Supplemental Digital Content 1
Figure S1. In-vitro suppression mediated by ex-vivo expanded BALB/c
CD4+CD25highCD62Lhigh Tregs. (A) Foxp3+ staining before (gray) and after (black) Treg
expansion for 7-10 days. (B and C) FACS analysis of expanded Tregs stained with antiCD4, anti-CD25 and anti-CD62L. (D and E) Suppressive capacity of Tregs in MLR of
C57BL/6 CD4+ T-cells stimulated against irradiated BALB/c splenocytes. (D)
Proliferation of CFSE labeled effector cells was measured by FACS analysis in the
presence (black) or absence (dotted line) of Tregs at a 1:1 cell ratio. A typical experiment
showing FACS analysis of CFSE labeling in H2Kb+ effector T cells, with gating by
7AAD for viable cells. (E) Summary of the results of three experiments comparing CFSE
labeling in the presence or absence of Tregs or CD4+CD25- T effector cells. The bars
indicate mean value ± SD, *** P≤0.001 (Student’s t-test), N=3.
Figure S2. Histological appearance of E42 pig pancreatic implants, 8 weeks after
transplantation. Representative H&E staining of the implants in (A) NOD-SCID mice and
(B-C) C57BL/6 mice treated with debulking agents, Rapamycin, and FTY720 with (B),
or without (C) infusion of 2x106 BALB/c Tregs.
Materials and Methods:
Purification and ex-vivo expansion of Tregs and Teffs. Mouse auxiliary, mesenteric
and inguinal lymph nodes were harvested, and single cell suspensions were obtained by
passage through a wire mesh. The cells were collected in PBS. Cells were first enriched
for the CD25+ population using anti-CD25 PE antibody (hybridoma 7D4, Miltenyi
Biotec, Germany), followed by incubation with anti-PE MicroBeads and positive
selection on a MACS separation column (Miltenyi Biotec). For Tregs purification, the
cells were then stained with anti-CD4 APC and anti-CD62L FITC-conjugated antibodies
(clones RM4-5, MEL-14 respectively; BD Biosciences/Pharmingen, CA, USA), followed
by cell sorting gated for triple (CD4+CD25highCD62Lhigh) positive population using a
FACSAria Sorter. CD4+CD25- Teffs were obtained from the CD25- fraction using antiCD4 magnetic beads (MACS) (Miltenyi Biotec). The enriched Tregs or Teffs were
activated with mouse CD3/CD28 magnetic Dynabeads (Invitrogen Dynal, Norway) (at a
ratio of 1 bead/cell) and cultured in RPMI complete tissue culture medium (CTCM)
supplemented with recombinant human interleukin 2 (rhIL-2; 100U/ml; Amgen, CA).
Cells were expanded 20- 40 fold within 7-10 days.
Cryopreservation and thawing of Tregs. After expansion, Tregs were cryopreserved
using DMSO at a final concentration of 8% in FCS, stored at -80oC. The cells were
thawed quickly by incubation in a 37oC water bath for 1-2 minutes and seeded for 2-4
hours in CTCM supplemented with 100U/ml rhIL-2.
Evaluation of the suppressive potential of Tregs in-vitro by mixed lymphocyte
culture. Fresh CD4+, CD8+ or a mixture of both (2x105) C57BL/6 (H-2Kb) responder
cells were first labeled with 5µM CFSE and stimulated with irradiated (20Gy) whole
spleen BALB/c (H-2Dd) cells in flat-bottom 96 well plates at a responder/stimulator ratio
of 1:2, supplemented with CD3/CD28 beads at a ratio of 1/400 cells. The expanded
Treg/Teff population was added at the different ratios. After 4 days, the proliferation of
the live H-2Kb cells (dead cells were gated out with 7AAD, BD Biosciences/Pharmingen)
was measured by FACScan™(BD), based on CFSE dilution. The index of suppression
was
1
calculated
using
the
following
formula:
Number of proliferat ing cells in the assessed well with Tregs
Number of proliferat ing cells in the control well w / o Tregs
Evaluation of the suppressive potential of Tregs in-vivo. Single cell suspensions were
prepared from lymph nodes, liver, and spleen harvested from transplanted mice. Isolated
cells were stained with anti-H-2Dd, anti-CD8 and anti-CD4 (BD Pharmingen). FACS
analysis was performed upon fixed time counts of each sample.
Results:
Tregs purification and ex-vivo expansion
Since there is no selective expansion protocol for Tregs and the common protocols used
can also trigger Teffector cells (Teffs), it was important to use, as a starting population,
highly purified preparations of Tregs with minimal contamination of Teffs. To that end,
lymph node cell preparations were sorted by gating on CD4+ and CD25high cells. This
gating afforded 93% purity of Foxp3+ Tregs. However, upon ex-vivo expansion, the
residual contaminating Teffs were found to gradually increase in abundance and became
dominant. Thus, we resorted to further purification of the initial cell preparation by using
triple gating on CD4+CD25highCD62Lhigh cells, leading to 98% purity of Foxp3+ Tregs
(Fig.S1a). Polyclonal expansion using CD3/CD28 antibody-coated beads in the presence
of high dose recombinant human IL-2 retained CD25 and CD62L high expression;
however, it was associated with some reduction of the Foxp3+ cell purity. Therefore, the
ex-vivo expansion was limited to 7-10 days, by which time the proliferating cells had
expanded 20-40 fold, and the percentage of Foxp3+ cells remained at 90-95% (Fig.S1a,b).
In-vitro suppression mediated by expanded Tregs
To define the suppressive activity of the Tregs preparation, we initially attempted to test a
xenogeneic MLR, measuring the response of C57BL/6 splenocytes against porcine
splenocytes. However, as the proliferation signal was insignificant, the efficacy of Tregs
prior to transplantation was verified in MLR comprising C57BL/6 responders and Balb/c
stimulators. Proliferation of the responder T-cells in the presence or absence of Tregs was
monitored 4 days after initiation of MLR using labeling with CFSE. As can be seen in
Fig.S1c, strong suppressive activity was exhibited by the Tregs on alloreactive CD4 + Tresponder cells, reducing the percentage of proliferating cells from 80±0.35% to
16±1.1%.
In contrast, addition of BALB/c CD4+CD25- Teffs did not induce any
suppression. The expanded BALB/c Tregs were able to suppress proliferation in a dose
dependent manner up to a suppressor:responder ratio of 1:50 (Fig.S1d).
The same suppressive effect was observed in the presence of CD8+ responder T-cells or
when using a mixed population of CD4+ and CD8+ T-cells, exhibiting a reduction of
proliferating cells from 80±5.1% to 30±1.3% and from 85±5.2% to 14±2.8%,
respectively. Collectively, these results demonstrate that Tregs retain their suppressive
activity and remain functional following ex-vivo expansion.