U.S. patent application number 12/743680 was filed with the patent office on 2011-02-24 for modulation of the immune response.
Invention is credited to Francisco J. Quintana, Howard Weiner.
Application Number | 20110044902 12/743680 |
Document ID | / |
Family ID | 40668061 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110044902 |
Kind Code |
A1 |
Weiner; Howard ; et
al. |
February 24, 2011 |
Modulation of the Immune Response
Abstract
Methods for identifying compounds that modulate the generation
of regulatory T cells (Treg) in vivo and in vitro, i.e., compounds
that act on the transcription factors that increase or decrease
expression of Foxp3.
Inventors: |
Weiner; Howard; (Brookline,
MA) ; Quintana; Francisco J.; (Jamaica Plain,
MA) |
Correspondence
Address: |
FISH & RICHARDSON P.C. (BO)
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
40668061 |
Appl. No.: |
12/743680 |
Filed: |
November 10, 2008 |
PCT Filed: |
November 10, 2008 |
PCT NO: |
PCT/US08/83016 |
371 Date: |
August 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60989309 |
Nov 20, 2007 |
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61070410 |
Mar 21, 2008 |
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Current U.S.
Class: |
424/9.1 ;
424/173.1; 424/93.71; 435/375; 435/7.24; 514/365; 514/410; 514/415;
514/452; 530/389.6; 548/201; 548/418; 548/504; 549/359 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 29/00 20180101; A61K 31/357 20130101; A61K 31/407 20130101;
G01N 33/5088 20130101; A61P 3/10 20180101; C12N 2501/38 20130101;
A61K 2035/122 20130101; A61K 35/17 20130101; A61P 25/00 20180101;
A61P 37/06 20180101; A61K 39/3955 20130101; C12N 2501/60 20130101;
A61K 31/427 20130101; A61P 37/02 20180101; C12N 5/0636 20130101;
A61K 31/4045 20130101; A61K 31/405 20130101; A61K 31/135 20130101;
A61K 31/137 20130101 |
Class at
Publication: |
424/9.1 ;
435/375; 424/93.71; 435/7.24; 548/418; 549/359; 548/504; 548/201;
530/389.6; 514/452; 514/365; 514/410; 514/415; 424/173.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C12N 5/0783 20100101 C12N005/0783; A61K 35/12 20060101
A61K035/12; G01N 33/566 20060101 G01N033/566; C07D 487/04 20060101
C07D487/04; C07D 319/14 20060101 C07D319/14; C07D 209/16 20060101
C07D209/16; C07D 417/06 20060101 C07D417/06; C07K 16/28 20060101
C07K016/28; A61K 31/357 20060101 A61K031/357; A61K 31/427 20060101
A61K031/427; A61K 31/407 20060101 A61K031/407; A61K 31/404 20060101
A61K031/404; A61K 39/395 20060101 A61K039/395; A61P 37/02 20060101
A61P037/02; A61P 25/00 20060101 A61P025/00 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Grant
Nos. AI435801, AI043458, and NS38037 awarded by the National
Institutes of Health. The Government has certain rights in the
invention.
Claims
1. A composition comprising a ligand that binds specifically to an
aryl hydrocarbon receptor (AHR) transcription factor, linked to a
biocompatible nanoparticle.
2. The composition of claim 1, wherein the ligand is a small
molecule that competes for binding to the AHR competitively with
2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and activates
AHR-dependent signaling.
3. The composition of claim 1, wherein the ligand is 2,3,7,8
tetrachlorodibenzo-p-dioxin (TCDD).
4. The composition of claim 1, wherein the ligand is tryptamine
(TA).
5. The composition of claim 1, further comprising a monoamine
oxidase inhibitor such as tranylcypromine.
6. The composition of claim 1, wherein the ligand is
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE).
7. The composition of claim 1, wherein the ligand is 6
formylindolo[3,2 b]carbazole (FICZ).
8. The composition of claim 1, further comprising an antibody that
selectively binds to an antigen present on a T cell, a B cell, a
dendritic cell, or a macrophage.
9. The composition of claim 8, wherein the antibody is linked to
the biocompatible nanoparticle.
10. A method for increasing the number of CD4/CD25/Foxp3-expressing
T regulatory (Treg) cells in a population of T cells, the method
comprising: contacting the population of cells with a sufficient
amount of a composition comprising one or more AHR ligands selected
from the group consisting of 2,3,7,8 tetrachlorodibenzo-p-dioxin
(TCDD), tryptamine (TA), and
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE), wherein the ligand is linked to a biocompatible
nanoparticle, and optionally evaluating the presence and/or number
of CD4/CD25/Foxp3-expressing cells in the population; wherein the
method results in an increase in the number and/or activity of
regulatory T cells (Treg).
11. The method of claim 10, wherein the population of T cells
comprises naive T cells or CD4+CD62 ligand+ T cells.
12. The method of claim 10, further comprising administering the
Treg cells to a subject suffering from an autoimmune disorder, in
an amount sufficient to improve or ameliorate a symptom of the
disorder.
13. The method of claim 10, wherein the population of T cells is in
a living mammalian subject.
14. The method of claim 13, wherein the subject has an autoimmune
disorder.
15. The method of claim 10, wherein the autoimmune disorder is
multiple sclerosis.
16. The method of claim 13, comprising administering the one or
more ligands orally.
17. The method of claim 13, comprising administering the one or
more ligands intravenously.
18. A method of identifying a candidate compound that increases
generation or activity of regulatory T cells (Treg), the method
comprising: providing a cell expressing a reporter construct
comprising a binding sequence for the Aryl Hyrocarbon Receptor
(AHR) in a mammalian Foxp3 promoter sequence, wherein said binding
sequence is operably linked to a reporter gene, for example a
reporter gene selected from the group consisting of luciferase,
green fluorescent protein, and variants thereof; contacting the
cell with a test compound; and evaluating an effect of the test
compound on expression of the reporter gene, wherein a test
compound that increases or decreases expression of the reporter
gene is a candidate compound that modulates generation of Treg.
19. The method of claim 18, further comprising measuring expression
of the reporter construct in the presence of a known AHR ligand
selected from the group consisting of 2,3,7,8
tetrachlorodibenzo-p-dioxin (TCDD), tryptamine (TA), and
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE), or a compound that binds to the AHR competitively therewith;
determining whether the candidate compound competes for binding to
the AHR with the known compound; and selecting the candidate
compound if it binds the AHR competitively with the known
compound.
20. A method of identifying a candidate compound that modulates
generation of regulatory T cells (Treg), the method comprising:
providing a living zebrafish; contacting the zebrafish with a test
compound; evaluating an effect of the test compound on Foxp3
expression in the zebrafish, wherein a test compound that increases
or decreases expression of Fox-3 in the zebrafish is a candidate
compound that modulates generation of Treg.
21. The method of claim 5, wherein the zebrafish comprises a
luciferase reporter construct encoding a human, murine or zebrafish
Foxp3 gene.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. Nos. 60/989,309, filed on Nov. 20, 2007,
and 61/070,410, filed on Mar. 21, 2008, the entire contents of
which are hereby incorporated by reference.
TECHNICAL FIELD
[0003] This invention relates to methods and compositions for
increasing the number and/or activity of regulatory T cells (Tregs)
in vivo and in vitro.
BACKGROUND
[0004] Regulatory T cells (Treg) control the autoreactive
components of the immune system. Consequently, Treg dysfunction is
linked to severe autoimmunity, and compounds that increase Treg
numbers or activity are expected to be useful in the treatment of
autoimmune disorders such as multiple sclerosis.
[0005] Treg cells are a specialized subset of T cells involved in
the control of pathogenic autoimmunity (Sakaguchi et al., Arm. Rev.
Immunol., 22:531-562, 2004. The importance of Treg for
immunoregulation is highlighted by the immune disorders that result
from Treg depletion with antibodies (Sakaguchi et al., J. Immunol.
155, 1151-64 (1995)); as a result of the thymectomy of 3 day old
newborns (Sakaguchi et al., J Exp Med. 156, 1565-76 (1982)); or
treatment with diphtheria toxin in transgenic mice with a
Treg-restricted expression of the diphtheria toxin receptor (Kim et
al., Nat Immunol. 8, 191-7 (2007)). In addition, Treg deficiencies
have been described in several autoimmune diseases such as multiple
sclerosis (Viglietta et al., J. Exp. Med. 199, 971-9 (2004)),
rheumatoid arthritis (Ehrenstein et al., J Exp Med. 200, 277-85
(2004)), diabetes (Brusko et al., Diabetes. 54, 1407-14 (2005);
Lindley et al., Diabetes. 54, 92-9 (2005)), and lupus (Mudd et al.,
Scand. J. Immunol. 64(3):211-218 (2006)).
SUMMARY
[0006] The present invention is based, at least in part, on the
discovery that transcription factors capable of modulating (e.g.,
increasing or decreasing) the expression and/or activity of the
Foxp3 gene provide useful targets for therapeutic immunomodulation.
Accordingly, the present invention provides, inter alia,
compositions and methods for the prevention or treatment of
diseases caused by an abnormal (e.g., autoimmune) or absent (e.g.,
including insufficient) immune response.
[0007] In one aspect, the present invention features compositions
including a ligand that binds specifically to an aryl hydrocarbon
receptor (AHR) transcription factor, linked to a biocompatible
nanoparticle. The ligand can be, e.g., a small molecule that
competes for binding to the AHR competitively with 2,3,7,8
tetrachlorodibenzo-p-dioxin (TCDD) and activates AHR-dependent
signaling. In some embodiments, the ligand is 2,3,7,8
tetrachlorodibenzo-p-dioxin (TCDD), tryptamine (TA),
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE), or 6-formylindolo[3,2-b]carbazole (FICZ).
[0008] In some embodiments, the composition also includes an
inhibitor of degradation of the ligand, e.g., a monoamine oxidase
inhibitor such as tranylcypromine. The inhibitor can be present on
(i.e., linked to) the same nanoparticles, linked to different
nanoparticles (of the same or different types) or free in solution.
In some embodiments, the methods and compositions described herein
include the use of a ligand that binds specifically to an aryl
hydrocarbon receptor (AHR) transcription factor, and an inhibitor
of degradation thereof, e.g., tryptamine and tranylcypromine,
wherein neither is linked to a nanoparticle.
[0009] In some embodiments, the composition also includes an
antibody that selectively binds to an antigen present on a T cell,
a B cell, a dendritic cell, or a macrophage. The antibody can be
present on (i.e., linked to) the same nanoparticles, linked to
different nanoparticles (of the same or different types) or free in
solution.
[0010] In a further aspect, the invention features methods for
increasing the number or activity of CD4/CD25/Foxp3-expressing T
regulatory (Treg) cells in a population of T cells. The methods
include contacting the population of cells with a sufficient amount
of a composition comprising one or more AHR ligands selected from
the group consisting of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD),
tryptamine (TA), and
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE), wherein the ligand is linked to a biocompatible
nanoparticle, and optionally evaluating the presence and/or number
of CD4/CD25/Foxp3-expressing cells in the population. The method
results in an increase in the number and/or activity of regulatory
T cells (Treg).
[0011] In some embodiments, the initial population of T cells
includes one or both of naive T cells or CD4.sup.+CD62 ligand.sup.+
T cells. The population of T cells can be isolated, i.e., in vitro,
or in a living mammalian subject, e.g., a subject who has an
autoimmune disorder, e.g., multiple sclerosis. In embodiments where
the T cells are in a living subject, the methods can include
administering the one or more ligands orally, mucosally, or
intravenously.
[0012] In some embodiments, Treg cells generated or activated using
a method described herein are administered to a subject suffering
from an autoimmune disorder, in an amount sufficient to improve or
ameliorate a symptom of the disorder.
[0013] Also provided herein are methods for identifying candidate
compounds that increase generation or activity of regulatory T
cells (Treg). The methods include providing a cell expressing a
reporter construct comprising a binding sequence for the Aryl
Hyrocarbon Receptor (AHR) in a mammalian Foxp3 promoter sequence,
wherein said binding sequence is operably linked to a reporter
gene, for example a reporter gene selected from the group
consisting of luciferase, green fluorescent protein, and variants
thereof; contacting the cell with a test compound; and evaluating
an effect of the test compound on expression of the reporter gene.
A test compound that increases or decreases expression of the
reporter gene is a candidate compound that modulates generation of
Treg.
[0014] The methods can optionally include measuring expression of
the reporter construct in the presence of a known AHR ligand
selected from the group consisting of TCDD, tryptamine, and (ITE),
or a compound that binds to the AHR competitively therewith;
determining whether the candidate compound competes for binding to
the AHR with the known compound; and selecting the candidate
compound if it binds the AHR competitively with the known
compound.
[0015] In one aspect, the present invention provides methods of
identifying candidate compounds that modulate the generation of
regulatory T cells (Treg). These methods include providing a cell
expressing a reporter construct containing a binding sequence for a
transcription factor operably linked to a reporter gene. Suitable
binding sequences for inclusion in the reporter construct include
NKX22, AHR, EGR1, EGR2, EGR3, NGFIC, and Delta EF1. The cell is
then contacted with a test compound, and the effect of the test
compound on expression of the reporter gene is evaluated. A test
compound that increases or decreases expression of the reporter
gene is a candidate compound that modulates generation of Treg.
[0016] In another aspect, the present invention provides methods of
identifying candidate compounds that modulate generation of
regulatory T cells (Treg). These methods include providing a living
zebrafish, e.g., a zebrafish embryo, e.g., 30 minutes after the egg
is laid; contacting the zebrafish with a test compound, e.g., by
putting the test compound in water in which the zebrafish is living
or microinjecting the compound into an embryo; and evaluating an
effect of the test compound on Foxp3 expression in the zebrafish,
wherein a test compound that increases or decreases expression of
Fox-3 in the zebrafish is a candidate compound that modulates
generation of Treg.
[0017] In a further aspect, the present invention provides
compositions comprising transcription factor ligands capable of
promoting increased expression, activity, or both of a Foxp3
gene.
[0018] In yet another aspect, the present invention provides
methods for increasing the numbers of Treg in a population of T
cells. These methods include contacting the cell with one or more
transcription factor ligands, e.g., selected from the group
consisting of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD),
tryptamine (TA), and
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE), wherein the method results in an increase in the number
and/or activity of regulatory T cells (Treg). In some embodiments,
the methods include determining levels of Foxp3 expression in the
cells.
[0019] In an additional aspect, the present invention provides
methods for increasing the numbers of Treg in a patient. These
methods include administering one or more transcription factor
ligands to a patient selected for treatment, e.g., 2,3,7,8
tetrachlorodibenzo-p-dioxin (TCDD), tryptamine (TA), and/or
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE), wherein the method results in an increase in the number
and/or activity of regulatory T cells (Treg).
[0020] As used herein, "treatment" means any manner in which one or
more of the symptoms of a disease or disorder are ameliorated or
otherwise beneficially altered. As used herein, amelioration of the
symptoms of a particular disorder refers to any lessening, whether
permanent or temporary, lasting or transient of the symptoms, that
can be attributed to or associated with treatment by the
compositions and methods of the present invention.
[0021] The terms "effective amount" and "effective to treat," as
used herein, refer to an amount or a concentration of one or more
of the compositions described herein utilized for a period of time
(including acute or chronic administration and periodic or
continuous administration) that is effective within the context of
its administration for causing an intended effect or physiological
outcome.
[0022] The term "patient" is used throughout the specification to
describe an animal, human or non-human, rodent or non-rodent, to
whom treatment according to the methods of the present invention is
provided. Veterinary and non-veterinary applications are
contemplated. The term includes, but is not limited to, mammals,
e.g., humans, other primates, pigs, rodents such as mice and rats,
rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and
goats. Typical patients include humans, farm animals, and domestic
pets such as cats and dogs.
[0023] The term gene, as used herein refers to an isolated or
purified gene. The terms "isolated" or "purified," when applied to
a nucleic acid molecule or gene, includes nucleic acid molecules
that are separated from other materials, including other nucleic
acids, which are present in the natural source of the nucleic acid
molecule. An "isolated" nucleic acid molecule, such as an mRNA or
cDNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized.
[0024] An "isolated" or "purified" polypeptide, peptide, or protein
is substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. "Substantially free" means
that the preparation of a selected protein has less than about 30%,
(e.g., less than 20%, 10%, or 5%) by dry weight, of non-selected
protein or of chemical precursors. Such a non-selected protein is
also referred to herein as "contaminating protein". When the
isolated therapeutic proteins, peptides, or polypeptides are
recombinantly produced, it can be substantially free of culture
medium, i.e., culture medium represents less than about 20%, (e.g.,
less than about 10% or 5%) of the volume of the protein
preparation. The invention includes isolated or purified
preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry
weight.
[0025] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0026] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0027] FIG. 1A is a bar graph of proliferative response to MT or
ConA of splenocytes from six-month old zebrafish, 14 days after
immunization with MT or PBS in IFA. Results are presented as the
mean cpm+s.d. of triplicates.
[0028] FIG. 1B-1D are bar graphs of expression of CD3 (1B), IL-17
(1C) and IFNg (1D) in six month old zebrafish 14 or 28 days after
immunization with zebrafish brain homogenate (zCNS) or PBS in CFA,
as measured by real time PCR (mean+s.d. of triplicates).
[0029] FIG. 1E is a sequence comparison of putative FoxP3 genes of
zebrafish, human and mouse. The stars indicate identity, dashes
were introduced for optimal alignment. The zinc finger, leucine
zipper and forkhead domains are highlighted with a blue, green or
red box, respectively.
[0030] FIG. 1F is a bar graph of zFoxp3 expression in 293T cells
cotransfected with constructs coding for His-labeled zFoxp3 and
Renilla-labeled Foxp3. The results are normalized for the total
amount of luciferase before precipitation (mean+s.d. of
triplicates).
[0031] FIG. 1G is a radial gene tree showing the Foxp1, Foxp2,
Foxp3 and Foxp4 proteins in mammals and fish, where the Ciona
intestinalis Foxp sequence is the outgroup. The branch lengths are
proportional to the distance between the sequences. Mm, Mus
musculus; Hs, Homo sapiens; Dr, Danio rerio; Ga, Gasterosteus
aculeatus (stickleback); Ci, Ciona intestinalis.
[0032] FIG. 2A is a pair of bar graphs of 293T cells co-transfected
with reporter constructs coding for luciferase under the control of
a NF-kB or NFAT responsive promoters, and p65 NF-kB (top graph) or
NFAT (bottom graph) in the presence of vectors coding for zFoxp3,
Foxp3 or control (empty vector). Luciferase activity was normalized
to the renilla activity of a co-transfected control (mean+s.d. of
triplicates)
[0033] FIG. 2B is a pair of Western blots of 293T cells
co-transfected with His-tagged zFoxp3, Foxp3 and NF-kB (top graph)
or HA-flagged NFAT (bottom graph) and immunoprecipitated with
antibodies to His antibodies. The precipitates were resolved by
PAGE-SDS and detected by western blot with antibodies to NF-kB or
HA antibodies.
[0034] FIG. 2C is a set of four graphs of MACS-purified
CD4.sup.+CD25.sup.-T-cells that were transduced with a bicistronic
retrovirus coding for GFP and zFoxp3 or an empty control
retrovirus, and the GFP.sup.+ population was analyzed for the
surface expression of (from left to right) CD25, GITR, CD152 and
CD4.
[0035] FIGS. 2D(i)-(iii) and 2E(i)-(iii) are bar graphs of
MACS-purified CD4.sup.+CD25.sup.- T-cells transduced with a
bicistronic retrovirus coding for GFP and zFoxp3, Foxp3 or an empty
control retrovirus. The GFP.sup.+ population was analyzed for its
proliferation, IL-2 and IFNg secretion upon activation with plate
bound antibodies to CD3 (mean cpm or pg/ml+s.d. in triplicate
wells) and (e) its suppressive activity on the proliferation and
IL-2 and IFNg secretion of mouse CD4.sup.+CD25.sup.- T-cells
activated with plate-bound antibodies to CD3 (mean cpm or
pg/ml+s.d. in triplicate wells).
[0036] FIG. 3A is a bar graph of expression of zFoxP3 determined by
real time PCR in zebrafish monocytes, lymphocytes and erythrocytes
sorted by FACS (mean+s.d. of triplicates).
[0037] FIG. 3B is a list of the conserved AHR binding site (CABS)
on zebrafish, human and mouse Foxp3 sequence indicated and
highlighted in yellow.
[0038] FIG. 3C is a pair of bar graphs of FoxP3 (left) and AHR
(right) expression in FACS sorted CD4.sup.+Foxp3:GFP.sup.+ and
CD4.sup.+Foxp3:GFP.sup.- T cells as measured by real time PCR
(mean+s.d. of triplicates normalized to GAPDH expression).
[0039] FIG. 3D is a bar graph of zFoxp3 expression 72 hours after
TCDD was added to the water of three-day post-fertilization
zebrafish embryos, as determined by real time PCR (mean+s.d. of
triplicates normalized to GAPDH expression).
[0040] FIG. 3E is a bar graph of frequency of CD4.sup.+FoxP3.sup.+
T cells in the CD4.sup.+ T-cell population as determined in the
draining lymph nodes by FACS from naive C57BL/6J mice 11 days after
after administration of 1 mg/mouse TCDD or corn oil as control, and
10 days after the mice were immunized (or not) with 100 mg/mouse of
MOG.sub.35-55/CFA (mean+s.d. of three mice).
[0041] FIG. 3F is a bar graph of proliferation of purified
CD4.sup.+ T cells stimulated with plate bound antibodies to CD3 in
the presence of different concentrations of TCDD for 72 hours. Cell
proliferation is indicated as cpm+s.d. in triplicate wells
(***P<0.0001, one-way ANOVA, n=3).
[0042] FIG. 3G is a set of three FACS plots of CD4.sup.+Foxp3:GFP T
cells in the CD4.sup.+ T-cell population from Foxp3.sup.gfp knock
in mice stimulated with plate bound antibodies to CD3 and CD28 for
5 days in the presence of normal media (control, left panel) TCDD
(middle panel) or TGFb1 (right panel).
[0043] FIG. 3H is a bar graph of Foxp3:GFP CD4+ T-cells positive
for the donor-specific marker CD90.2, isolated and analyzed by FACS
from host mice that received FACS-purified CD4.sup.+Foxp3:GFP.sup.-
2D2 T cells from CD90.2 Foxp3.sup.gfp knock in donor mice treated
with 1 mg/mouse of TCDD or corn oil as control and then immunized
with 100 mg/mouse MOG.sub.35-55/CFA. The results are presented as
the mean+s.d., five mice were included per group. *P<0.02,
unpaired t-test.
[0044] FIG. 3I Sequences corresponding to non-evolutionary
conserved AHR-binding sites (NCABS)-1, -2 and -3.
[0045] FIG. 3J is a schematic representation of the foxp3 gene.
Arrows indicate location of PCR primers used in ChIP assays, exons
are depicted in red, with their number indicated below them.
[0046] FIG. 3K is a bar graph of activation of the transcription of
Renilla luciferase-tagged foxp3 (BACFoxp3:Ren) by expression in
EL-4 cells of mouse AHR or a constitutively activated TGF receptor
II. Renilla activity was normalized to the luciferase activity of a
co-transfected control (mean+s.d. of triplicates).
[0047] FIG. 3L is a bar graph of ChIP analysis of the interaction
of AHR with NCABS and CABS in foxp3 and cyp1a1 in CD4+ T cells
treated with TCDD. (c) AHR, CYP1A1 and Foxp3 expression measured by
real time PCR on CD4+Foxp3:GFP- T cells (GFP-), CD4+Foxp3:GFP+ Treg
(GFP+) and CD4+Foxp3:GFP+ Treg treated with resveratrol for 5 h
(GFP++R) (mean+s.d. of triplicates normalized to GAPDH
expression).
[0048] FIG. 3M is a bar graph of ChIP analysis of the interaction
of AHR to the CABS and NCABS in foxp3 and cyp1a1 in thymic
CD4.sup.+ T cells from TCDD.sup.- or control-treated mice.
[0049] FIGS. 3N(i)-(iii) are bar graphs of AHR (N(i)), CYP1A1
(N(ii)) and Foxp3 (N(iii)) expression measured by real time PCR on
CD4+Foxp3:GFP- T cells (GFP-), CD4.sup.+Foxp3:GFP.sup.+ Treg
(GFP.sup.+) and CD4.sup.+Foxp3:GFP.sup.+ Treg treated with
resveratrol for 5 h (GFP.sup.++R) (mean+s.d. of triplicates
normalized to GAPDH expression).
[0050] FIG. 3O is a bar graph of the effect of AHR-inactivation
with resveratrol on the suppressive activity of CD4.sup.+
Foxp3:GFP.sup.+ Treg that were FACS-sorted from naive Foxp3gfp
mice, assayed using CD4.sup.+ Foxp3:GFP.sup.- cells activated with
antibodies to CD3 as effector T cells in the presence of
resveratrol. Cell proliferation is indicated as cpm+s.d. in
triplicate wells.
[0051] FIG. 3P is a bar graph of MOG.sub.35-55-specific suppressive
activity of Treg purified from TCDD or control-treated mice,
assayed using CD4.sup.+ Foxp3:GFP.sup.- 2D2 T cells. Cell
proliferation is indicated as cpm+s.d. in triplicate wells.
[0052] FIG. 3Q is a bar graph of suppressive activity of natural
Treg, or Treg induced with TGF.beta.1 (TGFb1) or TCDD (TCDD). Cell
proliferation is indicated as cpm+s.d. in triplicate wells.
[0053] FIG. 3R is a bar graph showing the effect of AHR activation
with TCDD on the proliferation of CD4.sup.+ Foxp3:GFP.sup.+ Treg
and CD4.sup.+ Foxp3:GFP.sup.- T cells that were FACS-sorted from
naive Foxp3gfp mice. Cell proliferation is indicated as cpm+s.d. in
triplicate wells.
[0054] FIG. 3S is a bar graph of the effect of AHR-activation with
TCDD on the suppressive activity of CD4.sup.+ Foxp3:GFP.sup.+ Treg
that were FACS-sorted from naive Foxp3gfp mice, assayed using
CD4.sup.+ Foxp3:GFP.sup.- cells activated with antibodies to CD3 as
effector T cells in the presence of resveratrol. Cell proliferation
is indicated as cpm+s.d. in triplicate wells.
[0055] FIG. 4A is a line graph showing the effect on EAE of TCDD,
or oil as control, administered ip to C57BL/6 mice. EAE was induced
24 hours later by immunization with MOG.sub.35-55/CFA. The course
of EAE in these mice is shown as the mean EAE score+s.e.m.
(P<0.001, two-way ANOVA, n=6).
[0056] FIG. 4B is a line graph showing the effect on EAE of TCDD,
or oil as control, administered ip to C57BL/6 wild type or AHR-mt
mice. EAE was induced 24 hours later by immunization with
MOG.sub.35-55/CFA. The course of EAE in these mice is shown as the
mean EAE score+s.e.m. (P<0.001, two-way ANOVA, n=10).
[0057] FIGS. 4C-D are bar graphs of the proliferative response to
MOG.sub.35-55 (4C) or antibodies to CD3 (4D) of lymph node cells
taken from TCDD or control treated animals 10 days after
immunization with MOG.sub.35-55/CFA. Cell proliferation is
indicated as cpm+s.d. in triplicate wells.
[0058] FIGS. 4E(i)-(iii) are bar graphs of cytokine secretion
(expressed as pg/ml) triggered by MOG.sub.35-55 in lymph node cells
taken from TCDD or control treated animals 10 days after
immunization with MOG.sub.35-55/CFA.
[0059] FIGS. 4F(i)-(ii) are bar graphs showing the decreased
frequency of CD4.sup.+IL17.sup.+ and CD4.sup.+IFNg.sup.+ T cells
associated to the inhibition of EAE by AHR activation with TCDD.
Draining lymph node cells were isolated from TCDD or control
treated mice 10 days after immunization with MOG.sub.35-55/CFA,
activated with MOG.sub.35-55, and stained for intracellular Foxp3,
IL-17 or IFNg. Data represent the mean percentage of cytokine.sup.+
cells within the effector CD4.sup.+Foxp3.sup.- T cell
population+s.d., five mice were included per group. *P<0.04,
unpaired t-test.
[0060] FIGS. 4G-I are bar graphs showing that AHR activation by
TCDD inhibits CNS inflammation, demyelination and axonal loss.
Briefly, quantification of the cellular infiltrate, demyelination
and axonal loss on the spinal cord of TCDD-treated and control
mice. Spinal cords were taken on day 19 after EAE induction and
stained with hematoxylin & eosin, luxol fast blue or silver
stain to quantify the cellular infiltrate (g), demyelination (h)
and axonal loss (i), respectively. The effect of TCDD-treatment was
analyzed using Student's t-test.
[0061] FIG. 5A is a bar graph illustrating the effects on EAE of
TCDD, or oil as control, administered ip to C57BL/6 mice. EAE was
induced 24 hours later by immunization with MOG.sub.35-55/CFA. The
frequency of CD4.sup.+FoxP3.sup.+ T cells in the spleen CD4.sup.+
T-cell population was determined 21 days after EAE induction by
FACS (mean+s.d. of five mice). *P<0.02, unpaired t-test.
[0062] FIG. 5B is a bar graph illustrating the proliferative
response to MOG.sub.35-55 of CD4.sup.+CD25.sup.- lymph node cells
taken from TCDD or control treated animals 10 days after
immunization with MOG.sub.35-55/CFA. Cell proliferation is
indicated as cpm+s.d. in triplicate wells.
[0063] FIG. 5C is a line graph of EAE scores in mice treated with
CD4.sup.+ or CD4.sup.+CD25.sup.- T cells (5.times.10.sup.6) that
were purified from TCDD or control treated mice 10 days after
immunization with MOG.sub.35-55/CFA. After 1 day, EAE was induced
in the recipient mice with MOG.sub.35-55/CFA. The course of EAE in
these mice is shown as the mean EAE score+s.e.m. (P<0.001,
two-way ANOVA, n=4).
[0064] FIG. 5D is a bar graph showing the proliferative response of
lymph node cells taken from TCDD-treated animals 10 days after
immunization with MOG.sub.35-55/CFA, activated in vitro with
MOG.sub.35-55 in the presence of blocking antibodies to IL-4,
IL-10, TGFb or isotype control. Cell proliferation is indicated as
cpm+s.d. in triplicate wells.
[0065] FIG. 5E is a line graph of EAE in naive wild type (WT) or
dominant negative TGFbRII mice injected with CD4.sup.+ T cells
(5.times.10.sup.6) purified from TCDD or control treated mice 10
days after immunization with MOG.sub.35-55/CFA. After 1 day, EAE
was induced in the recipient mice with MOG.sub.35-55/CFA. The
course of EAE in these mice is shown as the mean EAE score+s.e.m.
(P<0.001, two-way ANOVA, n=4).
[0066] FIG. 5F is a bar graph of proliferation to MOG.sub.35-55 of
CD4.sup.+Foxp3:GFP.sup.- lymph node cells from TCDD.sup.- or
control-treated Foxp3gfp mice, (cpm+s.d. in triplicate wells).
[0067] FIG. 5G is a bar graph of the recall cytokine response to
MOG.sub.35-55 of CD4.sup.+Foxp3:GFP.sup.- lymph node cells taken
from TCDD or control treated Foxp3gfp mice 10 days after
immunization with MOG.sub.35-55/CFA. Cytokine secretion is
expressed as pg/m in triplicate wells. FIG. 5H is a line graph of
clinical EAE scores. TCDD-treated mice showed a significant delay
in the onset of EAE (P=0.03, Student's t-test, n=9).
[0068] FIG. 5I is a pair of FACS plots from draining lymph node
cells recovered on day 18, stimulated with PMA/ionomycin and
stained for CD4 and intracellular IL-17 and IFN.gamma.. The numbers
in the quadrants show percentages of cytokine positive cells in the
CD4.sup.+Foxp3:GFP.sup.- T cell gate. Treatment with TCDD led to a
significant decrease in the frequency of CD4.sup.+ IL-17.sup.+ T
cells (p=0.03, Student's t-test, n=4).
[0069] FIGS. 6A-C are bar graphs showing that endogenous AHR
ligands control EAE development. 6A, the frequency of
CD4.sup.+Foxp3.sup.+ T cells in the CD4.sup.+ T-cell population was
determined by FACS in the blood of wild type C57BL/6 and AHR-mt
mice, and is presented as the mean+s.d. (n=5-11, p<0.03 t-test).
6B, EAE was induced in wild type C57BL/6J and AHR-mt mice by
immunization with MOG.sub.35-55/CFA. The course of EAE is shown as
the mean EAE score +s.e.m. (p<0.001, two-way ANOVA, n=6-8. 6C,
ITE (100 mg/mouse), TA (100 mg/mouse) or PBS as a control were
administered on daily basis to C57BL/6 mice. One day after the
first administration, EAE was induced by immunization with
MOG.sub.35-55/CFA. The course of EAE is shown as the mean EAE score
+s.e.m. (p<0.001, two-way ANOVA, n=9).
[0070] FIGS. 7A-B show the results of phylogenetic footprinting for
the identification of putative TFBS. The genomic sequences of
human, mouse, rat, dog and zebrafish Foxp3 were analyzed by
phylogenetic footprinting. 7A presents a phylogenetic tree; Tree
distances are in # of substitutions per 1 kb. 7B is a graph
illustrating the dynamic visualization of the location of putative
TFBS conserved between human (SEQ ID NO: 1) and zebrafish (SEQ ID
NO: 2).
[0071] FIGS. 8A-E and 9A-E are bar graphs showing expression levels
of transcription factors in cells transfected with Foxp3. FIGS.
8A-E show an increase in FOXP3 (8A) and NKX2.2 (8B), and a decrease
in EGR1 (8C), EGR2 (8D), and EGR3 (8E) in transfected cells. FIGS.
9A-D show an increase in NKX2.2, and a decrease in EGR1, EGR2, and
EGR3 expression in Foxp3 transfected cells at days 3 and 6.
[0072] FIG. 10 is a list of the binding sites of NKX22, EGR1, EGR2,
EGR3, NGFIC and Delta EF1 in the mouse Foxp3 gene, relative to the
numbering of the gene as shown in GenBank Acc. No.
NT.sub.--039700.6.
[0073] FIGS. 11 A-G are a list of all the AHR binding sites on the
mouse Foxp3 gene, GenBank Acc. No. NT.sub.--039700.6.
[0074] FIGS. 12A-F is the genomic sequence of the zebrafish Foxp3,
NW.sub.--644989.1.
[0075] FIG. 13 is a bar graph showing the effect of the monoamine
oxidase inhibitor Tranylcypromine on the suppression of EAE by TA.
C57BL/6 mice (4-7/group) were treated with TA or TA and
Tranylcypromine (INH), EAE was induced and the mice were monitored
for the development of EAE.
[0076] FIG. 14 is a bar graph of the expression of a renilla-tagged
mouse foxp3 locus on zebrafish embryos in the presence of
increasing amounts of TCDD.
[0077] FIGS. 15A-B are bar graphs of IP- and oral-ITE suppression
of EAE. EAE was induced in B6 mice (n=10), the mice were treated
daily with ITE (200 .mu.g/mouse) or vehicle administered orally or
intraperitoneally, and the mice were scored for EAE development on
daily basis.
[0078] FIGS. 16 and 17 are FACS plots (16) and bar graphs (17)
showing the induction of FoxP3.sup.+ T.sub.reg by IP administration
of ITE. EAE was induced in B6 mice (n=10), the mice were treated
with ITE or vehicle, and T.sub.reg levels were analyzed by FACS on
splenocytes at day 17 after EAE induction.
[0079] FIGS. 18 and 19 are FACS plots (18) and bar graphs (19)
showing the induction of FoxP3.sup.+ T.sub.reg by oral
administration of ITE. EAE was induced in B6 mice (n=10), the mice
were treated with ITE or vehicle, and T.sub.reg levels were
analyzed by FACS on splenocytes at day 17 after EAE induction.
[0080] FIGS. 20A and B are FACS plots (20A) and bar graphs (20B)
showing the: induction of FoxP:GFP3.sup.+ T.sub.reg by IP
administration of ITE to FoxP3.sup.gfp mice. B6 mice (n=3), the
mice were treated with ITE or vehicle, immunized with
CFA/MOG.sub.35-55 and T.sub.reg levels were analyzed by FACS on
splenocytes 10 days after immunization.
[0081] FIGS. 21A and B are FACS plots (21A) and bar graphs (21B)
showing the: induction of FoxP:GFP3.sup.+ T.sub.reg by IP
administration of ITE to FoxP3.sup.gfp mice. B6 mice (n=3), the
mice were treated with ITE or vehicle, immunized with
CFA/MOG.sub.35-55 and T.sub.reg levels were analyzed by FACS on
blood 10 days after immunization
[0082] FIGS. 22A and B are line graphs showing that IP-ITE
suppresses the recall response to MOG. EAE was induced in B6 mice
(n=3), the mice were treated with ITE or vehicle, and the recall
response to MOG.sub.35-55 (22A) or .alpha.CD3 (22B) on splenocytes
was analyzed at day 17 after EAE induction.
[0083] FIG. 22C is a set of six bar graphs of cytokine expression
in the same cells as in 22A and B.
[0084] FIGS. 23A and B are a line graph (23A) and a set of six bar
graphs (23B) showing that IP-ITE interferes with the generation of
T.sub.H1 and T.sub.H17 cells. EAE was induced in B6 mice (n=3), the
mice were treated with ITE or vehicle, and the induction of
T.sub.H1 and T.sub.H17 cells was followed at day 17 after EAE
induction by FACS and by ELISA.
[0085] FIGS. 24A and B are FACS plots (24A) and bar graphs (24B)
showing that oral-ITE decreases the recall response to MOG. EAE was
induced in B6 mice (n=3), the mice were treated with ITE or
vehicle, and the recall response to MOG.sub.35-55 (left panels) or
.alpha.CD3 (right panels) on splenocytes was analyzed at day 17
after EAE induction.
[0086] FIGS. 25A and B are FACS plots IP-ITE increases the
Treg:Teff ratio of MOG.sub.35-55 specific T cells. FoxP3.sup.gfp
mice (n=3) were immunized with MOG.sub.35-55 and treated with ITE
(lower panels of 25A) or vehicle (upper panels of 25A), and the
frequency of MOG.sub.35-55-specific Treg and Teff cells was
followed by FACS at day 10 after immunization.
[0087] FIG. 26 is a set of four line graphs showing that IP-ITE
suppresses the CD4+ T cell response to MOG.sub.35-55. FoxP3.sup.gfp
mice (n=3) were immunized with MOG.sub.35-55 and treated with ITE
or vehicle, and the recall response of sorted T cell populations
was analyzed at day 10 after immunization.
[0088] FIGS. 27A and B are bar graphs showing that IP-ITE
potentiates MOG.sub.35-55-specific T.sub.reg activity.
FoxP3.sup.gfp mice (n=3) were immunized with MOG.sub.35-55 and
treated with ITE or vehicle, FoxP3:GFP.sup.+ T.sub.reg were
FACS-sorted 10 days after immunization and their suppressive
activity was evaluated using 2D2 FoxP3:GFP.sup.- T cells as
responders. FIG. 27C is a bar graph showing that this effect could
be inhibited with antibodies blocking antibodies to TGFb1.
[0089] FIG. 28 is a pair of line graphs showing that CD4.sup.+ T
cells can transfer the protection against EAE induced by IP and
oral-ITE. T cells were sorted out from EAE B6 mice treated with
vehicle or ITE on day 20 after disease induction, 3 million cells
were transferred to naive B6 mice (n=5) and EAE was induced in the
recipients 2 days after the cell transfer.
[0090] FIG. 29 is a set of six FACS plots showing modulation of APC
populations by IP-ITE. EAE was induced in B6 mice (n=3), the mice
were treated with ITE or vehicle, and APC were analyzed by FACS on
splenocytes at day 17 after EAE induction.
[0091] FIG. 30 is a line graph showing that weekly administration
of ITE fails to suppress EAE development. EAE was induced in B6
mice (n=10) and the mice were treated daily or weekly with 200
.mu.g/mouse of ITE or vehicle.
[0092] FIG. 31 is a schematic diagram of gold nanoparticles for
AHR-ligand delivery.
[0093] FIG. 32 is a pair of graphs showing the functionality of
gold nanoparticles containing AHR-ligands. Nanoparticles were
evaluated for their ability to activate the luciferase activity of
an AHR reporter cell line.
[0094] FIG. 33 is a bar graphs showing modulation of EAE by
AHR-ligand nanoparticles. EAE was induced in B6 mice (n=5), the
mice were treated with nanoparticles weekly starting from day 0,
and the animals were followed for signs of EAE.
[0095] FIG. 34 is a set of nine FACS plots showing induction of
FoxP3.sup.+ Treg by nanoparticle-mediated delivery of ITE. EAE was
induced in B6 mice (n=5), the mice were treated with nanoparticles
weekly starting from day 0, and T.sub.reg levels were analyzed by
FACS on splenocytes at day 22 after EAE induction.
[0096] FIGS. 35A and B show nanoparticle-mediated delivery of ITE
suppresses the recall response to MOG. EAE was induced in B6 mice
(n=5), the mice were treated with ITE or vehicle, and the recall
response to MOG.sub.35-55 (35A, top) or .alpha.CD3 (35A, bottom) on
splenocytes was analyzed at day 22 after EAE induction. FIG. 35B
shows the cytokine response in the same cells.
[0097] FIG. 36 is a set of FACS plots showing induction of human
CD4+FoxP3+ T cells by TCDD. CD4.sup.+ CD62L.sup.+ CD45RO.sup.- T
cells were isolated by FACS and differentiated in vitro for 5 days
with antibodies to CD3 and CD28 in the presence of TCDD 100 nM or
TGF.beta.1 2.5 ng/ml or both.
[0098] FIG. 37 is a set of four FACS plots demonstrating
heterogeneity in the induction of human CD4.sup.+ FoxP3.sup.+ T
cells by TCDD. CD4.sup.+ CD62L.sup.+ CD45RO.sup.- T cells were
isolated by FACS and differentiated in vitro for 5 days with
antibodies to CD3 and CD28 in the presence of TCDD 100 nM or
TGF.beta.1 2.5 ng/ml or both.
[0099] FIG. 38 is a pair of bar graphs showing activation of human
T cells in the presence of TCDD induces suppressive T cells.
CD4.sup.+ CD62L+ CD45RO.sup.- T cells were isolated by FACS and
differentiated in vitro for 5 days with antibodies to CD3 and CD28
in the presence of TCDD or TGF.beta.1 2.5 ng/ml or both, and after
repurification by FACS, CD4.sup.+ CD25.sup.High and CD4.sup.+
CD25.sup.Low T cells were assayed for their suppressive activity on
non-treated effector T cells activated with antibodies to CD28 and
CD3.
[0100] FIG. 39 is a: FoxP3 expression by in vitro differentiated
human T cells. CD4.sup.+ CD62L.sup.+ CD45RO.sup.- T cells were
isolated by FACS and differentiated in vitro for 5 days with
antibodies to CD3 and CD28 in the presence of TCDD 100 nM or
TGF.beta.1 2.5 ng/ml or both, and FoxP3 expression was analyzed by
real-time PCR on CD25.sup.High or CD25.sup.Low sorted CD4 T
cells.
[0101] FIG. 40 is a pair of bar graphs showing AHR expression by in
vitro differentiated human T cells. CD4.sup.+ CD62L.sup.+
CD45RO.sup.- T cells were isolated by FACS and differentiated in
vitro for 5 days with antibodies to CD3 and CD28 in the presence of
TCDD 100 nM or TGF.beta.31 2.5 ng/ml or both, and AHR expression
was analyzed by real-time PCR on CD25.sup.High or CD25.sup.Low
sorted CD4 T cells.
[0102] FIG. 41 is a pair of bar graphs showing IL-10 production by
in vitro differentiated human T cells. CD4.sup.+ CD62L.sup.+
CD45RO.sup.- T cells were isolated by FACS and differentiated in
vitro for 5 days with antibodies to CD3 and CD28 in the presence of
TCDD 100 nM or TGF.beta.1 2.5 ng/ml or both, and IL-10 production
was analzyed by real-time PCR on CD25.sup.High or CD25.sup.Low
sorted CD4 T cells.
[0103] FIG. 42 is a bar graph showing the suppressive activity of
human CD4.sup.+ CD25.sup.High T cells induced with TCDD is
dependent on IL-10. CD4.sup.+ CD62L.sup.+ CD45RO.sup.- T cells were
isolated by FACS and differentiated in vitro for 5 days with
antibodies to CD3 and CD28 in the presence of TCDD or TGF.beta.1
2.5 ng/ml or both, and after re-purification by FACS, CD4.sup.+
CD25.sup.High T cells were assayed for their suppressive activity
on non-treated effector T cells activated with antibodies to CD28
and CD3 in the presence of blocking antibodies to IL-10.
DETAILED DESCRIPTION
[0104] Because of the importance of the central role Tregs play in
immunomodulation, characterization of the pathways and
identification of compounds capable of modulating these pathways,
e.g., to promote the generation (e.g., differentiation of cells to
or towards) Treg cells or that promote increased activity of Tregs
is important for the treatment of, e.g., autoimmunity, infections
and cancer.
[0105] The present invention provides, inter alia, compositions and
methods useful for therapeutic immunomodulation.
[0106] Accordingly, the present invention is based, at least in
part, on the discovery that modulation of the AhR by compounds
described herein can be used to modulate (e.g., increase or
decrease the number and/or activity of) immunomodulatory cells in
vitro and in vivo.
[0107] In some embodiments, the present invention is based on the
identification of the ligand-activated transcription factor aryl
hydrocarbon receptor (AHR) as a Foxp3 dependent regulator of Treg
differentiation (e.g., generation) and/or activity in vitro and in
vivo. Also described herein are ligands of a transcription factor
(e.g., AHR) that cause increased Treg expression and/or activity.
More specifically, the data presented herein demonstrates the use
of AHR-specific ligands, e.g., the high affinity AHR ligand
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), tryptamine (TA), and/or
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE), to promote an increase in the number and/or activity of Treg
immunomodulatory cells, which will be useful to suppress the immune
response in the treatment of diseases or disorders caused by an
abnormal (e.g., an excessive, elevated, or inappropriate) immune
response, e.g., an autoimmune disease or disorder. Surprisingly,
effective doses of TCDD can be administered intravenously or
orally.
[0108] Other potentially useful AHR transcription factor ligands
are described in Denison and Nagy, Ann. Rev. Pharmacol. Toxicol.,
43:309-34, 2003, and references cited herein, all of which are
incorporated herein in their entirety. Other such molecules include
planar, hydrophobic HAHs (such as the polyhalogenated
dibenzopdioxins, dibenzofurans, and biphenyls) and PAHs (such as
3-methylcholanthrene, benzo(a)pyrene, benzanthracenes, and
benzoflavones), and related compounds. (Denison and Nagy, 2003,
supra). Nagy et al., Toxicol. Sci. 65:200-10 (2002), described a
high-throughput screen useful for identifying and confirming other
ligands. See also Nagy et al., Biochem. 41:861-68 (2002). In some
embodiments, those ligands useful in the present invention are
those that bind competitively with TCDD, TA, and/or ITE.
[0109] In some embodiments, the present invention provides methods
useful for identifying transcription factors (e.g.,
ligand-activated transcription factors) and/or ligands (e.g.,
ligands capable of promoting an increased association between a
ligand-activated transcription factor and Foxp3) capable of
modulating (e.g., increasing or decreasing) Foxp3 expression or
activity.
Therapeutic Sequences
[0110] As stated above, the present invention includes the
identification of specific transcription factor binding sites in
the Foxp3 gene. These binding sites include, i.e., NKX22, AHR,
EGR1, EGR2, EGR3, NGFIC, and Delta EF1. As described herein,
manipulating activity and/or levels of those TFs can alter
expression of Foxp3, and thus modulate (e.g., promote) generation
and/or increased activity of Treg in vivo and in vitro. Compounds
that modulate the activity and/or levels of those TFs to increase
generation and/or activity of Treg are useful, e.g., in the
treatment of disorders in which it is desirable to decrease an
aberrant immune response, e.g., autoimmune diseases.
[0111] Sequences useful in the methods described herein include,
but are not limited to, e.g., NKX22, AHR, EGR1, EGR2, EGR3, NGFIC
and Delta EF1 sequences, and TF binding sequences therefore, all of
which are known in the art. In some embodiments, the methods
include the use of nucleic acids or polypeptides that are at least
80% identical to a human NKX22, AHR, EGR1, EGR2, EGR3, NGFIC, or
Delta EF1 sequence, e.g., at least 80%, 85%, 90%, or 95% identical
to a human sequence as described herein.
[0112] To determine the percent identity of two sequences, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in one or both of a first and a second amino acid
or nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). The length
of a reference sequence aligned for comparison purposes is at least
60%, e.g., at least 70%, 80%, 90%, 100% of the length of the
reference sequence. The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0113] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In the present methods, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm
which has been incorporated into the GAP program in the GCG
software package (available on the world wide web at www.gcg.com),
using a Blossum 62 scoring matrix with a gap penalty of 12, a gap
extend penalty of 4, and a frameshift gap penalty of 5.
[0114] Active fragments of TFs useful in the methods described
herein are those fragments that bind to the same DNA sequence
(e.g., promoter sequence) that the full-length TF binds to, and has
at least 30% of the transcription initiating activity of the
full-length TF, e.g., at least 40%, 50%, 60%, 70%, 80%, 90% or more
of the activity of the full-length protein, on the same promoters
and the same genes as the full-length protein.
Foxp3
[0115] At least in some species, Treg differentiation and function
is driven by the transcription factor Foxp3 (Fontenot et al., Nat.
Immunol., 4:330-336, 2003; Hori et al., Science, 299:1057-61,
2003). Foxp3 may also be important for human Treg; mutations in
Foxp3 have been linked to various immunological conditions (e.g.,
autoimmune conditions), for example, autoimmune syndrome immune
dysregulation, polyendocrinopathy, and enteropathy X-linked (IPEX)
(Chatila et al., J. Clin. Invest., 106:R75-81 (2000); Gavin et al.,
Proc. Natl. Acad. Sci. U.S.A., 103: 6659-64 (2006)). In humans,
Foxp3-negative Tregs have also been described, see, e.g., Roncarolo
and Gregori, Eur J Immunol. 38, 925 (2008).
[0116] Exemplary human Foxp3 mRNA sequences are known in the art
and include Genbank Acc. No. NM.sub.--014009.2; the amino acid
sequence of the protein is Genbank Acc. No. NP.sub.--054728.2. The
sequence of the human Foxp3 gene can be found at NC.sub.--000023.9;
the mouse gene is at NT.sub.--039700.6. The Foxp3 promoter has been
identified and sequenced, see, e.g., Mantel et al., J. Immunol. 176
(6): 3593 (2006). All of the binding sites for AHR in the mouse
Foxp3 gene are highlighted, e.g., in FIGS. 11A-G; the binding sites
for the other TFs are identified in FIGS. 10A-B.
Transcription Factors That Increase Transcription of Foxp3
[0117] As described herein, NKX22, AHR, and Delta EF1 increase
transcription of Foxp3. Therefore, compounds that increase levels
and/or activity of these TFs would increase the generation and/or
activity of Treg. Conversely, compounds that decrease levels and/or
activity of these TFs would be expected to decrease generation of
Tregs, thereby increasing the immune response.
AHR
[0118] Exemplary human AhR mRNA sequences are known in the art and
include Genbank Acc. No. NM.sub.--001621.3; the amino acid sequence
of the protein is Genbank Acc. No. NP.sub.--001612.1. Active
fragments of AhR are DNA binding fragments with transcription
activity, and contain at least one PAS region, e.g., amino acids
122-224 or 282-381 of NP.sub.--001612.1. Consensus recognition
sequences that bind AhR include the sequence TNGCGTG.
DeltaEF1
[0119] Exemplary human DeltaEF1 mRNA sequences are known in the art
and include Genbank Acc. No. NM.sub.--030751.3; the amino acid
sequence of the protein is Genbank Acc. No. NP.sub.--110378.2.
Consensus recognition sequences that bind DeltaEF1 include the
sequences CACCT and CACCTG (Sekido et al., Genes Cells 2:771-783
(1997)).
NKX2.2
[0120] Exemplary human NKX2.2 mRNA sequences are known in the art
and include Genbank Acc. No. NM.sub.--002509.2; the amino acid
sequence of the protein is Genbank Acc. No. NP.sub.--002500.1.
Consensus recognition sequences that bind NKX2.2 include the
sequences ACTTGAT and T(T/C)AAGT(A/G)(C/G)TT (Watada et al., Proc.
Natl. Acad. Sci. U.S.A. 97 (17):9443-9448 (2000))
Transcription Factors That Decrease Transcription of Foxp3
[0121] As described herein, EGR1, EGR2, EGR3, and NGFIC (EGR4)
decrease transcription of Foxp3. Therefore, compounds that increase
levels and/or activity of these TFs would decrease generation of
Tregs, thereby increasing the immune response. Conversely,
compounds that decrease levels and/or activity of these TFs would
be expected to increase generation of Tregs, reducing the immune
response.
EGR1
[0122] The sequence of human egr1 protein is available in the
GenBank database at Accession No. NP.sub.--001955.1; the mRNA is at
Accession No. NM.sub.--001964.2. Additional information regarding
egr1 can be found on the internet at ncbi.nlm.nih.gov, in the
UniGene database at UniGene Hs.326035, and in the Entrez Gene
database at GeneID: 1958. Consensus recognition sequences that bind
EGR1 include the sequence 5'GCG(G/T)GGGCG3' (Nakagama et al., Mol.
Cell. Biol., 15 (3):1489-1498 (1995)).
[0123] Active fragments of egr1 include those portions of the
protein that bind DNA, e.g., one or more of the two C2H2 type
DNA-binding zinc fingers (see, e.g., Sukhatme et al., 1988, supra),
e.g., amino acids 338-362 and/or 368-390 of GenBank Acc. No.
NP.sub.--001955.1. Exemplary active fragments are described in
Huang et al., Cancer Res. 1995; 55 (21):5054-5062, and in Jain et
al., J. Biol. Chem. 1996; 271 (23):13530-6.
[0124] Inhibitors of egr-1 are described in WO2007/118157.
EGR2
[0125] The sequence of human egr2 protein is available in the
GenBank database at Accession No. NP.sub.--000390.2; the mRNA is at
Accession No. NM.sub.--000399.2.
[0126] Consensus recognition sequences that bind EGR2 include the
sequences GCGGGGGCG and T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase
letters indicate bases selected less frequently) (Swirnoff and
Milbrandt, Mol. Cell. Biol. 15:2275-2287 (1995)).
EGR3
[0127] The sequence of human egr3 protein is available in the
GenBank database at Accession No. NP.sub.--004421.2; the mRNA is at
Accession No. NM.sub.--004430.2. The gen
[0128] Consensus recognition sequences that bind EGR3 include the
sequences GCGGGGGCG and T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase
letters indicate bases selected less frequently) (Swirnoff and
Milbrandt, Mol. Cell. Biol. 15:2275-2287 (1995)).
NGFIC (EGR4)
[0129] Exemplary human NGFIC mRNA sequences are known in the art
and include Genbank Acc. No. NM.sub.--001965.2; the amino acid
sequence of the protein is Genbank Acc. No. NP.sub.--001956.2. See
Crosby et al., Mol Cell Biol. 11 (8):3835-41 (1991).
[0130] Consensus recognition sequences that bind NGFIC include the
sequences GCGGGGGCG and T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase
letters indicate bases selected less frequently) (Swirnoff and
Milbrandt, Mol. Cell. Biol. 15:2275-2287 (1995)).
Methods of Identifying Compounds That Modulate Expression, Levels
Or Activity of One Or More of NKX22, AHR, EGR1, EGR2, EGR3, NGFIC
And Delta EF1
[0131] A number of methods are known in the art for evaluating
whether a compound alters expression, levels or activity of one or
more of NKX22, AHR, EGR1, EGR2, EGR3, NGFIC, and/or Delta EF1.
[0132] Methods of assessing expression are well known in the art
and include, but are not limited to, Northern analysis,
ribonuclease protection assay, reverse transcription-polymerase
chain reaction (RT-PCR), real time PCR, and RNA in situ
hybridization (see, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3.sup.rd Ed., Cold Spring Harbor Laboratory
Press (2001)). Levels of peptides can be monitored by, e.g.,
Western analysis, immunoassay, or in situ hybridization. Activity,
e.g., altered promoter binding and/or transcription activity, can
be determined by, e.g., electrophoretic mobility shift assay, DNA
footprinting, reporter gene assay, or a serine, threonine, or
tyrosine phosphorylation assay. In some embodiments, the effect of
a test compound on expression, level or activity is observed as a
change in glucose tolerance or insulin secretion of the cell, cell
extract, co-culture, explant, or subject. In some embodiments, the
effect of a test compound on expression, level, or activity of one
or more of NKX22, AHR, EGR1, EGR2, EGR3, NGFIC, and/or Delta EF1,
is evaluated in a transgenic cell or non-human animal, or explant,
tissue, or cell derived therefrom, having altered glucose tolerance
or insulin secretion, and can be compared to a control, e.g.,
wild-type animal, or explant or cell derived therefrom.
[0133] The effect of a test compound on expression, level, or
activity can be evaluated in a cell, e.g., a cultured mammalian
cell, a pancreatic beta cell, cell lysate, or subject, e.g., a
non-human experimental mammal such as a rodent, e.g., a rat, mouse,
or rabbit, or a cell, tissue, or organ explant, e.g., pancreas or
pancreatic cells.
[0134] In some embodiments, the ability of a test compound to
modulate level, expression or activity of one or more of NKX22,
AHR, EGR1, EGR2, EGR3, NGFIC and/or Delta EF1 is evaluated in a
knockout animal, or other animal having decreased expression,
level, or activity of one or more of NKX22, AHR, EGR1, EGR2, EGR3,
NGFIC and/or Delta EF1 conditional knockout transgenic animal.
[0135] In some embodiments, the ability of a test compound to
modulate, e.g., increase or decrease, e.g., permanently or
temporarily, expression from one or more of NKX22, AHR, EGR1, EGR2,
EGR3, NGFIC, and/or Delta EF1 promoter can be evaluated by, e.g., a
routine reporter (e.g., LacZ or GFP) transcription assay. For
example, a cell or transgenic animal whose genome includes a
reporter gene operably linked to an NKX22, AHR, EGR1, EGR2, EGR3,
NGFIC and/or Delta EF1 promoter can be contacted with a test
compound; the ability of the test compound to increase or decrease
the activity of the reporter gene or gene product is indicative of
the ability of the compound to modulate expression of the TF. In
another example, a cell or transgenic animal whose genome includes
a reporter gene operably linked to a promoter comprising a
recognition sequence for one of those TFs, e.g., all or a portion
of the Foxp3 promoter comprising recognition sequences for one of
those TFs, can be contacted with a test compound; the ability of
the test compound to increase or decrease the activity of the
reporter gene or gene product is indicative of the ability of the
compound to modulate activity of the TF.
[0136] The test compound can be administered to a cell, cell
extract, explant, or subject (e.g., an experimental animal)
expressing a transgene comprising an NKX22, AHR, EGR1, EGR2, EGR3,
NGFIC, and/or Delta EF1 promoter or recognition sequence fused to a
reporter such as GFP or LacZ (see, e.g., Nehls et al., Science,
272:886-889 (1996), and Lee et al., Dev. Biol., 208:362-374 (1999),
describing placing the beta-galactosidase reporter gene under
control of the whn promoter). Enhancement or inhibition of
transcription of a transgene, e.g., a reporter such as LacZ or GFP,
as a result of an effect of the test compound on the promoter or
factors regulating transcription from the promoter, can be used to
assay an effect of the test compound on transcription of one or
more of the TFs identified herein. Reporter transcript levels, and
thus promoter activity, can also be monitored by other known
methods, e.g., Northern analysis, ribonuclease protection assay,
reverse transcription-polymerase chain reaction (RT-PCR) or RNA in
situ hybridization (see, e.g., Cuncliffe et al., Mamm Genome,
13:245-252 (2002); Sambrook et al., Molecular Cloning: A Laboratory
Manual, 3.sup.rd Ed., Cold Spring Harbor Laboratory Press (2001)).
Test compounds can also be evaluated using a cell-free system,
e.g., an environment including a promoter-reporter transgene (e.g.,
an ARNT promoter-LacZ transgene), transcription factors binding the
promoter, a crude cell lysate or nuclear extract, and one or more
test compounds (e.g., a test compound as described herein), wherein
an effect of the compound on promoter activity is detected as a
color change.
[0137] In one embodiment, the screening methods described herein
include the use of a chromatin immunoprecipitation (ChIP) assay, in
which cells, e.g., pancreatic beta cells, expressing one or more of
the TFs identified herein, are exposed to a test compound. The
cells are optionally subjected to crosslinking, e.g., using UV or
formaldehyde, to form DNA-protein complexes, and the DNA is
fragmented. The DNA-protein complexes are immunoprecipitated, e.g.,
using an antibody directed to one or more of the TFs identified
herein. The protein is removed (e.g., by enzymatic digestion) and
analyzed, e.g., using a microarray. In this way, changes in binding
of the transcription factor to its target genes can be evaluated,
thus providing a measure of activity of the TFs identified
herein.
Test Compounds
[0138] Test compounds for use in the methods described herein are
not limited and can include crude or partially or substantially
purified extracts of organic sources, e.g., botanical (e.g.,
herbal) and algal extracts, inorganic elements or compounds, as
well as partially or substantially purified or synthetic compounds,
e.g., small molecules, polypeptides, antibodies, and
polynucleotides, and libraries thereof.
[0139] A test compound that has been screened by a method described
herein and determined to increase expression, levels, or activity
of one or more of the TFs described herein can be considered a
candidate compound for the treatment of a disorder treatable with
immune therapy (i.e., by increasing or decreasing control of the
immune response by increasing or decreasing levels of Treg), e.g.,
cancer, or an autoimmune disorder. A candidate compound that has
been screened, e.g., in an in vivo model of a disorder treatable
with immune therapy, e.g., cancer, or an autoimmune disorder, and
determined to have a desirable effect on the disorder, e.g., on one
or more symptoms of the disorder, can be considered a candidate
therapeutic agent. Candidate therapeutic agents, once screened and
verified in a clinical setting, are therapeutic agents. Candidate
therapeutic agents and therapeutic agents can be optionally
optimized and/or derivatized, and formulated with physiologically
acceptable excipients to form pharmaceutical compositions.
Methods of Treatment
[0140] As described above, the present invention is based, at least
in part, on the identification of useful targets for therapeutic
immunomodulation. Accordingly, the present invention provides
compositions and methods for treating a patient (e.g., a human)
with an immunological condition Immunological conditions that will
benefit from treatment using the present invention include those
diseases or disorders caused by an autoimmune response or an absent
or insufficient immune response.
Autoimmunity
[0141] Autoimmunity is presently the most common cause of disease
in the world and is the third most prevent disease in the U.S.
Autoimmune conditions that may benefit from treatment using the
compositions and methods described herein include, but are not
limited to, for example, Addison's Disease, alopecia, ankylosing
spondylitis, antiphospholipid syndrome, autoimmune hemolytic
anemia, autoimmune hepatitis, autoimmune oophoritis, Bechet's
disease, bullous pemphigoid, celiac disease, chronic fatigue immune
dysfunction syndrome (CFIDS), chronic inflammatory demyelinating
polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid,
cold agglutinin disease, CREST Syndrome, Crohn's disease, diabetes
(e.g., type I), dysautonomia, endometriosis, eosinophilia-myalgia
syndrome, essential mixed cryoglobulinemia, fibromyalgia,
syndrome/fibromyositis, Graves' disease, Guillain Barre syndrome,
Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic
thrombocytopenia purpura (ITP), inflammatory bowel disease (IBD),
lichen planus, lupus, Meniere's disease, mixed connective tissue
disease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus,
pernicious anemia, polyarteritis nodosa, polychondritis,
polymyalgia rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's
phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid
arthritis, sarcoidosis, scleroderma, Sjogren's syndrome,
spondyloarthropathy, stiff-man syndrome, Takayasu arteritis,
temporal arteritis/giant cell arteritis, thyroid disease,
ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's
granulomatosis.
[0142] As described herein, a patient with one or more autoimmune
conditions can be treated by increasing the number of Treg cells
and/or the activity of Treg cells in the patient using, e.g., a
therapeutically effective amount of one or more transcription
factors (e.g., a ligand-activated transcription factor such as AHR)
and/or one or more transcription factor ligands (e.g., TCDD,
tryptamine (TA), and/or
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE)) that are capable of promoting an increase in the expression
and/or activity of Foxp3, and thereby promoting an increase in the
number or activity of Treg cells in vitro and/or in vivo.
[0143] In some embodiments, the methods include administering
(e.g., to a population of T cells or to a subject) a composition
comprising a nucleic acid encoding a transcription factor as
described herein, e.g., e.g., NKX22, AHR, EGR1, EGR2, EGR3, NGFIC
and/or Delta EF1. The nucleic acid can be in an expression vector,
e.g., a modified viral vector such as is known in the art, e.g., a
lentivirus, retrovirus, or adenovirus. Methods for using these
vectors in cell or gene therapy protocols are known in the art. For
cell therapy methods, it is desirable to start with a population of
T cells taken from the subject to be treated.
[0144] In some embodiments, the methods include administering a
composition comprising a ligand that activates a transcription
factor described herein, e.g., the AHR receptor. In some
embodiments, the ligand is co-administered with one or more
inhibitors of its degradation, e.g., tryptamine together with a
monoamine oxidase inhibitor, e.g., tranylcypromine. The inhibitor
can be administered in the same or in a separate composition. Thus
the invention also includes compositions comprising tryptamine and
an inhibitor of its degradation, e.g., a MAOI, e.g.,
tranylcypromine.
[0145] In some embodiments, a patient in need of treatment can be
administered a pharmaceutically effective dose of one or more
ligands capable of promoting an increase in the expression and/or
activity of Foxp3 and thereby promoting an increase in the number
or activity of Treg cells in vitro and/or in vivo (e.g., TCDD,
tryptamine (TA), and/or
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE)).
[0146] Alternatively or in addition, a population of cells capable
of differentiation into Treg cells (e.g., naive T cells and/or
CD4.sup.+CD62 ligand.sup.+ T cells) can be contacted with a
transcription factor ligand capable of promoting increase in Foxp3
expression and/or activity (e.g., TCDD, tryptamine (TA), and/or
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE)) in vitro, thereby effectively promoting an increase in the
number of Treg cells in the population. Alternatively or in
addition, a population of cells containing Treg cells (e.g.,
isolated Treg cells (e.g., 100%) or a population of cells
containing at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% Treg
cells) can be contacted with a transcription factor ligand capable
of promoting an increase in Foxp3 expression and/or activity (e.g.,
TCDD, tryptamine (TA), and/or
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE)), thereby effectively promoting an increase in the activity
of the Treg cells in the population. Alternatively or in addition,
the cells can be contacted with an expression vector, e.g., a viral
vector such as a lentivirus, retrovirus, or adenovirus, comprising
a nucleic acid encoding a transcription factor described herein,
e.g., NKX22, AHR, EGR1, EGR2, EGR3, NGFIC and Delta EF1. In some
embodiments, the cells are also activated, e.g., by contacting them
with an effective amount of a T cell activating agent, e.g., a
composition of one or both of anti-CD3 antibodies and anti-CD28
antibodies. One or more cells from these populations can then be
administered to the patient alone or in combination with one or
more ligands capable of promoting an increase in the expression
and/or activity of Foxp3 and thereby promoting an increase in the
number or activity of Treg cells in vitro and/or in vivo (e.g.,
TCDD, tryptamine (TA), and/or
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE).
Patient Selection
[0147] The compositions and methods described herein are of
particular use for treating a patient (e.g., a human) that would
benefit from therapeutic immunomodulation (e.g., a patient in need
of a suppressed immune response). The methods include selecting a
patient in need of treatment and administering to the patient one
or more of the compositions described herein. A subject in need of
treatment can be identified, e.g., by their medical
practitioner.
[0148] In some embodiments, the methods include determining
presence and/or levels of autoantibodies to an autoantigen specific
for the disease, e.g., the presence and/or levels of autoantibodies
to an autoantigen listed in Table 1 or 2. The results can be used
to determine a subject's likelihood or risk of developing the
disease; subjects can be selected for treatment using a method
described herein based on the presence and/or levels of
autoantibodies.
Validation of Treatment/Monitoring Treatment Efficacy
[0149] During and/or following treatment, a patient can be assessed
at one or more time points, for example, using methods known in the
art for assessing severity of the specific autoimmune disease or
its symptoms, to determine the effectiveness of the treatment. In
some embodiments, levels of autoantibodies to an autoantigen
specific for the disease can also be monitored, e.g., levels of
autoantibodies to an autoantigen listed in Table 1 or 2; a decrease
(e.g., a significant decrease) in levels of autoantibodies would
indicate a positive response, i.e., indicating that the treatment
is successful; see, e.g., Quintana et al., Proc. Natl. Acad. Sci.
U.S.A., 101 (suppl. 2):14615-14621 (2004). Treatment can then be
continued without modification, modified to improve the progress or
outcome (e.g., increase dosage levels, frequency of administration,
the amount of the pharmaceutical composition, and/or change the
mode of administration), or stopped.
Administration
[0150] A therapeutically effective amount of one or more of the
compositions described herein can be administered by standard
methods, for example, by one or more routes of administration,
e.g., by one or more of the routes of administration currently
approved by the United States Food and Drug Administration (FDA;
see, for example world wide web address
fda.gov/cder/dsm/DRG/drg00301.htm), e.g., orally, topically,
mucosally, intravenously or intramuscularly.
[0151] In some embodiments, one or more of the ligands described
herein can be administered orally with surprising
effectiveness.
Pharmaceutical Formulations
[0152] A therapeutically effective amount of one or more of the
compositions (e.g., including, but not limited to, one or more of
the small molecule ligands, for example TCDD, tryptamine (TA),
and/or 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl
ester (ITE)) described herein can be incorporated into
pharmaceutical compositions suitable for administration to a
subject, e.g., a human. Such compositions typically include the
composition and a pharmaceutically acceptable carrier. As used
herein the language "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances are known. Except insofar as any
conventional media or agent is incompatible with the active
compound, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions, e.g., an inhibitor of degradation of the
ligand.
[0153] In some embodiments, the composition can also include an
autoantigen, e.g., an autoantigen listed in Table 1 or 2, or
another autoantigen known in the art to be associated with an
autoimmune disease.
[0154] A pharmaceutical composition can be formulated to be
compatible with its intended route of administration. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0155] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0156] Sterile injectable solutions can be prepared by
incorporating the composition (e.g., an agent described herein) in
the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0157] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, PRIMOGEL.TM. (sodium carboxymethyl starch), or corn
starch; a lubricant such as magnesium stearate or STEROTES.TM. ; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0158] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known, and include,
for example, for transmucosal administration, detergents, bile
salts, and fusidic acid derivatives. Transmucosal administration
can be accomplished through the use of nasal sprays or
suppositories. For transdermal administration, the active compounds
are formulated into ointments, salves, gels, or creams as generally
known in the art.
[0159] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0160] Nucleic acid molecules can be inserted into vectors and used
as gene therapy vectors. Gene therapy vectors can be delivered to a
subject by, for example, intravenous injection, local
administration (see U.S. Pat. No. 5,328,470) or by stereotactic
injection (see e.g., Chen et al., PNAS 91:3054-3057, 1994). The
pharmaceutical preparation of the gene therapy vector can include
the gene therapy vector in an acceptable diluent, or can include a
slow release matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g. retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
[0161] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration. In one aspect, the pharmaceutical compositions can
be included as a part of a kit.
[0162] Generally the dosage used to administer a pharmaceutical
compositions facilitates an intended purpose for prophylaxis and/or
treatment without undesirable side effects, such as toxicity,
irritation or allergic response. Although individual needs may
vary, the determination of optimal ranges for effective amounts of
formulations is within the skill of the art. Human doses can
readily be extrapolated from animal studies (Katocs et al., Chapter
27 In: "Remington's Pharmaceutical Sciences", 18th Ed., Gennaro,
ed., Mack Publishing Co., Easton, Pa., 1990). Generally, the dosage
required to provide an effective amount of a formulation, which can
be adjusted by one skilled in the art, will vary depending on
several factors, including the age, health, physical condition,
weight, type and extent of the disease or disorder of the
recipient, frequency of treatment, the nature of concurrent
therapy, if required, and the nature and scope of the desired
effect(s) (Nies et al., Chapter 3, In: Goodman & Gilman's
[0163] "The Pharmacological Basis of Therapeutics", 9th Ed.,
Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).
AHR Ligand-Nanoparticles
[0164] As demonstrated herein, compositions comprising
nanoparticles linked to AHR ligands are surprisingly effective in
delivering the ligand, both orally and by injection, and in
inducing the Treg response in living animals. Thus, the invention
further includes compositions comprising AHR ligands linked to
biocompatible nanoparticles, optionally with antibodies that target
the nanoparticles to selected cells or tissues.
AHR Transcription Factor Ligands
[0165] AHR-specific ligands, e.g., the high affinity AHR ligand
2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), tryptamine (TA),
and/or 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl
ester (ITE), promote an increase in the number and/or activity of
Treg immunomodulatory cells, which will be useful to suppress the
immune response in the treatment of diseases or disorders caused by
an abnormal (e.g., an excessive, elevated, or inappropriate) immune
response, e.g., an autoimmune disease or disorder.
[0166] Other potentially useful AHR transcription factor ligands
are described in Denison and Nagy, Ann Rev. Pharmacol. Toxicol.,
43:309-34, 2003, and references cited herein, all of which are
incorporated herein in their entirety. Other such molecules include
planar, hydrophobic HAHs (such as the polyhalogenated
dibenzo-pdioxins, dibenzofurans, and biphenyls) and PAHs (such as
3-methylcholanthrene, benzo(a)pyrene, benzanthracenes, and
benzoflavones), and related compounds. (Denison and Nagy, 2003,
supra). Nagy et al., Toxicol. Sci. 65:200-10 (2002), described a
high-throughput screen useful for identifying and confirming other
ligands. See also Nagy et al., Biochem. 41:861-68 (2002). In some
embodiments, those ligands useful in the nanoparticle compositions
are those that bind competitively with TCDD, TA, and/or ITE.
Biocompatible Nanoparticles
[0167] The nanoparticles useful in the methods and compositions
described herein are made of materials that are (i) biocompatible,
i.e., do not cause a significant adverse reaction in a living
animal when used in pharmaceutically relevant amounts; (ii) feature
functional groups to which the binding moiety can be covalently
attached, (iii) exhibit low non-specific binding of interactive
moieties to the nanoparticle, and (iv) are stable in solution,
i.e., the nanoparticles do not precipitate. The nanoparticles can
be monodisperse (a single crystal of a material, e.g., a metal, per
nanoparticle) or polydisperse (a plurality of crystals, e.g., 2,3,
or 4, per nanoparticle).
[0168] A number of biocompatible nanoparticles are known in the
art, e.g., organic or inorganic nanoparticles. Liposomes,
dendrimers, carbon nanomaterials and polymeric micelles are
examples of organic nanoparticles. Quantum dots can also be used.
Inorganic nanoparticles include metallic nanoparticle, e.g., Au,
Ni, Pt and TiO2 nanoparticles. Magnetic nanoparticles can also be
used, e.g., spherical nanocrystals of 10-20 nm with a Fe2+ and/or
Fe3+ core surrounded by dextran or PEG molecules. In some
embodiments, colloidal gold nanoparticles are used, e.g., as
described in Qian et al., Nat. Biotechnol. 26 (1):83-90 (2008);
U.S. Pat. Nos. 7,060,121; 7,232,474; and U.S. P.G. Pub. No.
2008/0166706. Suitable nanoparticles, and methods for constructing
and using multifunctional nanoparticles, are discussed in e.g.,
Sanvicens and Marco, Trends Biotech., 26 (8): 425-433 (2008).
[0169] In all embodiments, the nanoparticles are attached (linked)
to the AHR ligands described herein via a functional groups. In
some embodiments, the nanoparticles are associated with a polymer
that includes the functional groups, and also serves to keep the
metal oxides dispersed from each other. The polymer can be a
synthetic polymer, such as, but not limited to, polyethylene glycol
or silane, natural polymers, or derivatives of either synthetic or
natural polymers or a combination of these. Useful polymers are
hydrophilic. In some embodiments, the polymer "coating" is not a
continuous film around the magnetic metal oxide, but is a "mesh" or
"cloud" of extended polymer chains attached to and surrounding the
metal oxide. The polymer can comprise polysaccharides and
derivatives, including dextran, pullanan, carboxydextran,
carboxmethyl dextran, and/or reduced carboxymethyl dextran. The
metal oxide can be a collection of one or more crystals that
contact each other, or that are individually entrapped or
surrounded by the polymer.
[0170] In other embodiments, the nanoparticles are associated with
non-polymeric functional group compositions. Methods are known to
synthesize stabilized, functionalized nanoparticles without
associated polymers, which are also within the scope of this
invention. Such methods are described, for example, in Halbreich et
al., Biochimie, 80 (5-6):379-90, 1998.
[0171] In some embodiments, the nanoparticles have an overall size
of less than about 1-100 nm, e.g., about 25-75 nm, e.g., about
40-60 nm, or about 50-60 nm in diameter. The polymer component in
some embodiments can be in the form of a coating, e.g., about 5 to
20 nm thick or more. The overall size of the nanoparticles is about
15 to 200 nm, e.g., about 20 to 100 nm, about 40 to 60 nm; or about
60 nm.
Synthesis of Nanoparticles
[0172] There are varieties of ways that the nanoparticles can be
prepared, but in all methods, the result must be a nanoparticle
with functional groups that can be used to link the nanoparticle to
the binding moiety.
[0173] For example, AHR ligands can be linked to the metal oxide
through covalent attachment to a functionalized polymer or to
non-polymeric surface-functionalized metal oxides. In the latter
method, the nanoparticles can be synthesized according to a version
of the method of Albrecht et al., Biochimie, 80 (5-6): 379-90,
1998. Dimercapto-succinic acid is coupled to the nanoparticle and
provides a carboxyl functional group. By functionalized is meant
the presence of amino or carboxyl or other reactive groups that can
be used to attach desired moieties to the nanoparticles, e.g., the
AHR ligands described herein or antibodies.
[0174] In another embodiment, the AHR ligands are attached to the
nanoparticles via a functionalized polymer associated with the
nanoparticle. In some embodiments, the polymer is hydrophilic. In a
specific embodiment, the conjugates are made using oligonucleotides
that have terminal amino, sulfhydryl, or phosphate groups, and
superparamagnetic iron oxide nanoparticles bearing amino or carboxy
groups on a hydrophilic polymer. There are several methods for
synthesizing carboxy and amino derivatized-nanoparticles. Methods
for synthesizing functionalized, coated nanoparticles are discussed
in further detail below.
[0175] Carboxy functionalized nanoparticles can be made, for
example, according to the method of Gorman (see WO 00/61191).
Carboxy-functionalized nanoparticles can also be made from
polysaccharide coated nanoparticles by reaction with bromo or
chloroacetic acid in strong base to attach carboxyl groups. In
addition, carboxy-functionalized particles can be made from
amino-functionalized nanoparticles by converting amino to carboxy
groups by the use of reagents such as succinic anhydride or maleic
anhydride.
[0176] Nanoparticle size can be controlled by adjusting reaction
conditions, for example, by varying temperature as described in
U.S. Pat. No. 5,262,176. Uniform particle size materials can also
be made by fractionating the particles using centrifugation,
ultrafiltration, or gel filtration, as described, for example in
U.S. Pat. No. 5,492,814.
[0177] Nanoparticles can also be treated with periodate to form
aldehyde groups. The aldehyde-containing nanoparticles can then be
reacted with a diamine (e.g., ethylene diamine or hexanediamine),
which will form a Schiff base, followed by reduction with sodium
borohydride or sodium cyanoborohydride.
[0178] Dextran-coated nanoparticles can also be made and
cross-linked, e.g., with epichlorohydrin. The addition of ammonia
will react with epoxy groups to generate amine groups, see Hogemann
et al., Bioconjug. Chem. 2000. 11 (6):941-6, and Josephson et al.,
Bioconjug. Chem., 1999, 10 (2):186-91.
[0179] Carboxy-functionalized nanoparticles can be converted to
amino-functionalized magnetic particles by the use of water-soluble
carbodiimides and diamines such as ethylene diamine or hexane
diamine.
[0180] Avidin or streptavidin can be attached to nanoparticles for
use with a biotinylated binding moiety, such as an oligonucleotide
or polypeptide. See e.g., Shen et al., Bioconjug. Chem., 1996, 7
(3):311-6. Similarly, biotin can be attached to a nanoparticle for
use with an avidin-labeled binding moiety.
[0181] In all of these methods, low molecular weight compounds can
be separated from the nanoparticles by ultra-filtration, dialysis,
magnetic separation, or other means. The unreacted AHR ligands can
be separated from the ligand-nanoparticle conjugates, e.g., by size
exclusion chromatography.
[0182] In some embodiments, colloidal gold nanoparticles are made
using methods known in the art, e.g., as described in Qian et al.,
Nat. Biotechnol. 26 (1):83-90 (2008); U.S. Pat. Nos. 7,060,121;
7,232,474; and U.S. P.G. Pub. No. 2008/0166706.
[0183] In some embodiments, the nanoparticles are pegylated, e.g.,
as described in U.S. Pat. Nos. 7,291,598; 5,145,684; 6,270,806;
7,348,030, and others.
Antibodies
[0184] In some embodiments, the nanoparticles also include
antibodies to selectively target a cell. The term "antibody," as
used herein, refers to full-length, two-chain immunoglobulin
molecules and antigen-binding portions and fragments thereof,
including synthetic variants. A typical full-length antibody
includes two heavy (H) chain variable regions (abbreviated herein
as VH), and two light (L) chain variable regions (abbreviated
herein as VL). The term "antigen-binding fragment" of an antibody,
as used herein, refers to one or more fragments of a full-length
antibody that retain the ability to specifically bind to a target.
Examples of antigen-binding fragments include, but are not limited
to: (i) a Fab fragment, a monovalent fragment consisting of the VL,
VH, CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent
fragment comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) a Fd fragment consisting of the VH and
CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains
of a single arm of an antibody, (v) a dAb fragment (Ward et al.,
Nature 341:544-546 (1989)), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded
for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. Science 242:423-426 (1988); and Huston et al. Proc.
Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single chain
antibodies are also encompassed within the term "antigen-binding
fragment."
[0185] Production of antibodies and antibody fragments is well
documented in the field. See, e.g., Harlow and Lane, 1988.
Antibodies, A Laboratory Manual. Cold Spring Harbor, New York: Cold
Spring Harbor Laboratory. For example, Jones et al., Nature 321:
522-525 (1986), which discloses replacing the CDRs of a human
antibody with those from a mouse antibody. Marx, Science
229:455-456 (1985), discusses chimeric antibodies having mouse
variable regions and human constant regions. Rodwell, Nature
342:99-100 (1989), discusses lower molecular weight recognition
elements derived from antibody CDR information. Clackson, Br. J.
Rheumatol. 3052: 36-39 (1991), discusses genetically engineered
monoclonal antibodies, including Fv fragment derivatives, single
chain antibodies, fusion proteins chimeric antibodies and humanized
rodent antibodies. Reichman et al., Nature 332: 323-327 (1988)
discloses a human antibody on which rat hypervariable regions have
been grafted. Verhoeyen, et al., Science 239: 1534-1536 (1988),
teaches grafting of a mouse antigen binding site onto a human
antibody.
[0186] In the methods described herein, it would be desirable to
target the compounds to T cells, B cells, dendritic cells, and/or
macrophages, therefore antibodies selective for one or more of
those cell types can be used. For example, for T cells, anti-CXCR4,
anti-CD28, anti-CD8, anti-TTLA4, or anti-CD3 antibodies can be
used; for B cells, antibodies to CD20, CD19, or to B-cell receptors
can be used; for dendritic cell targeting, exemplary antibodies to
CD11c, DEC205, MHC class I or class II, CD80, or CD86 can be used;
for macrophages, exemplary antiboduies to CD11b, MHC class I or
class II, CD80, or CD86 can be used. Other suitable antibodies are
known in the art.
Kits
[0187] The present invention also includes kits. In some
embodiments the kit comprise one or more doses of a composition
described herein. The composition, shape, and type of dosage form
for the induction regimen and maintenance regimen may vary
depending on a patients requirements. For example, dosage form may
be a parenteral dosage form, an oral dosage form, a delayed or
controlled release dosage form, a topical, and a mucosal dosage
form, including any combination thereof.
[0188] In a particular embodiment, a kit can contain one or more of
the following in a package or container: (1) one or more doses of a
composition described herein; (2) one or more pharmaceutically
acceptable adjuvants or excipients (e.g., a pharmaceutically
acceptable salt, solvate, hydrate, stereoisomer, and clathrate);
(3) one or more vehicles for administration of the dose; (5)
instructions for administration. Embodiments in which two or more,
including all, of the components (1)-(5), are found in the same
container can also be used.
[0189] When a kit is supplied, the different components of the
compositions included can be packaged in separate containers and
admixed immediately before use. Such packaging of the components
separately can permit long term storage without loosing the active
components' functions. When more than one bioactive agent is
included in a particular kit, the bioactive agents may be (1)
packaged separately and admixed separately with appropriate
(similar of different, but compatible) adjuvants or excipients
immediately before use, (2) packaged together and admixed together
immediately before use, or (3) packaged separately and admixed
together immediately before use. If the chosen compounds will
remain stable after admixing, the compounds may be admixed at a
time before use other than immediately before use, including, for
example, minutes, hours, days, months, years, and at the time of
manufacture.
[0190] The compositions included in particular kits of the present
invention can be supplied in containers of any sort such that the
life of the different components are optimally preserved and are
not adsorbed or altered by the materials of the container.
[0191] Suitable materials for these containers may include, for
example, glass, organic polymers (e.g., polycarbonate and
polystyrene), ceramic, metal (e.g., aluminum), an alloy, or any
other material typically employed to hold similar reagents.
Exemplary containers may include, without limitation, test tubes,
vials, flasks, bottles, syringes, and the like.
[0192] As stated above, the kits can also be supplied with
instructional materials. These instructions may be printed and/or
may be supplied, without limitation, as an electronic-readable
medium, such as a floppy disc, a CD-ROM, a DVD, a Zip disc, a video
cassette, an audiotape, and a flash memory device. Alternatively,
instructions may be published on a internet web site or may be
distributed to the user as an electronic mail.
EXAMPLES
[0193] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
Cloning And Characterization of Zebrafish Foxp3
[0194] The zebrafish is an experimental model of vertebrate
development; as described herein, it can also be used as an
immunogenic model. This example describes the cloning and
characterize of the zebrafish (Danio rerio) functional homologue of
mammalian Foxp3 (herein termed zFoxp3).
Identification of zFoxp3
[0195] To investigate whether Foxp3-dependent immunoregulatory
mechanisms operate in the zebrafish, we searched the zebrafish
genome for a Foxp3 homologue, which we termed zFoxp3 (FIG. 1E). A
phylogenetic analysis placed zFoxp3 in a sub-tree together with
mammalian and other fish orthologous predictions, suggesting that
zFoxp3 is the zebrafish ortholog for mammalian Foxp3 (FIG. 1F). In
mammals Foxp3 is located in a well-conserved synteny block. Indeed,
we found several orthologous genes between mammalian chromosome X
and zebrafish chromosome 8 in the region where zFoxp3 is located
(suv39h1, cacna1s, tspyl2, wasp), strengthening the likelihood of
zFoxp3 being the fish ortholog of Foxp3.
[0196] The accession numbers for the amino acid sequences used in
the gene tree analysis are as follows: Danio rerio Foxp1a Q08BX8
BC124513; Foxp1b Q2LE08 NM.sub.--001039637; Foxp2 Q4JNX5
NM.sub.--001030082; Foxp3 annotated (EST CK028390); Foxp4
annotated. Homo sapiens: Foxp1 Q9H334 NM.sub.--001012505, Foxp2
015409 NM.sub.--148899, Foxp3 Q9BZS1 NM.sub.--014009, Foxp4 Q8IVH2
NM.sub.--138457; Mus musculus: Foxp1 P58462 NM.sub.--053202, Foxp2
P58463 NM.sub.--053242, Foxp3 Q99JB6 NM.sub.--054039, Foxp4 Q9DBY0
NM.sub.--028767; Ciona intestinalis Foxp Q4H3H6. The amino acid
sequence of the apparent stickleback orthologues of Foxp1, Foxp2,
Foxp3 and Foxp4 were obtained from Ensembl.
Cloning zFoxp3
[0197] zFoxp3 was cloned from cDNA prepared from zebrafish kidney
by using a TOPO.RTM. PCR cloning kit (Invitrogen, Calif., USA)
according to the manufacturer's instructions.
Characterization of Foxp3
[0198] The amino acids (aa) predicted to mediate the interaction of
the forkhead domain with DNA (Stroud et al., Structure. 14, 159-66
(2006)) or the transcription factor NFAT (Wu et al., Cell 126,
375-87 (2006)) in mammalian Foxp3 are conserved in zFoxp3, as well
as aa found to be mutated in humans with impaired Foxp3 activity
(Ziegler, Annu Rev Immunol. 24, 209-26 (2006)) (FIG. 1E). The zinc
finger/leucine zipper domain is important for the homodimerization
of Foxp3 and its transcriptional regulatory activities (Chae et
al., Proc Natl Acad Sci USA 103, 9631-6 (2006)). To study the
ability of zFoxp3 to dimerize, we designed a pull-down assay using
His-tagged zFoxp3 and a renilla luciferase-tagged Foxp3
(Foxp3-Ren). 293 cells were transfected as described (Bettelli et
al., Proc Natl Acad Sci USA 102, 5138-43 (2005)) and the cells were
analyzed after 24 or 48 hours with the dual luciferase assay kit
(New England Biolabs, Ipswich, Mass.) (cells were lysed, and zFoxp3
was pulled-down with Ni-Agarose and the renilla luciferase activity
in the pellet was quantified). Tk-Renilla was used for
standardization. Alternatively, the transfected cells were lysed
and immuno-precipitation was carried out as described (Bettelli et
al., Proc Natl Acad Sci USA 102, 5138-43 (2005)); hemagglutinin
(HA) labeled NFAT and NF-kB were detected with anti-HA and anti-P65
antibodies obtained from Santa Cruz Biotechnology (Santa Cruz,
Calif., USA).
[0199] As shown in FIG. 1G, zFoxp3 pulled-down Foxp3-Ren indicating
that zFoxp3 can homodimerize. Hence, zFoxp3 has structural features
common to mammalian Foxp3.
[0200] Foxp3 can physically interact with NF-kB and NFAT to
down-regulate their transcriptional activities (Wu et al., (2006),
supra; Bettelli et al., (2005), supra). As shown in FIG. 2a, zFoxp3
interfered with the activation of NFAT and NF-KB responsive
promoters. This effect was stronger for NF-.kappa.B.
Co-immunoprecipitation experiments showed that zFoxp3 interacts
both with NF-.kappa.B and NFAT. In agreement with the reduced
inhibitory effect of Foxp3 on NFAT-driven reporters (see FIG. 2a),
the zFoxp3-NFAT interaction was weaker (see FIG. 2b). These results
suggest that zFoxp3 can directly interact with NFAT and NF-.kappa.B
to interfere with their transcriptional activities.
[0201] MSCV GFP-RV retroviral DNA plasmids were transfected into
the Phoenix packaging cell line and 72 hours later the
retrovirus-containing supernatants were collected. MACS-purified
CD4+ T cells were activated 24 hours later with plate-bound
antibodies to CD3 and CD28, and infected by centrifugation (45
minutes at 2000 rpm) with retrovirus-containing supernatant
supplemented with 8 .mu.g/ml Polybrene (Sigma-Aldrich) and
recombinant human IL-2 (25 units/ml).
[0202] Cells were cultured in serum-free X-VIVO 20.TM. media
(BioWhittaker, Walkersville, Md., USA) for 72 hours. During the
last 16 hours, cells were pulsed with 1 .mu.Ci of
[.sup.3H]thymidine (PerkinElmer, Waltham, Mass., USA) followed by
harvesting on glass fiber filters and analysis of incorporated
[.sup.3H]thymidine in a beta-counter (1450 Microbeta, Trilux,
PerkinElmer). Alternatively, culture supernatants were collected 48
after activation and the cytokine concentration was determined by
ELISA using antibodies for IFN-.gamma., IL-17 , IL-4, IL-10 from BD
Biosciences and antibodies to TGF-.beta. from R&D Systems. For
suppression assays, MACS purified CD4.sup.+CD25.sup.- T cells from
naive C57BL/6 mice (1-5.times.10.sup.4 cells/well) were stimulated
with antibodies to CD3 and C57BL/6 irradiated spleen cells
(0.3-1.5.times.10.sup.4 cells/well) for 3 days in the presence of
different ratios of CD4.sup.+GFP.sup.+ retrovirus-transduced T
cells.
[0203] Retroviral transduction of zFoxp3 into mouse T cells led to
the up-regulation of surface molecules associated with Treg
function such as CD25, CTLA-4 and GITR (see FIG. 2c). Moreover,
ectopic expression of zFoxp3 in mouse T cells led to a significant
decrease in their proliferation and cytokine secretion upon
activation with antibodies to CD3 (see FIG. 2d). Moreover, zFoxp3
transduced T cells could inhibit the activation of other T cells,
both in terms of T cell proliferation and of cytokine secretion, in
a dose dependent manner (see FIG. 2e). In summary, expression of
zFoxp3 in mouse T cells induced a Treg-like phenotype. These data
suggest that zFoxp3 is a functional homologue of mammalian Foxp3,
and that Foxp3 is capable of promoting a Treg like phenotype.
[0204] Western blot studies of zebrafish tissues identified a Foxp3
cross-reactive protein in thymus, kidney and spleen compatible with
the predicted size of zFoxp3. The expression of zFoxp3 was then
analyzed by real-time PCR on FACS-sorted lymphocytes,
myelomonocytes and erythrocytes(Traver et al., Nat Immunol 4,
1238-46 (2003)). RNA was extracted from cells using RNAeasy columns
(Qiagen, Valencia, Calif., USA), complementary DNA was prepared as
recommended (Bio-Rad Laboratories, Hercules, Calif., USA) and used
as template for real time PCR. The expression of Foxp3 was
quantified with specific primers and probes (Applied Biosystems,
Foster City, Calif., USA) on the GeneAmp 5500 Sequence Detection
System (Applied Biosystems). Expression was normalized to the
expression of the housekeeping gene, GAPDH.
[0205] As shown in FIG. 3a, zFoxp3 expression was restricted to the
lymphocyte fraction. This observation is consistent with the
expression pattern of mammalian Foxp3 and supports the conservation
of the regulatory mechanisms of gene expression that control tissue
specificity.
Example 2
Identification of Transcription Factor Binding Sites In Foxp3
[0206] The elements regulating gene expression in genomic DNA are
under selective pressure, and therefore are more conserved than the
surrounding nonfunctional sequences. Phylogenetic footprinting is a
method based on the analysis of sequence conservation between
orthologous genes from different species to identify regions of DNA
involved in the regulation of gene expression. Once identified,
these conserved regions can be analyzed with TFBS detection
algorithms to generate a list of putative TFBS.
[0207] We performed a phylogenetic footprinting analysis aimed at
identifying regulatory regions within the zebrafish, mouse and
human Foxp3 gene (Ovcharenko et al., Genome Res 15, 184-94 (2005)).
The inclusion of distant species like the zebrafish is highly
informative because it facilitates the identification of conserved
regulatory sequences amidst DNA regions that were not subjected to
any selective pressure (Ovcharenko et al., Genome Res 15, 184-94
(2005)). The Mulan server (mulan.dcode.org) was used to perform a
phylogenetic footprinting analysis of Foxp3. Mulan brings together
different algorithms in a web-based user-friendly interface:
programs for the rapid identification of local sequence
conservation connected to the multiTF/TRANSFAC database for the
detection of evolutionarily conserved TFBS in multiple alignments.
FIGS. 7A-B show the results obtained using the sequences of Foxp3
in rat, mouse, dog, human and zebrafish. Putative TFBS were found
for 6 transcription factors, all of them known to be expressed and
functional in T cells: NKX22, AHR, EGR1, EGR2, EGR3, NGFIC and
Delta EF1. These TF identified by phylogenetic footprinting are
other potential regulators of Foxp3 expression and Treg
development.
Example 3
Adaptive Cellular Immunity And Foxp3-Dependent Immunoregulation In
Zebrafish
[0208] The adaptive cellular immune response of 6 month old
zebrafish immunized intraperitoneally (ip) with heat killed M.
tuberculosis (MT) or PBS in incomplete Freund's adjuvant (IFA) was
studied. As shown in FIG. 1A, spleen cells prepared 14 days after
immunization with MT or PBS proliferated in response to stimulation
with Concanavalin A (ConA), but only cells taken from MT-immunized
fish proliferated upon activation with MT.
[0209] Another group of six month old zebrafish were anesthetized
with 0.02% tricaine (Sigma-Aldrich) and immunized i.p. with 10
.mu.l/fish of zebrafish brain homogenate (zCNS) emulsified in
complete Freund's adjuvant (CFA). As shown in FIGS. 1b-d, this
resulted in the accumulation of CD3, IFNg and IL-17 expressing
cells in the brain.
[0210] These results demonstrated that zebrafish can mount adaptive
antigen-specific cell-mediated immune and autoimmune responses.
[0211] C. elegans and D. melanogaster have been extremely useful
for the identification of the genes governing innate immunity
(Lemaitre et al., Nat Rev Immunol 4, 521-7 (2004)). These
experimental models, however, lack an adaptive immune system and
therefore cannot be used to study vertebrate-specific immune
processes. The zebrafish harbors both innate and adaptive immune
systems with functional macrophages (Davis et al., Immunity 17,
693-702 (2002)), B cells (Danilova et al., Proc Natl Acad Sci USA
99, 13711-6 (2002)) and T cells (Danilova et al., Dev Comp Immunol
28, 755-67 (2004); Langenau et al., Proc Natl Acad Sci USA 101,
7369-74 (2004)). Taking together the presence of basic components
of the adaptive immune system (Langenau et al., Nat Rev Immunol 5,
307-17 (2005)) with the experimental advantages offered by the
zebrafish for the realization of large scale genetic and chemical
screens (Lieschke et al., Nat Rev Genet. 8, 353-67 (2007)), the
zebrafish can serve as an experimental model for the study of
pathways controlling adaptive immune processes such as Treg
development.
Example 4
AHR Controls Foxp3 Expression And Treg Generation
[0212] Using the methods described above, a conserved binding site
for the aryl hydrocarbon receptor (AHR) was identified in the
genomic sequence of Foxp3 (see FIGS. 3b and 3j), which was termed
the conserved AHR binding site (CABS). A similarly located
regulatory sequence controls the expression of the AHR-regulated
cytochrome P4501A2 (CYP1A2). In addition, three non-evolutionary
conserved AHR-binding sites (NCABS) were identified in the zFoxp3
promoter (termed NCABS-1, -2, and -3) (see FIGS. 3i and 3j).
[0213] First, Foxp3 expression was measured in mouse Treg isolated
from Foxp3.sup.gpf knock in mice. Foxp3.sup.gpf knock in mice have
a GFP reporter inserted in the Foxp3 gene, producing GFP in
Foxp3.sup.+ Treg, which facilitates the identification and FACS
sorting of GFP:Foxp3.sup.+ Treg (Bettelli et al., Nature 441, 235-8
(2006)).
[0214] CD4+ T cells were purified from Foxp3gfp knock in mice using
anti-CD4 beads (Miltenyi, Auburn, Calif., USA) and sorted
(FACSAria.TM. cell sorter, BD Biosciences) into naive
CD4.sup.+Foxp3:GFP.sup.- or CD4.sup.+Foxp3:GFP.sup.+ T cells.
CD4.sup.+Foxp3:GFP.sup.- T cells were stimulated with plate bound 1
.mu.g/m1 of anti-CD3 (145-2C11, eBioscience) and 2 .mu.g/ml of
anti-CD28 (37.51, eBioscience) for 5 days, supplemented with
recombinant IL-2 (50 U/ml) at day 2 and 4, and analyzed by FACS at
day 5 for their differentiation into CD4.sup.+Foxp3:GFP.sup.4 Treg.
TGF.beta.1 (2.5 ng/ml) was used as a positive control.
[0215] Higher levels of AHR expression were detected on FACS-sorted
CD4.sup.+GFP:Foxp3.sup.+ Treg than in CD4.sup.+GFP:Foxp3.sup.- T
cells (see FIG. 3c), highlighting a possible link between AHR and
Foxp3 expression. The relationship between AHR and Foxp3 was then
further analyzed using RT-PCR.
[0216] Briefly, CD4+ T cells were purified from Foxp3gfp knock in
mice as described above. RNA was then extracted using RNAeasy
columns (Qiagen, Valencia, Calif., USA). Complementary DNA was
prepared as recommended (Bio-Rad Laboratories, Hercules, Calif.,
USA) and used as template for real time PCR. The expression of
Foxp3 was quantified with specific primers and probes (Applied
Biosystems, Foster City, Calif., USA) on the GeneAmp 5500 Sequence
Detection System (Applied Biosystems). Expression was normalized to
the expression of the housekeeping genes, GAPDH or actin.
[0217] Data generated using RT-PCR corroborated the observed
association between AHR and Foxp3. In addition, CYP1A1 expression,
a AHR responsive gene, was also observed in
CD4.sup.+GFP:Foxp3.sup.+ Treg cells. Furthermore, as shown in FIG.
3n, treatment of the cells with the AHR antagonist resveratol
resulted in a significant decrease in both Foxp3 and CYP1A1
expression levels (P<0.0023 and P<0.0235, respectively).
Decreases in the suppressive activity was also noted in resveratol
treated cells (FIG. 3o). Together, these results strongly suggest
that the detected AHR is functional.
[0218] To investigate whether AHR directly controls Foxp3
expression, we used a bacterial artificial chromosome that
contained the entire foxp3 locus tagged with a
[0219] Renilla luciferase reporter after the ATG start codon. More
specifically, we used the RP23-267C15 BAC clone, which contains 200
kb of mouse genomic DNA, including the entire locus of the Foxp3
gene. A Renilla cDNA cassette was the cloned immediately after the
ATG start codon of Foxp3 gene by homologous recombination using the
Red recombineering system contained in the DY 380 bacteria strain.
The final construct was designated BACFoxp3:Ren.
[0220] As shown in FIG. 3k, cotransfection of BACFoxp3:Ren with a
construct coding for mouse AHR resulted in a significant
up-regulation of Renilla activity (p<0.01), similar to that
achieved with a constitutively activated TGF.beta.3 receptor II.
This observation demonstrates that AHR is capable of directly
controlling Foxp3 expression.
[0221] Chromatin immunoprecipitation (ChIP) was then applied to
analyze the interaction of AHR with the CABS and NCABS shown in
FIGS. 3b and 3i, respectively.
[0222] Briefly, cells were treated for 90' with TCDD, fixed with 1%
formaldehyde for 15 minutes and quenched with 0.125 M glycine.
Chromatin was isolated and sheared to an average length of 300-500
bp by sonication. Genomic DNA (input) was prepared by treating
aliquots of chromatin with RNase, proteinase K and heat for
de-crosslinking, followed by ethanol precipitation. AHR-bound DNA
sequences were immuno-precipitated with an AHR-specific antibody
(Biomol SA-210). Crosslinks were reversed by incubation overnight
at 65.degree. C., and ChIP DNA was purified by phenol-chloroform
extraction and ethanol precipitation. Quantitative PCR reactions
were then performed using the following primer pairs:
TABLE-US-00001 Cyp1a1-845 F: aggctcttctcacgcaactc (SEQ ID NO: ) and
Cyp1a1-845 R: ctggggctacaaagggtgat; (SEQ ID NO: ) Foxp3
(NCAB-1)-2269 F: agctgcccattacctgttag (SEQ ID NO: ) and Foxp3
(NCAB-1)-2269 R: ggaggtctgcatggatcttag; (SEQ ID NO ) Foxp3
(NCAB-2)-1596 F: gccttgtcaggaaaaactctg (SEQ ID NO: ) and Foxp3
(NCAB-2)-1596 R: gtcctcgatttggcacagac; (SEQ ID NO ) Foxp3
(NCAB-3)-800 F: cttgcccttcttggtgatg (SEQ ID NO ) and Foxp3
(NCAB-3)-800 R: ttgtgctgagtgccctgac; (SEQ ID NO ) Foxp3 (CAB)
+13343 F: gctttgtgcgagtggagag (SEQ ID NO ) and Foxp3 (CAB) +13343
R: agggattggagcacttgttg. (SEQ ID NO )
[0223] The Untr6 region in chromosome 6 located at
chr6:120,258,582-120,258,797 was amplified as a control using Untr6
F: tcaggcatgaaccaccatac (SEQ ID NO) and Untr6 R:
aacatccacacgtccagtga (SEQ ID NO).
[0224] Experimental Ct values were converted to copy numbers
detected by comparison with a DNA standard curve run on the same
PCR plates. Copy number values were then normalized for primer
efficiency by dividing by the values obtained using Input DNA and
the same primer pairs. Error bars represent standard deviations
calculated from the triplicate determinations.
[0225] ChIP analysis of the interaction of AHR with CABS and NCABS
in Foxp3 and CYP1A1 was then performed in control and
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; a high affinity AHR
ligand) treated CD4.sup.+ T cells and T cells isolated from
mice.
[0226] As shown in FIG. 31, treatment of CD4+ T cells with TCDD
increased AHR binding to the CABS and NCABS-2 (p<0.05). This
up-regulation was comparable to that detected in the promoter of
the AHR-regulated gene cytochrome P4501A1 (CYP1A1) gene. No
significant increase in AHR binding was seen in NCABS-1, NCABS-3 or
the control sequence UTR6. As shown in FIG. 3m, similar results
were obtained when CD4 T cells were purified from TCDD treated
mice. These data suggest that AHR controls Foxp3 expression.
[0227] TCDD was also used to characterize the functional
relationship between AHR and Foxp3. Treatment of 3-day
post-fertilization zebrafish embryos with TCDD led to a
dose-dependent increase in zFoxp3 expression, suggesting that the
conserved AHR binding site in the zFoxp3 sequence is functional
(see FIG. 3D).
[0228] We then studied the effect of AHR activation on mouse Treg
numbers. Naive C57BL/6 mice were treated with TCDD (1 mg/mouse, ip)
and immunized 24 hours later with MOG.sub.35-55 in CFA. Draining
lymph nodes were prepared 10 days later and CD4.sup.+Foxp3.sup.+
Treg were quantified by FACS. Administration of TCDD led to a small
increase in the number of CD4.sup.+Foxp3.sup.+ Treg (see FIG. 3E).
Moreover, a single administration of TCDD followed by immunization
with MOG.sub.35-55 led to a significant increase in the number of
the CD4.sup.+Foxp3.sup.+ T cells (see FIG. 3E). Furthermore, the
CD4.sup.+Foxp3:GFP.sup.+ T cells expanded in vivo by TCDD
administration and MOG.sub.35-55 immunization were functional and
showed increased MOG.sub.35-55-specific suppressive activity (see
FIG. 3P).
[0229] To rule out any direct cytotoxic or pro-apoptotic effect of
TCDD on effector T cells, purified mouse CD4.sup.+CD25.sup.- T
cells were activated in vitro with antibodies to CD3 in the
presence of TCDD. Incubation with TCDD did not increase T cell
apoptosis as measured by annexin-FITC staining and did not decrease
the proliferative response (see FIG. 3F). Taken together, these
data suggest that AHR controls Foxp3 expression and Treg expansion
both in zebrafish and in mice.
[0230] To establish if TCDD triggered the conversion of
CD4.sup.+Foxp3.sup.- T cells into new Foxp3.sup.+ Treg cells, FACS
sorted CD4.sup.+Foxp3:GFP.sup.- T cells were activated in vitro
with antibodies to CD3 and CD28 in the presence of TCDD, and the
generation of CD4.sup.+Foxp3:GFP.sup.+ Treg was followed by FACS.
TGFb1 was used as a positive control. As shown in FIG. 3G, TCDD
triggered the conversion of approximately 13% of the cells in
culture into CD4.sup.+Foxp3:GFP.sup.+ Treg. Additionally, as shown
in FIG. 3, CD4.sup.+Foxp3:GFP.sup.+ Treg induced by TCDD showed a
suppressive activity similar to that of Treg induced in vitro with
TGF.beta.1 or CD4.sup.+Foxp3:GFP.sup.+ Treg sorted from naive
Foxp3gpf mice.
[0231] Thus, AHR activation by the high affinity AHR ligand TCDD
can trigger the conversion of CD4.sup.+Foxp3.sup.-T cells into
functional CD4.sup.+GFP.sup.+ Treg. As shown in FIGS. 3r and 3s,
treatment with the AHR antagonist resveratrol (50 .mu.M) interfered
with the induction of Treg by TGF.beta.1 and TCDD, but had a
stronger effect on the Treg conversion triggered by TCDD (p=0.0053,
FIG. 1h). CD4.sup.+Foxp3:GFP.sup.+ Treg purified from naive mice
did not proliferate and did not show increased suppressive activity
upon stimulation with antibodies to CD3 and CD28 and TCDD. These
observations suggest that AHR is more important for the
differentiation of new Treg than for the activity of established
Treg.
[0232] To investigate if new Treg could also be generated in vivo
following TCDD administration, we transferred
CD4.sup.+Foxp3:GFP.sup.- 2D2 T cells from CD90.2 donors into wild
type CD90.1 recipients. CD4.sup.+Foxp3:GFP.sup.- 2D2 T cells
express a MOG.sub.35-55-specific T cell receptor. The recipients
were administered 1 .mu.g/mouse TCDD and were immunized 2 days
later with MOG.sub.35-55. CD4.sup.+Foxp3:GFP.sup.+ CD90.2 T cells
(donor cells that underwent conversion into Treg upon treatment
with TCDD) were then quantified by FACS. As shown in FIG. 3h, TCDD
promoted a significant (p<0.02, unpaired t-test, n=5) conversion
of CD4.sup.+Foxp3:GFP.sup.- CD90.2 donor T cells into Treg
cells.
[0233] Thus, the increase in the frequency of Treg that follows
activation of AHR with TCDD is due, at least in part, to the
conversion of CD4.sup.+Foxp3:GFP.sup.- T cells into
CD4.sup.+Foxp3:GFP.sup.+ Treg.
Example 5
AHR Activation By TCDD Suppresses EAE
[0234] To analyze the functionality of the Treg cells induced by
AHR activation, we studied the effect of TCDD on EAE
development.
[0235] C57BL/6 mice were given a single intraperitoneal (ip) dose
of TCCD, and one day later EAE was induced by immunization with
MOG.sub.35-55 in CFA. TCDD was also administered orally (1
.mu.g/mouse) to determined whether an effective dose of this ligand
can be delivered via oral administration and whether this dose is
capable of reducing EAE development. EAE was induced by injecting
the mice subcutaneously with 100 ml of the MOG.sub.35-55 peptide
(MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO:)) in complete Freund adjuvant
oil. In addition, the mice received 150 ng of pertussis toxin
(Sigma-Aldrich) ip on days 0 and 2. Clinical signs of EAE were
assessed according to the following score: 0, no signs of disease;
1, loss of tone in the tail; 2, hind limb paresis; 3, hind limb
paralysis; 4, tetraplegia; 5, moribund.
[0236] As shown in FIG. 4a and Table 3, ip administered TCDD had a
dose-dependent effect on the clinical signs of EAE. 1 .mu.g/mouse
markedly inhibited the clinical signs of EAE (p<0.001; n=6).
TABLE-US-00002 TABLE 3 TCDD Treatment Suppresses EAE Mean day of
onset Mean maximum Treatment Incidence (Mean .+-. standard score
(.mu.g per mouse) (positive/total) deviation (SD)) (mean .+-. SD)
Control 42/49 (87%) 13.6 .+-. 2.8 2.4 .+-. 1.4 TCDD 1 .mu.g 4/40
(10%) 21.8 .+-. 1.5* 0.2 .+-. 0.6* TCDD 0.1 .mu.g 5/5 (100%) 17.0
.+-. 1.2 3.1 .+-. 0.5 TCDD 0.01 .mu.g 7/7 14.1 .+-. 2.9 2.7 .+-.
0.6 Mice treated with corn oil (control) or TCDD (ip) were
immunized with MOG.sub.35-55 peptide in CFA and monitored for EAE
development. Statistical analysis was performed by comparing groups
using one-way analysis of variance. *P < 0.0001.
[0237] As shown in FIGS. 4G-4I, IP administered TCDD also reduced
the histopathological signs of EAE. In addition, orally
administered of 1 .mu.g/mouse of TCDD, one day before EAE
induction, also prevented EAE development (p<0.001, two-way
ANOVA, n=10). This observation suggests that an effective does of
TCDD can be administered orally.
[0238] To confirm that the effects on EAE were mediated by the
activation of AHR, we used C57BL/6 mice carrying the d allele of
the ahr gene (AHR-d mice). This allele codes for a mutant AHR with
a 10 fold reduction in its affinity for TCDD and other ligands
(Okey et al., Mol Pharmacol. 35, 823-30 (1989)) due to mutations in
its ligand binding sites. The administration of TCDD (1
.mu.g/mouse) to AHR-d mice did not increase the levels of
CD4.sup.+Foxp3.sup.+ Treg in AHR-mt mice, and did not inhibit the
progression of EAE, as shown in FIG. 4B and Table 4.
TABLE-US-00003 TABLE 4 TCDD Treatment of AHR-d Mice Mean day of
Incidence onset Mean maximum Treatment (positive/total) (Mean .+-.
SD) score (mean .+-. SD) WT control 12/14 (86%) 13.9 .+-. 1.9.sup.
2.4 .+-. 1.4 AHR-d + TCDD 9/11 (82%) 17.3 .+-. 3.0.sup..dagger. 2.2
.+-. 1.4 WT + TCDD .sup. 1/10 (10%).sup.# 21 0.2 .+-. 0.6* C57BL/6
(WT) and AHR-d mice treated with corn oil (control) or TCDD (1
.mu.g/mouse) were immunized with MOG.sub.35-55 peptide in CFA and
monitored for EAE development. Statistical analysis was performed
by comparing groups using one-way analysis of variance. *P<
0.001 vs WT control group and P < 0.01 vs AHR-d TCDD group;
.sup..dagger.P = 0.0046 vs WT control group; .sup.#P = 0.0005 vs WT
control group, P = 0.0019 vs AHR-d TCDD group.
[0239] Taken together, these results show that TCDD-dependent AHR
activation can inhibit or suppress the development and/or
progression of EAE. The data presented in Example 3 indicate that
this effect is due to the TCDD-dependent AHR activation that
promotes the induction of functional Treg.
[0240] Antigen microarrays were then used to study the antibody
response to myelin in mice that did not develop EAE as consequence
of AHR activation by TCDD. The antigens listed in Table 1 were
spotted onto Epoxy slides (TeleChem, Sunnyvale, Calif., USA) as
described (Quintana et al., Proc Natl Acad Sci USA 101 Suppl 2,
14615-21 (2004)). Antigens were spotted in replicates of 6, the
microarrays were blocked for 1 h at 37.degree. C. with 1% bovine
serum albumin, and incubated for 2 hours at 37.degree. C. with a
1:100 dilution of the test serum in blocking buffer. The arrays
were then washed and incubated for 45 min at 37.degree. C. with
goat anti-mouse IgG Cy3-conjugated detection antibodies (Jackson
ImmunoResearch Labs, West Grove, Pa., USA). The arrays were scanned
with a ScanArray 4000.times. scanner (GSI Luminomics, Billerica,
Mass., USA). Antigen reactivity was defined by the mean intensity
of binding to the replicates of that antigen on the microarray. Raw
data were normalized and analyzed using the GeneSpring software
(Silicon Genetics, Redwood City, Calif., USA) with the
non-parametric Wilcoxon-Mann-Whitney test, using the Benjamini and
Hochberg method with a false discovery rate (FDR) of 0.05 to
determine significance. The samples were clustered using a pairwise
average linkage algorithm based on Spearman's rank correlation as a
distance measure.
[0241] The microarrays consisted of a collection of 362 CNS-related
autoantigens including tissue lysates, recombinant proteins,
peptide libraries spanning the whole sequence of myelin proteins
and lipids found in the central and peripheral nervous system, a
complete list of the antigens used is provided in Table 1.
TABLE-US-00004 TABLE 1 362 CNS-Related Autoantigens Heat Shock
Proteins (HSP) 27 kDa Heat Shock Protein 32 kDa Heat Shock Protein
40 kDa Heat Shock Protein 47 kDa Heat Shock Protein 60 kDa Heat
Shock Protein 60 kDa Heat Shock Protein peptide: aa 106-125; aa
1-20; aa 121-140; aa 136-155; aa 151-170; aa 16-35; aa 166-185; aa
181-199; aa 195-214; aa 210-229; aa 225-244; aa 240-259; aa
255-275; aa 271-290; aa 286-305; aa 301-320; aa 31-50; aa 316-335;
aa 331-350; aa 346-365; aa 361-380; aa 376-395; aa 391-410; aa
406-425; aa 421-440; aa 436-455; aa 451-470; aa 466-485; aa 46-65;
aa 481-500; aa 496-515; aa 511-530; aa 526-545; aa 541-560; aa
556-573; aa 61-80; aa 76-95; or aa 91-110 65 kDa Heat Shock Protein
M. tuberculosis 70 kDa Heat Shock Protein 70 kDa Heat Shock Protein
peptide aa 106-125; aa 1-20; aa 121-140; aa 136-155; aa 151-170; aa
16-35; aa 166-185; aa 181-199; aa 195-214; aa 210-229; aa 225-244;
aa 240-259; aa 255-275; aa 271-290; aa 286-305; aa 301-320; aa
31-50; aa 316-335; aa 331-350; aa 346-365; aa 361-380; aa 376-395;
aa 391-410; aa 406-425; aa 421-440; aa 436-455; aa 451-470; aa
466-485; aa 46-65; aa 481-500; aa 496-515; aa 511-530; aa 526-545;
aa 541-560; aa 556-575; aa 571-590; aa 586-605; aa 601-620; aa
616-635; aa 61-80; aa 631-640; aa 76-95; or aa 91-110 71 kDa Heat
Shock Protein M. tuberculosis 90 kDa Heat Shock Protein GroEL CNS
2',3'-cyclic nucleotide 3'-phosphodiesterase peptide aa 106-125; aa
1-20; aa 121-140; aa 136-155; aa 151-170; aa 16-35; aa 166-185; aa
181-200; aa 196-215; aa 211-230; aa 226-245; aa 241-260; aa
256-275; aa 271-290; aa 286-305; aa 301-320; aa 31-50; aa 316-335;
aa 331-350; aa 346-365; aa 361-380; aa 376-395; aa 391-410; aa
406-421; aa 46-65; aa 61-80; aa 76-95; or aa 91-110 Acetyl
Cholinesterase ADAM-10 alpha-Cristallin beta-Cristallin bovine
Myelin Basic Protein Brain Extract I Brain Extract II Brain Extract
III Glial Filament Acidic Protein Research Diagnostic guinea pig
Myelin Basic Protein human Myelin Basic Protein Myelin-Associated
Oligodendrocytic Basic Protein peptide aa 106-125; aa 1-20; aa
121-140; aa 136-155; aa 151-170; aa 16-35; aa 166-185; aa 181-200;
aa 31-50; aa 46-65; aa 61-80; aa 76-95; aa 91-110; aa 106-125; aa
1-20; aa 121-140; aa 136-155; or aa 151-170 Myelin/oligodendrocyte
glycoprotein peptide aa 16-35; aa 166-185; aa 181-200; aa 196-215;
aa 211-230; aa 226-247; aa 31-50; aa 35-55; aa 46-65; aa 61-80; aa
76-95; or aa 91-110 murine Myelin Basic Protein Myelin Associated
Glycoprotein Myelin Basic Protein peptide aa 104-123; aa 11-30; aa
113-132; aa 1-20; aa 121-138; aa 124-142; aa 138-147; aa 141-161;
aa 143-168; aa 155-178; aa 26-35; aa 31-50; aa 41-60; aa 51-70; aa
61-80; aa 71-92; aa 84-94; aa 89-101; aa 173-186; or aa 93-112
Myelin Protein 2 peptide aa 106-125; aa 1-20; aa 121-132; aa 16-35;
aa 31-50; aa 46-65; aa 61-80; aa 76-95; or aa 91-110 Neurofilament
160 kd Neurofilament 200 kd Neurofilament 68 kd Neuronal Enolase
Nicastrin NMDA receptor NOGO Olygodendrocyte-Specific Protein
peptide aa 106-125; aa 1-20; aa 121-140; aa 136-155; aa 151-170; aa
16-35; aa 166-185; aa 181-199; aa 195-217; aa 31-50; aa 46-65; aa
61-80; aa 76-95; or aa 91-110 Proteolipid Protein Proteolipid
Protein peptide aa 100-119; aa 10-29; aa 110-129; aa 1-19; aa
125-141; aa 137-150; aa 137-154; aa 150-163; aa 151-173; aa
158-166; aa 161-180; aa 178-191; aa 180-199; aa 190-209; aa 20-39;
aa 205-220; aa 215-232; aa 220-239; aa 220-249; aa 248-259; aa
250-269; aa 265-277; aa 35-50; aa 40-59; aa 50-69; aa 65-84; aa
80-99; or aa 91-110 Retinol Binding Protein S100beta protein Assay
Designs Super Oxide Dismutase Synuclein, beta Synuclein, gamma
Tissue Amydgala Amydgala AD Brain lysate Brain Tissue Membrane
Cerebellar pedunculus Cerebral meninges Corpus Callosum Corpus
Callosum AD Diencephalon Fetal brain Frontal lobe Frontal lobe AD
Hippocampus Hippocampus AD Insula Occipital lobe Occipital lobe AD
Olfactory region Optic Nerve Parietal lobe Parietal lobe AD Pons
Pons AD Postcentral gyrus Postcentral gyrus AD Precentral gyrus
Precentral gyrus AD Spinal cord Temporal lobe Temporal lobe AD
Thalamus Thalamus AD Amyloid beta AD related Amyloid beta 10-20
Amyloid beta peptide 1-12; 12-28; 1-23; 1-38; beta 17-40; 25-35; or
34-42 Amyloid bri protein precursor 227 Amyloid DAN Protein
Fragment 1-34 Amyloid Precursor Protein Amyloid protein no AB
component Secreted amyloid precursor protein (SAP) beta Tau isoform
variant 0N3R Tau isoform variant 1N3R Tau isoform variant 0N4R Tau
isoform variant 2N3R Tau phospho Ser412 Tau phospho Ser441 Tau
phospho Thr181 Tau Protein human Lipids (.+-.)9-HODE Cayman
Chemical 1 Palmitoyl-2-(5'oxo-Valeroyl)-sn-Glycero-3-
Phosphocholine 15a-hydroxycholestene 15-ketocholestane
15-ketocholestene 1-Palmitoil-2-(9'oxo-Nonanoyl)-sn-Glycero-3-
Phosphocholine 1-Palmitoil-2-Azelaoyl-sn-Glycero-3-Phosphocholine
1-Palmitoil-2-Glutaroyl-sn-Glycero-3-Phosphocholine 5
.alpha.-cholestane-3 .beta.,15 .alpha.-diol 9(S)-HODE Cayman
Chemical Asialoganglioside-GM1 Asialoganglioside-GM2 Brain
ceramides Brain D-erythrosphingosine Brain
lysophosphatidylethanolamine Brain L-.alpha.-lysophosphatidylserine
Brain L-.alpha.-phosphatidylcholine Brain
L-.alpha.-phosphatidyl-ethanolamine Brain
L-.alpha.-phosphatidylserine Brain polar lipid extract Brain
sphingomyelin Brain sulfatide Brain total lipid extract Cardiolipin
Ceramide Ceramide 1-phosphate Cholesterol Disialogaglioside-GD1B
Disialogaglioside-GD2 Disialoganglioside GD1a Disialoganglioside
GD3 Fucosyl-GM1 Galactocerebrosides Ganglioside Mixture
Ganglioside-GM4 Gangliotetraosylceramide asialo-GM1 HDL
Hexacosanoic acid (26) Hydroxy fatty acid ceramide Isoprostane F2 I
Lactocerebrosides Lactosylceramide LDL Lipid A, diphosphoryl from
Salmonella enterica Lipopolysaccharides from Escherichia coli
Lipopolysaccharides from Pseudomona aeruginosa Lipopolysaccharides
from Salmonella enterica Lyso-GM1 Monosialoganglioside GM1
Monosialoganglioside GM2 Monosialoganglioside GM3
N-Hexanoyl-D-sphingosin Non-hydroxy fatty acid ceramide
Phosphatidylinositol-4 phosphate Squalene Sulfatides Tetracosanoic
acid (24) Tetrasialoganglioside-GQ1B TNPAL Galactocerebroside Total
brain gangliosides Total cerebroside Trisialoganglioside GT1a
Trisialoganglioside-GT1B
[0242] The control of EAE by AHR activation correlated with a
significant decrease in IgG serum antibodies to 97 myelin antigens,
which are listed in Table 2.
TABLE-US-00005 TABLE 2 Specificity of IgG Antibodies Showing a
Significant (FDR < 0.05) Downregulation in TCDD-Treated Mice
Antigen FDR 70 kDa. Heat Shock Protein peptide aa 331-350 1.78E-05
60 kDa. Heat Shock Protein peptide aa 255-275 0.00547 60 kDa. Heat
Shock Protein peptide aa 13-35 0.00547 32 kDa. Heat Shock protein
0.00547 Myelin Basic Protein peptide aa 138-147 0.00547 Proteolipid
Protein peptide aa 1-19 0.00547 Proteolipid Protein peptide aa
161-180 0.00547 Proteolipid Protein peptide aa 10-29 0.00547 60
kDa. Heat Shock Protein peptide aa 1-20 0.0055 70 kDa. Heat Shock
Protein peptide aa 61-80 0.0055 Ceramide 0.0055 Myelin-Associated
Oligodendrocytic Basic Protein peptide aa 91-110 0.0055 Proteolipid
Protein peptide aa 137-150 0.0055 NOGO 0.00557
Olygodendrocyte-Specific Protein peptide aa 76-95 0.00557
b-Cristallin 0.0058 Myelin-Associated Oligodendrocytic Basic
Protein peptide aa 121-140 0.00703 60 kDa. Heat Shock Protein
peptide aa 225-244 0.00708 Myelin Basic Protein peptide aa 113-132
0.00925 Olygodendrocyte-Specific Protein peptide aa 46-65 0.00925
Myelin Protein 2 peptide aa 91-110 0.00925 Myelin-Associated
Oligodendrocytic Basic Protein peptide aa 151-170 0.0093
Myelin/oligodendrocyte glycoprotein peptide aa 31-50 0.0093 NT-3
0.0093 Proteolipid Protein peptide aa 40-59 0.0116 70 kDa. Heat
Shock Protein peptide aa 421-440 0.0118 Myelin Basic Protein
peptide aa 173-186 0.0125 70 kDa. Heat Shock Protein peptide aa
121-140 0.0132 2',3'-cyclic nucleotide 3'-phosphodiesterase peptide
aa 391-410 0.0132 Olygodendrocyte-Specific Protein peptide aa
136-155 0.0132 Olygodendrocyte-Specific Protein peptide aa 106-125
0.0134 70 kDa. Heat Shock Protein peptide aa 136-155 0.0141
2',3'-cyclic nucleotide 3'-phosphodiesterase peptide aa 406-421
0.0141 Myelin-Associated Oligodendrocytic Basic Protein peptide aa
166-185 0.0143 Myelin Protein 2 peptide aa 1-20 0.0143 Myelin
Protein 2 peptide aa 76-95 0.0144 Proteolipid Protein peptide aa
125-141 0.0144 Proteolipid Protein peptide aa 178-191 0.0144 40
kDa. Heat Shock Protein 0.0145 2',3'-cyclic nucleotide
3'-phosphodiesterase peptide aa 106-125 0.0158
Olygodendrocyte-Specific Protein peptide aa 195-217 0.0174
2',3'-cyclic nucleotide 3'-phosphodiesterase peptide aa 240-259
0.0187 70 kDa. Heat Shock Protein peptide aa 76-95 0.0194
Proteolipid Protein peptide aa 265-277 0.0194 Myelin Basic Protein
peptide aa 89-101 0.0199 Myelin Basic Protein peptide aa 71-92
0.0199 Myelin-Associated Oligodendrocytic Basic Protein peptide aa
16-35 0.0199 Proteolipid Protein peptide aa 265-277 0.0199 60 kDa.
Heat Shock Protein peptide aa 46-65 0.0241 70 kDa. Heat Shock
Protein peptide aa 166-185 0.0241 2',3'-cyclic nucleotide
3'-phosphodiesterase peptide aa 151-170 0.0241 2',3'-cyclic
nucleotide 3'-phosphodiesterase peptide aa 376-395 0.0241 Myelin
Basic Protein peptide aa 11-30 0.0241 Myelin/oligodendrocyte
glycoprotein peptide aa 211-230 0.0241 Proteolipid Protein peptide
aa 265-277 0.0241 70 kDa. Heat Shock Protein peptide aa 181-199
0.0242 Olygodendrocyte-Specific Protein peptide aa 31-50 0.0242
Proteolipid Protein peptide aa 265-277 0.0242
Myelin/oligodendrocyte glycoprotein peptide aa 91-110 0.0249 Optic
Nerve lysate 0.0249 2',3'-cyclic nucleotide 3'-phosphodiesterase
peptide aa 361-380 0.0258 Lactosylceramide 0.0258 Myelin Protein 2
peptide aa 31-50 0.0258 Myelin Basic Protein peptide aa 1-20 0.028
NMDA receptor 0.0285 CNF 0.0289 2',3'-cyclic nucleotide
3'-phosphodiesterase peptide aa 136-155 0.0292 Myelin Basic Protein
peptide aa 141-161 0.0298 70 kDa. Heat Shock Protein peptide aa
406-425 0.0307 2',3'-cyclic nucleotide 3'-phosphodiesterase peptide
aa 210-229 0.0307 Galactocerebrosides 0.0307 Myelin/oligodendrocyte
glycoprotein peptide aa 46-65 0.0307 Proteolipid Protein peptide aa
150-163 0.0307 Proteolipid Protein peptide aa 265-277 0.0307
Proteolipid Protein peptide aa 80-99 0.0307 60 kDa. Heat Shock
Protein peptide aa 210-229 0.0323 Proteolipid Protein peptide aa
137-154 0.0324 2',3'-cyclic nucleotide 3'-phosphodiesterase peptide
aa 1-20 0.0337 2',3'-cyclic nucleotide 3'-phosphodiesterase peptide
aa 225-244 0.0337 Myelin-Associated Oligodendrocytic Basic Protein
peptide aa 61-80 0.0337 Proteolipid Protein peptide aa 158-166
0.0337 Ceramide 1 phosphate 0.0346 Myelin-Associated
Oligodendrocytic Basic Protein peptide aa 136-155 0.0369 Myelin
Basic Protein peptide aa 155-178 0.0379 Myelin/oligodendrocyte
glycoprotein peptide aa 106-125 0.0392 Proteolipid Protein peptide
aa 180-199 0.0408 Myelin Protein 2 peptide aa 121-132 0.0413 Myelin
Basic Protein peptide aa 104-123 0.0419 70 kDa. Heat Shock Protein
0.0421 Non h fatty acid ceramide 0.0421 Myelin-Associated
Glycoprotein 0.0452 Myelin Basic Protein peptide aa 143-168 0.0452
2',3'-cyclic nucleotide 3'-phosphodiesterase peptide aa 91-110
0.047 2',3'-cyclic nucleotide 3'-phosphodiesterase peptide aa
181-199 0.0476 70 kDa. Heat Shock Protein peptide aa 255-275 0.0486
Brain ceramides 0.0486 Myelin Protein 2 peptide aa 46-65 0.0496
[0243] To further characterize the suppression of EAE by AHR
activation we studied the activity of myelin specific T cells
induced by vaccination with MOG.sub.35-55/CFA in TCDD-treated mice.
TCDD-treated mice showed a suppressed recall proliferative response
to the MOG.sub.35-55 peptide, however no differences were seen upon
activation with antibodies to CD3 (see FIGS. 4c-d).
[0244] In addition, cells were stimulated in culture medium
containing 100 .mu.g/ml MOG.sub.35-55 for 2 days or with PMA (50
ng/ml) (Sigma-Aldrich) and ionomycin (1 nM) (Calbiochem, San Diego,
Calif., USA) for 4 hours, Golgistop (BD Biosciences) was added to
the culture during the last 4 hours. After staining of surface
markers, cells were fixed and permeabilized using Cytofix/Cytoperm
and Perm/Wash buffer from BD Biosciences according to the
manufacturer's instructions. All antibodies to cytokines
(IFN-gamma, IL-17, IL-10) including the corresponding isotype
controls were obtained from BD Biosciences. Cells were incubated
(1:100) at 25.degree. C. for 20 min and washed twice in Perm/Wash
before analysis. Data were acquired on a FACSCalibur (BD
Biosciences) and analyzed with FlowJo software (Tree Star, Ashland,
Oreg., USA). When compared to the draining lymph node cells from
control animals, cells from TCDD-treated mice secreted higher
amounts of TGFb1 and lower amounts of IFNg and IL-17 upon
activation with MOG.sub.35-55 (see FIG. 4e); we did not detect
significant amounts of IL-4 or IL-10. Moreover, AHR activation with
TCDD led to a decrease in the frequency of CD4.sup.+IL-17.sup.+ and
CD4.sup.+IFNg.sup.+ T cells in the draining lymph nodes (see FIG.
4F).
[0245] These data suggest that AHR activation interferes with the
generation of the encephalitogenic T cell response.
Example 6
Treg Induced By AHR Activation Suppress EAE By A TGFb1-Dependent
Mechanism
[0246] The inhibition of the development of EAE by AHR activation
with TCDD was associated with a significant increase in the
frequency of CD4.sup.+Foxp3.sup.+ T cells (see FIG. 5A). To
identify the mechanism responsible for the decreased proliferation
to MOG.sub.35-55 in TCDD-treated animals shown in FIG. 4D, the
CD4.sup.+CD25.sup.+ Treg population was depleted with magnetic
beads. Treg depletion recovered the recall response to
MOG.sub.35-55 in immunized mice treated with TCDD (see FIG. 5B),
suggesting that the suppression observed in FIG. 4D resulted from
the activity of the TCDD-induced Treg (see FIG. 5A). Moreover,
protection from EAE could be transferred to wild type naive animals
by the transfer of 5.times.10.sup.6 CD4.sup.+ T cells from
TCDD-treated mice, but not with cells isolated from vehicle-treated
mice (p<0.001, two-way ANOVA, n=4; FIG. 5C). The control of the
pathogenic T cell response was mediated by CD4.sup.+CD25.sup.+
Treg, their depletion abrogated the protective effect of the
transferred cells (p<0.001, two-way ANOVA, n=4; FIG. 5C).
[0247] Further characterization revealed that effector
CD4.sup.+Foxp3:GFP.sup.- T cells purified from TCDD-treated mice
showed normal proliferation (FIG. 5F), but significantly decreased
secretion of IL-17 and IFN.gamma. upon activation with
MOG.sub.35-55 (see FIG. 5G).
[0248] To confirm that the protective effect of TCDD on EAE was
Treg mediated we depleted the natural Treg with antibodies to CD25
prior to TCDD treatment. The difference between undepleted and
depleted cell populations are shown in FIG. 5H. TCDD-treated mice
showed a faster rebound in their Treg numbers (P<0.04 at day 7)
(see FIG. 5H), concomitant with a significant delay in the onset of
EAE (P<0.03) and a significant reduction in IL-17+CD4+ T cells
in the draining lymph nodes (P<0.03; see FIGS. 51 and 5J).
Moreover, the transfer of 5.times.10.sup.6 CD4.sup.+ T cells from
TCDD-treated mice significantly inhibited the development of EAE,
as shown in FIG. 5h and Table 5. This protective effect was lost
when CD4.sup.+CD25.sup.+ T cells were depleted, see FIG. 5h and
Table 5. Together, these data suggest that AHR activation by TCDD
results in the generation of CD4+Foxp3+ Treg that control the
encephalitogenic response.
TABLE-US-00006 TABLE 5 EAE Suppression in Treg Depleted Cell
Populations Mean day Incidence of onset Mean maximum Treatment
(positive/total) (Mean .+-. SD) score (mean .+-. SD) CD4.sup.+
control 7/7 (100%) 12.3 .+-. 1.9 2.7 .+-. 1.0 CD4.sup.+ TCDD 3/6
(57%) 13.3 .+-. 0.6 0.7 .+-. 0.8* CD4.sup.+CD25.sup.- TCDD 3/4
(75%) 11.0 .+-. 0.0 2.6 .+-. 1.8 Naive C57BL/6 mice received
CD4.sup.+ or CD4.sup.+CD25.sup.- T cells (5 .times. 10.sup.6)
purified from TCDD or control treated mice 10 days after
immunization with MOG.sub.35-55/CFA. 24 hours later EAE was induced
in the recipient mice with MOG.sub.35-55/CFA, and the mice were
monitored for EAE development. Statistical analysis was performed
by comparing groups using one-way analysis of variance. *P <
0.05 vs CD4.sup.+ control group.
[0249] TGFb1 has been linked to the suppressive activity of Treg in
vitro and in vivo (Li et al., Annu Rev Immunol. 24, 99-146 (2006)).
To assess the role played by TGFb1 in the inhibition of the recall
response to MOG.sub.35-55 by Treg (see FIGS. 4d and 5b), we
activated lymph node cells from TCDD treated mice in the presence
of blocking antibodies to IL-4, IL-10, TGFb1, or an isotype-matched
control. FIG. 5d shows that incubation with antibodies to TGFb1,
but not to IL-4 or IL-10 could recover the recall response to
MOG.sub.35-55.
[0250] To analyze the role played by TFGb1 in vivo in the control
of EAE, we transferred CD4.sup.+ T cells from TCDD treated mice
into naive mice expressing a dominant negative variant of the TGFb
receptor II on their T cells; T cells from these mice are
unresponsive to the immunosuppressive effects of TGFb1 (Gorelik et
al., Immunity. 12, 171-81 (2000)). As shown in FIG. 5E, transferred
Treg cells could control EAE in wild type mice but not in mice
harboring T cells unresponsive to TGFb1 (p<0.001, two-way ANOVA,
n=4). Thus, Treg induced by the activation of AHR with TCDD inhibit
the progression of EAE by a TGFb1-dependent mechanism.
Example 7
Endogenous AHR Ligands Control Treg Development In Vivo
[0251] The observations described herein regarding the control of
Treg development by AHR activation suggest that endogenous AHR
ligands participate in immune regulation. In support of this, we
have demonstrated that naive AHR-d mice harbor lower levels of
CD4.sup.+Foxp3.sup.+ T cells (p<0.03, t-test; see FIG. 6A), and
higher levels of CD4.sup.+CD25.sup.+ Foxp3.sup.+ T cells. In
addition, we have demonstrated that these cells develop a
significantly stronger EAE, which is characterized by an earlier
disease onset and a higher clinical score (p<0.001, two-way
ANOVA, n=6-8; see FIG. 6B).
[0252] Several endogenous AHR ligands are described in the art
.sup.28. Based on our results, AHR ligands such as TCDD could be
useful in the control of Treg development. Our data additionally
demonstrate that AHR ligands such as TCDD can be used to suppress
the development and/or progression of EAE. Clearly, such technology
would also be useful in the modulation of other immunological
disorders such as autoimmune disorders. Two additional endogenous
high affinity ligands for AHR are tryptamine (TA) (Heath-Pagliuso
et al., Biochemistry. 37, 11508-15 (1998)), a derivative of
tryptophan (Trp) catabolism and
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE) (Song et al., Proc Natl Acad Sci USA. 99, 14694-9 Epub 2002
Oct. 30 (2002)), a molecule isolated from the lung. Interestingly,
no toxicity has been reported for these AHR ligands in vivo,
probably as a result of their short half-life (Henry et al., Arch
Biochem Biophys. 450, 67-77 Epub 2006 Mar 3 (2006)). To confirm the
physiologic relevance of AHR activation for the control of Treg
activity, we tested the effect of TA and ITE on EAE. Based on the
short half-life of these molecules we administered them on a daily
basis. The administration of ITE, but not TA, led to a significant
reduction on EAE severity (p<0.001, two-way ANOVA, n=9; see FIG.
6c), likely due to rapid degradation of TA. This observation
suggests that endogenous AHR ligands participate in the control of
inflammation under physiological conditions.
[0253] Together, these results indicate that modulation of Foxp3
expression by modulating activity of a transcription factor that
binds to Foxp3 can be used to affect Treg and control of the immune
response in vivo.
Example 8
Expression Levels of Transcription Factors In Foxp3 Knock-In
Mice
[0254] Mouse Treg and non-Treg were isolated from Foxp3gpf knock in
mice, mRNA was prepared and Foxp3 (see FIG. 8A), NKX2.2 (see FIG.
8B), EGR1 (see FIG. 8C), EGR2 (see FIG. 8D) and EGR3 (see FIG. 8E)
expression was quantified by real time PCR. Foxp3gpf knock in mice
have a GFP reporter inserted in the Foxp3 gene, producing GFP in
Foxp3.sup.+ Treg and therefore facilitating the identification and
FACS sorting of GFP:Foxp3.sup.+ Treg.
[0255] GFP.sup.-CD4.sup.+ T cells were isolated from Foxp3gpf knock
in mice, and then were activated in vitro with antibodies to CD3
and CD28 in the presence of TGF.beta.1 to induce Treg
differentiation in vitro. mRNA was prepared at the beginning of the
experiment and after 3 or 6 days in culture, and the expression of
Foxp3 (see FIG. 9A), NKX2.2 (see FIG. 9B), EGR1 (see FIG. 9C), EGR2
(see FIG. 9D) and EGR3 (see FIG. 9E) expression was quantified in
the Foxp3:GFP.sup.+CD4.sup.+ T cells by real time PCR.
[0256] These results indicate that expression of Foxp3 and the
transcription factors EGR1, EGR2, EGR3 and NKX2.2 is correlated.
Taken together, the reported effects that these TFs exert on the
regulation of gene expression, the identification of TF binding
sites on the Foxp3 gene, and the correlation between the expression
of these TF and Fox3, suggest that EGR1, EGR2, EGR3 and NKX2.2 play
a role in the regulation of Foxp3 expression and the generation of
Treg.
Example 9
Combination Treatment Using Tryptamine (TA)
[0257] As shown above, TA is rapidly degraded in vivo by monoamine
oxidase inhibitors. As shown in FIG. 13, when combined with the
monoamine oxidase inhibitor trans-2-Phenylcyclopropylamine
hydrochloride (Tranylcypromine), TA effectively suppresses EAE
suppression.
[0258] This observation suggests that TA is a TCDD-like ligand
that, when used in combination with a monoamine oxidase inhibitor,
can be used as a transcription factor ligand for promoting an
increase in the number and/or activity of Treg.
Example 10
Modified Screening Assays
[0259] A modified zebrafish based screening assay was established
by microinjecting fertilized zebrafish eggs with a BAC construct
encoding the complete mouse Foxp3 locus, with a renilla reporter
inserted after the Foxp3 methionine start codon (ATG). Six days
after microinjection, renilla activity was determined in total
zebrafish lysates. As shown in FIG. 14, murine Foxp3 was expressed
in the microinjected fish as determined by renilla luciferase
activity. The activity increased in the presence of TCDD in a
dose-dependent manner.
[0260] These data suggest that zebrafish lines encoding murine
Foxp3 can be used to screen for small molecules that increase or
decrease Foxp3 expression levels.
Example 11
AHR Activation With Its Non-Toxic Ligand ITE Induces Functional
Treg
[0261] The ligand-activated transcription factor aryl hydrocarbon
receptor (AHR) is a regulator of zebrafish, mouse and human Foxp3
expression and T.sub.reg differentiation (Quintana et al., Nature
23, 23 (2008)). AHR activation by its ligand
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced T.sub.reg that
suppressed experimental autoimmune encephalomyelitis (EAE) by a
TGFb1-dependent mechanism. These findings identify AHR as a
therapeutic target of interest for the management of autoimmune
disorders, but its therapeutic exploitation is limited by the
well-characterized toxic features of TCDD (Baccarelli et al.,
Environ Health Perspect. 110, 1169 (2002)).
[0262] Several endogenous AHR ligands have been isolated, among
them tryptophan derivatives like tryptamine (TA) (Heath-Pagliuso et
al., Biochemistry. 37, 11508 (1998))and the mucosal associated
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester
(ITE) depicted in FIG. 1 (Song et al., Proc Natl Acad Sci USA. 99,
14694 (Nov. 12, 2002)). Notably, although ITE and TA have been
shown to be high affinity AHR ligands, they do not display the
toxic effects reported by TCDD (Heath-Pagliuso et al., (1998),
supra; Henry et al., Arch Biochem Biophys. 450, 67 (2006)). As
demonstrated herein, the non-toxic AHR ligand ITE administered
intraperitoneally, orally or with pegylated gold nanoparticles can
be used to induce functional T.sub.reg.
[0263] To analyze the feasibility of using ITE to activate AHR in
vivo in a therapeutic setup, we studied the effect of ITE on EAE
development. EAE was induced on naive C57BL/6 mice and ITE (200
mg/mice) was administered orally or intraperitoneally on daily
basis. ITE administration, either orally or intraperitoneally,
resulted in a significant delay on EAE development and a
significant reduction of EAE clinical score (FIGS. 15A-B). Thus,
AHR activation by ITE induces functional T.sub.reg that can control
EAE.
[0264] To study the mechanism by which the ITE-induced T.sub.reg
control EAE we studied the ability of AHR activation by ITE to
induce T.sub.reg. We treated naive C57BL/6 mice with ITE (200
mg/mouse administered ip, daily) and immunized them with 100
mg/mouse of MOG.sub.35-55 in CFA. Spleens were prepared 10 days
later and CD4.sup.+FoxP3.sup.+ T.sub.reg were quantified by FACS.
Administration of ITE led to a significant increase in the number
of CD4.sup.+FoxP3.sup.+ T.sub.reg (FIGS. 16 and 17). Notably, this
increase resulted from the expansion of both CD25.sup.+ and
CD25.sup.- CD4.sup.+FoxP3.sup.+ T.sub.reg, but did lead to
significant alterations in the levels of LAP.sup.+ regulatory T
cells (FIGS. 16 and 17). Thus AHR activation by ITE results in the
expansion of the CD4.sup.+FoxP3.sup.+ T.sub.reg compartment.
[0265] To confirm the lack of toxicity of ITE, we administered it
intraperitoneally for 14 days, 200 mg/mouse and studied the blood
levels of biochemical indicators of liver function induction.
Hepatocites are known to express high levels of AHR, thus toxic
effects of AHR activation are manifested in the liver. Table 6
shows that at day 14 we did not detect any significant difference
in the biochemical indicators of liver function, confirming the
lack of toxicity in ITE.
TABLE-US-00007 TABLE 6 ITE Administration Does Not Result in Liver
Toxicity TEST Units Control ITE Reference Range ALT/GPT U/L 16 .+-.
2 19 .+-. 4 0-54 AST/GOT U/L 79 .+-. 29 94 .+-. 17 9-74 Alkaline
Phosphatase U/L 75 .+-. 8 55 .+-. 11 36-300 Total Bilirubin mg/dL 0
.+-. 0 0 .+-. 0 0.1-1.2 Direct Bilirubin mg/dL 0 .+-. 0 0 .+-. 0
0.0-0.8 Total Protein g/dL 6 .+-. 0 5 .+-. 0 4.4-8.0 Albumin g/dL 2
.+-. 0 3 .+-. 0 2.9-5.4 Globulin g/dL 3 .+-. 0 3 .+-. 0 2.0-4.0
[0266] To study the feasibility of administering ITE orally to
activate AHR and induce functional T.sub.reg we treated naive
C57BL/6 mice with ITE (200 mg/mouse administered orally, daily) and
immunized them with 100 mg/mouse of MOG.sub.35-55 in CFA. Spleens
were prepared 10 days later and CD4.sup.+FoxP3.sup.+ T.sub.reg were
quantified by FACS. Administration of ITE led to a significant
increase in the number of CD4.sup.+FoxP3.sup.+ T.sub.in (FIGS. 18
and 19). Notably, this increase resulted from the expansion of both
CD25.sup.+ and CD25.sup.- CD4.sup.+FoxP3.sup.+ T.sub.reg, but did
lead to significant alterations in the levels of LAP.sup.+
regulatory T cells (FIGS. 18 and 19). Thus ITE can be administered
orally to activate AHR and expand the CD4.sup.+FoxP3.sup.+
T.sub.reg compartment.
[0267] To confirm the ability of AHR activation by ITE to expand
the T.sub.reg compartment we used Foxp3.sup.gpf knock in mice.
Foxp3.sup.gpf knock in mice have a GFP reporter inserted in the
Foxp3 gene, producing a GFP:Foxp3 fusion protein that facilitates
the identification and FACS sorting of GFP:FoxP3.sup.+ T.sub.reg
(Bettelli et al., Nature 441, 235 (2006)). Foxp3.sup.gpf knock in
mice were treated with ITE (200 mg/mouse administered ip, daily)
and immunized with 100 mg/mouse of MOG.sub.35-55 in CFA. Ten days
later and CD4.sup.+FoxP3:GFP.sup.+ T.sub.reg were quantified by
FACS. Administration of ITE led to a significant increase in the
number of CD4.sup.+FoxP3:GFP.sup.+ T.sub.reg in blood and spleen
(FIGS. 20A-B and 21A-B). Thus AHR activation by ITE results in the
expansion of the CD4.sup.+FoxP3.sup.+T.sub.reg compartment.
[0268] We then studied the effect of ITE administration on the
encephalitogenic response against myelin. EAE was induced on naive
C57BL/6 mice and ITE (200 mg/mice) was administered orally or
intraperitoneally on daily basis. Ten days after vaccination with
MOG.sub.35-55/CFA, ITE-treated mice showed a suppressed recall
proliferative response to the MOG.sub.35-55 peptide (FIG. 22); no
differences were seen upon activation with antibodies to CD3 (FIG.
22). When compared to the splenocytes from control animals,
CD4.sup.+ T cells from ITE-treated mice secreted higher amounts of
TGFb1 and IL-10 and lower amounts of IL2, IL6, IFNg and IL17 upon
activation with MOG.sub.35-55 (FIG. 22). Similar results were
observed on the recall response to MOG.sub.35-55 of mice treated
with orally administered ITE (FIG. 23).
[0269] To confirm the suppressive effects of AHR activation on the
generation of T cells secreting IFNg and IL17, IFNg.sup.+ and
IL17.sup.+ CD4.sup.+ T cells were quantified by FACS in the
draining lymph nodes ten days after footpad immunization and
intraperitoneal administration of ITE (200 mg/mice). AHR activation
with ITE led to a decrease in the frequency of CD4.sup.+IL17.sup.+
and CD4.sup.+IFNg.sup.+ T cells (FIG. 24). In accordance with these
results, we found a significant reduction in the secretion of IL-17
and IFNg by lymph node cells from ITE-treated mice activated with
MOG.sub.35-55 or aCD3.
[0270] To investigate the effect of AHR activation by ITE on the
frequency of MOG.sub.35-55 specific T.sub.reg and effector T cells
(T.sub.eff), Foxp3.sup.gpf knock in mice were immunized with
MOG.sub.35-55/CFA, treated daily with intreaperitoneal ITE (200
mg/mice) and MOG.sub.35-55-specific T.sub.reg
(CD4.sup.+FoxP3:GFP.sup.+) and T.sub.eff (CD4.sup.+FoxP3:GFP.sup.-)
were analyzed by FACS using recombinant MHC class II tetramers
containing MBP.sub.35-55 or the control peptide TMEV.sub.70-86. AHR
activation with ITE led to a decrease in the frequency of
MOG.sub.35-55-specific T.sub.eff and to a concomitant increase in
the frequency of MOG.sub.35-55-specific T.sub.reg, reducing the
MOG.sub.35-55-specific T.sub.eff/T.sub.reg ratio by half (FIG.
25).
[0271] To investigate the active suppression of
MOG.sub.35-55-specific Teff by Treg, Foxp3.sup.gpf knock in mice
were immunized with MOG.sub.35-55/CFA, treated daily with
intreaperitoneal ITE (200 mg/mice) and the recall response to
MOG.sub.35-55 and a mitogenic antibody to CD3 antibody was studied
on FACS-sorted CD4.sup.+ T cells and CD4.sup.+FoxP3:GFP.sup.-
T.sub.eff. Purified CD4+ T cells from ITE-treated mice showed a
suppressed response to MOG.sub.35-55 but not to anti-CD3 (FIG. 26).
This suppressed response to MOG.sub.35-55 was lost upon removal of
the CD4.sup.+FoxP3:GFP.sup.+ T.sub.reg (FIG. 26). To further
analyze the MOG.sub.35-55-specific suppressive activity of the
CD4.sup.+FoxP3:GFP.sup.+ T.sub.reg, they were cocultured at
different ratios and assayed for the suppression of MOG.sub.35-55
or anti-CD3-triggered proliferation of CD4.sup.+FoxP3:GFP.sup.-
T.sub.eff form 2D2 mice, which harbor a TCR specific for
MOG.sub.35-55. CD4.sup.+FoxP3:GFP.sup.+ T.sub.reg, from ITE-treated
mice displayed an increased MOG.sub.35-55-specific suppressive
activity, which could be inhibited with antibodies blocking
antibodies to TGFb1 (FIGS. 27A-C). All in all, these data suggests
that, similarly to what we have described for TCDD, AHR activation
by ITE results in the expansion of antigen-specific
CD4.sup.+FoxP3.sup.+ T.sub.reg that suppress the encephalitogenic
response in a TGFb1-dependent manner.
[0272] To demonstrate that the effect of ITE on EAE was mediated by
T.sub.reg, we purified CD4.sup.+ T cells from mice protected from
EAE by oral or intraperitoneal administration of ITE 14 days after
EAE induction. Protection from EAE could be transferred to wild
type naive animals by the transfer of 5 10.sup.6 CD4.sup.+ T cells
ITE-treated mice, but not with cells isolated from vehicle-treated
mice (FIG. 28). The control of the pathogenic T cell response was
mediated by CD4.sup.+CD25.sup.+ T.sub.reg, their depletion from the
transferred population abrogated the protective effect of the
transferred cells. Thus, the T.sub.reg induced by the activation of
AHR with ITE inhibit the progression of EAE.
[0273] AHR is known to be expressed by antigen presenting cells
(APC) such as dendritic cells (CD11c.sup.+) and macrophages
(CD11b.sup.+) (Vorderstrasse and Kerkvliet, Toxicol Appl Pharmacol.
171, 117 (2001); Laupeze et al., J Immunol. 168, 2652 (2002);
Hayashi et al., Carcinogenesis. 16, 1403 (1995); Komura et al., Mol
Cell Biochem. 226, 107 (2001)). To analyze the effects that AHR
activation by ITE might have on different APC populations, which
can potentially influence the generation of T.sub.eff and T.sub.reg
cells, we studied the effect of ITE and TCDD treatment on MHC class
II expression by as dendritic cells (CD11c.sup.+) and macrophages
(CD11b.sup.+). C57BL/6 mice were treated with ITE (200 mg/mouse
administered ip, daily) or TCDD (1 mg/mouse administered ip on day
0) and immunized them with 100 mg/mouse of MOG.sub.35-55 in CFA.
Spleens were prepared 10 days later and MCH class I expression was
investigated on CD11b.sup.+ and CD11c.sup.+ cells by FACS.
Administration of ITE or TCDD resulted in a significant decrease in
CD11c.sup.+ MHC class II expression, which was concomitant with a
significant increase in CD11b.sup.+ MHC class II expression (FIG.
29). Since CD11c.sup.+ MHC-II.sup.+ and CD11b.sup.+ MHC-II.sup.+
have been recently linked to the induction of T.sub.eff and
T.sub.reg, respectively, these results suggest that changes in the
different APC populations might contribute to the immunomodulatory
effects of AHR activation by ITE.
Example 12
Administration of ITE-Loaded Nanoparticles Induces Functional
Treg
[0274] As noted above, administration of a single dose of 1
mg/mouse of the AHR ligand TCDD could prevent the development of
EAE. To achieve similar effects on diasease progression, 200
mg/mouse of ITE have to be administered daily throughout the
experiment. ITE is a tryptophan derivative which is thought to have
a short half-life in vivo as a result of the activity of specific
enzymes. Indeed, administration of ITE at weekly intervals, instead
of daily, results in a complete loss of tis protective effects on
EAE (FIG. 30).
[0275] Gold colloid has been in use for over 50 years in the
treatment of rheumatoid arthritis, these gold colloid nanoparticles
have been shown to have little to no long-term toxicity or adverse
effects (Paciotti et al., Drug Deliv. 11, 169 (2004)). Due to their
small size (10-100nm diameter), gold colloid nanoparticles have
large surface areas on which multiple small proteins or other
molecules can be conjugated (Paciotti et al., Drug Deliv. 11, 169
(2004)). The PEGylation of gold colloid nanoparticles greatly
enhances the overall stability of the molecule to which it is
covalently bonded (Qian et al., Nat Biotechnol. 26, 83 (2008)).
Moreover, recently it has been shown that PEGylated) gold colloid
nanoparticles can be linked to specific antibodies to target them
to specific cell types (Qian et al., Nat Biotechnol. 26, 83
(2008)). Thus, to increase the half-life of ITE and to facilitate
its targeting to specific cell types, we constructed polyethylene
glycol coated (PEGylated) gold colloid nanoparticles loaded with
AHR ligands (FIG. 31).
[0276] PEGylated gold colloid nanoparticles carrying the AHR
ligands FICZ, ITE or TCDD showed a typical spectrum of optical
absorption (FIG. 32). Moreover, FICZ, ITE or TCDD-loaded
nanoparticles activated luciferase expression on an AHR-reporter
cell line to levels similar to those achieved by 10 nM TCDD.
[0277] To investigate the in vivo functionality of AHR-ligand
loaded nanoparticles we induced EAE on naive C57BL/6 mice and
treated them, starting at day 0, weekly with 45 femtomoles of
nanoparticles. Similarly to what we have described in our previous
experiments, treatment with TCDD resulted in a complete suppression
of EAE, while the AHR ligand FICZ worsened the disease (FIG. 33).
Weekly administration of ITE-loaded nanoparticles resulted in a
significant inhibition of EAE development (FIG. 34). Thus, the
administration of ITE using nanoparticles augments its suppressive
effect on EAE (compare FIGS. 31 and 33)
[0278] To study the effect of ITE-loaded nanoparticles on the
T.sub.reg compartment we induced EAE on naive C57BL/6 mice and
treated them, starting at day 0, weekly with 45 femtomoles of
nanoparticles. Spleens were prepared 21 days after EAE induction
and CD4.sup.+FoxP3.sup.+ T.sub.reg were quantified by FACS.
Administration of ITE-loaded nanoparticles led to a significant
increase in the number of CD4.sup.+FoxP3.sup.+ T.sub.reg (FIG. 34);
this increase resulted from the expansion of both CD25.sup.+ and
CD25.sup.- CD4.sup.+FoxP3.sup.+ T.sub.reg (FIG. 34). Thus
ITE-loaded nanoparticles can be used to activate AHR and expand the
T.sub.reg compartment.
[0279] To study the mechanism by which the ITE-loaded nanoparticles
control EAE we studied the activity of myelin specific T cells. We
induced EAE on naive C57BL/6 mice and treated them, starting at day
0, weekly with 45 femtomoles of nanoparticles. Spleens were
prepared 21 days after EAE was induced and analyzed for their
recall response to MOG.sub.35-55 and anti-CD3. Mice treated with
ITE-loaded nanoparticles showed a suppressed recall proliferative
response to the MOG.sub.35-55 peptide (FIG. 35); no differences
were seen upon activation with antibodies to CD3 (FIG. 35). When
compared to the splenocytes from control animals, CD4.sup.+ T cells
from mice treated with ITE-loaded nanoparticles secreted higher
amounts of TGFb1 and IL-10 and lower amounts of IL2, IL6, IFNg and
IL17 upon activation with MOG.sub.35-55 (FIG. 35).
Example 13
Induction of Functional Human Regulatory T Cells By AHR
Activation
[0280] To investigate the potential of AHR targeting for the
induction of human T.sub.reg we activated purified naive CD4.sup.+
CD62L.sup.+ CD45RO.sup.- T cells for healthy donors for 5 days with
antibodies to CD3 and CD28 in the presence of TCDD 100 nM or TGFb1
2.5 ng/ml or both. T cell activation in the presence of TCDD
resulted in the induction of CD4.sup.+ FoxP3.sup.+ T cells in some
(FIG. 36) but not all human samples (FIG. 37).
[0281] To study the functionality of the putative human T.sub.reg
induced in the presence of TCDD we studied their suppressive
activity of CD4.sup.+ CD25.sup.High and CD4.sup.+ CD25.sup.Low T
cells following 5 days of activation in the of TCDD or TGFb1 2.5
ng/ml or both. Activation in the presence of TGFb1 did not result
in the induction of suppressive T cells (FIG. 38). However,
activation in the presence of TCDD led to the generation of both
CD4.sup.+ CD25.sup.High and CD4.sup.+ CD25.sup.low functional human
regulatory T cells, as shown by their ability to inhibit the
proliferation of responder T cells (FIG. 38). This effect of TCDD
was amplified in the presence of TGFb1, as shown by the increased
suppressive activity of the T cells generated under these
conditions (FIG. 38).
[0282] To investigate the mechanism mediating the suppressive
activity of the TCDD-induced T.sub.reg, we analyzed them by real
time PCR for the expression of several genes that have been
previously linked to the suppressive function of T.sub.reg. FoxP3
expression was significantly up-regulated upon activation in the
presence of TCDD and TGFb1 (FIG. 39), however it was also induced
buy TGFb1 alone, suggesting that FoxP3 expression does not
correlate with the induction of suppressive function via AHR
activation. This is confirmed by the marginal induction of FoxP3
expression triggered by AHR activation with TCDD in the absence of
TGFb1 (FIG. 39), although these cells expressed low levels of FoxP3
were suppressive in co-culture assays (FIG. 38). TGFb1 also
up-regulated AHR expression levels several fold over the basal
levels observed on T cells, however the AHR expression levels also
did not correlate with the induction of suppressive activity as
shown in co-culture assays (FIG. 40). Strikingly, TCDD treated
expressed increased levels of IL-10, which where complete inhibited
by TGFb1 (FIG. 41). Accordingly, IL-10-specific blocking antibodies
could interfere with the suppressive activity of T.sub.reg induced
with TCDD, but not the of those induced with TGFb1 and TCDD (FIG.
42). Thus, TCDD-induced CD4.sup.+CD25.sup.High T cells are
FoxP3.sup.- regulatory cells whose suppressive activity is
mediated, at least partially, via IL-10, resembling the phenotype
of type 1 T.sub.reg (Roncarolo et al., Immunol Rev. 212, 28 (2006);
Roncarolo and Gregori, Eur J Immunol. 38, 925 (2008)).
Other Embodiments
[0283] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *
References