Naturally Occurring Regulatory T Cells Regulate the Differentiation of
Pro-inflammatory Th17 Cells through PI3K-dependemt Mechanisms

Introduction
Helper T cells (Th cells) are playing central roles in every immune reaction; they
connect innate immunity (e.g. phagocytosis by macrophages) to adaptive immunity (e.g.
antibody production from activated B cells). Their depletion in HIV infection results ina severe immunodeficiency. There are three famous lineages of Th cells. All of them
derive from naive CD4-positive T cells through antigen presentation from dendritic cells
in lymph nodes, and their lineages are determined by the combination of cytokines
released from dendritic cells during antigen presentation (Figure 1). Th1 cells
differentiate in the presence of IL-12 and produce interferon-γ (IFN-γ) to activate
macrophages. Th2 cells are induced by IL-4, activating B cells through CD40/CD40L
interaction to produce antibodies. Th17 cells were identified in 2003 as a new lineage
producing IL-17 and activated by IL-23 (Aggarwal et al., 2003; Murphy et al., 2003),
and they are now changing our understandings of adaptive immunity, which has long
been investigated based on the Th1/Th2 theory published in 1986 (Mosmann et al.,1986). Th17 cells are now said to be important in the induction of inflammation.
Besides these Th cells, induced regulatory T cells (iTregs) arise from naive
CD4-positive T cells when only TGF-β is released from surrounding mesenchymes

1(Chen et al., 2003).
Th17 cells are now particularly spotlighted not only because they are newly
found, but also because they are highly pathogenic. Th17 cells differentiate from naive
CD4-positive T cells in the presence of IL-6 and TGF-β (Bettelli et al., 2006). During
differentiation they secret IL-21 in an autocrine fashion to proliferate (Nurieva et al.,2007), and IL-23 secreted from mesenchymal cells contributes to their further
maturation. Th17 cells are important in the induction of inflammatory reactions, andtheir long-lasting activation results in a variety of chronic inflammatory disorders such
as Crohn’s disease and rheumatoid arthritis (Langrish et al., 2005). Correspondingly,
large amount of Th17 cell infiltration has been detected in the tissues of those who
suffer from chronic inflammatory disorders (Pène et al., 2008), and experimentally, oral
dose of anti-IL-23 Ab could ameliorate the severity of experimental autoimmune
encephalomyelitis in mice (Chen et al., 2006).
Interestingly, the differentiation of proinflammatory Th17 cells andanti-inflammatory iTregs from naive CD4-positive T cells share a lot of mechanisms(Figure 2). Both need TGF-β from mesenchyme, which activate the phosphorylation of
Smad2 and Smad3 in these cells, followed by the induction of both Foxp3 and RORγt.
In this condition containing only TGF-β, Foxp3 directly binds to RORγt and suppresses
its transcriptional effect, and iTregs mainly differentiate from naive CD4-positive T
cells (Zhou et al., 2008; Ichiyama et al., 2008; Yang et al., 2008). Therefore, Foxp3

2

knockout mice cannot produce regulatory T cells (Hori et al., 2003). When IL-6 isadded to this condition, JAK2 associating with IL-6 receptors begins phosphorylation of
STAT3, and phosphorylated STAT3 dimerize in cytosol and translocate to nucleus,
where they further activate the transcription of RORγt. Therefore, in the Th-17
condition (IL-6 + TGF-β), RORγt/Foxp3 balance shift toward a RORγt-dominant state,polarizing naive CD4-positive T cells into Th17 cells. Actually, the knockout of RORγt
and mutations in STAT3 depleted the differentiation of Th17 cells (Ivanov et al., 2006;
Milner et al., 2008). There is also a report that RORα is also important in the
differentiation of Th17 cells, but its role and the problem whether they interact with
Foxp3 like seen in RORγt are still largely unknown (Yang et al., 2008).
During antigen presentation, signals from T cell receptors (TCRs) and fromco-stimulatory molecules CD28 are also essential for further maturation of naive
CD4-positive T cells. In this process, phosphoinositide 3-kinase (PI3K) becomes
activated in the downstream of these molecules. PI3K is a molecule that has a variety ofimportant roles in many kinds of cell types in our bodies. PI3K consists of catalytic
domain and regulatory domain, and the combination of catalytic domain p110δ andregulatory domain p85α is mainly expressed in immune cells (Koyasu, 2003); forinstance, PI3K is important in the determination of IL-12/IL-10 balance secreted fromdendritic cells (Ohtani et al., 2008; Weichhart et al., 2008; Fukao et al., 2002). The
activation of PI3K in the downstream of TCR and CD28 results in the up-regulation of3

many signal cascades. In many cells, PI3K phosphorylates Akt, which then results infollowing phosphorylation of mammalian target of rapamycin (mTOR) and glycogensynthase kinase 3 (GSK3). These mTOR and GSK3 have been reported tophosphorylate STAT3 (Kusaba et al., 2004; Baurel and Jope, 2008), which is one of the
key molecules in the induction of proinflammatory Th17 cells as described above.
Given all these facts, I used p85α knockout mouse to investigate the roles of PI3K in the
differentiation of Th17 cells in vitro.

Materials and Methods
Mice
Wild-type BALB/c mice (WT), Balb/c p85α+/- (KO) and p85α-/- (HT) mice were used inaccordance with the guidelines of the Institutional Animal Care and Use Committee of
Keio University School of Medicine.
Isolation of naive CD4-positive T cells
For negative sorting, splenocytes was incubated with biotinylated anti-CD8α Ab,anti-B220 Ab, anti-DX5 Ab, anti-MAC-1 Ab, anti-CD11c Ab and anti-TER119 Ab.
The cells were then incubated with magnetic beads coupled with streptavidin andnegatively sorted on autoMACS (miltenyibiotec). The negative fraction was treatedwith biotinylated anti-CD62L Ab and then streptavidin-magnetic beads again, followedby positive sorting by autoMACS. The purity of CD44-CD62L+CD4+ T cells (naive4CD4-positive T cells) was >90%. When CD4+CD62L+CD25- T cells were especiallyrequired, we further negatively sorted CD4+CD25+ T cells using anti-CD25 Ab,
magnetic beads coupled with streptavidin, and autoMACS.
Differentiation into Th1 and Th17 cells
1x106 cells were cultured for three to four days in RPMI1640 containing 10% FCS withplate-bound anti-CD3ε Ab (10μg/ml) and soluble anti-CD28 Ab (1μg/ml). For Th1 or
Th17 cell differentiation, IL-12 (10ng/ml), or IL-6 (30ng/ml), TGF-β (3ng/ml), IL-21(100ng/ml) and IL-23 (10ng/ml) were added, respectively.
Intracellular cytokine staining
Cultured cells were stimulated by PMA (50ng/ml) and ionomycin (1μg/ml) for twohours followed by brefeldin A (5μg/ml) treatment for another two hours. Cytokine
staining was performed using Intra Prep (Beckman Coulter) following manufacture’s
instruction. For Foxp3 staining, Foxp3 Staining Buffer Set (eBioscience) was used.
Cells were stained by FITC-labeled anti-IFN-γ Ab (2.5μg/ml) and PE-labeledanti-IL-17A Ab (1μg/ml) and analyzed by FACS. In another experiment, cells were
stained by FITC-labeled anti-IL-17A Ab (1μg/ml), Alexa-Fluor-labeled anti-IL-17F Ab(1μg/ml), and PE-labeled anti-Foxp3 Ab (1μg/ml).
ELISA
Supernatant of cultured medium was harvested and the concentration of each cytokine
was analyzed by Quantikine Mouse IFN-γ and Mouse IL-17 (R&D systems).

5Western blotting
Cell lysates at 0, 30, and 90 minutes after stimulation for Th17 cell differentiation wereseparated by SDS-PAGE and transferred to PVDF membrane. After the treatment by
TBS-T containing 5% skimmed milk, membrane was treated with primary Abs: rabbit
anti-pAkt Ab, rabbit anti-Akt Ab, rabbit anti-pTyr705 STAT3 Ab, rabbit anti-pSer727
STAT3 Ab, rabbit anti-STAT3 Ab, mouse anti-pERK2 Ab, rabbit anti-ERK2 Ab, andrabbit anti-c-Fos Ab. After washed by TBS-T containing 5% skimmed milk, membrane
was further treated by secondary Ab (HRP-conjugated anti-rabbit-IgG Ab or
HRP-conjugated anti-mouse-IgG Ab). Each band was detected by LAS3000 (Fuji) with
an ECL detection kit. For the detection of pAkt, Akt, and c-Fos, ECL advance (GEHealthscience) was adopted.
RT-PCR
Total RNA at 0, 24, 36, and 48 hours after stimulation for Th17 cell differentiation was
extracted, and cDNA was synthesized. For PCR reaction, the following probes wereused: RORγt, 5’-ACC TCC ACT GCC AGC TGT GTG CTG TC-3’ and 5’-TCA TTTCTG CAC TTC TGC ATG TAG ACT GTC CC-3’; Foxp3, 5’-CAC CCA GGA AAG
ACA GCA ACC-3’ and 5’-GCA AGA GCT CTT GTC CAT TGA-3’: IL-17F, 5’-CAG
CAG CGA TCA TCC CTC AAA G-3’ and 5’-CAG GAC CAG GAT CTC TTC
CTG-3’.

6

Results
CD4+CD62L+ T cells from KO mice showed impaired ability for Th17 differentiation.
In order to compare the abilities of naive CD4-positive T cells from KO and HT mice todifferentiate into Th17 cells, I first sorted CD4+CD62L+ T cells from spleens of thesemice, and the cells were cultured in both Th1- and Th17-cell conditions. After threedays, cells were harvested, stained for IFN-γ and IL-17, and analysed using FACS. In
the FACS analysis, CD4+CD62L+ T cells from KO mouse showed a decreased rate of
Th17 cell differentiation, which was <50% of the one marked by the CD4+CD62L+ T
cells from HT mouse (Figure 3A).
Next I examined the concentration of secreted IFN-γ and IL-17 in the culture
medium on Day 3 after stimulation using ELISA. In Th1-cell condition, theconcentrations of IL-17 in the culture medium were strikingly low both in the cells fromKO and HT mice, while high amount of IFN-γ secretion was marked in both, however,
little difference in the concentration of secreted IFN-γ was detected between the cells
from KO and HT mice (Figure 4A). In the Th17-cell condition, low IFN-γ concentrationand high IL-17 concentration in both culture medium was detected as expected, andIL-17 concentration in the medium containing cells from KO mouse was nearly only the
one-third of that from HT mouse (Figure 4B), which corresponds to the result that Th17
cell differentiation is suppressed in CD4+CD62L+ T cells sorted from KO mice.

7

No difference was detected in the phosphorylation level of STAT3 between
CD4+CD62L+ T cells from KO mice and HT mice.
Given the result that p85α depletion impaired Th17 cell differentiation, I next
investigated the phosphorylation level of STAT3 at Tyr705 and Ser727 because previous
studies had shown that the activation of mTOR and GSK3 in the downstream of PI3K
and Akt had increased the phosphorylation level of STAT3 (Kusaba et al., 2004; Baurel
and Jope, 2008). I sorted CD4+CD62L+ T cells from spleens of both KO and HT mice,and then stimulation for Th17 cell differentiation was added to both. At 0, 30, and 90
minutes after stimulation, cell lysates were harvested and examined the amount ofproteins listed in Figure 5 by western blotting.
As a result, I ascertained the phosphorylation of Akt was strongly inhibited in
CD4+CD62L+ T cells from KO mouse; however, there were no differences between
CD4+CD62L+ T cells from KO and HT mice in the phosphorylation level of STAT3 atboth Tyr705 and Ser727. Therefore, impaired differentiation of Th17 cells in
CD4+CD62L+ T cells from KO mice was not due to a decreased phosphorylation levelof STAT3 in cytosol.
On the other hand, I could detect that the phosphorylation level of Erk wasslightly suppressed in CD4+CD62L+ T cells from KO mouse, in which the expression
level of c-Fos was also largely decreased.

8CD4+CD62L+ T cells from KO mouse rather showed RORγt-dominant mRNA
expression pattern.
Given the results of recent studies that Foxp3 directly inhibits RORγt and that thebalance between RORγt and Foxp3 is important in the differentiation of both Th17 andiTreg cells (Zhou et al., 2008; Ichiyama et al., 2008; Yang et al., 2008), I next
performed RT-PCR analysis for RORγt, Foxp3, IL-17A, and IL-17F mRNA expressions
in CD4+CD62L+ T cells from KO and HT mice. CD4+CD62L+ T cells were sorted andwhole mRNA was harvested at the point of 0, 24, 48, and 72 hours after the stimulation
for Th17 cell polarization. Surprisingly, CD4+CD62L+ T cells from KO mouse revealed
lower Foxp3 mRNA level and elevated RORγt mRNA level compared to thecounterparts from HT mouse (Figure 6). Therefore, from the present our understandingsof Th17 cell differentiation, CD4+CD62L+ T cells from KO mouse themselves haverather a tendency to differentiate into Th17 cells, with their RORγt/Foxp3 balanceshifted strongly toward a RORγt-dominant state.
On the other hand, the amount of IL-17A and IL-17F mRNA expression wereboth lower in CD4+CD62L+ T cells from KO mouse than these cells from HT mouse;
therefore, these results strongly implies the presence of other mechanisms controllingthe differentiation of Th17 cells besides RORγt/Foxp3 balance.

Little difference in the ability for Th17 differentiation was detected between

9CD4+CD62L+CD25- T cells from KO mice and HT mice.
There is 10 to 20% of CD4+CD25+ naturally occurring regulatory T cells (nTregs) in the
population of CD4+CD62L+ T cells, hence I negatively sorted the CD4+CD25+ T cell
population from CD4+CD62L+ T cells both from KO and HT mice and differentiatedthese newly obtained CD4+CD62L+CD25- T cells into Th17 cells. Three days later,
cultured cells were stained for IL-17A, IL-17F, and Foxp3 and compared by FACS
analysis (Figure 3B).
Because nTregs were depleted from the cultured population, twofold increase in
the amount of IL-17Α expressing cells was detected in the cultured cell population from
HT mouse as expected, and surprisingly, the difference in the rate of Th17 celldifferentiation between KO and HT seen in CD4+CD62L+ cells vanished. Since ourlaboratory previously observed that there was little difference in the amount of
CD4+CD25+ T cell population between KO and HT mice (data not shown), it can beassumed that the ability of nTregs in KO mice to suppress Th17 cell differentiation
would have been elevated due to the p85α depletion.

Discussion
In this experiment, impaired STAT3 phosphorylation during Th17 cell differentiation
due to p85α depletion could not be detected, although there are many molecularbiological studies concluding that mTOR and GSK3 in the downstream of PI3K and Akt

10

could increase the phosphorylation level of STAT3 (Kusaba et al., 2004; Baurel and
Jope, 2008). Hence, the impaired Th17 cell differentiation in KO mouse couldn’t be
explained by an alteration in phosphorylation level of STAT3. Instead, I could detect aslight decrease in pErk level and a large drop in c-Fos expression level; hence, now it
can be assumed that the amount of transcriptional factor AP-1, which has also importantroles in immune system, would be lower in KO mice during Th17 cell differentiation.
New phenotype in Th17 cell differentiation and in chronic inflammation could beobserved with a c-Fos-depleted mouse.
In another study, it was reported that weaker TCR signaling and inhibition of
PI3K/Akt pathway resulted in elevated Foxp3 mRNA expression and increased
differentiation of iTregs (Sauer et al., 2008). Actually, I could get the same result in
iTreg differentiation; however, when it comes to Th17 cell differentiation, depletion of
p85α contrary decreased the level of Foxp3 mRNA expression and elevated the level ofRORγt mRNA expression. Hence, the mechanism of impaired Th17 cell differentiation
after p85α depletion also can not be explained by impaired expression of RORγt.
For an explanation of this impairment, the existence of nTregs in the cultured
population and their interaction with naive CD4-positive T cells now seem to beimportant. In this study, the effect of p85α depletion on Th17 cell differentiation
vanished when CD4+CD25+ T cells (nTregs) were removed from the cultured
population, implying the importance of PI3K signaling in the function of nTregs. The

11

roles of PI3K in the differentiation and function of both Th17 cells and nTregs have notbeen investigated well, although it is a well-known fact that PI3K has a lot of roles in
immune cells. The suppressive function of nTregs on effective Th cells is mainly
mediated by the expression of CTLA4 and the secretion of IL-10 (Jonuleit et al., 2001;
Wing et al., 2008), therefore the ability of KO mice to express CTLA4 and to produce
IL-10 should be examined as a next step, although there is an ambivalent report thatp110δ-/- mice revealed an impaired ability in IL-10 secretion during IL-2 stimulation(Patton et al., 2006). There is also another possibility that naive CD4-positive T cells
from KO mice are more susceptive to the suppressive function of nTregs. Hence, thesusceptivity of naive CD4-positive T cells from KO and HT mice to CTLA4 and IL-10
expressed by nTregs should also be compared. What is more, our laboratory’s datasuggest that depletion of p85α shows little difference in Th1 and Th2 differentiation;
hence, the distinctive role of nTregs on the differentiation of Th17 cells must also
investigated.
This nTreg’s particular suppression of Th17 cell differentiation and proliferation
seems to have a clinical significance. Now, the treatment of chronic inflammatory
disorders is highly dependent on the use of immunosuppressants such as corticosteroids
and methotrexate. These regimens frequently result in compromised immunities.
Recently, antibody drugs such as anti-TNFα antibody (infliximab) have been developed
as more potent and side-effect-free regimens, however, they are still very expensive.

12

Now that it is widely accepted that extreme activation of Th17 cells is important in thepathogenesis of these chronic inflammatory disorders and nTregs are necessary to
alleviate these long-lasting immune reactions, targeting PI3K/Akt pathway to modulate
the balance between nTreg function and Th17 cell differentiation could serve as a strong
or an alternative regimen in the future.

Conclusion
Depletion of p85α resulted in an impaired ability for Th17 cell differentiation fromnaive CD4-positive T cells, and this was observed only when nTregs were co-cultured
with naive CD4-positive T cells. From the results of previous studies and thisexperiment, it could be assumed that this impairment in Th17 cell differentiation would
be achieved by either or both of following two mechanisms: an elevated function ofnTregs to suppress the differentiation and proliferation of Th17 cells, and an elevatedsusceptivity of Th17 cells to these nTreg functions.

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cell

differentiationbyantagonizing

Figures

Figure 1 | Antigen presentation and differentiation of Th cells.

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Figure 2 | Intracellular signaling cascades in the down stream of cytokine
receptors.

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Figure 3 | Results of FACS analysis. (A) Impaired Th17 cell differentiation in
CD4+CD62L+ T cells of KO mouse. CD4+CD62L+ T cells from KO and HT mice were
sorted and differentiated into Th1 and Th17 cells. After three days, cells were stained for
IL-17A and IFN-γ. (B) No impairment in Th17 cell differentiation was detected when
CD4+CD62L+CD25- T cells from KO and HT mice were compared.
CD4+CD62L+CD25- T cells from KO and HT mice were sorted and differentiated into
Th17 cells. After three days of culture, they were stained for Foxp3 and IL-17A or
IL-17F.

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Figure 4 | Results of ELISA. (A) Little difference in IFN-γ secretion from Th1 cells
differentiated from CD4+CD62L+ T cells of KO and HT mice. (B) Impairment in IL-17
secretion from Th17 cells differentiated from CD4+CD62L+ T cells of KO mouse.

Figure 5 | Results of western blotting. The phosphorylation level of Akt was strongly
suppressed in CD4+CD62L+ T cells from KO; however, there ware no differences in thephosphorylation level of STAT3 at both Tyr705 and Ser727 between these cells fromKO and HT.

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Figure 6 | Results of RT-PCR. The Foxp3/RORγt balance in the CD4+CD62L+ T cells
from KO was rather RORγt-dominant.

24