Expansion, phenotype and suppressive capacity of naïve Treg
expanded with IL-2 or a combination of IL-2 and IL-7.
We then compared two methods for expansion of highly purified
(Supplementary figure 2 A, B and C) sorted
CD45RA+CD62L+ Treg
(Treg-n/Treg-scm). A standard method (StM: aanti CD3/CD28 microbeads at
1:1 ratio in the presence of 100UI of rhIL-2) was compared to the IL-7
method (IL-7M) similar to the StM but with the addition of 10ng/ml of
rhIL-7. After 14 days Treg expanded more with the StM (fold expansion:
68, 61-93) than with the IL-7M (fold expansion: 49, 47-59,P =0.021) resulting in a 28% decrease in the final cell yield
using the IL-7M (Figure 3A). Surface phenotype analysis showed that Treg
expanded with the IL-7M were highly enriched in
CD45RA+CD62L+CD95+cells (Figure 2B). Dividing Treg into subsets according to the
expression of CD45RA, CD62L and CD95 we observed that Treg expanded with
the two methods showed a similar percentage (median, IQR) of Treg-n (StM
5.4, 3.4-8.1 vs IL-7M 3.5, 2.4-5.5, p=0.234), Treg-cm (StM 39, 29.7-45.7
vs IL-7M 46, 40.1-57, p=0.458) and Treg-emra (StM 1.1, 0.8-1.2 vs IL-7M
1, 0.3-1.2, p=0.88) after 14 days of expansion (Figure 2C). Treg
expanded with the IL-7M were highly enriched in Treg-scm (StM 4.3, 2.6-6
vs IL-7M 23.2, 17.9-31, p=0.0078) and have less Treg-em (StM 51,
40.5-63.5 vs IL-7M 23.5, 11.7-39, p=0.0375) (Figure 2C). After 14 days
Treg cultures were magnetically depleted of anti CD3/CD28 microbeads and
cultured for additional 3 days in the absence of cytokines (resting
period). After resting expanded Treg showed a preservation of the
relative percentages of Treg subsets after resting. Treg expanded with
the StM or the IL-7M methods were tested in a suppression assay against
CD8+ T cells, showing a significant reduction in the suppressive
activity of Treg expanded with the IL-7M compared to Treg expanded with
the StM (Figure 2D). Since we have shown that IL-7 can reduce the Treg
suppressive activity but the suppressive function is re-established once
IL-7 is removed, we tested Treg after a 3 day resting period, which
resulted in a full recovery of the suppressive function after removal of
IL-7. To determine whether the kinetic of proliferation can impact on
the differences in the phenotype of expanded Treg we analyzed the
phenotype of proliferating Treg according the dilution of CFSE after 5
days of expansion. Treg expanded with the StM showed a homogeneous
proliferation pattern in which the large majority of Treg performed
several cell cycles and lost the expression of CD45RA (Figure 3E and
3F). Treg expanded with the IL-7M can be divided in two groups, of which
one performed multiple cell cycles and lost CD45RA, and a second one
which performed a limited number of cell cycles and preserves the
expression of CD45RA. Sorted Treg that performed more than two cell
cycles from both the StM and the IL-7M were committed to differentiate
predominantly into Treg-cm and Treg-em (Figure 3G). On the other hand,
sorted Treg that performed less that two cell cycles from both the StM
and the IL-7M were committed to differentiate into Treg-n and Treg-scm.
These results suggest that a population of slowly proliferating Treg
that is predominant in the IL-7M (but very scarce in the StM) is
responsible for the abundance of Treg with a stem cell memory phenotype
found in the final Treg product after 14 days of culture. Taken together
these data suggest that the presence of IL-7 reduces the proliferation
rate (and the final cell yield) but preserves a poorly differentiated
phenotype. Moreover, the reduced suppressive function can be recovered
after a brief culture in the absence of IL-7.