Retinoic acid suppresses IL-17 production and pathogenic activity of γδ T cells in CNS autoimmunity.

TL;DR: The findings demonstrate that the anti‐inflammatory properties of RA are mediated in part by suppressing STAT3‐mediated activation of cytokine production and cytokine receptor expression in γδ T cells, which suppresses their ability to activate Th17 cells.

Abstract: Retinoic acid (RA) in the steady state enhances induction of Foxp3(+) regulatory T (Treg) cells and inhibits differentiation of Th1 and Th17 cells, thereby maintaining tolerance, but can in inflammatory conditions promote effector Th1 and Th17 cells that mediate inflammation. IL-17-producing γδ T cells have recently been shown to have a major pathogenic role in autoimmune diseases. Here, we examined the immunomodulatory effects of RA on γδ T cells. We found that RA had a dramatic suppressive effect on IL-17A and IL-17F production by γδ T cells stimulated with IL-1β and IL-23. RA suppressed RORγt, IL-1R and IL-23R expression in γδ T cells. Treatment of mice with RA suppressed IL-17 production by γδ T cells in vivo. Furthermore, treatment of T cells with RA attenuated their ability to induce disease in experimental autoimmune encephalomyelitis (EAE), a murine model for multiple sclerosis. This was associated with a reduction in the number of central nervous system-infiltrating γδ T cells, but also CD4(+) T cells that produced IL-17A, IL-17F or GM-CSF. Interestingly, treatment of γδ T cells with RA or removal of γδ T cells from a bulk population of T cells significantly reduced their capacity to induce EAE, demonstrating a critical role for γδ T cells in promoting pathogenic Th17 cells. Our findings demonstrate that the anti-inflammatory properties of RA are mediated in part by suppressing STAT3-mediated activation of cytokine production and cytokine receptor expression in γδ T cells, which suppresses their ability to activate Th17 cells.

Summary

RA inhibits IL-17 production by γδ T cells in vitro and in vivo

• T cells (Th17 cells) have been shown to have pathogenic roles in a range of autoimmune diseases, including MS and EAE where they promote inflammation.
• It has previously been reported that retinoids can attenuate development of EAE.
• This may explain the overall reduction in IFN-γ production detected by ELISA on cultures of mixed LN and spleen cells stimulated with MOG, IL-1β and IL-23 .

RA mediates immunomodulatory activity largely through its effect on γδ T cells

• Spleen and lymph nodes cells from mice immunized with MOG and complete Freund's adjuvant were depleted of γδ T cells, or left un-separated, before being stimulated for 3 days in Th17-polarizing conditions and then transferred to recipient animals.
• The γδ T cells were stimulated for 6 h with IL-1β and IL-23, in the presence of RA or its vehicle, before being washed thoroughly and cultured with the γδ T-celldepleted fraction.
• The combined cells were then cultured with IL-1β, IL-23 and MOG for 3 days and transferred to recipient mice.
• The frequency and absolute number of CD4 +.
• T cells producing IL-17A, IFN-γ and GM-CSF was reduced in the brains of the mice injected with the cells containing RA-treated γδ T cells .

RA inhibits the IL-1β and IL-23 pathway in γδ T cells

• The authors found that, within 3 h of culture with IL-1β and IL-23, RA inhibited the expression of the receptors subunits for IL-1β and IL-23, which transduce the signals to promote IL-17A and IL-17F expression in γδ T cells .
• Interestingly, stimulation of purified γδ T cells with IL-12 and IL-18, which are more potent stimuli for IFN-γ production, induced low concentration of IL-17A, which was significantly enhanced by RA, while the production of IL-17A induced by IL-1β and IL-23 was inhibited by RA .
• T cells expressing cell surface receptors for IL-1β and IL-23 .
• The activation of the IL-1β/IL-23 pathway is known to involve the phosphorylation of STAT3, which induces Th17-type cytokine in IL-17-producing cells.
• The authors found that treatment of cells from MOG-immunized mice with RA inhibited STAT3 phosphorylation .

DISCUSSION

• The authors demonstrate that RA significantly inhibits the function of IL-17A-producing γδ T cells, impairing their proliferation, cytokine production and their pathogenic activity in vivo in the EAE model.
• In addition, the authors show that under steady state conditions and during inflammation, RA also inhibits cytokine production by Th17 cells, which are considered to be the primary pathogenic T cells in EAE and other autoimmune diseases.
• 28 There is convincing evidence from animal models that Th17 cells and more recently IL-17-secreting γδ T cells have crucial pathogenic roles in EAE and other autoimmune diseases.
• T cells or even when γδ T cells are cultured with IL-12 and IL-18, the classical stimuli for IFN-γ production.
• The authors found that while RA induces the expression of TGF-β during EAE, the production of IL-10 is decreased (unpublished observations), indicating that RA does not protect against disease by inducing Treg cells.

METHODS Mice

• C57BL/6 mice (Harlan, Bicester, UK) were bred under specific pathogen free conditions and maintained according to European Union regulations.
• All experiments were performed under license from the Health Products Regulatory Authority and with approval from the Trinity College Dublin Animal Research Ethics Committee.
• The mice were co-housed for every EAE experiment.

Induction and assessment of EAE by adoptive transfer

• Donor mice were immunized subcutaneously with 100 μg of MOG 35-55 peptide (GenScript, Piscataway, NJ, USA) emulsified in complete Freund's adjuvant containing 4 mg ml − 1 (0.4 mg per mouse) heat-killed Mycobacterium tuberculosis (Chondrex, Redmond, WA, USA).
• After 10 days, the mice were killed, their brachial, axillary and inguinal lymph nodes and spleens were collected and a single cell suspension was prepared.
• In some experiments, γδ T cells were purified by magnetic-activated cell sorting prior to the culture and either depleted from the culture or added back to the rest of the cells .
• The cells were then stimulated for 3 days with MOG, IL-1β and IL-23.
• The animals were weighed and monitored for signs of EAE daily.

Isolation and FACS analysis of brain cells

• Mice were killed and perfused with phosphate-buffered saline, and their brains were isolated.
• The concentrations of the cytokines IL-17A, IL-17F and IFN-γ in culture supernatants were quantified by ELISA.

Flow cytometry

• Cells were washed before being incubated with a live/dead stain and then surface-stained with antibodies specific for CD3, CD4, CD8, δTCR, Vγ1, Vγ4, IL-1R1, IL-23R and CCR6.
• The cells were washed, fixed and permeabilized using 2% PFA (Pierce, Walthman, MA, USA) or the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA), which allow the staining of intranuclear proteins.
• The cells were stained in 0.5% saponin (Sigma-Aldrich) or permeabilization buffer containing antibodies directed against IL-17A, IL-17F, IFN-γ, GM-CSF, RORγt, Tbet and Ki67.
• Cells were analyzed using a flow cytometer LSRFortessa (BD) and the data were analyzed with FloJo software.

Reverse transcription-PCR

• RNA was extracted from the cells using TRIzol (Invitrogen, Grand Island, NY, USA) and reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA).
• Rara, il17a, il17f, rorc, il1r1 and il23r expression were quantified by RT-PCR using commercially available primers and an ABI PRISM7500 Sequence Detection System (Applied Biosystems).
• The amount of each cytokine was determined by normalization to 18S rRNA.

Western blots

• Supernatants were assayed for protein concentration using the Pierce 660 nm protein assay and diluted to the same protein concentration with lysis buffer.
• Samples were run on a 10% SDS-PAGE gel, transferred to polyvinylidene difluoride and analyzed by immunoblot using polyclonal rabbit antibody raised against mouse phospho-STAT3 (Cell Signaling, Danvers, MA, USA), mouse monoclonal antibody raised against beta actin (Abcam, Cambridge, MA, USA), anti-mouse IgG, HRP conjugate (Promega, Fitchburg, WI, USA) and anti-rabbit IgG, HRP conjugate (Dako, Carpinteria, CA, USA).

Statistics

• Statistical analysis was performed using GraphPad Prism 5.
• Analysis of variance with Bonferroni post-test or Student t test were used to compare statistical differences of means between groups.

Figures

Figure 5 Depletion of γδ T cells attenuates EAE induced by T-cell transfer. Spleen and lymph node cells from MOG-immunized mice were depleted of γδ T cells or un-depleted. Cells were cultured for 3 days with MOG, IL-1β +IL-23, and then 15×106 cells were transferred to naive mice. (a) IL-17A, IL-17F and IFN-γ concentration measured by ELISA in supernatants the cultures after 3 days. (b) Representative FACS plots of IL-17A, RORγt, IFN-γ and Tbet expression in T helper cells at the end of the 3-day culture. (c) EAE clinical scores of recipient mice. Data are representative of two independent experiments and are shown as mean+s.d. (a) or mean± s.e.m. (c). Statistics were calculated using two-tailed unpaired t-test (a) or a two-way analysis of variance with Bonferroni post-test (c). *Po0.05, **Po0.01 and ***Po0.001.

Figure 1 RA impairs IL-17A but not IFN-γ production by γδ T cells and CD4+ T cells. (a, b) Spleen cells from naive mice were cultured for 3 days with medium or anti-CD3 and in combination with IL-1β and IL-23 and in the presence or absence of RA. (a) Representative FACS plots of IL-17A expression by γδ T cells and CD4+ T cells. (b) Mean absolute numbers of IL-17A+ and IFN-γ+ γδ T cells and CD4+ T cells in the cultures. (c–f) γδ T cells purified from lymph nodes of naive mice were cultured for 3 days with IL-1β+IL-23, with or without RA. (c) IL-17A, IL-17F and IFN-γ concentration measured by ELISA in the supernatants of purified γδ T cells cultured for 3 days with IL-1β+IL-23, with or without RA. (d) IL-17A and IL-17F concentration measured by ELISA in the supernatants of purified Vγ1+ or Vγ4+ γδ T cells cultured for 3 days with IL-1β+IL-23, with or without RA. (e) Relative expression of rara in CD27+ and CD27− γδ T cells purified by FACS. (f, g) Representative FACS plots (f) and mean frequencies (g) of Vγ1+ and Vγ4+ T cells expressing IL-17A and RORγt after 3 days of culture with IL-1β+IL-23, with or without RA. Data are representative of three independent experiments. Bars are mean+s.d.; *Po0.05, **Po0.01 and ***Po0.001, two-tailed unpaired t-test.

Figure 6 Selective treatment of γδ T cells with RA decreases the production of IL-17A and IL-17F during EAE and alters the capacity of T cells from MOG-immunized mice to induce EAE by cell transfer. Spleen and lymph node cells from MOG-immunized mice were isolated and the γδ T cells purified and cultured separately with RA or its vehicle for 6 h before being put back in culture with the rest of the cells. The total cells containing either the vehicle- or the RA-treated γδ T cells were then cultured with MOG, IL-1β and IL-23. After 3 days of culture, 10×106 cells were transferred to naive mice. (a) IL-17A and IL-17F expression as measured by ELISA in the supernatants at the end of the 3-day culture. (b) Mean absolute numbers of IL-17A+, IL-17F+ and IFN-γ+ Vγ4+ γδ T cells at the end of the 3-day culture. (c) Representative FACS plots indicating the proportion of IL-17A+ Vγ4+ and IL-17A+ Vγ4− γδ T cells at the end of the 3-day culture. (d) EAE clinical scores of recipient mice. (e) Representative FACS plots indicating the proportion of IL-17A+ CD4+ T cells in the brains of the recipient mice on day 25 post transfer. (f) Absolute numbers of infiltrating IL-17A+, IFN-γ+ and GM-CSF+ CD4+ T cells in the brains of the recipient mice on day 25 post transfer. Data are representative of two independent experiments. Bars are mean+s.d. (a, b), mean± s.e.m. (d) or mean+s.e.m. (f). Statistics are performed as and two-tailed unpaired t-test (a–c and f) or two-way analysis of variance with Bonferroni post-test (d); *Po0.05, **Po0.01 and ***Po0.001.

Figure 2 RA inhibits IL-17 production by γδ T cells and CD4+ T cells in vivo. (a, b) Representative FACS plots (a) and absolute numbers (b) of IL-17A+ and IFN-γ+ γδ T cells and CD4+ T cells isolated from peritoneal cavity 18 h after injection of RA or vehicle only (dimethyl sulfoxide) and re-stimulation with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A. Bars are mean +s.e.m.; *Po0.05 and ***Po0.001, two-tailed unpaired t-test.

Figure 3 RA suppresses IL-17 production and RORγt expression in CD4+ T cell and γδ T cell and impairs Vγ4+ T-cell proliferation under inflammatory conditions. Mice were immunized subcutaneously with MOG emulsified in complete Freund’s adjuvant. Ten days after immunization, lymph node and spleen cells were cultured together for 3 days with MOG, IL-1β and IL-23 and either RA or vehicle. (a) IL-17A, IL-17F and IFN-γ production was quantified in supernatants by ELISA at the end of the 3-day culture. (b) Representative FACS plots of the IL-17A+ and IFN-γ+ CD4+ T cells and IL-17A+ Vγ4+ γδ T cells at the end of the 3-day culture. (c) Mean absolute numbers of IL-17A+ and IFN-γ+ CD4+ T cells and γδ T cells at the end of the 3-day culture. (d, e) Relative expression of il17a, il17f (d) and rorc (e) mRNA measured by RT-PCR on CD4+ T cells and γδ T cells after 3 days of culture. (f) Representative histogram, mean frequencies and mean fluorescence intensity quantifying Ki67 expression in Vγ4+ T cells at the end of the 3-day culture. (g) Absolute number of Vγ4+ γδ T cells after 3 days of culture. Bars are mean+s.d. *Po0.05, **Po0.01 and ***Po0.001 (two-tailed unpaired t-test).

Figure 7 RA impairs the IL-1β and IL-23 pathway in γδ T cells but does not alter cytokine production induced by IL-12 and IL-18. (a) Relative expression of il1r1 and il23r mRNA measured by RT-PCR in γδ T cells purified from naive mice and cultured for 3 h with IL-1β+IL-23, with or without RA. (b, c) γδ T cells purified from lymph nodes of naive mice were cultured for 24 h with IL-1β+IL-23 or IL-12+IL-18, and with or without RA. Mean frequencies (b) and corresponding FACS plots (c) of IL-17A+ Vγ4+ CCR6+ γδ T cells and IFN-γ+ γδ T cells in the culture. (d) Cells from MOG-immunized mice were cultured with vehicle, MOG or MOG+RA for 24 h and absolute numbers of IL-17A+ γδ T cells and CD4 T cells were determined by FACS. (e–g) Cells from MOG-immunized mice were cultured for 3 days with MOG, IL-1β and IL-23 and either RA or its vehicle. (e) Relative expression of il1r1 and il23r mRNA measured by RT-PCR on γδ T cells sorted at the end of the 3-day culture. (f, g) Representative FACS plots (f) and mean frequencies (g) of IL-1R1 and IL-23R expression on Vγ4+ T cells after 3 days of culture. (h) Western blots showing the effect of RA on STAT3 phosphorylation (pSTAT3) with actin as a control after 24, 48 and 72 h of culture (V: vehicle). Bars are mean+s.d.; *Po0.05, **Po0.01 and ***Po0.001, two-tailed unpaired t-test.

Figure 4 Treatment of T cells from MOG-immunized mice with RA suppresses their ability to induce EAE by adoptive transfer. Donor mice were immunized with MOG+complete Freund’s adjuvant and after 10 days, spleen and lymph node cells were cultured for 3 days with MOG, IL-1β+IL-23 and RA or vehicle and then 15×106 cells were transferred to naive mice. (a) EAE clinical scores in recipient mice. (b) Absolute number of CD3+ T cells detected in the brains of the recipient mice 16 days post transfer. Representative FACS plots (c) and mean absolute numbers (d) of infiltrating IL-17A+, IFN-γ+, IL-17F+ and GMCSF+ CD4+ and γδ T cells in the brains of the recipient mice 16 days post transfer and re-stimulated ex vivo for 5 h with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A. Data are representative of three independent experiments. Data shown as mean± s.e.m. (a) or mean+s.e.m. (b, d) and statistics were calculated using a two-way analysis of variance with Bonferroni post-test (a) or two-tailed unpaired t-test (b–d). *Po0.05, **Po0.01 and ***Po0.001.

Full text

ORIGINAL ARTICLE
Retinoic acid suppresses IL-17 production and
pathogenic activity of γδ T cells in CNS autoimmunity
Mathilde Raverdeau
1
, Conor J Breen
2
, Alicja Misiak
1
and Kingston HG Mills
1
Retinoic acid (RA) in the steady state enhances induction of Foxp3
+
regulatory T (Treg) cells and inhibits differentiation of Th1
and Th17 cells, thereby maintaining tolerance, but can in inflammatory conditions promote effector Th1 and Th17 cells that
mediate inflammation. IL-17-producing γδ T cells have recently been shown to have a major pathogenic role in autoimmune
diseases. Here, we examined the immunomodulatory effects of RA on γδ T cells. We found that RA had a dramatic suppressive
effect on IL-17A and IL-17F production by γδ T cells stimulated with IL-1β and IL-23. RA suppressed RORγt, IL-1R and
IL-23R expression in γδ T cells. Treatment of mice with RA suppressed IL-17 production by γδ T cells in vivo. Furthermore,
treatment of T cells with RA attenuated their ability to induce disease in experimental autoimmune encephalomyelitis (EAE),
a murine model for multiple sclerosis. This was associated with a reduction in the number of central nervous system-infiltrating
γδ T cells, but also CD4
+
T cells that produced IL-17A, IL-17F or GM-CSF. Interestingly, treatment of γδ T cells with RA or
removal of γδ T cells from a bulk population of T cells significantly reduced their capacity to induce EAE, demonstrating a
critical role for γδ T cells in promoting pathogenic Th17 cells. Our findings demonstrate that the anti-inflammatory properties of
RA are mediated in part by suppressing STAT3-mediated activation of cytokine production and cytokine receptor expression in
γδ T cells, which suppresses their ability to activate Th17 cells.
Immunology and Cell Biology (2016) 94, 763–773; doi:10.1038/icb.2016.39
At the border between innate and adaptive immunity, γδ Tcells
express T-cell receptor (TCR) that recognize non-major histo-
compatibility complex-restricted antigens, but also express receptors
for cytokines and Toll-like receptors. These cells are rapidly activated
upon inflammation, stress or infection, provide early sources of
cytokines such as IFN-γ, IL-17 and IL-21, and enhance immune
responses, for instance by influencing antigen-specificCD4
+
Tcells.
1–3
In experimental autoimmune encephalomyelitis (EAE), a mouse
model for multiple sclerosis (MS), the role of γδ Tcells,and
particularly of IL-17-producing γδ T cells has been the subject of
debate, although the consensus view is that they have a pathogenic role
in this disease.
1,4–6
γδ T cells secrete IL-17 in response to stimulation
with IL-23 and IL-1β,IL-1α or IL-18, without TCR engagement.
3,7
During development of EAE, IL-17
+
γδ T cells and in particular the
Vγ4subtypeofγδ T cells are the first cells to produce IL-17 and are
found at high numbers in the brains of the mice early in the course of
disease.
2,3,8
Moreover, mice lacking functional γδ T cells (TCRδ
− / −
mice) have delayed and attenuated EAE and the production of IL-17A
by autoantigen-specific Th17 cells is reduced in these mice, indicating
that γδ T cells enhance the pathogenicity of CD4
+
T cells in this
model.
3,9
Activated γδ T cells also antagonize the anti-inflammatory
activity of regulatory T (Treg) cells and inhibit their differentiation,
thereby enhancing the activity of effector T cells that mediate the
development of EAE.
2
Retinoic acid (RA), the active metabolite of vitamin A, is a powerful
immunomodulator, which, in the steady state, promotes immune
tolerance by enhancing Foxp3
+
Treg cells and inhibiting the differ-
entiation of Th17 cells.
10
However, in inflammatory conditions,
RA can promote effector Th1 and Th17 and thereby enhance
inflammation.
11,12
AM80, a selective agonist of the RA nuclear
receptor-α (RARα) has been shown to have a protective effect in
the early stages of EAE by inhibiting the differentiation of pathogenic
Th17 cells.
12–15
Administered at later stages of the disease, AM80
delays the recovery from EAE by inducing the apoptosis and
differentiation of immature myeloid-derived suppressor cells, a cell
population that exerts immunosuppressive effects on effector T cells.
16
RA has also been shown to protect mice from EAE by inhibiting the
maturation of dendritic cells and monocytes thereby impairing their
antigen presentation function.
17
We have previously reported that RA has a protective effect in a
mouse model of colitis by promoting IL-22 production by γδ Tcells
and type 3 innate lymphoid cells (ILC3).
18
Given the important role of
IL-17A-producing γδ T cells in the development of EAE, we addressed
the hypothesis that RA may be protective in EAE through modulating
the function of γδ T cells. We found that RA significantly inhibited
1
Immune Regulation Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland and
2
Department of
Biology, Maynooth University, Maynooth, Kildare, Ireland
Correspondence: Professor KHG Mills, Immune Regulation Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin,
Dublin 2, Ireland.
E-mail: kingston.mills@tcd.ie
Received 2 February 2016; revised 12 April 2016 ; accepted 13 April 2016; accepted article preview online 19 April 2016; advance online publication, 10 May 2016
Immunology and Cell Biology (2016) 94, 763– 773
&
2016 Australasian Society for Immunology Inc. All rights reserved 0818-9641/16
www.nature.com/icb

IL-17A and IL-17F production by γδ TcellsandCD4
+
Tcells
stimulated with IL-1β and IL-23, but the effect on γδ T cells was
more dramatic. Furthermore, treatment of γδ TcellswithRA
significantly inhibited the capacity of T cells from myelin
oligodendrocyte glycoprotein (MOG)-immunized mice to induce
EAE by adoptive transfer. In addition, RA impaired the differentiation
of IL-17A-expressing γδ T cells by inhibiting RORγtexpressionand
downregulating expression of receptors for IL-1β and IL-23.
RESULTS
RA inhibits IL-17 production by γδ Tcellsin vitro and in vivo
We have previously demonstrated that γδ T cells secrete IL-17 and
IFN-γ following stimulation with IL-1β and IL-23 without TCR
activation, whereas CD4
+
memory T cells secrete IL-17 following
stimulation with IL-1β and IL-23 in the presence of anti-CD3.
3,19
Here, we examined the effect of RA on cytokine production by γδ
T cells. We found that RA suppressed IL-17 production by both γδ
T cells within a bulk population of spleen cells stimulated with IL-1β
and IL-23 (Figures 1a and b). IL-1β and IL-23 also induced IL-17
production by CD4
+
T cells but only when co-stimulated with anti-
CD3 and this was also inhibited by co-culture with RA (Figures 1a
and b). We then examined the effect of RA on purified γδ Tcellsor
Vγ1
+
or Vγ4
+
γδ T cells from lymph nodes of naive mice. Stimulation
of γδ T cells with RA alone did not change basal cytokine production
(Figure 1c). Stimulation of γδ T cells with IL-1β and IL-23 induced
high concentrations of IL-17A and IL-17F, mainly by Vγ4
+
Tcells
(Figures 1c–e), as well as IFN-γ (Figure 1c). The addition of RA
significantly reduced the production of IL-17A and IL-17F (Figures 1c,
d and f). In contrast, RA did not affect the production of IFN-γ by γδ
T cells (Figure 1c and data not shown). The expression of the nuclear
receptor for RA, RARα, was 34 times higher in the IL-17-producing γδ
T cells (CD27
−
)whencomparedwithγδ T cells that express IFN-γ
(CD27
+
) (Figure 1e). Interestingly, the expression of the transcription
factor RORγt, required for the production of IL-17, was impaired by
0
2
4
6
8
Vehicle
RA
IL-17A IL-17F IFN-γ
***
ns
***
IL-1β + IL-23
IL-1β + IL-23 + RA
Cytokine (ng/ml)
0
10
20
30
40
50
*
Veh
RA
IL-1β + IL-23
IL-1β + IL-23 + RA
*
*
RORγt
+
γδ T cells (%)
0
1
2
3
4
5
Vγ1
+
***
***
Vγ4
+
Vγ1
+
Vγ4
+
Vγ1
+
Vγ4
+
Vehicle IL-1β + IL-23 IL-1β + IL-23 + RA
IL-17A (ng/ml)
0
1
2
3
***
IL-17F (ng/ml)
0.0
0.5
1.0
1.5
2.0
**
Vehicle
RA
IL1-β + IL-23
IL1-β + IL-23 + RA
IL-17A
+
γδ T cells (x10
3
)
0
1
2
3
**
**
+ Anti-CD3
IL-17A
+
CD4
+
T cells (x10
3
)
CD27
+
CD27
-
0
10
20
30
40
50
*
Rara mRNA (RE)
RORγt
IL-17A
Vγ4
+
γδ T cellsVγ1
+
γδ T cells
0.2
5.2
5.5
1.805.3
0.3
24.9
25.6
8.1021.3
Vehicle
RA
Vehicle
RA
13.5 1.7
δ TCR
Vehicle
γδ T cells
RA
IL-17A
1.3
CD4
Vehicle
CD4
+
T cells
RA
0.4
Figure 1 RA impairs IL-17A but not IFN-γ production by γδ T cells and CD4
+
Tcells.(a, b) Spleen cells from naive mice were cultured for 3 days with
medium or anti-CD3 and in combination with IL-1β and IL-23 and in the presence or absence of RA. (a) Representative FACS plots of IL-17A expression by
γδ T cells and CD4
+
Tcells.(b) Mean absolute numbers of IL-17A
+
and IFN-γ
+
γδ TcellsandCD4
+
T cells in the cultures. (c–f) γδ T cells purified from
lymph nodes of naive mice were cultured for 3 days with IL-1β+IL-23, with or without RA. (c) IL-17A, IL-17F and IFN-γ concentration measured by ELISA in
the supernatants of purified γδ T cells cultured for 3 days with IL-1β+IL-23, with or without RA. (d) IL-17A and IL-17F concentration measured by ELISA in
the supernatants of purified Vγ1
+
or Vγ4
+
γδ T cells cultured for 3 days with IL-1β+IL-23, with or without RA. (e) Relative expression of rara in CD27
+
and
CD27
−
γδ Tcellspurified by FACS. (f, g) Representative FACS plots (f) and mean frequencies (g)ofVγ1
+
and Vγ4
+
T cells expressing IL-17A and RORγt
after 3 days of culture with IL-1β+IL-23, with or without RA. Data are representative of three independent experiments. Bars are mean+s.d.; *Po0.05,
**Po0.01 and ***Po0.001, two-tailed unpaired t-test.
Immunomodulatory effects of RA on γδ Tcells
M Raverdeau et al
764
Immunology and Cell Biology

RA in γδ T cells stimulated or not with IL-1β and IL-23, and
particularly in Vγ4
+
T cells (Figures 1f and g).
We next assessed the effect of RA on cytokine production by γδ
TcellsandCD4
+
Tcellsin vivo. The production of IL-17A by γδ
T cells was strongly inhibited in mice injected with RA when
compared with mice injected with vehicle (Figures 2a and b). The
production of IL-17A by CD4
+
T cells was also affected in the mice
injected with RA but to a lesser extent when compared with the effect
on γδ T cells (Figures 2a and b). In contrast, RA did not affect the
production of IFN-γ by γδ T cells or CD4
+
Tcells(Figure2b).
RA inhibits IL-17 production by CD4 and γδ T cells from mice with
EAE
IL-17A-producing γδ TcellsandCD4
+
T cells (Th17 cells) have been
shown to have pathogenic roles in a range of autoimmune diseases,
including MS and EAE where they promote inflammation. It has
previously been reported that retinoids can attenuate development of
EAE.
15,17
However, these studies did not examine the effect of RA on
γδ T cells. Having shown that RA impairs IL-17A production mainly
by γδ TcellsbutalsobyCD4
+
T cells, we examined its effect on γδ
TcellsandCD4
+
T cells from mice with EAE. Mice were immunized
with the myelin antigen MOG emulsified in complete Freund’s
adjuvant and 10 days later their spleens and draining lymph nodes
cells were isolated and cultured with MOG, IL-1β and IL-23 to drive
the differentiation of IL-17-producing γδ T cells and Th17 cells, and in
the presence of RA or vehicle control. The production of IL-17A,
IL-17F and IFN-γ measured in the supernatant of the cultures by
ELISA was strongly attenuated by addition of RA (Figure 3a). We next
examined more closely the effect of RA on cytokine production by γδ
TcellsandCD4
+
T cells using intracellular cytokine staining and
fluorescence-activated cell sorting (FACS) analysis (Figures 3b and c)
or by RT-PCR performed on γδ TcellsandCD4
+
T cells sorted at the
end of the culture (Figure 3d). We found that RA inhibited IL-17A
principally in γδ T cells and particularly in the Vγ4
+
γδ Tcells,but
also in CD4
+
Tcells(Figures3b–d). The expression of IL-17F was also
strongly reduced by RA in both γδ TcellsandCD4
+
Tcells
(Figure 3d). Interestingly, the number of IFN-γ
+
γδ T cells was
unchanged and the number of IFN-γ
+
CD4
+
T cells was increased by
RA (Figures 3b and c). However, assessment of other IFN-γ-producing
cell types by FACS indicated that RA impaired IFN-γ expression in
CD8
+
T cells (data not shown). This may explain the overall reduction
in IFN-γ production detected by ELISA on cultures of mixed LN and
spleen cells stimulated with MOG, IL-1β and IL-23 (Figure 3a).
We also assessed the effect of RA on expression of the gene coding
for RORγt by RT-PCR in γδ TcellsandCD4
+
T cells purified from
the 3-day cultures and found that the expression of rorc was strongly
reduced by RA in both cell types (Figure 3e). We also found that the
proliferation of γδ T cells (but not CD4
+
T cells) and more specifically
of the Vγ4
+
γδ T cells was impaired by treatment with RA leading to a
reduced number of Vγ4
+
γδ T cells in the culture (Figures 3f and g
and data not shown). Collectively, these findings demonstrated that
RA suppresses IL-17 production by γδ T cells and Th17 cells and
affects the polarization of these cells. Our data also show that RA has
the most pronounced inhibitory effect on activation and proliferation
of IL-17-secreting Vγ4
+
γδ T cells.
RA suppresses the pathogenic function of γδ TcellsandCD4
+
T cells in the EAE model
Having shown that RA inhibits IL-17A and IL-17F production
principally by γδ T cell but also by CD4
+
T cells from MOG-
immunized mice, we examined the effect of RA on the effector and
pathogenic function of these cells in EAE by adoptive transfer to naive
mice. Spleen and lymph nodes cells from mice immunized with MOG
and complete Freund’s adjuvant were cultured under Th17-polarizing
conditions (MOG, IL-1β and IL-23) in the presence of RA or a vehicle
control. After 3 days, the cells were washed thoroughly and transferred
to naive recipient mice (15 × 10
6
cells per mouse). The mice injected
with the vehicle-treated cells developed signs of EAE by day 7
(Figure 4a). In contrast, the onset of EAE was delayed by several days
and the clinical signs of disease were strongly attenuated in mice
injected with the RA-treated cells (Figure 4a). An examination of
infiltrating lymphocytes in the brains of the mice 16 days after the cell
transfer revealed that the number of CD3
+
Tcellsinfiltrating the brains
was greatly reduced in the recipients of the RA-treated cells when
compared with the mice injected with the control cells (Figure 4b).
Moreover, the number of CD4
+
Tcellsandγδ Tcellsandthe
production of the cytokines IL-17A, IL-17F, IFN-γ and GM-CSF by
these cells were significantly lower in the brains of the animals injected
with the RA-treated cells when compared with the control group
(Figures 4c and d). Thus, RA decreased the production of IFN-γ,
IL-17A and IL-17F induced by IL-1β and IL-23 and particularly the
production of IL-17A and IL-17F by γδ T cells and Th17 (Figure 3),
which resulted in a reduced number of pathogenic γδ T cells and CD4
+
0
2
4
6
CD4
***
*
γδ
Vehicle
RA
IL-17A
+
cells (x10
3
)
0
10
20
30
ns
ns
CD4
γδ
IFN-γ
+
cells (x10
3
)
IFN-γ
IL-17A
γδ T cells
CD4
+
T cells
Vehicle
51.1
12.9
17.7
0.117.40.9
0.9
20.7
28.8
0.10.60.1
51.1
17.4
RA
Vehicle
RA
Figure 2 RA inhibits IL-17 production by γδ T cells and CD4
+
T cells in vivo.
(a, b) Representative FACS plots (a)andabsolutenumbers(b) of IL-17A
+
and
IFN-γ
+
γδ TcellsandCD4
+
T cells isolated from peritoneal cavity 18 h after
injection of RA or vehicle only (dimethyl sulfoxide) and re-stimulation with
phorbol 12-myristate 13-acetate, ionomycin and brefeldin A. Bars are mean
+s.e.m.; *Po0.05 and ***Po0.001, two-tailed unpaired t-test.
Immunomodulatory effects of RA on γδ Tcells
M Raverdeau et al
765
Immunology and Cell Biology

Tcellsinfiltrating the brains and a consequent attenuation of the
clinical signs of EAE (Figure 4).
RA mediates immunomodulatory activity largely through its effect
on γδ Tcells
As the impact of RA seemed particularly striking on γδ Tcells
(Figures 1, 2 and 3), we examined more closely the effect of RA on the
pathogenic function of these cells. We first evaluated the importance
of γδ T cells in our EAE model by performing an adoptive transfer of
cells from MOG-immunized mice depleted of γδ T cells. Spleen and
lymph nodes cells from mice immunized with MOG and complete
Freund’s adjuvant were depleted of γδ Tcells,orleftun-separated,
before being stimulated for 3 days in Th17-polarizing conditions and
then transferred to recipient animals. Depletion of γδ Tcellspriorto
culture reduced the production of IL-17A, IL-17F and IFN-γ by the
spleen and lymph node cells (Figure 5a). Intracellular cytokine staining
and FACS showed that depletion of γδ T cells prior to culture
impaired the expansion of Tbet
+
and RORγt
+
CD4
+
T cells (Th1 and
0
2
4
6
8
10
***
IL-17F (ng/ml)
0
20
40
60
80
100
***
IFN-γ (ng/ml)
0
2
4
6
8
***
Vehicle RA
IL-17A (ng/ml)
IL-17A IFN-γ
0
5
10
15
20
ns
***
γδ T cells (x10
3
)
IL-17A IFN-γ
0
5
10
15
20
**
*
Vehicle RA
CD4
+
T cells (x10
3
)
CD4
γδ
0.0
0.5
1.0
1.5
***
***
**
il17f mRNA (RE)
CD4
γδ
0.0
0.5
1.0
1.5
***
***
***
Vehicle RA
il17a mRNA (RE)
CD4
γδ
0.0
0.5
1.0
1.5
**
***
**
Vehicle
RA
Rorc mRNA (RE)
Veh RA
0.0
0.5
1.0
1.5
2.0
***
Vγ4
+
γδ T cells (x10
5
)
Vehicle RA
CD4
+
T cells
2.9 0.4
2.0 4.6
0.72.3
IFN-γ
IL-17A
γδ T cells
3.3 1.0
19.4
29.4
4.5
33.5
Vehicle RA
Vγ4
IL-17A
Ki67
Vγ4
+
T cells
RA (MFI : 13,3 x 10
3
)
Vehicle (MFI : 27,5 x 10
3
)
90.4
75.4
75.4
Figure 3 RA suppresses IL-17 production and RORγt expression in CD4
+
Tcellandγδ T cell and impairs Vγ4
+
T-cell proliferation under inflammatory
conditions. Mice were immunized subcutaneously with MOG emulsified in complete Freund’s adjuvant. Ten days after immunization, lymph node and spleen
cells were cultured together for 3 days with MOG, IL-1β and IL-23 and either RA or vehicle. (a) IL-17A, IL-17F and IFN-γ production was quantified in
supernatants by ELISA at the end of the 3-day culture. (b) Representative FACS plots of the IL-17A
+
and IFN-γ
+
CD4
+
T cells and IL-17A
+
Vγ4
+
γδ Tcells
at the end of the 3-day culture. (c) Mean absolute numbers of IL-17A
+
and IFN-γ
+
CD4
+
T cells and γδ T cells at the end of the 3-day culture. (d, e)
Relative expression of il17a, il17f (d) and rorc (e) mRNA measured by RT-PCR on CD4
+
T cells and γδ T cells after 3 days of culture. (f) Representative
histogram, mean frequencies and mean fluorescence intensity quantifying Ki67 expression in Vγ4
+
T cells at the end of the 3-day culture. (g) Absolute
number of Vγ4
+
γδ T cells after 3 days of culture. Bars are mean+s.d. *Po0.05, **Po0.01 and ***Po0.001 (two-tailed unpaired t-test).
Immunomodulatory effects of RA on γδ Tcells
M Raverdeau et al
766
Immunology and Cell Biology

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
1
2
3
4
5
Vehicle
RA
***
**
* * **
****
**
Days post transfer
Clinical scores
Vehicle RA
0
2
4
6
8
*
CD3
+
T cells (x 10
4
)
IL-
17A
IL-
17
F
γ
IFN
-
G
M-C
S
F
0
5
10
15
20
25
30
Vehicle
RA
** * *
CD4
+
T cells (x10
3
)
IL-17A
IL
-17
F
γ
IF
N
-
GM-CSF
0
5
10
15
**
*****
γδ T cells (x10
2
)
35.8
4.4
9.5
1.5
27.8 3.3
IL-17A
CD3
γδ T cells
Vehicle RA
CD4
+
T cells
63.7 22.5
IFN-γ
Vehicle RA
IL-17FGM-CSF
14.534.5
4.210.4
9.3 3.7
14.9 3.7
Figure 4 Treatment of T cells from MOG-immunized mice with RA suppresses their ability to induce EAE by adoptive transfer. Donor mice were immunized
with MOG+complete Freund’s adjuvant and after 10 days, spleen and lymph node cells were cultured for 3 days with MOG, IL-1β+IL-23 and RA or vehicle
and then 15 × 10
6
cells were transferred to naive mice. (a) EAE clinical scores in recipient mice. (b)AbsolutenumberofCD3
+
T cells detected in the brains
of the recipient mice 16 days post transfer. Representative FACS plots (c) and mean absolute numbers (d)ofinfiltrating IL-17A
+
,IFN-γ
+
,IL-17F
+
and GM-
CSF
+
CD4
+
and γδ T cells in the brains of the recipient mice 16 days post transfer and re-stimulated ex vivo for 5 h with phorbol 12-myristate 13-acetate,
ionomycin and brefeldin A. Data are representative of three independent experiments. Data shown as mean ± s.e.m. (a) or mean+s.e.m. (b, d) and statistics
were calculated using a two-way analysis of variance with Bonferroni post-test (a)ortwo-tailedunpairedt-test (b–d). *Po0.05, **Po0.01 and ***Po0.001.
Immunomodulatory effects of RA on γδ Tcells
M Raverdeau et al
767
Immunology and Cell Biology