Supplemental Figure 1. IL-4, but not IL-2 suppresses IL-21-induced secretion of GzmB
by BCR-engaged B cells.
Purified B cells (> 99.5%) from healthy subjects were cultured on GzmB-specific 96-well
ELISpot plates at 1x105 cells per well with anti-BCR and IL-2, IL-4, or IL-21 as indicated.
After 16 h plates were developed and dots counted. Results from three different donors are
shown.
Supplemental Figure 2. IL-21 does not sensitize B cells to a GzmB-inducing effect of IL4 and IL-10.
B cells from 4 healthy subjects were isolated and cultured for 16 h in presence or abscence of
IL- 21 (50 ng/ml), IL-4 (500 U/ml), IL-10 (50 ng/ml) and anti-BCR (6.5 µg/ml). Brefeldin A
was added for four more hours, cells were harvested, fixed, permeabilized, stained for
intracellular GzmB and analyzed by FACS. (A) Zebra plots show GzmB expression in B cells
from one representative donor after 20 h stimulation with the indicated agents. Gates indicate
GzmB+ B cells. (B) Bar graphs show average percentages of GzmB+ B-cells from four
individuals. Error bars indicate SEM.
Supplemental Figure 3. GzmB+ B cells display an activated immunophenotype.
Purified B cells from healthy donors were cultured for 2 days in the presence or absence of
IL-21 and anti-BCR. Brefeldin A was added for 4 more hrs, cells were harvested, fixed,
permeabilized and stained. Expression of various surface markers in untreated B cells and in
IL-21/anti-BCR-treated GzmB+ or GzmB- B cells was determined by FACS analysis.
Histograms illustrate fluorescence intensity distributions for different surface antigens as
indicated. Median fluorescence intensities of isotype controls were set to 10 (dashed vertical
lines). Data are representative of at least 3 independent experiments.
Supplemental Figure 4. Recent vaccination of healthy subjects sensitizes B cells to IL21-dependent GzmB expression induced by corresponding viral antigens.
B cells from healthy subjects before and 2 weeks after vaccination against TBEV (n=8),
rabies (n=4) or hepatitis B (n=2) were purified and cultured for 16 h in the presence or
absence of IL-21 and increasing concentrations of the corresponding viral vaccines or the
control with Al(OH)3 carrier (n=4) as indicated. Brefeldin A was added for 4 more h, B cells
were harvested, fixed, permeabilized, stained for GzmB and analyzed by FACS. (A) Gated
populations within zebra plots indicate GzmB+ B cells from one representative donor before,
2 weeks after first and 2 weeks after second vaccination against TBEV. (B) Line graphs show
average frequencies of GzmB+ B cells relative to stimulation with IL-21 alone. Standard
concentrations used were 10 ng/ml for the TBEV vaccine, 0.5 mIU/ml for the rabies vaccine,
100 ng/ml for the hepatitis B vaccine and 1.5 µg/ml for the Al(OH)3 carrier control. Error
bars indicate SEM, * indicate p < 0.05, ** indicate p < 0.005.
Supplemental Figure 5. ARH-77 cells express enzymatically active GzmB.
ARH-77 cells were cultured for 36 h in the presence of IL-21 (50 ng/ml). Brefeldin A was
added to a final concentration of 1 µg/ml and cells were cultured for 4 h. Cells were harvested
and spinning disk confocal microscopy was performed after staining with Cell Mask deep
red membrane stain (red) and either anti-GzmB biotin and streptavidin-Alexa Fluor 488
(green, left image) or GranToxiLux fluorogenic GzmB substrate (green-yellow, right
image). Two representative GzmB+ ARH-77 cells are shown.
Supplemental Table 1. B cell lines vary considerably in their potential to secrete GzmB
in response to IL-21.
Various B cell lines and three non-B cell lines were cultured at 37ºC on GzmB-specific 96well ELISpot plates at 1x105 cells per well with IL-21 (50 ng/ml) and anti-BCR (0.13 µg/ml)
as indicated. After 16 h, plates were developed and dots counted. Shown are average results
from at least three independent experiments for each cell line. EBV = positive for EpsteinBarr virus antigens, ‘-’ = no GzmB secretion, ‘+’ = low GzmB secretion, ‘++’ = moderate
GzmB secretion, ‘+++’ = strong GzmB secretion, ‘++++’ = very strong GzmB secretion.
Supplemental Video 1. Video illustrates granular distribution and secretion process of
active GzmB in ARH-77 cells.
ARH-77 cells were cultured for 36 h in the presence of IL-21 (50 ng/mL). Brefeldin A was
added to a final concentration of 1 µg/ml and cells were cultured for 4 h. Cells were harvested
and spinning disk confocal microscopy was performed after staining with GranToxiLux
fluorogenic GzmB substrate (green) and Cell Mask deep red membrane stain (red). Threedimensional view of one representative GzmB+ ARH-77 cell is shown.
Supplemental Video 2. Time lapse video shows delivery of enzymatically active GzmB
from ARH-77 cells to HeLa cells.
ARH-77 cells were pre-incubated for 24 h in complete medium (1x106/mL) in the presence of
IL-21 (50 ng/mL). 1x105 anti-CD19 PerCP-stained ARH-77 cells (purple) were added to
ibiTreat chamber slides containing 2.5x104 immobilized HeLa cells stained with Cell Mask
deep red membrane dye (red). Cells were cultured for another 16 h in the incubator, then
GranToxiLux fluorogenic GzmB substrate (green) was added and spinning disk confocal
microscopy was performed for 3 h. Video was deconvolved using ImageJ software.
Supplemental Video 3. Time lapse video shows two GzmB+ ARH-77 cells attacking a
HeLa cell.
Anti-CD19 PerCP-stained ARH-77 cells were pre-incubated for 36 h in complete medium
(1x106/mL) in the presence of IL-21 (50 ng/mL). 1x105 ARH-77 cells were washed and
plated on ibiTreat chamber slides together with 2.5x104 HeLa cells stained with Cell Mask
deep red membrane dye (red). Finally, GranToxiLux fluorogenic GzmB substrate (greenyellow) was added and spinning disk confocal microscopy was performed for 14 h. Since
PerCP staining was bleaching within a few hours, for discrimination reasons the observation
was started in an area with ARH-77 cells that were highly positive for active GzmB (greenyellow). Video was deconvolved using ImageJ software.