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. 2016 Mar 15;7(11):12163-75.
doi: 10.18632/oncotarget.7512.

Human Naïve Regulatory T-cells Feature High Steady-State Turnover and Are Maintained by IL-7

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Free PMC article

Human Naïve Regulatory T-cells Feature High Steady-State Turnover and Are Maintained by IL-7

Susana L Silva et al. Oncotarget. .
Free PMC article

Abstract

Naïve FoxP3-expressing regulatory T-cells (Tregs) are essential to control immune responses via continuous replenishment of the activated-Treg pool with thymus-committed suppressor cells. The mechanisms underlying naïve-Treg maintenance throughout life in face of the age-associated thymic involution remain unclear. We found that in adults thymectomized early in infancy the naïve-Treg pool is remarkably well preserved, in contrast to conventional naïve CD4 T-cells. Naïve-Tregs featured high levels of cycling and pro-survival markers, even in healthy individuals, and contrasted with other circulating naïve/memory CD4 T-cell subsets in terms of their strong γc-cytokine-dependent signaling, particularly in response to IL-7. Accordingly, ex-vivo stimulation of naïve-Tregs with IL-7 induced robust cytokine-dependent signaling, Bcl-2 expression, and phosphatidylinositol 3-kinase (PI3K)-dependent proliferation, whilst preserving naïve phenotype and suppressive capacity. Altogether, our data strongly implicate IL-7 in the thymus-independent long-term survival of functional naïve-Tregs, and highlight the potential of targeting the IL-7 pathway to modulate Tregs in different clinical settings.

Keywords: IL-7; Immune response; Immunity; Immunology and Microbiology Section; human regulatory T-cells; naïve regulatory T-cells; regulatory T-cell homeostasis; thymectomy.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Preservation of the naïve-Treg compartment following thymus removal
Circulating naïve-Treg and naïve-Tconv counts; as well as % of cycling-cells, and Bcl-2 MFI within these subsets in healthy and thymectomized adults; each symbol represents one individual; bars represent median; significant P-values of comparisons within each subset are shown.
Figure 2
Figure 2. High steady-state turnover of the naïve-Treg compartment
A. Bcl-2 MFI and % of cycling-cells, within circulating CD4 T-cell subsets; each dot represents one individual; bars represent median; significant P-values are shown. B. % of cycling-cells within naïve-Tregs and naïve-Tconvs from matched blood and tonsil samples (each symbol represents one child: 2years/open, 6years/grey, and 7years/black).
Figure 3
Figure 3. Ex-vivo evidence for ongoing naïve-Treg response to IL-7
A. Representative histogram of ex-vivo pSTAT5 levels in circulating naïve-Tregs and naïve-Tconvs, with graph showing pSTAT5 MFI within these and counterpart memory CD4 T-cell subsets; comparison of pSTAT5 levels only revealed significant differences between naïve-Tregs and all the other subsets (* P < 0.05; n = 6). B. Analysis of pSTAT5 MFI within gated naïve-Tregs upon in-vitro stimulation with increasing concentrations of IL-7 or IL-2; each symbol represents one individual. C. IL-7Rα MFI within naïve-Tregs analyzed ex-vivo and after 24 hour-culture without serum or supplemented with 40% autologous serum alone or with IL-7 (n = 6); as well as with IL-2, anti-IL-2 blocking monoclonal antibody, or isotype-control (n = 3); * P < 0.05 as compared to ex-vivo levels.
Figure 4
Figure 4. Evidence of naïve-Treg response to IL-7 while preserving their naïve and suppressive phenotype
A. Representative contour-plots of FoxP3 and CD25 expression within total naïve CD4 T-cells ex-vivo (dØ) and upon 13 day-culture with IL-7 or IL-2. B. Illustrative histogram demonstrating absence of FoxP3 induction in purified naïve-Tconvs following 13 day-culture with IL-7. C. Cell recovery upon 13 day-culture with IL-7 or IL-2 of total naïve CD4 T-cells (n = 11), purified naïve-Tregs (n = 3) or purified naïve-Tconvs (n = 3); graph shows median and interquartile range or range (purified populations). E.-F. Analysis of naïve-Tregs and naïve-Tconvs ex-vivo and post-13 day-culture of naïve CD4 T-cells with IL-7 or IL-2 showing representative contour-plots of CD31/CD45RA expression (D), and graphs of MFI of CD31 (D), CD25 (E), and IL-7Rα (F); each dot represents one individual; bars represent median; comparisons done within each subset; significant P-values are shown. G. Illustrative histograms of CTLA-4, Helios, HLA-DR, CD39, Tim-3, PD-1 and PD-L1 expression within naïve-Tregs and naïve-Tconvs ex-vivo and post-13 day-culture with IL-7 or IL-2 (one/3-14). H. IL-2, IL-4, IL-17 and IFN-γ production after short-term PMA/Ionomycin stimulation ex-vivo and post-13 day-culture with IL-7 or IL-2 (one/3). I. Illustrative histograms of CFSE expression within naïve-Tconvs cultured alone or with naïve-Tregs pre-incubated with medium alone, IL-7 or IL-2 (numbers represent % of cells that divided at least once); graph shows % of suppression of naïve-Tconv proliferation in 3 individuals.
Figure 5
Figure 5. IL-7 induced naïve-Treg survival and proliferation
A.-D. Analysis of naïve-Tregs and naïve-Tconvs ex-vivo (dØ) and post 13 day-culture of purified naïve CD4 T-cells with IL-7 or IL-2 or IL-7 plus anti-IL-2 blocking monoclonal antibody: (A) Representative contour-plots of Bcl-2 and Ki-67 expression; graphs show Bcl-2 MFI (B) and % of cycling-cells (C), bars represent median; significant P-values of comparisons within each subset are shown; (D) Contour-plots illustrating the impact of IL-7 or IL-2 on EdU incorporation by naïve-Tregs (final 12 hours-culture, n = 2). E. Illustrative histograms of Bcl-2 and Ki-67 expression within gated FoxP3+ or FoxP3 cells ex-vivo and post 7 day-culture of purified CD4SP thymocytes with IL-7 or IL-2 (one/3). F. Impact of the PI3K-inhibitor (LY294002), mTOR-inhibitor (rapamycin), or vehicle control (DMSO), on naïve CD4 T-cells upon 7 day-culture with IL-7; graphs show % of naïve-Treg and change in cell recovery, Bcl-2 MFI and % of cycling-cells within naïve-Tregs and naïve-Tconvs, as compared to ex-vivo levels; each color represents one individual.

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