 days in the absence or presence of OT-II T reg cells at different cell-to-cell ratios (2:1 and 1:1). (D) Representative flow cytometry analysis of OT-I T cell maturation markers (CD62L, CD44, CD11a) after 5 days of stimulation with OVA_257-264 (0.1 pM) in the absence or presence of OT-II T reg cells at a cell-to-cell ratio of 2:1. (E) Representative flow cytometry analysis of CD4⁺ and CD8⁺ cell populations after 5 days of stimulation of OT-I splenocytes with OVA_257-264 (0.1 pM) in the absence (left) or presence of wild-type (middle) or OT-II (right) T reg cells at a cell-to-cell ratio of 2:1.