Figure 4
From: Monoclonal regulatory T cells provide insights into T cell suppression
(a) Supernatants from stimulated Treg cultures are not suppressive. Graph depicts mean proliferation index + /− SEM of OT-II Tconvs separately cultured in various concentrations of supernatant from separately stimulated B3K506 Treg cultures, n = 3. Representative histograms depict proliferation of OT-II Tconvs cultured in media containing 50% Treg supernatant (
) or in media alone (
). (b) Tregs do not affect CD86 expression on APCs or APC cell numbers. Top: Bar graph depicts % of CD86+ splenic B cell APCs from suppressive (with 3K peptide, 
) and non-suppressive (without 3K peptide, 
) co-cultures. Representative flow cytometry plots show CD86 expression on live, CD19+ CD4− splenic B cell APCs from these cultures at 72h. Numbers in the plots depict % of cells in each quadrant. Bottom: Graph shows mean number of live splenic B cell APCs + /− SD vs. time in suppressive vs. non-suppressive co-cultures at various times, n = 3. (c) B3K506 Tregs do not convert OT-II Tconvs into induced Tregs (iTregs). Representative flow cytometry plots show Foxp3 and CD4 expression on OT-II Tconvs from suppressive co-cultures at a 1Tconv/4Treg ratio at 72 h. Numbers in the plots depict % of cells in each quadrant or gate. (d) Suppression is enhanced when Treg and Tconv antigens are presented on the same APC. Left: Representative histograms show proliferation of CFSE labelled HA Tconvs co-cultured with B3K506 Tregs at a 1Tconv/4Treg ratio. HA Tconvs and B3K506 Tregs encounter their cognate ligand either on the same (top) or on separate (bottom) splenic B cell APCs (see diagram). Suppressive cultures (
) were compared to non-suppressive cultures where 3K peptide was omitted (
). Right: Graph shows mean % suppression (top) and mean proliferation index (bottom) + /−SD of HA-Tconvs co-cultured in the different conditions described in D, n = 4–5. (e) Left: Representative flow cytometry plots show CD25 expression on HA Tconvs in cultures described in D). Numbers in the plots represent % of cells in each quadrant. Right: Bar graph depicts mean % CD25+ HA-Tconvs + /−SD in these cultures at 72 h, n = 4.
