Figure 2
From: Monoclonal regulatory T cells provide insights into T cell suppression
(a) Stimulated B3K506 Tregs outcompete OT-II Tconvs, in vitro. Representative flow cytometry plots show % of OT-II Tconvs and B3K506 Tregs cocultured at a 1Tconv/4Treg ratio in suppressive (with 3K peptide) and non-suppressive (without 3K peptide) co-cultures at 72h. Numbers in the plots represent % of live cells in gate. Graphs show mean numbers + /− SD of live OT-II Tconvs (
) and B3K506 Tregs (
) from co-cultures described in A), n = 3. (b) Left: Graph depicts mean IFNγ (pg/ml) + /− SD in supernatants of suppressive (red) and non-suppressive (black) co-cultures at a 1Tconv/4Treg ratio, n = 3. Right: Representative flow cytometry plots show intracellular IFNγ staining of OT-II Tconvs in suppressive (
) vs. non-suppressive (
) co-cultures. (c) Left: Graph depicts mean IL-2 (pg/ml) + /− SD in supernatants of suppressive (
) and non-suppressive (
) co-cultures at a 1Tconv/4Treg ratio, n = 3. Right: Representative flow cytometry plots show GFP expression from eFluor labelled OT-II IL-2GFP reporter Tconvs in suppressive (
) vs. non-suppressive (
) co-cultures. (d) Experimental design of in vivo suppression assay. Dot plots show PBMCs from a B6 I-Ab Rag−/− host (recipient mouse) which had been grafted with skin from a B6 I-Abm12 Rag−/− donor mouse prior to T-cell transfer. (e) Skin from B6 I-Abm12 Rag−/− donor mouse was transplanted onto B6 I-Ab Rag−/− mice and allowed to heal for 14 days. The following day mice were injected with 2 × 10e4 ABM (I-Abm12 specific) Tconvs along with polyclonal B6 Foxp3EGFP Tregs (
) or monoclonal B3K506 Tregs (
and 
). An additional group (
) was injected with ABM Tconvs alone. One group (
) received 3K peptide (30 μg per mouse, i.p. every second day) until day 15. Graph shows % graft survival + /− SD vs. time. Each group contains 5 to 8 mice and results are pooled from 2 independent experiments. (f) Representative flow cytometry plots show ABM Tconvs and B3K506 Tregs from the draining LN of skin-grafted mice. Cells in upper plots are from a mouse which received 3K peptide and accepted the skin graft, while cells in lower plots are from a mouse which rejected it’s graft, despite receiving the Treg cognate peptide, 3K. Number in the plots depict % of cells in each gate/quadrant. (g) Representative flow cytometry plot showing ABM Tconvs and B3K506 Tregs from the draining LN of skin-grafted mice, which did not receive 3K peptide treatment and rejected their graft. Numbers in the plot depict % of cells in each gate/quadrant.
