A Kinetic and Dynamic Analysis of Foxp3 Induced in T Cells by TGF-β

Animals

Mice in which a GFP-Foxp3 fusion has been homologously inserted at the Foxp3 locus have been described (1) and were backcrossed more than five generations onto the C57BL/6J (Thy1.2⁺, CD45.2⁺) background before analysis. Male mice screened for GFP-Foxp3 were used for experimentation. Some GFP-Foxp3 mice were subsequently bred with CD45.1-congenic mice to obtain CD45.1⁺ GFP-Foxp3 mice. C57BL/6J mice and congenic CD45.1 (B6.SJL-Ptprc^a Pepc^b/BoyJ) and Thy1.1 (B6.PL-Thy1^a/CyJ) mice were purchased from The Jackson Laboratory. Experimentation was performed in accordance with institutional animal care and use procedures.

Media, reagents, and Abs

Media for T cell cultures was prepared as described earlier (28). Unconjugated anti-CD3, anti-CD28, and anti-CD16/CD32 Fc block (2.4G2) and fluorochrome-conjugated anti-CD4 (L3T4), anti-CTLA-4, and anti-CXCR4 were purchased from BD Pharmingen. All other Abs used and recombinant human TGF-β1 were purchased from eBioscience. Sulfate latex beads (Molecular Probes and Invitrogen Life Technologies) were coated with anti-CD3 (40 μg/ml) or anti-CD28 (40 μg/ml) or anti-CD3/CD28 (13.3:26.6 μg/ml) as per the manufacturers’ instruction.

Cell purification and cell culture

Lymph nodes (LN) and spleen cells were collected as described previously (28). CD4⁺Foxp3⁺ (nTreg) and CD4⁺Foxp3⁻ (non-Treg) cells were isolated by flow cytometric sorting on a MoFlo high-speed sorter (DakoCytomation) gating on CD4 and GFP (Foxp3) expression (28). Sorted cell purity ranged from 97 to 99%. nTreg were grown in medium supplemented with 1 ng/ml PMA, 200 ng/ml ionomycin, and 100 U/ml recombinant human IL-2 (rhIL-2; National Cancer Institute Biological Resources Branch Repository), which we found to optimally preserve Foxp3 expression. CD4⁺Foxp3⁻ cells were stimulated with antiCD3/CD28 beads at a cell:bead ratio of 1:1 and supplemented with 100 U/ml rhIL-2 with or without TGF-β (10 ng/ml) for 7–9 days to obtain iTreg or activated Foxp3⁻ cells, respectively. Cells were split into cytokine-containing medium, as needed, to prevent overcrowding.

Foxp3 regulation in CD4⁺Foxp3⁻ cells

Sorted CD4⁺Foxp3⁻ cells (2.5 × 10⁴ per well) were stimulated in 96-well plates in medium supplemented with anti-CD3/CD28-coated beads (cell:bead ratio of 1:1) and 100 U/ml rhIL-2, or as indicated. TGF-β (10 ng/ml) was added at day 0 or the indicated time after TCR stimulation. For Foxp3 reversion kinetics, cells were stimulated as described above and TGF-β was removed at the indicated time by removing the supernatant and washing with PBS three times before adding back IL-2-containing medium. To study Foxp3 up-regulation in memory T cells, CD4⁺CD44^highCD45Rb^lowFoxp3⁻ cells and control CD4⁺Foxp3⁻ cells were isolated by flow cytometric sorting, and 2 × 10⁴ cells/well cultured in 96-well plates in medium supplemented with anti-CD3/CD28-coated beads (cell:bead ratio of 1:1), 100 U/ml rhIL-2, with or without TGF-β (10 ng/ml). To prevent overcrowding in longer-term cultures, wells were examined and split every 3–4 days into medium supplemented with the same cytokines (IL-2 with or without TGF-β) as initially present. Quadruplicate samples for each condition were analyzed. Foxp3 analyses were performed every 24 h or as indicated by quantitative flow cytometry by measuring the GFP fluorescence.

PKH26 proliferation analysis

Naive flow cytometrically purified CD4⁺Foxp3⁻ cells were dye labeled using the PKH26 red fluorescent cell linker kit (Sigma-Aldrich) as per the manufacturer’s instructions, except that 1 × 10⁻⁶ M PKH26 dye was used for 1 × 10⁷ cells. Cells were cultured in medium supplemented with or without TGF-β (10 ng/ml) and analyzed at the indicated times by flow cytometry.

T cell proliferation suppression assay

Flow cytometrically purified CD4⁺CD25⁻ T cells from Thy1.1-congenic mice (responder cells) were mixed with either nTreg or iTreg at the indicated ratios and stimulated with anti-CD3/CD28 beads at a bead:cell ratio of 1:1. Cocultures established in quadruplicate were analyzed by flow cytometry at the indicated times. For analysis of revertant Foxp3⁻ iTreg populations, CD4⁺Foxp3⁻ cells were converted to iTreg and sorted to ∼99% purity as described above. These iTreg were recultured with or without TGF-β for 5 days, at which time the cells were again flow-sorted for Foxp3⁺ or Foxp3⁻ cells, respectively. The iTreg or revertant Foxp3⁻ cells were then evaluated for their ability to suppress proliferation of CFSE-labeled naive T cell responders as previously described (29). Because the iTreg express GFP, the fluorescence emission spectrum of which cannot readily be distinguished from CFSE, Thy1.1-disparate responder cells were used and analysis of CFSE levels in Thy1.1-gated cells was performed.

Cytokine production profile of stimulated nTreg, iTreg, and non-Treg

CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ cells were purified from GFP-Foxp3 knock-in mice and cultured as described above to obtain nTreg, iTreg, and Foxp3⁻ cells. At day 7, the expanded nTreg, iTreg, and non-Treg were flow cytometrically sorted to obtain ∼99% pure Foxp3⁺ or Foxp3⁻ populations. Five × 10⁴ cells were added to 96-well plates in 250 μl of medium in triplicate and restimulated with anti-CD3/CD28 Ab-coated beads at a 1:1 cell:bead ratio in the presence of 100 IU/ml rhIL-2. At 48 h, murine IL-2, IL-4, IL-5, IL-10, IL-17, IFN-γ, and TNF-α were measured in the cell culture supernatant by Bio-Plex according to the manufacturer’s instructions (Bio-Rad).

Migration and survival of iTreg after adoptive transfer

CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ cells were purified from CD45.1⁺ GFP-Foxp3 knock-in mice and cultured as described above to obtain CD45.1⁺ nTreg, CD45.1⁺ iTreg, and CD45.1⁺ Foxp3⁻ cells. At days 7–9, the expanded nTreg, iTreg, and non-Treg were flow-cytometrically sorted to obtain ∼99% pure Foxp3⁺ of Foxp3⁻ populations. Four × 10⁶ Foxp3⁺ nTreg, iTreg, or non-Treg were mixed with 1 × 10⁶ Thy1.1⁺ CD4⁺ T cells before adoptive transfer by retro-orbital injection into age- and sex-matched Thy1.1⁻CD45.1⁻ C57BL/6J hosts. The Thy1.1⁺CD4⁺ T cells were purified from a LN and splenic cell suspension from Thy1.1⁺-congenic mice by anti-CD4 magnetic bead separation using the MACS separation system according to the manufacturer’s instructions (Miltenyi Biotec). Two recipient mice for each condition and time point measured were sacrificed at days 2, 5, 12, and 28 after injection, and peripheral blood, LN, spleen, lung, liver, and bone marrow were isolated. Cells were collected by forced passage through a cell strainer. Lymphoid components of liver and lung cell suspensions were further purified by centrifugation over 37.5% Percoll. Cells were stained with the indicated Abs before flow cytometric analysis.

Flow cytometry

Cells were stained and analyzed on a FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences). Quantitative flow cytometry to determine total cell numbers was performed by enumerating all cells in a culture well. The presence of Foxp3 in cells from mice expressing the GFP-Foxp3 fusion was determined by measuring GFP fluorescence.

Statistics

Data were analyzed by ANOVA using JMP (SAS).

TGF-β collaborates with TCR stimulation to up-regulate Foxp3 in CD4⁺ cells

Previous studies (6, 7, 8, 30) have shown that TGF-β up-regulates Foxp3 in activated CD4⁺CD25⁻ T cells. To directly visualize this event, we analyzed the induction of Foxp3 in T cells from mice engineered to express a GFP-Foxp3 fusion protein. CD4⁺Foxp3⁻ T cells were purified, stimulated, and analyzed using quantitative flow cytometry. Similar to the results of others, TGF-β up-regulated Foxp3 in CD4⁺Foxp3⁻ T cells after CD3/CD28 stimulation, with conversion efficiencies of 50–90% routinely observed (Fig. 1⇓, A and B). Costimulation influenced the conversion. Total numbers of Foxp3⁺ cells were increased in the presence of anti-CD28. However, the percentage of Foxp3⁺ cells was equivalent in cultures stimulated with anti-CD3 in the presence or absence of anti-CD28, suggesting that costimulation acted by promoting the expansion of the cells rather than increasing the conversion efficiency (Fig. 1⇓B).

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FIGURE 1.

TGF-β collaborates with TCR stimulation but not costimulation to up-regulate Foxp3. CD4⁺Foxp3⁻ T cells were flow-cytometrically purified and 3 × 10⁴ distributed per well of a 96-well plate. The cells were stimulated with anti-CD3-, CD28-, or CD3/CD28 Ab-coated beads at a 1:1 cell:bead ratio. rhIL-2 and TGF-β were added at 100 U/ml and 10 ng/ml, respectively. On pretreatment day 0 or posttreatment day 5, wells were harvested and analyzed for expression of Foxp3 by quantitative flow cytometry. Mean pretreatment values for Foxp3⁻ and Foxp3⁺ T cells was 29,534 and 19, respectively, per well. A, Representative Foxp3 histogram plots are shown. B, Percent Foxp3⁺ cells is plotted. Mean absolute cell numbers are shown. C, Foxp3 is not up-regulated in memory cells. Cells were stimulated with anti-CD3/CD28-coated beads and IL-2 with or without TGF-β. Total cell counts on day 7 for the different populations are listed within parentheses. B and C, Mean ± SEM of quadruplicate samples are plotted.

Foxp3 up-regulation was dependent on the presence of both TGF-β- and TCR-specific signaling. As has been reported, in the absence of TGF-β, little or no up-regulation of Foxp3 was apparent (Fig. 1⇑, A and B, and not plotted; after anti-CD3 and anti-CD3/CD28 stimulation, day 5 Foxp3⁺/Foxp3⁻ cell counts were 212/95,567 and 128/141,065, respectively). In the absence of TCR stimulation, little conversion was also observed. This effect has been previously noted using RT-PCR analysis for Foxp3 (8). However, cell viability is extremely poor in the absence of TCR stimulation and an alternative explanation for this finding is that the Foxp3⁺ cells have impaired survival without TCR stimulation. Indeed, the total number of surviving T cells cultured with IL-2/TGF-β was only 1.1% of that observed in cultures also stimulated with anti-CD3/CD28. We found, however, that culture of CD4⁺Foxp3⁻ cells in the presence of anti-CD28 but not anti-CD3 greatly improved cell viability, with similar numbers of live cells present at day 5 as at the start of culture. Here too though, few of the cells (mean = 1.0%) up-regulated Foxp3. Therefore TCR but not CD28 signaling synergizes with TGF-β to drive Foxp3 expression. Foxp3 up-regulation was further restricted to the naive T cell population and was not increased in isolated CD4⁺CD44^highCD45Rb^lowFoxp3⁻ memory cells stimulated in the presence of TGF-β (Fig. 1⇑C).

TGF-β-induced Treg suppress the proliferation of CD4⁺ target cells

Earlier reports (7, 31) have shown iTreg, like nTreg, possess regulatory function and suppress CD4⁺ T cell proliferation in coculture experiments. We verified that our Foxp3⁺ iTreg were similarly capable of suppressing T cell expansion using quantitative flow cytometry (Fig. 2⇓) as well as proliferation analysis of CFSE-labeled responder cells (data not shown). In both studies, iTreg showed an efficiency similar to that of nTreg in suppressing T cell proliferation and expansion.

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FIGURE 2.

iTreg suppress the proliferation of CD4⁺ target cells. Thy1.1⁺CD4⁺CD25⁻ responder cells were mixed with congenic Thy1.1⁻ nTreg or iTreg in 96-well plates at the indicated ratios. Cells were stimulated with anti-CD3/CD28-coated beads at a cell:bead ratio of 1:1 for 3 days, and wells were then analyzed by quantitative flow cytometry for viable Thy1.1⁺ responder numbers. Mean ± SEM of quadruplicate samples are plotted. Data are representative of two independent experiments.

Similar cytokine profile of iTreg and nTreg

Because iTreg were as efficient as nTreg in suppressing T cell proliferation, we wanted to examine whether their cytokine production profiles were likewise comparable (Table I⇓). Naive CD4⁺ T cells were stimulated and expanded for 7 days without TGF-β or converted into iTreg with TGF-β. nTreg were likewise expanded. Foxp3⁺ (iTreg, nTreg) or Foxp3⁻ (non-Treg) cells were then flow-cytometrically sorted and stimulated. Both iTreg and nTreg demonstrated decreased production of most cytokines when compared with non-Treg, including IL-2, IL-4, IL-5, IFN-γ, and TNF-α. Although more IL-2 and IFN-γ was produced by the iTreg than nTreg, this was significantly diminished when compared with non-Treg. In contrast, IL-10 was strongly produced by both the iTreg and nTreg. Therefore, TGF-β-induced iTreg have a cytokine profile similar to that of nTreg, with strong expression of IL-10 and diminished expression of other effector cytokines.

Kinetics of Foxp3 expression after anti-CD3/CD28 and TGF-β treatment

Considering that memory T cells did not up-regulate Foxp3 in response to TGF-β, (Fig. 1⇑C), we were interested in defining the window period after activation during which T cells were susceptible to TGF-β. Indeed, a time dependence for the generation of regulatory cells using TGF-β has been previously reported (32, 33). To test for Foxp3 induction, we stimulated CD4⁺Foxp3⁻ cells with anti-CD3/CD28 and IL-2, supplementing with TGF-β at different time points after stimulation (Fig. 3⇓A). Two effects were notable. First, when TGF-β was provided at the time of TCR stimulation, up-regulation of Foxp3 protein only began after an ∼2-day delay. Interestingly, if TGF-β supplementation was provided at later time points after TCR stimulation, Foxp3 up-regulation was delayed by a similar ∼2-day period from the time TGF-β was administered. Second, treatment with TGF-β beginning up to 2 days after TCR stimulation had little impact on the ultimate percentage or number of Foxp3⁺ T cells in the culture. In contrast, cells treated with TGF-β on or after day 3 showed significantly (p < 0.01) diminished conversion into Foxp3⁺ cells. When treatment began on day 3, a peak conversion of only ∼20% of cells was observed compared with ∼80% with day 0 treatment. This difference did not result from an outgrowth of Foxp3⁻ T cells because total cell numbers were similar in the different treatment groups (Fig. 3⇓B). Indeed, quantitative analysis demonstrated that absolute numbers of Foxp3⁺ cells were significantly (p < 0.01) higher in the cells treated with TGF-β starting days 0–2 compared with the cells treated after day 2. Therefore, TGF-β/TCR stimulation has a limited window during which it can up-regulate Foxp3, and TGF-β supplementation leads to up-regulation of Foxp3 protein only after a significant (∼2-day) lag period.

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FIGURE 3.

Kinetics of Foxp3 expression after TGF-β induction. CD4⁺Foxp3⁻ cells (2.5 × 10⁴) were stimulated on day 0 with anti-CD3/CD28-coated beads in a 96-well plate at a cell:bead ratio of 1:1 in the presence of 100 U/ml rhIL-2. TGF-β (10 ng/ml) was added either at day 0 or at the time denoted. Sample wells were harvested on different days and analyzed by quantitative flow cytometry for Foxp3 expression by measuring GFP fluorescence. Wells were split every 3–4 days in medium with cytokine to prevent overcrowding. Mean ± SEM of percentage of Foxp3⁺ cells (A) and total cell numbers (B) of quadruplicate samples are plotted. Data are representative of three independent experiments.

TGF-β- and CD3-induced Foxp3 up-regulation is independent of cell cycling

Differentiation of naive T cells into Th1 and Th2 cell types occurs only after multiple rounds of cell cycling, an event believed to be required to relieve epigenetic repression of lineage-specific genes (34). After a T cell is stimulated through the TCR, it begins to cycle ∼2 days after stimulation, consistent with the time frame for TGF-β-mediated up-regulation of Foxp3 (Fig. 3⇑A). We were therefore interested whether Foxp3 up-regulation only occurred in T cells that had divided. To test this, we labeled CD4⁺Foxp3⁻ cells with the cell membrane-associated red fluorescent dye PKH26 and stimulated them with anti-CD3/CD28 with or without TGF-β (Fig. 4⇓). The fluorescence of PKH-26 is diminished with each cell division. In the absence of TGF-β, Foxp3 expression was not observed in divided or undivided cells. In contrast, Foxp3 was up-regulated in all cell populations treated with TGF-β, including those that had not divided. This demonstrates that Foxp3 up-regulation and iTreg development does not require cell cycling.

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FIGURE 4.

TGF-β-induced Foxp3 up-regulation is independent of cell cycling. Purified CD4⁺Foxp3⁻ cells were labeled with PKH26, washed, and stimulated with anti-CD3/CD28-coated beads at a cell:bead ratio of 1:1 in the presence of 100 U/ml rhIL-2 with or without 10 ng/ml TGF-β. Flow cytometric analysis of cells immediately after culture (day 0) and at days 2 and 3 are shown. Each condition was analyzed in triplicate with essentially identical results. Data are representative of two independent experiments.

Limited persistence of Foxp3 in iTreg after TGF-β withdrawal

To determine whether iTreg require continuous exposure to TGF-β to preserve Foxp3 expression, we stimulated CD4⁺Foxp3⁻ cells with anti-CD3/CD28 and TGF-β, and withdrew TGF-β at various time points after stimulation (Fig. 5⇓A). Foxp3 expression persisted at high levels in cells that received continuous TGF-β treatment for the 13 days of culture. In contrast, Foxp3 expression returned to baseline by day 13 in cells that had TGF-β removed after initial treatment. After TGF-β was withdrawn, Foxp3 persisted for another 3–4 days, at which point expression began to decrease (p = 0.013). Expression was virtually undetectable in the T cells by 9 days after TGF-β withdrawal. Cells that were exposed to <3 days of TGF-β lost Foxp3 at a much higher rate than those exposed for at least 3 days. Because total cell numbers in the wells were similar with or without withdrawal of TGF-β and total numbers of cells steadily increased with culture time (Fig. 5⇓B), the loss of Foxp3 seemed to be primarily due to the conversion of the Foxp3⁺ cells to Foxp3⁻ cells rather than cell death. This conversion was confirmed in separate experiments in which iTreg purified by flow cytometric sorting for GFP-Foxp3 and then cultured without exogenous TGF-β displayed a similar loss of Foxp3 (data not shown). When sorted Foxp3⁺ iTreg were allowed to lose Foxp3 expression, Foxp3 could not be reinduced by restimulation, even in the presence of TGF-β (data not shown). Therefore, Foxp3 can only be induced in a brief window as naive T cells differentiate into effector/memory cells. Foxp3 persistence is dependent on sustained TGF-β signaling.

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FIGURE 5.

Limited persistence of Foxp3 in iTreg after TGF-β withdrawal. Purified CD4⁺Foxp3⁻ cells were stimulated with anti-CD3/CD28-coated beads at a cell:bead ratio of 1:1 in the presence of 100 U/ml rhIL-2. No or 10 ng/ml TGF-β was added at day 0. TGF-β was removed by washing the cells at the indicated time. To prevent overcrowding during the extended culture, cells were split every 3–4 days into medium with IL-2 and with or without TGF-β. Cells were analyzed by quantitative flow cytometry at the indicated time points. Mean ± SEM of percentage of Foxp3⁺ cells (A) and total cell numbers (B) of quadruplicate samples are plotted. Data are representative of three independent experiments.

Loss of suppressive potency in iTreg that have down-regulated Foxp3

Considering that removal of TGF-β resulted in loss of Foxp3 in iTreg (Fig. 5⇑A), we were interested in whether loss of Foxp3 also leads to a loss of suppressive activity. To test this, TGF-β was either added to or excluded from cultures of flow-cytometrically purified Foxp3⁺ iTreg. Five days later, Foxp3⁺ or Foxp3⁻ cells from the respective cultures were flow-cytometrically isolated. The iTreg or revertant Foxp3⁻ cells were then added to naive, Thy1.1-disparate, CFSE-labeled T cells, and the proliferation of the naive population to anti-CD3/CD28 was measured by loss of CFSE (Fig. 6⇓). As in Fig. 2⇑, iTreg that retained Foxp3 strongly suppressed naive cell proliferation. In contrast, iTreg that lost Foxp3 showed a substantially reduced ability to suppress T cell proliferation. Thus, loss of Foxp3 expression is accompanied by a loss of suppressive activity.

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FIGURE 6.

Suppression of naive T cell proliferation by Foxp3⁻ revertant iTreg. Thy1.1⁻ iTreg were flow-cytometrically sorted for Foxp3⁺ cells 7 days after induction with TGF-β. These cells were recultured for 5 days with or without TGF-β. The cells grown with TGF-β were then sorted again for expression of Foxp3, whereas the revertant Foxp3⁻ cells were sorted from the population grown in the absence of TGF-β. Thy1.1⁺ naive T cells were CFSE labeled, mixed with the Foxp3⁺ or Foxp3⁻ populations at the indicated ratios, and stimulated with anti-CD3/CD28-coated beads. At 72 h, cultures were stained for Thy1.1, and CFSE expression on the Thy1.1⁺ population was analyzed by flow cytometry.

Migration and survival of iTreg after adoptive transfer

Our in vitro studies suggested that the persistence of iTreg is dependent upon exogenous TGF-β and that Foxp3 is lost within a several-day period after TGF-β removal. The transient nature of Foxp3 expression in iTreg implies that iTreg would not be suitable for immunotherapeutic application. Yet, studies have now documented that iTreg are effective in treating model alloimmune and autoimmune diseases (23, 24, 25). To determine whether iTreg persist in vivo, we adoptively transferred flow-cytometrically purified GFP-Foxp3⁺ iTreg derived from CD45.1⁺ mice into CD45.1⁻ congenic recipients and followed their migration and survival (Fig. 7⇓). As controls, equivalent numbers of either Foxp3⁻ cells from cultures stimulated in the absence of TGF-β or Foxp3⁺ nTreg from similarly expanded cultures were also transferred into naive mice. The CD45.1-congenic background permitted identification of the transferred cells regardless of their expression of GFP-Foxp3. To control for adoptive transfer efficiency among mice, control freshly purified Thy1.1⁺CD4⁺ cells were admixed at a 1:4 ratio with each of the adoptively transferred cell populations before transfer into congenic Thy1.1⁻CD45.1⁻ C57BL/6 hosts (Fig. 7⇓C).

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FIGURE 7.

Migration and survival of iTreg after adoptive transfer. A, Scheme for the preparation and adoptive transfer of iTreg, nTreg, or non-Treg. B, Flow cytometric analysis of expanded iTreg, nTreg, and non-Treg populations before flow purification. C, Flow cytometric analysis of adoptively transferred cell populations, which include purified CD4⁺Thy1.1⁺ injection controls and CD4⁺CD45.1⁺Thy1.1⁻Foxp3^{+ or −} experimental populations. Data are representative of three independent experiments.

At selected time points mice were sacrificed, cell suspensions were prepared from different organs, and the cells were analyzed by flow cytometry for CD4, Thy1.1, CD45.1, and Foxp3 expression. Similar numbers of Thy1.1⁺ cells were routinely observed in the recipient mice, indicating equivalent adoptive transfer efficiencies (data not shown). CD4⁺ cells were gated to distinguish the CD45.1⁺ transferred population and CD45.1⁻ host cells, and the transferred cells were then further segregated into Foxp3⁺ and Foxp3⁻ populations (Fig. 8⇓, A and B). Numbers of CD45.1⁺Foxp3⁺ or Foxp3⁻ cells were normalized to endogenous CD4⁺CD45.1⁻Thy1.1⁻ cell numbers and plotted (Fig. 8⇓C).

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FIGURE 8.

Migration and survival of iTreg, nTreg, and non-Treg after adoptive transfer. A, iTreg, nTreg, and non-Treg prepared as in Fig. 7⇑ were adoptively transferred by retro-orbital injection into C57BL/6J mice. After 2 days, the mice were sacrificed, and cells were isolated and stained with CD45.1, Thy1.1, and CD4. Sample flow cytometry plots from forward scatter/side scatter-gated LN cells are shown, demonstrating identification of the transferred CD4⁺CD45.1⁺ cells among endogenous CD45.1⁻ cells. Numbers in the right side quadrants indicate percentage of CD45.1⁺ and CD45.1⁻ cells among the total CD4⁺ population. B, Histogram plots of gated CD4⁺CD45.1⁺ cells (oval gates in A) analyzed for the expression of Foxp3 are shown. C, Mice were sacrificed at the indicated time points and analyzed for the presence of adoptively transferred cells. Numbers of adoptively transferred CD4⁺CD45.1⁺Foxp3⁺ or Foxp3⁻ cells were normalized to 25,000 host CD4⁺CD45.1⁻Thy1.1⁻ cells in the indicated organ and plotted. Mean ± SEM of two mice per condition are plotted. Data are representative of three independent experiments.

Transferred flow-cytometrically purified Foxp3⁺ iTreg largely disappeared within the first 2 days after adoptive transfer (Fig. 8⇑C). This seemed to result from down-modulation of Foxp3 since large numbers of CD4⁺CD45.1⁺Foxp3⁻ cells were simultaneously observed in several organs, including the spleen, liver, blood, and lung. Indeed, in support of this interpretation, numbers of Foxp3⁻ cells detected after transfer of purified Foxp3⁺ iTreg were similar to those observed after adoptive transfer of equal numbers of purified CD4⁺Foxp3⁻ non-Treg T cells. Transferred CD4⁺Foxp3⁻ cells were also found in similar locations as Foxp3⁻ former iTreg, specifically the spleen, liver, blood, and lung, within the first week after adoptive transfer. By 4 wk after transfer, virtually all of the Foxp3⁻ cells from either the iTreg or control non-Treg transfer had disappeared. One organ where Foxp3 was retained on the transferred iTreg was the LN. Indeed, of the organs analyzed at day 2 after iTreg transfer, greater numbers of transferred Foxp3⁺ cells then Foxp3⁻ cells were only detected within the LN.

By 2–4 wk after iTreg transfer, when the revertant Foxp3⁻ cells had largely disappeared, increasing numbers of transferred Foxp3⁺ cells were observed, particularly in the bone marrow and LN. Interestingly, only small numbers of Foxp3⁺ cells remained in the spleen, suggesting differential localization or inadequate support of Foxp3 expression at this site. Because the transferred CD45.1⁺ cells were ∼99% Foxp3⁺ (Fig. 7⇑C), this indicates that a subset of iTreg persisted and possibly expanded, surviving for at least 4 wk and primarily localizing within the bone marrow and LN.

The survival and localization of iTreg showed significant differences though also similarities with that of adoptively transferred preactivated nTreg. nTreg retained their Foxp3 to a much greater extent than iTreg. All organs analyzed showed a predominance of Foxp3⁺ cells after nTreg transfer, yet this was only observed for the LN after iTreg transfer. Similar to the Foxp3⁺ cohort after iTreg transfer, the nTreg persisted 4 wk after transfer and at late time points also resided predominantly in the LN and bone marrow. One additional feature of nTreg transfers was that despite the transfer of equivalent numbers of nTreg as non-Treg, fewer nTreg were detected in the different organs than non-Treg. For example ∼3,000 and 4,000 non-Treg were identified in the LN and spleen per 25,000 endogenous CD4⁺ T cells on day 2 after transfer, whereas ∼400 and 130 nTreg were detected, respectively. This may reflect an increased rate of death or decreased proliferation of the preactivated nTreg vs non-Treg after transfer, or alternatively differential migration to other organs not analyzed. In summary, transferred Foxp3⁺ iTreg yielded large numbers of Foxp3⁻ cells that entered the blood, lungs, and liver and then disappeared by 2–4 wk after transfer. A portion of iTreg, however, retained Foxp3. These persevered initially in the LN and then, as for nTreg, increasingly in the bone marrow. Thus, although many iTreg act in vivo as in vitro, losing Foxp3 after removal from TGF-β, a subset of these cells persist longer as Foxp3⁺ iTreg.

Transferred iTreg phenotypically resemble nTreg

nTreg constitutively express several T cell activation markers, including CD25 and glucocorticoid-induced TNFR family-related gene (GITR) (35). These markers were also nearly uniformly up-regulated on both iTreg and activated non-Treg before adoptive transfer (Table II⇓).

To determine whether expression of CD25 and GITR persisted after adoptive transfer, cells from mice receiving activated and expanded iTreg, nTreg, or non-Treg were analyzed for CD25 and GITR expression. Foxp3⁺ iTreg, like nTreg, retained CD25 and GITR expression in the LN (Fig. 9⇓, A and B) and all other organs analyzed (data not shown) throughout the 28-day time course of the experiment. In contrast, the majority of either transferred non-Treg or revertant iTreg-derived Foxp3⁻ cells lost CD25 and GITR expression within the first 2–5 days after transfer. The small fraction of nTreg that lost Foxp3 (Fig. 8⇑) also lost surface expression of CD25 and GITR (data not shown). This indicates that Foxp3⁺ iTreg phenotypically resemble nTreg in vivo, while the revertant Foxp3⁻ iTreg resemble Foxp3⁻ cells. Foxp3 protein expression is associated with the sustained expression of CD25 and GITR in vivo.

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FIGURE 9.

Transferred iTreg phenotypically resemble nTreg. CD4⁺CD45.1⁺ adoptively transferred iTreg, nTreg, and non-Treg were identified within the LN of recipient mice and analyzed for Foxp3 expression as in Figs. 7⇑ and ⇑8. These were additionally analyzed for surface expression of CD25 (A) or GITR (B). Percentage of CD4⁺CD45.1⁺ cells of the indicated cell type positive for CD25 or GITR is plotted. Data points are missing for some cell types/conditions at some time points due to an inadequate number of cells in the organs at those times for definitive expression analysis. Sample histogram plots from the day 2 time point of transferred iTreg populations in LN that lost Foxp3 (left) or retained Foxp3 (right) are also shown. Mean ± SEM of two mice per condition are plotted. Data are representative of two independent experiments.

It was interesting that the greatest concentration of transferred activated Foxp3⁺ iTreg or nTreg were found in the bone marrow and LN. Bone marrow localization of transferred nTreg has been previously shown to be associated with expression of the CXCR4 chemokine receptor, which is also expressed on other cell types localizing within the marrow (36). Indeed, blockade of CXCR4 can mobilize cells from the marrow into the blood (37). Analogously, nTreg expression of CD62L, a receptor for glycan ligands expressed on high endothelial venules, is associated with LN homing of nTreg as well as other T cells (38). To determine whether receptor expression correlates with tissue localization of iTreg, as it does for nTreg, we analyzed CXCR4 and CD62L expression on the transferred cells. At the time of transfer, <3% of iTreg expressed CXCR4 and only 26% expressed CD62L (Table II⇑). By 5 days after transfer, ∼90% of Foxp3⁺ iTreg or nTreg in bone marrow expressed CXCR4, while the same population in other organs showed very little CXCR4 expression (Fig. 10⇓A). Similar results were seen at later time points (data not shown). This is consistent with CXCR4 being the bone marrow homing receptor for iTreg, as for nTreg.

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FIGURE 10.

CXCR4 is preferentially expressed on adoptively transferred cells in the bone marrow and CD62L on cells in the LN. Data were acquired and analyzed as in Figs. 7⇑ and 8⇑. A, Percentage of indicated adoptively transferred populations positive for CXCR4 on day 5 after transfer is plotted. Sample histogram plots demonstrating CXCR4 expression on Foxp3⁺ iTreg in the LN or bone marrow (BM) are also shown. B, Percentage of the indicated adoptively transferred population positive for CD62L on day 5 or day 12 after transfer is plotted. Sample histogram plots demonstrating CD62L expression on Foxp3⁺ iTreg in the LN or bone marrow are also shown. Mean ± SEM of two mice per condition are plotted. Data are representative of two independent experiments. ND, No data.

At day 5, CD62L was discriminately expressed on cells within the LN; however, the differential expression was not as dramatic as for CXCR4 (Fig. 10⇑B). By day 12, segregation of cells based on CD62L expression was more prominent. Approximately 80% of LN Foxp3⁺ cells (both nTreg and iTreg) expressed CD62L at day 12 (Fig. 10⇑C), whereas only ∼10% of Foxp3⁺ cells in the bone marrow expressed CD62L. This indicates that the CD62L⁺Foxp3⁺ iTreg population preferentially localizes to LN. Thus, adoptively transferred iTreg segregated into populations with distinct localization patterns that correlate with the expression of specific chemokine receptors or adhesion molecules.