1. Induction of CD4+ T-cell anergy and apoptosis by activated human B cells
by Theresa Tretter, Ram K. C. Venigalla, Volker Eckstein, Rainer Saffrich, Serkan Sertel, Anthony D. Ho, and Hanns-Martin Lorenz
Blood
Volume 112(12):
December 1, 2008
©2008 by American Society of Hematology

2. Activated B cells mediate inhibition of Th-cell proliferation.
Activated B cells mediate inhibition of Th-cell proliferation. (A) Effect of differently activated B cells on CD4+ Th-cell proliferation: Equal amounts of B cells were added unstimulated, or after prestimulation for 3 days with SAC or αIg to Th cells and cCD3+ IL-2. [3H]TdR incorporation was measured after 4 days of culture. Mean plus or minus SEM of 3 representative experiments. (B) Separation of SAC-activated B cells into 2 major populations, according to cell size distribution and activation status. Shown is 1 representative sort gate for separation of small CD25− (R2 and R4 and not R6) and large CD25+ (R3 and R5 and not R6) B cells after prestimulation for 3 days
Theresa Tretter et al. Blood 2008;112:
©2008 by American Society of Hematology

3. The large CD25+ B cells mediate suppression of activated Th cells.
The large CD25+ B cells mediate suppression of activated Th cells. (A) Proliferation assay of cCD3-stimulated Th cells alone or in presence of lgB25+ or smB25− cells after 4 days in presence (■) or absence (▨) of IL-2. Bars represent mean of 9 independent experiments plus or minus SEM. (B) Proliferation of prestimulated () or freshly isolated (■) Th cells in presence of lgB25+ or smB25−B cells after 4-day culture with IL-2. Mean plus or minus SEM of 3 representative experiments. (C) Proliferation of Th cells in presence of different concentrations of lgB25+ cells, diluted in cell culture media (■) or in B25− cells (). Data expressed as mean plus or minus SEM of triplicates from one representative experiment.
Theresa Tretter et al. Blood 2008;112:
©2008 by American Society of Hematology

4. lgB25+ cells induce long lasting T-cell division arrest.
lgB25+ cells induce long lasting T-cell division arrest. (A) T cells were stimulated alone (rhombus), with unstimulated B cells (square), smB25− cells (cross), and lgB25+ cells (triangle) as usual in presence of cCD3 and IL-2. Data are expressed as mean plus or minus SEM of triplicates from 1 representative experiment of at least 3. (B) T-cell division after 3 and 6 days, measured by PKH-26 dilution. M1 relates to 100% PKH-26–positive cells at day 0. (C) MLR with autologous B and T cells (each 25 000) and allogeneic irradiated PBMCs (50 000) in presence of IL-2. Data are expressed as mean plus or minus SEM of triplicates from 1 representative experiment of at least 3 (symbols as in panel A)‏
Theresa Tretter et al. Blood 2008;112:
©2008 by American Society of Hematology

5. B cell–mediated Th-cell suppression is critically dependent on IL-2.
B cell–mediated Th-cell suppression is critically dependent on IL-2. (A-C) Th cells were incubated alone (■), with lgB25+ (▨) or smB25− (▤) cells in presence of cCD3 and different concentrations of IL-2 or without IL-2 (med). (A) [3H]TdR incorporation of T cells. Data are expressed as mean plus or minus SEM of triplicates from 1 representative experiment. (B) PKH-26 assay at day 6 (mean ± SEM of 3 independent experiments). (C) Blockade of IL-2Rαβ by addition of Abs directly to culture or by preincubation of lgB25+ cells (B*) alone; cultures provided with cCD3 and IL-2 (100 U/mL). Mean plus or minus SEM and P value of 5 independent experiments.
Theresa Tretter et al. Blood 2008;112:
©2008 by American Society of Hematology

6. T-cell inhibition requires cell contact and constant presence of activated B cells.
T-cell inhibition requires cell contact and constant presence of activated B cells. (A) PKH assay of Th cells stimulated alone or together with SAC-activated B cells, or separated by cell culture insert (T/Insert/B-SAC) after 3 and 6 days. (B) Surface expression of CD71, CD69, and CD25 on gated CD4+ T cells after 24-hour stimulation with cCD3/IL-2 in presence or absence of lgB25+ cells. (C) T cells prestimulated alone (T-prestim) or with lgB25+ cells (T-prestimB) for 48 hours and restimulated with IL-2 or IL-2+ lgB25+ cells. Data are expressed as mean plus or minus SEM of triplicates from 1 representative experiment. (D) Coculture of PKH-26+ responder T cells (Tresps) with Tprestim or with TprestimB for 24 or 48 hours. Tresps were used fresh () or prestimulated for 48 hours (■). PKH-26 was measured after 3 days.
Theresa Tretter et al. Blood 2008;112:
©2008 by American Society of Hematology

7. B cell–mediated Th-cell suppression involves induction of T-cell apoptosis.
B cell–mediated Th-cell suppression involves induction of T-cell apoptosis. (A) Annexin binding of PKH-26–labeled Th cells cultured alone or with lgB25+ cells + cCD3/IL-2 after different incubation times between 24 and 96 hours, 1 representative experiment of 4 independent experiments shown. (B) Annexin staining of T cells cultured alone or with lgB25+ or smB25− cells at different time points. Mean plus or minus SEM of 6 independent experiments shown. (C) Immunofluorescence microscopy after 24-hour incubation time; 40× magnification. Sixteen adjacent areas in a 4 × 4 array were imaged and stitched together. Images were acquired with respective filters for CD4-APC, CD19-FITC, and annexin-PE; overlay colors: green indicates B cells; blue, T cells; purple, Ax-positive T cells; and yellow, Ax-positive B cells. One representative area with T+ lgB25+ and T+ smB25− is shown.
Theresa Tretter et al. Blood 2008;112:
©2008 by American Society of Hematology

8. Activated B cells with inhibitory properties can be found in human tonsils.
Activated B cells with inhibitory properties can be found in human tonsils. (A) Representative example for phenotype of purified tonsillar B cells according to CD38 and IgD expression. Upper quadrant separates CD38+ from CD38++ B cells. Large cells gated on R5; small cells gated on R4; PI+ B cells (R2) gated out. (B) Sort gates for separation of IgD+ CD38++ B cells (R6 and R1 and not R2) and IgD+ CD38+ B cells (R7 and R1 and not R2); R1 and R2 refer to the gates of panel A. (C) Suppressor assay with freshly sorted autologous cell populations and CD3 alone or CD3+ IL-2. ■ indicate T cells alone; , T+ B-IgD+ CD38++; and ▨, T+ B-IgD+ CD38+. Data represent mean plus or minus SEM of tonsils from 3 different donors.
Theresa Tretter et al. Blood 2008;112:
©2008 by American Society of Hematology