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Cannabidiol (CBD) Induces Functional Tregs in Response to
Low-Level T Cell Activation
Saphala Dhital1, John V. Stokes2, Nogi Park2, Keun-Seok Seo2, and Barbara L.F. Kaplan1,*
1Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary
Medicine, Mississippi State University, Mississippi State, MS 39762, USA
2Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University,
Mississippi State, MS 39762, USA
Abstract
Many effects of the non-psychoactive cannabinoid, cannabidiol (CBD), have been described in
immune responses induced by strong immunological stimuli. It has also been shown that CBD
enhances IL-2 production in response to low-level T cell stimulation. Since IL-2, in combination
with TGF-β1, are critical for Treg induction, we hypothesized that CBD would induce
CD4+CD25+FOXP3+ Tregs in response to low-level stimulation. Low-level T cell stimulation
conditions were established based on minimal CD25 expression in CD4+ cells using suboptimal
PMA/Io (4 nM/0.05 μM, S/o), ultrasuboptimal PMA/Io (1 nM/0.0125 μM, Us/o) or soluble anti-
CD3/28 (400-800 ng each, s3/28). CBD increased CD25+FOXP3+ cells from CD4+, CD4+CD25+,
and CD4+CD25− T cells, as well as in CD4+ T cells derived from FOXP3-GFP mice. Most
importantly, the Us/o + CBD-induced CD4+CD25+ Tregs robustly suppressed responder T cell
proliferation, demonstrating that the mechanism by which CBD is immunosuppressive under low-
level T cell stimulation involves induction of functional Tregs.
Keywords
CD4+CD25+FOXP3+ Tregs; CBD; immunosuppression
*To whom correspondence should be addressed: Barbara L.F. Kaplan, Center for Environmental Health Sciences, Department of Basic
Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, USA, Tel 662-325-1113,
barbara.kaplan@msstate.edu.
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AUTHORSHIP
S. D. wrote the manuscript, designed and performed experiments. J. S. performed flow cytometry and reviewed the manuscript. N.P.
and K.S.S. performed proliferation assays, participated in data interpretation and experimental design, and reviewed the manuscript. B.
L. F. K. designed the study, wrote and reviewed manuscript, and performed experiments.
Compliance with Ethical Standards
The studies were carried out with approval from the Mississippi State University Institutional Animal Care and Use Committee
(IACUC) in accordance with AAALAC guidelines (IACUC protocol number 13-110 to BLFK). Euthanasia via cervical dislocation
was performed. This method is approved by the American Veterinary Medical Association for mice.
Conflict of Interest
The authors declare that they have no conflict of interest.
HHS Public Access
Author manuscript
Cell Immunol
. Author manuscript; available in PMC 2018 February 01.
Published in final edited form as:
Cell Immunol
. 2017 February ; 312: 25–34. doi:10.1016/j.cellimm.2016.11.006.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
1. Introduction
Cannabidiol (CBD) is a non-psychoactive compound derived from
Cannabis sativa
[1, 2].
Studies with CBD are important since evidence suggests it can be used as a therapeutic
agent for a variety of disease states [3]. For instance, CBD has exhibited anxiolytic,
antiemetic, anti-tumorigenic and immune suppressive actions [4]. Specifically, CBD has
been used for the management of seizures in severe epilepsy [3, 5]. CBD and its derivative
dimethylheptyl-CBD have demonstrated efficacy as anti-inflammatory agents [6-14] and
CBD also possesses anti-tumor activity [15, 16]. Moreover, in combination with the
psychoactive cannabinoid, Δ9-tetrahydrocannabinol (THC) (a cannabinoid combination
therapy known as Sativex®), CBD has been assessed for its efficacy to treat tumorigenic
pain [4, 17] or spasticity induced by multiple sclerosis [18].
Although there are multiple studies and clinical trials investigating the use of CBD for
immune-related diseases, its immunosuppressive mechanism is still unclear [19]. For
instance, none of the studies have considered how the magnitude of cellular activation might
alter CBD's effects. Studies such as these are important for many reasons. First, suboptimal
T cell stimulation has been shown to contribute to persistent diseases, such as
M.
tuberculosis
[20] or
T. cruzi
[21], so determination of the effects and mechanisms of CBD
under low-level stimulation conditions will contribute to information on its putative
therapeutic usefulness. Second, suboptimal T cell stimulation can be influenced by the
presence of optimal stimulation of a distinct antigen, in what has been termed extended
antigen priming [22], so studying low-level stimulation in the absence and presence of other
antigens is key to understanding complex immune responses. Third, our previous study
demonstrated that CBD either inhibited or enhanced IL-2 and IFN-γ production in response
to optimal or suboptimal T cell activation, respectively [23], demonstrating that cellular
activation dictates the CBD response. We were particularly interested in the consequences of
enhanced IL-2 production by CBD in response to low-level T cell activation since IL-2,
along with TGF-β1, are key components for inducing and maintaining CD4+CD25+FOXP3+
Tregs [24]. Thus, we hypothesized that CBD would induce CD4+CD25+FOXP3+ cells under
low-level stimulation of T cells. To address this hypothesis, we established low-level T cell
stimulation conditions based on minimal expression of CD25 in order to evaluate CBD-
induced CD25 and FOXP3 expression. Comparisons were made between naïve whole
splenocytes and purified CD4+ T cells, including assessment of the effect of CBD on low-
level stimulation of purified CD4+CD25+ (which likely contains a natural Treg population)
and CD4+CD25− T cells (potentially inducible Tregs). Finally, the functionality of CBD-
induced Tregs was evaluated via examination of their ability to suppress naïve responder T
cell proliferation. Together these data demonstrate that CBD induces functional
CD4+CD25+FOXP3+ Tregs under low-level stimulation conditions, suggesting that CBD
maintains its immunosuppressive actions regardless of magnitude of stimulation.
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2. Materials and Methods
2.1 CBD
CBD was provided by the National Institute on Drug Abuse. CBD was prepared as a 10 mM
solution in 99.5% pure ethanol and stored in aliquots at −80°C until use. All experiments
include a 0.1% ethanol vehicle (VH) control.
2.2 Mice
Specific pathogen free 5 - 8 week old C57BL/6 mice were purchased from Envigo
(Indianapolis, IN) and B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/J (FOXP3-GFP) mice were
purchased from Jackson Labs (Bar Harbor, ME). Mice were housed 3-5 per cage, at
22-24°C, 40-55% humidity and 12-hr light/dark light cycle. The studies were carried out
with approval from the Mississippi State University Institutional Animal Care and Use
Committee (IACUC) in accordance with AAALAC guidelines (IACUC protocol numbers
13-110 and 15-077 to BLFK). Euthanasia via cervical dislocation was performed. This
method is approved by the American Veterinary Medical Association for mice. All
experiments were conducted
in vitro
using primary mouse splenocytes from female mice.
Typically cells from 1-2 spleens were used for splenocyte experiments, and 3-4 spleens were
pooled for RNA studies, enriched or purified T cells, and induction of Tregs for use in the
functional assay.
2.3 Preparation of splenocyte cultures
Splenocytes were prepared as a single cell suspension by mechanical disruption in 1X RPMI
media (Gibco/Life Technologies, Grand Island, NY). Splenocytes were enumerated with a
Coulter Counter (Beckman Coulter, Indianapolis, IN). Splenocytes were cultured in
complete medium containing bovine calf serum (BCS), 1% penicillin/streptomycin and 50
μM 2-mercaptoethanol. Cells were cultured in 2% BCS-containing medium for overnight
cultures. For kinetic studies in which incubation periods included overnight cultures and
longer periods (i.e., cultures for 1, 3 or 5 days), 5% BCS-containing medium was used. In
some experiments, CD4+ T cells were enriched from splenocytes by negative selection using
a mouse T cell CD4 subset column kit (R&D Systems, Minneapolis, MN) according to the
manufacturer's instructions. Briefly, erythrocytes were lysed with ammonium-chloride-
potassium (ACK) buffer, and up to 2 × 108 splenocytes were incubated with an antibody
cocktail that allows for negative selection of CD4+ T cells. Cells were enriched on a
prewashed column for 10 min at RT and eluted. CD4-enriched cells were collected, washed
and adjusted in complete medium as required for assays. Cells were pretreated with CBD in
0.1% ethanol VH for 30 min then stimulated with suboptimal PMA/Io (4 nM/0.05 μM; S/o),
ultrasuboptimal PMA/Io (1 nM/0.012 μM; Us/o) or soluble anti-CD3/CD28 (400-800 ng
each; sCD3/28) for 1-5 days at 37°C at 5% CO2. CBD was not washed out prior to
stimulations in any experiment.
2.4 Purification of CD4+CD25+ Tregs and CD4+CD25− T cells
Cells were purified either from fresh naive splenocytes or following culture of splenocytes
with Us/o + CBD. For cultured cells, splenocytes (6 × 106 cells/ml, 6 ml/well) were seeded
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in a 6-well flat bottom plate and treated with CBD (10 μM) for 30 min followed by Us/o
stimulation for 5 days. CD4+CD25+ Tregs were isolated using the Mouse CD25 Regulatory
T cell Positive Selection Kit (Stemcell, Vancouver, BC, Canada) and CD4+CD25− T cells
were obtained using Mouse CD4+ T Cell Isolation Kit (Stemcell) according to the
manufacturer's protocol. Briefly, CD4+CD25+ cells were initially purified by positive
selection, followed by purification of the CD4+CD25− T cells from the decanted cells. The
purity of CD4+CD25+ and CD4+CD25− T cells was consistently higher than 80% as
assessed by immunofluorescence staining for viability, CD4 and CD25.
2.5 Immunofluorescent staining
Splenocytes (1× 106 cells/ml, 0.8 ml/well) were seeded in 48-well flat bottom plate and
treated with CBD (0.5 - 10 μM) or VH (0.1% ethanol) for 30 min. Cells were treated with
S/o, Us/o or CD3/28 stimulation for 1-5 days. In some studies, CD4-enriched cells,
CD4+CD25+ Tregs or CD4+CD25− T cells were used instead of, or in addition to,
splenocytes. For intracellular cytokine analysis cells were treated with Brefeldin A (BFA;
Biolegend) for the last 4 hr of culture to block protein release from the cell. Cells were
washed with PBS, centrifuged at 500 × g for 5 min at RT. Cells were then treated with
fixable viability dye (FVD; FITC or eFluor 780; BioLegend or eBioscience, San Diego, CA)
as appropriate for 30 min at RT. Cells were washed with FACS buffer [Hank's Balanced Salt
Solution (HBSS) with 1% bovine serum albumin (BSA), pH 7.3] and incubated with mouse
Fc block (purified anti-mouse CD16/CD32, clone 2.4G2, BD Biosciences, San Jose, CA) for
15 min at RT to prevent non-specific binding. Cells were stained with extracellular
antibodies (CD4 or CD25) for 30 min at RT, followed by fixation and permeabilization. For
IL-2, TGF-β1, IL-10 or FOXP3, cells were permeabilized using the FOXP3 staining kit
(eBioscience, San Diego, CA) according to the manufacturer's instructions. Following
extracellular staining, cells were washed, then incubated in fixation-permeabilization buffer
(eBioscience) for 30 min at RT in the dark. Cells were washed and incubated with antibodies
for intracellular markers for 30 min at RT. Immunofluorescent antibodies from BioLegend
were: CD4 (FITC clone GK15-5; PECy7 clone GK1.5; or PE clone RM4-4), CD25 (FITC
clone PC61), IL-2 (APC clone JES6-5H4), IL-10 (PeCy 7 clone JES5-16E3 or APC clone
JES5-16E3) and TGF-β1 (PE clone TW7-20B9). Antibodies purchased from eBioscience
were: CD25 (PE clone PC61.5) and FOXP3 (APC clone FJK-16s). For the FOXP3-GFP
cells, Sytox-Red (Invitrogen) was used to assess viability since these cells were analyzed
without fixation or permeabilization. Stained cells were assayed using a FACSCalibur flow
cytometer (BD Biosciences). Compensation was adjusted using single stain bead controls
and gating was performed using fluorescence minus one (FMO) controls for each
fluorochrome. Data were analyzed using FlowJo (FlowJo LLC, Ashland, OR). The gating
strategy was initial dead cell exclusion using FSC versus FVD or Sytox, inclusion of
lymphocytes using FSC versus SSC, then inclusion of CD4+ T cells using a histogram for
CD4. FOXP3 versus CD25 in live CD4 lymphocytes was then analyzed from a dot plot.
Representative gating strategies and viability percentages are provided in Supplemental Figs.
1-2.
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2.6 Enzyme linked immunosorbent assay (ELISA)
Splenocytes (1 × 106 cells/ml, 0.8 ml/well) were seeded in 48-well flat bottom plates and
treated with CBD (0.5 - 10 μM) or VH (0.1% ethanol) for 30 min. Cells were stimulated
with S/o, Us/o or CD3/28 stimulation for 1-5 days. Cytokine production in the culture
supernatant was assessed by ELISA. Wells were coated overnight at 4°C with 1.0 μg/ml
anti-mouse IL-2 (clone JES6-1A12, BioLegend) in coating buffer (0.1 M carbonate-
bicarbonate buffer, pH 9.6). Wells were washed three times each with 0.05% Tween-20 in
PBS (PBST, pH 7.4) and deionized water (DW), then blocked with 3% BSA in PBS for 1 h
at RT. After washing the wells, samples were added to each well and incubated for 1 h at RT.
Again after washing, wells were incubated with 1.0 μg/ml biotin-conjugated anti-mouse IL-2
(clone JES6-5H4, BioLegend) for 1 h at RT. Wells were washed and treated with 100 μl of
Avidin-conjugated with horseradish peroxidase (HRP-Avidin; 1:500, BioLegend) for 1 h at
RT in the dark. For color development, wells were washed then 100 μl of 3,3′,5,5 -
tetramethylbenzidine (TMB) substrate set (BioLegend) was added for 30 min followed by
the addition of 100 μl of 2 N sulfuric acid (H2SO4) to terminate the reaction. Optical density
(OD) was measured at 450 nm within 30 min of reaction termination. Quantification of
cytokine was performed by generation of a standard curve.
2.7 Reverse Transcriptase Quantitative Polymerase Chain Reaction (RTQPCR)
Splenocytes (5 × 106 cells/ml, 5 ml/well) were seeded in a 6-well flat bottom plate and
treated with CBD (10 μM) or VH (0.1% ethanol) for 30 min and treated with Us/o
stimulation for 1-5 days. Cells were collected and resuspended in 1 ml TRI Reagent (Sigma,
St. Louis, MO). RNA was isolated with RNeasy (Promega, Madison, WI) columns
following TRI reagent (St. Louis, MO) phase separation and nucleic acid precipitation.
Equal amounts of RNA were reverse transcribed using a high capacity cDNA reverse
transcription kit (Applied Biosystems, Foster City, CA). RT-QPCR amplification was
performed on an Mx3005P instrument (Stratagene) using a Taqman primer/probe for
Tgfb1
(Mm01178820_m1) or
Il10
(Mm00439614_m1) (Applied Biosystems) in a total reaction
volume of 20 μl. Relative gene expression in terms of fold-change values were determined
using the ΔΔCt method using18s rRNA as the internal reference and Day 1 VH as the
control [25].
2.8 Treg functional assay
CD4+CD25+ and CD4+CD25− T cells were purified from Us/o + CBD (10 μM)-treated
splenocytes for 5 days. Purified cells were treated with mitomycin C (MMC, Sigma) at 10
μg/106 cells for 2 hr and washed four times prior to use in the co-culture. Freshly isolated
naïve splenocytes (responder cells; 1 × 105 cells/well) were mixed with an equal number of
MMC-treated CD4+CD25+ and CD4+CD25− T cells. Cultures were then stimulated with
anti-CD3 plus anti-CD28 coated beads (Life Technologies) at 0.3 μl/well. Co-cultures were
incubated in a 96-well flat bottom plate in a final volume of 200 μl for 4 days. Cells were
treated with 1 μCi of titrated 3H-thymidine for the last 16 hr of culture. Cells were harvested
on glass fiber filters (Perkin Elmer) and 3H-thymidine incorporation (degree of proliferation)
was measured by scintillation counting.
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2.9 Statistical analysis
Statistical analysis was performed using GraphPad Prism 6 (GraphPad software, San Diego,
CA). Comparisons were performed by one-way or two-way ANOVA as appropriate. A post-
hoc test for multiple comparisons was performed and the significance test was determined as
p
< 0.05. Logarithmic transformation was conducted prior to statistical analysis for PCR
data using natural log (fold-change+1) for statistical analysis.
3. Results
3.1 Low-level PMA/Io stimulation as defined by CD25 expression
Previously we determined that cannabinoid effects differed depending on the magnitude to
which cells were stimulated. Specifically, S/o stimulation was defined as 4 nM PMA/0.05
μM Io based on the fact that no IL-2 was produced in response to S/o stimulation alone, and
CBD enhanced IL-2 production under these conditions [23]. Initially, this seemed
inconsistent with the notion that CBD is anti-inflammatory and immune suppressive [7, 11,
19, 26]. On the other hand, increased IL-2, in combination with TGF-β1, can drive Treg
development
in vitro
[27]. Thus, we hypothesized that CBD maintains its immune
suppressive actions under low-level stimulation conditions through induction of Tregs. We
first established low-level immunological stimulation conditions based on CD25 expression.
Using the previously-defined S/o conditions of 4nM PMA/0.05 μM Io in which little IL-2
was detected in the absence of CBD [23], CD25 was still readily induced following S/o
stimulation overnight (Fig. 1), indicating that CD25 is very sensitive to upregulation by
PMA/Io. Thus, a lower level of stimulation (1 nM PMA/0.0125 μM Io, designated Us/o) was
examined in order to identify a PMA/Io concentration that produced minimal expression of
CD25. Indeed, no CD25 was induced at Day 1 in response to Us/o stimulation. The
incubation time was also extended in order to allow time for Treg differentiation upon CBD
treatment in subsequent studies. The results confirmed that CD25 expression was only
modestly increased in response to Us/o stimulation at Day 5 (Fig. 1).
3.2 CBD increased CD25 and FOXP3 on CD4+ cells
3.2.1 CBD induced CD25 and FOXP3 in response to S/o and Us/o stimulation
—Initially, the effect of CBD on CD25 and FOXP3 after an overnight S/o stimulation was
conducted. CBD upregulated the expression of CD25 and FOXP3 on CD4+ cells (Fig. 2A).
In order to evaluate the possibility that CBD could induce Treg differentiation, as opposed to
transient CD25 upregulation, the effect of CBD over an extended time period in response to
the lower Us/o stimulation was examined. Indeed, increasing concentrations of CBD
resulted in an increased percentage of CD4+CD25+FOXP3+ cells as compared to VH in
response to Us/o stimulation for 5 days (Fig. 2B). A detailed analysis of gating strategies
revealed that CBD produces modest suppression on viable cells after S/o stimulation for 1
day and Us/o stimulation for 5 days (Supplemental Figs. 1-2). Interestingly, CD4 expression
was decreased by CBD after S/o stimulation for 1 day (Supplemental Fig. 1), but increased
by CBD after Us/o stimulation for 5 days (Supplemental Fig. 2). It is important to note that
we do not attribute the modest effect of CBD on viable cell percentages to direct
cytotoxicity, but to apoptosis or differential cell survival (especially in splenocyte cultures)
over the 5-day period.
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3.2.2 CBD modestly induced CD25 and FOXP3 in response to s3/28
stimulation—Next, the effect of CBD specifically on T cells was examined in response to
anti-CD3/CD28. It is important to note that we also wanted low-level stimulation with anti-
CD3/28, similar to our previous work in which IL-2 production was induced by low-level
anti-CD3/28 stimulation [23]. Thus we purposefully administered anti-CD3 in soluble form
(as opposed to plate-bound) and only used 400-800 ng each of anti-CD3 and anti-CD28. The
low-level stimulation conditions with anti-CD3/CD28 are designated as s3/28. As seen in
Fig. 2C, CBD also increased CD25 and FOXP3 expression on CD4+ cells at Day 5 in s3/28-
stimulated cells, albeit to a lower magnitude than that observed with Us/o stimulation.
3.2.3 CBD induced CD25 and FOXP3 in enriched CD4+ T cells—Given the
observation that FOXP3 expression was lower and only modestly induced by CBD in
response to s3/28 in whole splenocytes, the effect of CBD was determined using CD4-
enriched cells. CD4 enrichment increased the CD4+ population to ~76% from 25% in total
splenocytes. Treatment with CBD for 5 days resulted in a robust increase in the percentage
of the CD25+FOXP3+ population on CD4+ cells stimulated with Us/o and a modest increase
on CD4+ cells stimulated with sCD3/28 at 800 ng each (Fig. 3). The CBD-induced
CD25+FOXP3+ population was not increased over VH in response to s3/28 at 400 ng each,
but CD25 was robustly upregulated by CBD suggesting that CD25 is a very sensitive target
of CBD. It should be noted that CBD alone produced only a small increase in the
CD25+FOXP3+ population on CD4-enriched cells, indicating that CBD was more effective
at enhancing T cell signaling initiated by Us/o or s3/28, rather than producing robust
induction of CD25 and FOXP3 alone (Fig. 3).
3.2.4 CBD robustly induced CD25 and FOXP3 in FOXP3-GFP mice—To verify
that CBD induced FOXP3, splenocytes derived from GFP-FOXP3 mice were treated with
CBD or VH plus Us/o stimulation for 5 days. Us/o + CBD robustly increased the
CD25+FOXP3-GFP+ population (Fig. 4). These results also verify that CBD increased
FOXP3 expression, regardless of whether it was measured with a fluorescently-conjugated
antibody during intracellular staining or directly assessed using the FOXP3-GFP mice.
3.3 Effect of CBD on early IL-2 and TGF-β1 production
In light of the previous observation that CBD enhanced S/o-stimulated IL-2 production [23]
and, as shown here, CBD induced S/o- or Us/o-stimulated CD25 and FOXP3 expression in
CD4+ cells, the CBD-induced cytokine milieu was examined to determine if CBD could
induce the appropriate environment early to induce Treg differentiation. Thus, IL-2 and
TGF-β1 were examined after an overnight stimulation since these two cytokines have been
shown to promote Treg differentiation [27]. Cells were treated with CBD and stimulated
with S/o and Us/o for 1, 3, or 5 days and IL-2 production was assessed in the supernatants
by ELISA. CBD induced IL-2 production at all times in response to S/o stimulation, with a
peak response at Day 1. However, in response to Us/o stimulation, IL-2 production was not
detectable by ELISA at any time (Fig. 5A). However, CBD did induce IL-2 and TGF-β1,
plus a CD4+IL-2+TGF-β1+ population, in response to both S/o and Us/o stimulation after 1
day as assessed by intracellular staining (Fig. 5B and 5C). s3/28 also modestly induced IL-2
and TGF-β1 after 1 day (Fig. 5C). Together these results suggest that IL-2 and TGF-β1
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produced by CD4+ cells treated with CBD in response to S/o, Us/o and s3/28 stimulation
create a favorable microenvironment for the induction of CD4+CD25+FOXP3+ cells [24, 27,
28].
3.4 CBD elevated IL-10 production
Another hallmark of Tregs is IL-10 [29-31], so the effect of CBD on IL-10 production was
examined. In these and all subsequent studies, Us/o stimulation was used exclusively since it
induced the most robust CD25 and FOXP3 expression with CBD treatment by Day 5. IL-10
was significantly elevated in Us/o + CBD-treated total lymphocytes at Day 5 (Fig. 6A). We
also measured the FOXP3+IL-10+ percentage of CD4+ cells and found the percentage of
CD4+IL-10+ cells was modestly increased by CBD in Us/o-stimulated cells (Fig. 6B),
although most were FOXP3−. It should be noted that the IL-10+FOXP3+ results were more
variable as compared to other endpoints, which is reflected in the discrepancy between
percent gated numbers in the representative dot plot and the average provided above it. Next
the effect of CBD on
Il10
and
Tgfb1
mRNA gene expression in response to Us/o stimulation
was assessed for 1, 3 or 5 days. While CBD did not affect
Tgfb1
mRNA expression, CBD
significantly upregulated the expression of
Il10
mRNA at Day 5 in Us/o-stimulated cells
(Fig. 7A and 7B). It is important to note that the magnitude of CBD-induced
Il10
mRNA
expression at Day 5 was also variable. A comparison of 3 separate experiments indicated the
average increase was 67.1 ± 22.7 with at least a 2-fold increase in response to US/o + CBD
over Us/o + VH in all experiments. Despite this variability in the magnitude of CBD-
induced
Il10
expression, the consistent increase suggests that CBD-induced changes in
IL-10 are mediated at the transcriptional level.
3.5 CBD increased FOXP3 in both CD4+CD25− and CD4+CD25+ cells
To determine whether CBD increased FOXP3 on CD4+CD25− or CD4+CD25+ populations,
cells were purified directly from naïve splenocytes followed by treatment with CBD and
Us/o stimulation. CBD robustly converted CD4+CD25− T cells into CD4+CD25+FOXP3+
Tregs (Fig. 8). CBD also converted CD4+CD25+ into CD4+CD25+FOXP3+ Tregs. As a
comparison, the average conversion to CD4+CD25+FOXP3+ Tregs was 82% from
CD4+CD25− cells, 45% from CD4+CD25+ cells, and 44% from naïve splenocytes. These
data suggest CBD increased expression of CD25 and FOXP3 in both inducible and natural
Treg populations (at least as defined as CD4+CD25− or CD4+CD25+, respectively) in
response to low-level stimulation.
3.6 CBD-induced CD4+CD25+ and CD4+CD25− cells suppress responder cell proliferation
To assess the functionality of T cells induced by CBD, proliferation of fresh naïve responder
splenocytes as targets in the presence of putative Tregs was assessed. To stimulate the
responder splenocytes, anti-CD3 plus anti-CD28 bead (b3/28) stimulation was used (as
opposed to low-level soluble antibodies) since we wanted significant stimulation in order to
assess the ability of Us/o + CBD-induced cells to suppress proliferation. Us/o + CBD-
induced CD4+CD25+ or CD4+CD25− cells were purified after 5 days and mixed with b3/28-
stimulated responder splenocytes. Classical CD4+CD25+ Tregs robustly suppressed the
proliferation of naïve responder splenocytes. CD4+CD25− cells only modestly suppressed
b3/28-stimulated proliferation of naive responder splenocytes (Fig. 9).
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4. Discussion
Despite cannabinoid compounds exhibiting anti-inflammatory and immunosuppressive
effects, there have been many reports that they possess some immune stimulatory effects,
especially on cytokine production [23, 32-36]. Specifically, we previously demonstrated that
IL-2 was sensitive to differential regulation by cannabinoids that depended on the magnitude
to which the cells were activated [23, 32, 33]. In this study, we sought to determine the
effects of CBD on T cell function following low-level stimulation conditions and found that
CBD enhanced CD25 and FOXP3 on CD4+ cells. Most importantly, Us/o + CBD-induced
CD4+CD25+ Tregs robustly suppressed proliferation of naive responder splenocytes,
demonstrating for the first time that the mechanism by which CBD suppresses immune
function involves induction of functional Tregs in response to low-level T cell activation.
Initially we utilized S/o conditions, which were originally identified as conditions under
which little IL-2 was produced, and could be robustly enhanced with CBD [23]. While CBD
induced a CD4+CD25+FOXP3+ population in response to S/o overnight, the results also
showed that S/o conditions were not minimal for CD25 expression, suggesting that CD25 is
much more sensitive to PMA/Io than IL-2. Thus, we characterized CBD-induced IL-2
production in response to S/o and Us/o over an extended time course. While IL-2 was not
significantly detected by ELISA in response to Us/o stimulation, both IL-2 and TGF-β1
were detected intracellularly after 1 day suggesting CBD induced the appropriate cytokine
milieu for Treg development. Indeed, CBD induced CD25 and FOXP3 expression in CD4+
cells in response to Us/o stimulation after a 5-day culture and the effect was enhanced when
CD4 cells were enriched prior to culture initiation or with the increased sensitivity of the
FOXP3-GFP cells.
In order to verify the CBD-induced CD25 and FOXP3 expression on CD4+ cells response to
S/o or Us/o stimulation, cells were stimulated with s3/28 both in splenocytes and CD4-
enriched cells. We purposefully used both anti-CD3 and anti-CD28 in soluble form at ng
levels since we had previously demonstrated that use of anti-CD3 in soluble form (as
opposed to plate bound) resulted in low-level T cell stimulation [23]. The effect of CBD in
s3/28-stimulated cells was not as robust as in response to Us/o stimulation using
splenocytes, but CD25 and FOXP3 were robustly upregulated by s3/28 in CD4-enriched
cells. Previous studies demonstrated that CBD produced more robust IL-2 production in
response to S/o stimulation as opposed to soluble anti-CD3/28 antibodies [23]. The reason
that CBD produces more robust effects in response to PMA/Io as opposed to anti-CD3/28 is
not clear, but could involve different intracellular signals since PMA/Io bypasses the TCR.
Perhaps calcium signaling is involved since Io is a stronger inducer of intracellular calcium
than anti-CD3/28. Alternatively, it is possible that CBD requires stimulation of an accessory
cell (i.e., antigen presenting cell) for effective Treg induction that only gets stimulated with
PMA/Io, but not anti-CD3/28. It is clear from the study in which CBD alone was used that
CBD does require some kind of cellular stimulation for CD25 and FOXP3 induction.
CBD induced expression of CD25 and FOXP3, suggesting that CBD induced functional
Tregs. Indeed, CBD increased
Il10
mRNA and intracellular IL-10 and TGF-β1 in response
to Us/o stimulation. These results are consistent with another study in which CBD induced
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Il10
mRNA in anergy-inducing T cells, defined as CD4+CD25−LAG+CD69+ cells [8]. It was
interesting that while CBD induced
Il10
mRNA, it had no effect on
Tgfb1
mRNA,
suggesting that the mechanism by which CBD induced IL-10 was at the level of
transcription, while TGF- 1 upregulation might be post-translational. For instance, perhaps
CBD alters the ability of free TGF-β1 to be released from its latent form [37]. We also
determined that CBD induced FOXP3 in both CD4+CD25− and CD4+CD25+ cells, although
it was more effective at inducing FOXP3 in CD25− cells. Most importantly, Us/o + CBD-
induced CD4+CD25+ Tregs were functional as shown by their ability to robustly suppress
b3/28-stimulated naïve responder cell proliferation. Us/o + CBD-induced CD4+CD25− T
cells also modestly suppressed naïve responder T cell proliferation, although the possibility
that the CD4+CD25− cells were contaminated with a small proportion of CD4+CD25+ Tregs
exists. It should be noted that the intracellular IL-10 was detected predominantly in
CD4+FOXP3− cells, suggesting that the mechanism of suppression of responder splenocyte
proliferation is likely IL-10-independent and perhaps cell contact-dependent. Regardless, the
data do demonstrate robust suppressive function by Us/o + CBD-induced CD4+CD25+
Tregs, and suggests that CBD exhibits several mechanisms to suppress immunity, similar to
other reports for Tregs [38-40].
While this is the first report that CBD induced functional Tregs in response to low-level
stimulation, other reports have shown that cannabinoids induce Tregs. In response to
Concanavalin-A to induce hepatitis, THC increased
Foxp3
mRNA expression in liver and
increased the number of CD4+CD25+ cells and CD4+FOXP3+ cells in the liver [41]. THC
also increased FOXP3 expression in CD4+ T cells in the lung of staphylococcal enterotoxin
B-infused mice [42]. The synthetic cannabinoid agonist, WIN-55212-2, induced a
CD4+CD25+FOXP3+ population in the CNS as late as 110 days post EAE initiation [43]. A
CB2-selective agonist, O-1966 increased the proportion of CD4+CD25+FOXP3+ cells and
IL-10 production in a mixed lymphocyte response [44]. Finally, one study examined the
effect of CBD on Treg induction using encephalitogenic T cells restimulated with MOG-
loaded antigen presenting cells [8]. In contrast to our findings, CBD did not alter the
CD4+CD25+ population, but CBD did induce a CD69+LAG+ population in CD4+CD25−
cells, and increased expression of genes associated with anergy in CD4+ T cells [8]. In the
same study, CBD also induced
il10
mRNA expression in the T cells purified from the APC-
T cell co-culture [8].
Interestingly, another recent study has demonstrated that there exists a T cell activation
threshold for maximal induction of Tregs [45]. Specifically the authors identified a role for
the E3 ubiquitin ligase Cbl-b to control TCR strength, and found that CD25 and FOXP3
expression were maximal with relatively low concentrations of anti-CD3 antibody [45].
5. Conclusions
Immune responses to pathogens are complex, likely involving T cell populations that are
stimulated to various degrees by multiple antigens. Thus, an evaluation of the effects of a
putative immune modulator such as CBD is critical under low-level stimulation conditions.
These studies demonstrate for the first time that CBD maintains its immunosuppressive
activity under conditions of low-level stimulation through induction of functional Tregs.
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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS
Studies were funded by Mississippi State University College of Veterinary Medicine and NIH P20GM103646.
Abbreviations
BCS bovine calf serum
BFA brefeldin A
CBD cannabidiol
CB1cannabinoid receptor 1
CB2cannabinoid receptor 2
cpm counts per minute
DW deionized water
ELISA enzyme linked immunosorbent assay
FOXP3 forkhead box P3
FVD fixable viability dye
IFN interferon
IL interleukin
iTregs inducible T regulatory T cells
MMC mitomycin C
MOG myelin oligodendrocyte glycoprotein
PMA phorbol 12-myristate 13-acetate
RT-PCR reverse transcription-polymerase chain reaction
s3/28 soluble anti-CD3 plus anti-CD28 treatment
S/o suboptimal (4 nM phorbol ester/0.05 μM ionomycin)
TCR T cell receptor
TGF- 1 transforming growth factor-β1
THC Δ9-tetrahydrocannabinol
Us/o ultrasuboptimal (1 nM phorbol ester/0.012 μM ionomycin)
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VH vehicle
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Highlights
•CBD induces CD25 and FOXP3 on CD4+ T cells in response to Us/o, S/o and
s3/28 stimulation
•CBD is most effective in inducing CD4+CD25+FOXP3+ T cells in response to
Us/o
•Us/o + CBD increased FOXP3 expression on CD4+ cells derived from
FOXP3-GFP mice
•Us/o + CBD induced FOXP3 in pre-purified CD4+CD25− and CD4+CD25+ T
cells
•Us/o + CBD-induced CD4+CD25+ T cells suppressed responder cell
proliferation
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Fig. 1. Expression of CD25 in response to S/o and Us/o stimulation
Splenocytes were treated with S/o and Us/o stimulation and cultured for 1 or 5 days. Cells
were stained for viability, CD4 and CD25. Only CD25 is depicted here to emphasize that
Us/o stimulation produces low levels of CD25 as compared to S/o. Cells were gated on live
CD4+ cells and gating was set based on FMO controls. Histograms are representative of
triplicate results of at least three separate experiments for each stimulation. NA, naïve
(untreated).
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Fig. 2. Induction of CD4+CD25+FOXP3+ cells by CBD
Splenocytes were treated with CBD (0.5-10 μM) or VH (0.1% ethanol) for 30 min followed
by low-level stimulation. Cells were stained for viability, CD4, CD25 and FOXP3. Cells are
gated on live CD4+ cells. Numbers above plots are average percent gated numbers ± SE of
triplicate results for the quadrant. Dot plots are representative of triplicate results of at least
three separate experiments for each stimulation. (A) S/o stimulation for 1 day; (B) Us/o
stimulation for 5 days; (C) s3/28 for 5 days. *
p
< 0.05 as compared to VH within each
stimulation group. NA, naïve (untreated); Stim, stimulated.
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Fig. 3. Effect of CBD on CD25 and FOXP3 expression on CD4-enriched cells
CD4-enriched T cells were treated with CBD (10 μM) or VH (0.1% ethanol) for 30 min
followed by Us/o or s3/28 stimulation for 5 days. Cells were stained for viability, CD4,
CD25 and FOXP3. Cells are gated on live CD4+ cells. Numbers above plots are average
percent gated numbers ± SE of triplicate results for the quadrant. Dot plots are representative
of triplicate results of at least three separate experiments. *
p
< 0.05 as compared to VH
within each stimulation group. Stim, stimulated.
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Fig 4. CBD increased FOXP3-GFP
Splenocytes from FOXP3-GFP mice were treated with CBD (10 μM) or VH (0.1% ethanol)
for 30 min followed by Us/o stimulation for 5 days. Cells were stained for viability, CD4
and CD25 without fixation. Cells are gated on live CD4+ cells. Numbers above plots are
average percent gated numbers ± SE of triplicate results for the quadrant. Dot plots are
representative of triplicate results of at least two separate experiments. *
p
< 0.05 as
compared to VH.
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Fig. 5. Effect of CBD on IL-2 and TGF-β1 production
Splenocytes were treated with CBD (5 or 10 μM) or VH (0.1% ethanol) for 30 min followed
by low-level stimulation. (A) Cells were stimulated with S/o or Us/o stimulation for 1, 3 or 5
days. IL-2 was assessed by ELISA. Data are average of triplicate wells ± SD with *
p
< 0.05
as compared to VH within group; #
p
< 0.05 as compared to Day 1. (B, C) Cells were
stimulated with S/o (B) or Us/o or s3/CD28 (C) for 1 day. Cells were treated with BFA for
the last 4 hr of culture then stained for viability, CD4, IL-2 and TGF-β1. Numbers above
plots are average percent gated numbers ± SE of triplicate results for the quadrant. Dot plots
represent triplicate results of at least three separate experiments. Cells are gated on live
CD4+ cells. *
p
< 0.05 as compared to VH within each stimulation group. Stim, stimulated.
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Fig. 6. CBD increased IL-10 and FOXP3 expression
Splenocytes were treated with CBD (1-10 μM) or VH (0.1% ethanol) and Us/o stimulation
for 5 days. Cells were treated with BFA for the last 4 hr of culture then stained for viability
CD4, IL-10 and/or FOXP3. (A) Intracellular IL-10 in live lymphocytes. Data are average of
triplicate wells ± SD with *
p
< 0.05 as compared to VH. (B) Intracellular IL-10 and FOXP3
in CD4+ T cells. Numbers above plots are average percent gated numbers ± SE of triplicate
results for the quadrant. Dot plots represent triplicate results of at least three separate
experiments.
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Fig. 7. CBD upregulated the expression of Il10 mRNA
Splenocytes were treated with VH or CBD (10 μM) and Us/o stimulation for 1, 3 or 5 days.
Total RNA was isolated and quantitative RT-PCR was performed for (A)
Tgfb1
and (B)
Il10
mRNA. Data are relative gene expression as compared to Us/o + VH at Day 1 ± SD of
triplicate wells. Data are representative of triplicate results from at least three separate
experiments. *
p
< 0.05 as compared to expression at Day 1.
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Fig. 8. Induction of CD4+CD25+FOXP3+ cells from CD4+CD25− or CD4+CD25+ T cells by CBD
CD4+CD25+ Tregs and CD4+CD25− T cells from naive mouse splenocytes were purified
and treated with CBD (10 μM) plus Us/o stimulation for five days. Cells were stained for
viability, CD4, CD25 and FOXP3. Numbers above plots are average percent gated numbers
± SE of triplicate results for the quadrant. Cells are gated on live CD4+ cells. Dot plots are
representative of triplicate results from two separate experiments. Average conversion to
CD4+CD25+FOXP3+ cells from CD4+CD25− or CD4+CD25+ T cells was 82% or 45%,
respectively. Conversion from naive splenocytes was 44%. Conversion rates were calculated
as (percentage of CD25+FOXP3+ cells produced by CBD – percentage of CD25+FOXP3+
cells produced by VH)/percentage of CD25+FOXP3+ cells produced by CBD) X 100%. *
p
< 0.05 as compared to VH within each cell population.
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Fig. 9. Treg functional assay
Us/o + CBD-induced CD4+CD25+ and CD4+CD25− T cells were purified and treated with
MMC to prevent proliferation. Fresh responder splenocytes were activated with anti-CD3/28
beads (b3/28) and incubated with MMC-treated, Us/o + CBD-induced cells. The
effectiveness of MMC was assessed using fresh splenocytes treated with MMC followed by
b3/28 stimulation. Co-cultures were incubated for 4 days. 3H-thymidine was added for last
16 hr of culture. Cells were lysed and 3H-thymidine was collected on glass fiber filters and
counted using a scintillation counter. Data are average counts per minute (CPM) of
quadruplet wells ± SD. Results are representative of quadruplicate cultures from two
separate experiments. a,
p
< 0.05 as compared to b3/28-stimulated SPLC; b,
p
< 0.05 as
compared to MMC-treated CD4+CD25− T cells plus b3/28-stimulated SPLC.
Dhital et al. Page 24
Cell Immunol
. Author manuscript; available in PMC 2018 February 01.
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