U.S. patent application number 11/284418 was filed with the patent office on 2007-01-11 for methods and compositions for regulating t cell subsets by modulating transcription factor activity.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Laurie H. Glimcher, I-Cheng Ho.
Application Number | 20070009918 11/284418 |
Document ID | / |
Family ID | 24552580 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070009918 |
Kind Code |
A1 |
Glimcher; Laurie H. ; et
al. |
January 11, 2007 |
Methods and compositions for regulating T cell subsets by
modulating transcription factor activity
Abstract
Methods for modulating production of a T helper type 2
(Th2)-associated cytokine, in particular interleukin-4, by
modulating the activity of a transcription factor, in particular
the proto-oncoprotein c-Maf, that regulates expression of the
Th2-associated cytokine gene are disclosed. Methods for modulating
development of T helper type 1 (Th1) or T helper type 2 (Th2)
subsets in a subject using agents that modulate transcription
factor activity are also disclosed. The methods of the invention
can further involve use of agents that modulate the activity of
additional transcription factors that contribute to the regulation
of Th1- or Th2-associated cytokines, such as a Nuclear Factor of
Activated T cells (NF-AT) protein and/or an AP-1 family protein.
Compositions for modulating Th2-associated cytokine production,
recombinant expression vectors and host cells, as well as screening
assays to identify agents that modulate c-Maf activity, are also
disclosed.
Inventors: |
Glimcher; Laurie H.; (West
Newton, MA) ; Ho; I-Cheng; (Newton, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP
ONE POST OFFICE SQUARE
BOSTON
MA
02109-2127
US
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
24552580 |
Appl. No.: |
11/284418 |
Filed: |
November 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09248756 |
Feb 12, 1999 |
6967077 |
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11284418 |
Nov 21, 2005 |
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08636602 |
Apr 23, 1996 |
5958671 |
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09248756 |
Feb 12, 1999 |
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Current U.S.
Class: |
435/6.18 ;
435/455; 435/6.1; 435/7.1; 514/44R |
Current CPC
Class: |
C07K 14/5406 20130101;
C07K 14/82 20130101; A01K 2217/05 20130101; G01N 33/505 20130101;
A61K 38/1709 20130101; A61K 48/00 20130101; C07K 2319/00 20130101;
C07K 14/4702 20130101; A61P 37/00 20180101; C07K 14/52
20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 435/455; 514/044 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; A61K 48/00 20060101
A61K048/00 |
Goverment Interests
GOVERNMENT FUNDING
[0001] Work described herein was supported, at least in part, under
grant AI37833 awarded by the National Institutes of Health. The
U.S. government therefore may have certain rights in this
invention.
Claims
1. A method for modulating production of a T helper type 2
(Th2)-associated cytokine by a cell comprising contacting the cell
with an agent that modulates the expression or activity of a
transcription factor that regulates expression of a Th2-associated
cytokine gene such that production of the Th2-associated cytokine
by a cell is modulated.
2. The method of claim 1, wherein the agent acts intracellularly to
modulate the expression or activity of the transcription factor
that regulates expression of a Th2-associated cytokine gene.
3. The method of claim 1, wherein the transcription factor is a
member of the maf family.
4. The method of claim 3, wherein the transcription factor is
c-Maf.
5. The method of claim 1, wherein the Th2-associated cytokine is
interleukin-4.
6. The method of claim 1, further comprising contacting the cell
with a second agent that modulates the expression or activity of a
second transcription factor that contributes to regulating the
expression of a Th1- or Th2-associated cytokine gene.
7. The method of claim 6, wherein the second agent modulates the
expression or activity of a Nuclear Factor of Activated T
cells.
8. The method of claim 1, wherein production of a Th2-associated
cytokine by the cell is stimulated.
9. The method of claim 8, wherein the cell is a T helper type 1
(Th1) cell, a B cell or a nonlymphoid cell.
10. The method of claim 8, wherein the agent is a nucleic acid
molecule encoding a maf family protein, wherein the nucleic acid
molecule is introduced into the cell in a form suitable for
expression of the maf family protein in the cell.
11. The method of claim 1, wherein production of a Th2-associated
cytokine by the cell is inhibited.
12. The method of claim 11, wherein the cell is a Th2 cell.
13. The method of claim 11, wherein the agent is an intracellular
binding molecule.
14. The method of claim 1, further comprising administering the
cell to a subject to thereby modulate development of T helper type
1 (Th1) or T helper type 2 (Th2) cells in a subject.
15. A method for modulating development of T helper type 1 (Th1) or
T helper type 2 (Th2) cells in a subject comprising administering
to the subject an agent that modulates the activity of a
transcription factor that regulates expression of a Th2-associated
cytokine gene such that development of Th1 or Th2 cells in the
subject is modulated.
16. The method of claim 15, wherein the agent acts intracellularly
to modulate the expression or activity of the transcription factor
that regulates expression of a Th2-associated cytokine gene.
17. The method of claim 15, wherein the transcription factor is a
member of the maf family.
18. The method of claim 17, wherein the transcription factor is
c-Maf.
19. The method of claim 15, wherein the Th2-associated cytokine is
interleukin-4.
20. The method of claim 15, further comprising administering to the
subject a second agent that modulates the expression or activity of
a second transcription factor that contributes to regulating the
expression of a Th1- or Th2-associated cytokine gene.
21. The method of claim 20, wherein the second agent modulates the
expression or activity of a Nuclear Factor of Activated T
cells.
22. The method of claim 15, wherein production of a Th2-associated
cytokine by cells of the subject is stimulated.
23. The method of claim 15, wherein production of a Th2-associated
cytokine by cells of the subject is inhibited.
24. A recombinant expression vector comprising a nucleotide
sequence encoding a maf family protein operatively linked to
regulatory sequences that direct expression of the maf family
protein specifically in lymphoid cells.
25. The recombinant expression vector of claim 24, wherein the
regulatory sequences direct expression of the maf family protein
specifically in T cells.
26. The recombinant expression vector of claim 24, wherein the
regulatory sequences direct expression of the maf family protein
specifically in B cells.
27. A recombinant expression vector comprising a nucleotide
sequence encoding a maf family protein operatively linked to
regulatory sequences that direct expression of the maf family
protein specifically in hematopoietic stem cells.
28. A host cell into which a recombinant expression vector encoding
a maf family protein has been introduced, wherein the host cell is
a lymphoid cell.
29. The host cell of claim 28, which is a T cell.
30. The host cell of claim 28, which is a B cell.
31. A host cell into which a recombinant expression vector encoding
a maf family protein has been introduced, wherein the host cell is
a hematopoietic stem cell.
32. A method for identifying a compound that modulates the
expression or activity of a transcription factor that regulates
expression of a Th2-associated cytokine gene comprising: a)
preparing an indicator cell, wherein said indicator cell contains:
i) a recombinant expression vector encoding a transcription factor
that regulates expression of a Th2-associated cytokine gene; and
ii) a vector comprising regulatory sequences of the Th2-associated
cytokine gene operatively linked a reporter gene; b) contacting the
indicator cell with a test compound; c) determining the level of
expression of the reporter gene in the indicator cell in the
presence of the test compound; d) comparing the level of expression
of the reporter gene in the indicator cell in the presence of the
test compound with the level of expression of the reporter gene in
the indicator cell in the absence of the test compound; and e)
identifying a compound that modulates the expression or activity of
a transcription factor that regulates expression of a
Th2-associated cytokine gene.
33. A method for identifying a protein in a Th2 cell that interacts
with c-Maf comprising: a) providing a two hybrid assay including a
host cell which contains i) a reporter gene operably linked to a
transcriptional regulatory sequence; ii) a first chimeric gene
which encodes a first fusion protein, said first fusion protein
including c-Maf; iii) a library of second chimeric genes which
encodes second fusion proteins, the second fusion proteins
including proteins derived from Th2 cells; wherein expression of
the reporter gene is sensitive to interactions between the first
fusion protein, the second fusion protein and the transcriptional
regulatory sequence; b) determining the level of expression of the
reporter gene in the host cell; and c) identifying a protein in a
Th2 cell that interacts with c-Maf.
34. A method for identifying a compound that modulates the
interaction of a c-Maf protein with a maf response element (MARE)
of an IL-4 gene regulatory region, comprising: a) providing a c-Maf
protein and a DNA fragment comprising a MARE of an IL-4 gene
regulatory region; b) incubating the c-Maf protein and DNA fragment
in the presence of a test compound; c) determining the amount of
binding of the c-Maf protein to the DNA fragment in the presence of
the test compound; d) comparing the amount of binding of the c-Maf
protein to the DNA fragment in the presence of the test compound
with the amount of binding of the c-Maf protein to the DNA fragment
in the absence of the test compound; and e) identifying a compound
that modulates the interaction of a c-Maf protein with a MARE of an
IL-4 gene regulatory region.
Description
BACKGROUND OF THE INVENTION
[0002] CD4+ T helper cells are not a homogeneous population but can
be divided on the basis of cytokine secretion into at least two
subsets termed T helper type 1 (Th1) and T helper type 2 (Th2) (see
e.g., Mosmann, T. R. et al. (1986) J. Immunol. 136:2348-2357; Paul,
W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder, R. A. and
Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Th1 cells
secrete interleukin-2 (IL-2) and interferon-.gamma. (IFN-.gamma.)
while Th2 cells produce interleukin-4 (IL-4), interleukin-5 (IL-5),
interleukin-10 (IL-10) and interleukin-13 (IL-13). Both subsets
produce cytokines such as tumor necrosis factor (TNF) and
granulocyte/macrophage-colony stimulating factor (GM-CSF). In
addition to their different pattern of cytokine expression, Th1 and
Th2 cells are thought to have differing functional activities. For
example, Th1 cells are involved in inducing delayed type
hypersensitivity responses, whereas Th2 cells are involved in
providing efficient "help" to B lymphocytes and stimulating
production of IgG1 and IgE antibodies.
[0003] There is now abundant evidence that the ratio of Th1 to Th2
cells is highly relevant to the outcome of a wide array of
immunologically-mediated clinical diseases including autoimmune,
allergic and infectious diseases. For example, in experimental
leishmania infections in mice, animals that are resistant to
infection mount predominantly a Th1 response, whereas animals that
are susceptible to progressive infection mount predominantly a Th2
response (Heinzel, F. P., et al. (1989) J. Exp. Med. 169:59-72;
Locksley, R. M. and Scott, P. (1992) Immunoparasitology Today
1:A58-A61). In murine schistosomiasis, a Th1 to Th2 switch is
observed coincident with the release of eggs into the tissues by
female parasites and is associated with a worsening of the disease
condition (Pearce, E. J., et al. (1991) J. Exp. Med. 173:159-166;
Grzych, J-M., et al. (1991) J. Immunol. 141:1322-1327; Kullberg, M.
C., et al. (1992) J. Immunol. 148:3264-3270). Many human diseases,
including chronic infections (such as with human immunodeficiency
virus (HIV) and tuberculosis) and certain metastatic carcinomas,
also are characterized by a Th1 to Th2 switch (see e.g., Shearer,
G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43;
Clerici, M and Shearer, G. M. (1993) Immunology Today 14:107-111;
Yamamura, M., et al. (1993) J. Clin. Invest. 91:1005-1010; Pisa,
P., et al. (1992) Proc. Natl. Acad. Sci. USA 89:7708-7712; Fauci,
A. S. (1988) Science 239:617-623). Furthermore, certain autoimmune
diseases have been shown to be associated with a predominant Th1
response. For example, patients with rheumatoid arthritis have
predominantly Th1 cells in synovial tissue (Simon, A. K., et al.
(1994) Proc. Natl. Acad. Sci. USA 91:8562-8566) and experimental
autoimmune encephalomyelitis (EAE) can be induced by autoreactive
Th1 cells (Kuchroo, V. K., et al. (1993) J. Immunol.
151:4371-4381).
[0004] The ability to alter or manipulate ratios of Th1 and Th2
subsets requires an understanding of the mechanisms by which the
differentiation of CD4 T helper precursor cells (Thp), which
secrete only IL-2, choose to become Th1 or Th2 effector cells. It
is clear that the cytokines themselves are potent Th cell inducers
and form an autoregulatory loop (see e.g., Paul, W. E. and Seder,
R. A. (1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994)
Ann. Rev. Immunol. 12:635-673). Thus, IL-4 promotes the
differentiation of Th2 cells while preventing the differentiation
of precursors into Th1 cells, while IL-12 and IFN-.gamma. have the
opposite effect. One possible means therefore to alter Th1:Th2
ratios is to increase or decrease the level of selected cytokines.
Direct administration of cytokines or antibodies to cytokines has
been shown to have an effect on certain diseases mediated by either
Th1 or Th2 cells. For example, administration of recombinant IL-4
or antibodies to IL-12 ameliorate EAE, a Th1-driven autoimmune
disease (see Racke; M. K. et al. (1994) J. Exp. Med. 180:1961-1966;
and Leonard, J. P. et al. (1995) J. Exp. Med. 181:381-386), while
anti-IL-4 antibodies cure the Th2-mediated parasitic disease,
Leishmania major (Sadick, M. D. et al. (1990) J. Exp. Med.
171:115-127). However, as therapeutic options, systemic
administration of cytokines or antibodies may have unwanted side
effects and, accordingly, alternative approaches to manipulating
Th1/Th2 subsets are still needed.
[0005] The molecular basis for the tissue-specific expression of
IL-4 in Th2 cells, or any T cell cytokine, has remained elusive.
One possibility is the presence of repressor proteins that
selectively silence cytokines. Transcriptional silencing has been
well documented for bacteria, yeast and mammalian genes. Examples
include E. coli thermoregulation genes (Goransson, M. et al. (1990)
Nature 344:682-685), yeast .alpha.2 mating type genes (Keleher, C.
A. et al. (1988) Cell 53:927-936) and mammalian MHC class I and
TcR.alpha. genes (Weisman, J. D. and Singer, D. S. (1991) Mol.
Cell. Biol. 11:4228-4234; Winoto, A. and Baltimore, D. (1989) Cell
59:649-655). Indeed, early experiments involving injection of IL-2
genomic DNA into Xenopus oocytes suggested the existence of a
repressor protein for IL-2 in resting versus activated T cell
extracts (Mouzaki, A. et al. (1991) EMBO J. 10:1399-1406). These
studies suggested that the absence of IL-2 production in resting T
cells was due to proteins that silenced the transcription of IL-2
by interacting with negative elements in the IL-2 promoter.
[0006] A second possibility is the existence of Th selective
transactivators. A family of four related transcription factors
called Nuclear Factor of Activated T cells (NF-AT), plays a key
role in the regulation of cytokine gene expression (see e.g.,
Emmel, E. A. et al. (1989) Science 246:1617-1620; Flanagan, W. M.
et al. (1991) Nature 352:803-807; Jain, J. et al. (1993) Nature
365:352-355; McCaffrey, P. G. et al. (1993) Science 262:750-754;
Rao, A. (1994) Immunol. Today 15:274-281; Northrop, J. P. et al.
(1994) Nature 369:497). However, NF-AT family members can bind to
and transactivate the promoters of multiple cytokine genes
including IL-2 and IL-4 (Rooney, J. et al. (1995) Immunity
2:545-553; Szabo, S. J. et al. (1993) Mol. Cell. Biol.
13:4793-4805; Flanagan, W. M. et al. (1991) Nature 352:803-807;
Northrop, J. P. et al. (1994) Nature 369:497). Thus, they are not
likely to be responsible for directing Th1- or Th2-specific
cytokine transcription. Most, if not all, NF-AT binding sites in
cytokine promoter regulatory regions are accompanied by nearby
sites that bind auxiliary transcription factors, usually members of
the AP-1 family. It has been shown that NF-AT and AP-1 proteins
bind coordinately and cooperatively and are required for full
activity of the IL-2 and IL-4 promoters. Different AP-1 proteins,
specifically c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, have been
shown to bind to these sites (Rao, A. et al. (1994) Immunol. Today
15:274-281; Jain, J. et al. (1993) Nature 365:352-355; Boise, L. H.
et al. (1993) Mol. Cell. Biol. 13:1911-1919; Rooney, J. et al.
(1995) Immunity 2:545-553; Rooney, J. et al. (1995) Mol. Cell.
Biol. 15:6299-6310). However, none of these AP-1 proteins is
expressed in a Th1- or Th2-specific manner and there is no evidence
for the differential recruitment of AP-1 family members to the IL-2
or IL-4 composite sites (Rooney, J. et al. (1995) Mol. Cell. Biol.
15:6299-6310). Thus, neither NF-AT proteins nor the AP-1 family
members c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D can account for
the tissue-specific transcription of IL-4 in Th2 cells.
SUMMARY OF THE INVENTION
[0007] This invention pertains to methods for regulating Th1 or Th2
subsets by modulating the activity of a transcription factor that
regulates expression of a Th2-specific cytokine gene. As described
further herein, it has now been discovered that the tissue-specific
expression of IL-4 in Th2 cells is not due to a repressor protein
but rather to a Th2-specific transactivator protein. The
proto-oncogene c-maf has now been demonstrated to be responsible
for the tissue-specific expression of the Th2-associated cytokine
interleukin-4. Moreover, ectopic expression of c-maf in cells other
than Th2 cells (e.g, Th1 cells, B cells and non-lymphoid cells)
leads to activation of the IL-4 promoter and, under appropriate
conditions, production of endogenous IL-4.
[0008] Accordingly, in one aspect the invention provides a method
for modulating production of a T helper type 2 (Th2)-associated
cytokine by a cell. The method involves contacting the cell with an
agent that modulates the activity of a transcription factor that
regulates expression of a Th2-associated cytokine gene such that
production of the Th2-associated cytokine by a cell is modulated.
In particular, the agents of the invention act intracellularly to
modulate the activity of a transcription factor that regulates
expression of a Th2-associated cytokine gene. Preferably, the
transcription factor is a member of the maf family. Most
preferably, the transcription factor is c-Maf. The Th2-associated
cytokine modulated in the method is preferably interleukin-4. In
one embodiment, production of the Th2-associated cytokine (e.g.,
IL-4) is stimulated, for example in a cell that does not normally
express the cytokine (such as a Th1 cell or a B cell). A variety of
agents can be used to stimulate cytokine production, including a
nucleic acid molecule encoding a maf family protein that is
introduced into and expressed in the cell and chemical agents that
enhance the expression or activity of an endogenous maf family
protein in the cell. In another embodiment, production of a
Th2-associated cytokine by a cell (e.g., a Th2 cell) is inhibited.
A variety of agents can be used to inhibit cytokine production,
including antisense nucleic acid molecules that are complementary
to a maf family gene, intracellular antibodies that bind maf family
proteins (e.g., in the cell nucleus), inhibitory forms of maf
family proteins (e.g., dominant negative forms) and chemical agents
that inhibit the expression or activity of an endogenous maf family
protein in the cell. Cytokine production by the cell can be
modulated in vitro or in vivo. In one embodiment, a cell is
contacted with a modulating agent in vitro and then is administered
to a subject to thereby regulate the development of Th1 and/or Th2
subsets in the subject.
[0009] In another aspect, the invention provides methods for
regulating the development of Th1 or Th2 subsets in a subject. In
addition to the embodiment discussed above wherein ex vivo modified
cells are administered to the subject, in another embodiment, these
methods involve direct administration to the subject of an agent
that modulates the activity of a transcription factor (e.g., a maf
family member) that regulates expression of a Th2-associated
cytokine gene (e.g., IL-4) such that development of Th1 or Th2
cells in the subject is modulated.
[0010] The methods of the invention can further involve the use of
additional agents that modulate the activity of additional
transcription factors that contribute to regulating the expression
of Th1- or Th2-associated cytokines. Preferred additional agents
are those which modulate the activity of a Nuclear Factor of
Activated T cells (NF-AT) protein. Thus, in one embodiment, a
stimulatory method of the invention can involve the use of a first
agent that stimulates the expression and/or activity of a maf
protein and a second agent that stimulates the expression and/or
activity of an NF-AT protein. Similarly, an inhibitory method of
the invention can involve the use of a first agent that inhibits
the expression and/or activity of a maf protein and a second agent
that inhibits the expression and/or activity of an NF-AT protein.
Alternatively or additionally, the modulatory methods of the
invention can involve the use of additional agents that modulate
the activity of an AP-1 family protein.
[0011] The modulatory methods of the invention can be used to
manipulate Th1:Th2 ratios in a variety of clinical situations. For
example, inhibition of Th2 formation may be useful in allergic
diseases, malignancies and infectious diseases whereas enhancement
of Th2 formation may be useful in autoimmune diseases and organ
transplantation.
[0012] Another aspect of the invention pertains to compositions
that are useful for modulating the production of a Th2-associated
cytokine by a cell and/or for modulating the development of Th1 or
Th2 subsets in a subject. In one embodiment, these compositions
include recombinant expression vectors that encode a maf family
protein, wherein the maf-encoding sequences are operatively linked
to regulatory sequences that direct expression of the maf family
protein in a specific cell type, such as lymphoid cells (e.g., T
cells or B cells) or hematopoietic stem cells. In another
embodiment, these compositions include host cells, such as host
lymphoid cells (e.g., host T cells or host B cells) or host
hematopoietic stem cells, into which a recombinant expression
vector encoding a maf family protein has been introduced.
[0013] Yet another aspect of the invention pertains to screening
assays for identifying compounds that modulate the activity of a
transcription factor that regulates expression of a Th2-associated
cytokine gene. In one type of screening assay, an indicator cell
which contains both 1) a recombinant expression vector encoding a
transcription factor that regulates expression of a Th2-associated
cytokine gene and 2) a vector comprising regulatory sequences of
the Th2-associated cytokine gene operatively linked a reporter gene
is used to identify compounds that modulate the expression and/or
activity of the transcription factor. In another embodiment, a
screening assay of the invention identifies proteins from Th2 cells
that form a protein-protein interaction with a transcription factor
(e.g., c-Maf) that regulates expression of a Th2-associated
cytokine gene. In yet another embodiment, a screening assay of the
invention identifies compounds that modulate the interaction of
c-Maf with a maf response element (MARE) in the promoter of a
Th2-associated cytokine gene (e.g., a MARE in the IL-4
promoter).
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A is a schematic of the cell fusion approach used to
demonstrate that cytokine expression is not due to a repressor
[0015] FIG. 1B is a reverse transcriptase-polymerase chain reaction
(RT-PCR) analysis of Il-2 and IL-4 cytokine, and control
.beta.-actin, mRNA expressed by an unfused Th1 clone (D1.1), an
unfused Th2 clone (D10), Th1 and Th2 homokaryons and Th1-Th2
heterokaryons.
[0016] FIG. 2A is a Northern blot analysis depicting expression of
an isolated cDNA clone in Th1 cells, Th2 cells or B lymphoma cells.
A control probe specific for GAPDH was used to show equal loading
of RNA.
[0017] FIG. 2B is a Northern blot analysis depicting upregulated
expression of the isolated cDNA clone during in vitro
differentiation of normal naive spleen cells into Th2 cells. Total
RNA was isolated from cells harvested at the indicated time points.
Culture supernatant at the appropriate dilution was measured for
cytokine (IL-10) production by ELISA to determine differentiation
into the Th1 or Th2 lineage.
[0018] FIG. 3A is a bar graph depicting transactivation of the IL-4
promoter by c-Maf in a Th1 clone (AE7). AE7 cells were
cotransfected with a wild-type IL-4 CAT reporter construct and
either a control plasmid (pMEX-NeoI), a c-Maf expression plasmid
(pMEX-Maf) or a
c-Fos expression plasmid (pMEX-c-Fos). Half of each sample was
stimulated 24 hours after transfection with antibodies to CD3. All
samples were harvested 48 hours after transfection and relative CAT
activities were determined.
[0019] FIG. 3B is a photograph of a thin layer chromotography plate
depicting the relative CAT activity in M12 B lymphoma cells
cotransfected with a wild-type IL-4 CAT reporter construct and
either two control plasmids (pMEX-NeoI and pREP.sub.4), a c-Maf
expression plasmid and a control plasmid (pMEX-Maf and pREP4), a
c-Fos expression plasmid and a control plasmid (pMEX-c-Fos and
pREP4), a c-Jun expression plasmid and a control plasmid
(pMEX-c-Jun and pREP4), a control plasmid and an NF-ATp expression
plasmid (pMEX-NeoI and pREP-NFATp), a c-Maf expression plasmid and
an NFATp expression plasmid (pMEX-Maf and pREP-NFATp) or a c-Fos
expression plasmid and an NF-ATp expression plasmid (pMEX-c-Fos and
pREP-NFATp). Half of each sample was stimulated 24 hours after
transfection with PMA and ionomycin. All samples were harvested 48
hours after transfection and relative CAT activities were
determined.
[0020] FIG. 4 is a bar graph depicting endogenous production of
IL-4 in M12 cells by ectopic expression of c-Maf and NF-ATp. Cells
stably transfected with the indicated control or expression
plasmids were either unstimulated or stimulated with PMA and
ionomycin for 24 hours. 200 .mu.l of supernatant from each sample
was subjected to ELISA for cytokine quantitation.
[0021] FIG. 5A is a photograph of a DNAse I footprint gel of the
IL-4 promoter performed using nuclear extracts from Th2 (D10,
CDC35) or Th1 (AE7, S53) clones harvested at the indicated time
points after stimulation with anti-CD3 antibodies, which depicts a
Th2-specific footprint immediately downstream of the putative MARE
site in the IL-4 promoter. Two Th2-specific hypersensitive residues
on the non-coding strand of the IL-4 promoter are indicated by *.
Five lanes of a DNAse I digestion of the IL-4 promoter probe
(without nuclear extract) and a Maxam-Gilbert A/G ladder were run
next to the DNAse I treated samples.
[0022] FIG. 5B is a schematic representation of the proximal
regulatory region of the murine IL-4 promoter. The top portion
shows the primary sequence of the murine IL-4 promoter. The numbers
indicated are relative to the start site of transcription at +1.
Asterisks denote the Th2-specific hypersensitive residues seen on
DNAse I footprint. The bottom portion shows the sequence of the
wild type (-59 to -28) oligonucleotide and the 4 bp mutants used in
EMSA and the functional assays shown in FIGS. 6 and 7. Altered
nucleotides are shown in lowercase bold and correspond to the
numbering system shown in the top portion.
[0023] FIG. 6 is a photograph of an electrophoretic mobility shift
assay (EMSA) demonstrating that c-Maf but not c-Jun binds to the
proximal IL-4 promoter and forms a complex with NF-ATp. EMSA was
performed using the indicated proteins and labeled double-stranded
oligonucleotides.
[0024] FIG. 7A is a bar graph (top) and a photograph of a thin
layer chromotography plate (bottom) depicting the relative CAT
activity in M12 cells co-transfected with a c-Maf expression vector
and either the wild-type IL-4 CAT reporter construct or one of the
4 bp mutants shown in FIG. 5B, demonstrating that transactivation
of the IL-4 promoter by c-Maf maps to the MARE and Th2-specific
footprint. The average of three independent experiments and one
representative experiment are shown in the top and bottom portions,
respectively.
[0025] FIG. 7B is a photograph of an EMSA, performed using
recombinant c-Maf, the IL-4 promoter (-59 to -27) probe and the
indicated unlabeled double-stranded oligonucleotides as
competitors, demonstrating that binding of recombinant c-Maf to the
IL-4 promoter maps to the MARE and Th2-specific footprint.
DETAILED DESCRIPTION OF THE INVENTION
[0026] This invention pertains to methods and compositions for
regulating T cell subsets by modulating transcription factor
activity, as well as to screening assays for identifying compounds
that can modulate such transcription factor activity. The invention
is based, at least in part, on the discovery that Th2-specific
expression of the interleukin-4 gene does not result from the
action of a specific repressor protein (as shown in Example 1) but
rather from the action of a specific transactivator protein. As
described further herein, the transcription factor responsible for
Th2-specific expression of the interleukin-4 gene has now been
identified as the c-Maf proto-oncoprotein, which is selectively
expressed in differentiating and mature Th2 cells and absent from
Th1 cells (see Example 2). Ectopic expression of c-Maf in cells
that do not normally express it (such as Th1 cells and B cells)
leads to transactivation of the IL-4 promoter (see Example 3) and,
under appropriate conditions, to production of endogenous IL-4 (see
Example 4). Moreover, a protein present in nuclear extracts of Th2
cells, but not Th1 cells, footprints the IL-4 promoter in the
region of a maf response element (MARE) (see Example 5) and
recombinant c-Maf binds to the IL-4 promoter in vitro (see Example
6). The ability of c-Maf to transactivate IL-4 maps to the MARE and
Th2-specific footprint in the IL-4 promoter (see Example 7).
[0027] The maf family of proteins is a sub-family of AP-1/CREB/ATF
proteins. The v-maf oncogene was originally isolated from a
spontaneous musculoaponeurotic fibrosarcoma of chicken and
identified as the transforming gene of the avian retrovirus, AS42
(Nishizawa, M. et al. (1989) Proc. Natl. Acad. Sci. USA
86:7711-7715). V-maf encodes a 42 kd basic region/leucine zipper
(b-zip) protein with homology to the c-fos and c-jun oncogenes. Its
cellular homologue, the c-maf proto-oncogene has only two
structural changes in the coding region from v-maf (Kataoka, K. et
al. (1993) J. Virol. 67:2133-2141). The maf family includes c-maf,
mafB, a human retina-specific gene Nrl (Swaroop, A. et al. (1992)
Proc. Natl. Acad. Sci. USA 89:266-270), mafK, mafF, mafG and p18.
The latter four, majK, mafF, mafG and p18, each encode proteins
that lack the amino terminal two thirds of c-Maf that contains the
transactivating domain (Fujiwara, K. T. et al. (1993) Oncogene
8:2371-2380; Igarashi, K. et al. (1995) J. Biol. Chem.
270:7615-7624; Andrews, N. C. et al. (1993) Proc. Natl. Acad. Sci.
USA 90:11488-11492; Kataoka, K. et al. (1995) Mol. Cell. Biol.
15:2180-2190) and are referred to herein as "small" mafs. C-maf and
maf family members form homodimers and heterodimers with each other
and with Fos and Jun, consistent with the known ability of the AP-1
proteins to pair with each other (Kerppola, T. K. and Curran, T.
(1994) Oncogene 9:675-684; Kataoka, K. et al. (1994) Mol. Cell.
Biol. 14:700-712). The DNA target sequence to which c-Maf
homodimers bind, termed the c-Maf response element (MARE), is a 13
or 14 bp element which contains a core TRE (T-MARE) or CRE (C-MARE)
palindrome respectively. Prior to the present invention, little was
known about the function of maf family members, although c-Maf has
been shown to stimulate transcription from the Purkinje
neuron-specific promoter L7 (Kurscher, C. and Morgan, J. I. (1994)
Mol. Cell. Biol. 15:246-254) and Nrl has been shown to drive
expression of the QR1 retina-specific gene (Swaroop, A. et al.
(1992) Proc. Natl. Acad. Sci. USA 89:266-270). The small mafs have
been shown to function as repressors of .alpha. and .beta.-globin
transcription when bound as homodimers but are essential as
heterodimeric partners with the erythroid-specific factor p45NF-E2
to activate globin gene transcription (Kataoka, K. et al. (1995)
Mol. Cell. Biol. 15:2180-2190; Igarashi, K. et al. (1994) Nature
367:568-572). MafK overexpression has been shown to induce
erythroleukemia cell differentiation (Igarashi, K. et al. (1995)
Proc. Natl. Acad. Sci. USA 92:7445-7449). However, prior to the
present invention, there have been no reports implicating c-maf or
maf family members in the regulation of genes expressed in lymphoid
cells or in cytokine gene expression in any tissue.
[0028] Various aspects of the present invention are described in
further detail in the following subsections.
I. Modulation of Th2-Associated Cytokine Production
[0029] As discussed above, the transcription factor responsible for
the Th2-specific expression of the interleukin-4 gene has now been
identified as the c-Maf proto-oncogene. Modulation of the
expression and/or activity of c-Maf, therefore, provides a means to
regulate the production of interleukin-4. Since IL-4 itself serves
an autoregulatory function in the development of Th2 cells (see
e.g., Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder,
R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673), and
thus production of IL-4 can lead to the production of additional
Th2-associated cytokines such as IL-5, IL-10 and IL-13 through
further Th2 differentiation, modulation of c-Maf expression and/or
activity provides a general approach for modulating production of
Th2-associated cytokines.
[0030] Accordingly, the invention provides a method for modulating
production of a Th2-associated cytokine by a cell. This method
involves contacting the cell with an agent that modulates the
activity of a transcription factor that regulates expression of a
Th2-associated cytokine gene such that production of the
Th2-associated cytokine by a cell is modulated. In particular, the
modulatory agents of the invention are characterized by acting
intracellularly to modulate the activity of a transcription factor.
Preferably, the transcription factor is a member of the maf family
and most preferably is c-Maf. As used herein, the term
"Th2-associated cytokine" is intended to refer to a cytokine that
is produced preferentially or exclusively by Th2 cells rather than
by Th1 cells. Preferably the Th2-associated cytokine is
interleukin-4. As used herein, the term "contacting" (i.e.,
contacting a cell with an agent) is intended to include incubating
the agent and the cell together in vitro (e.g., adding the agent to
cells in culture) and administering the agent to a subject such
that the agent and cells of the subject are contacted in vivo. As
used herein, the various forms of the term "modulation" are
intended to include stimulation (e.g., increasing or upregulating a
particular response or activity) and inhibition (e.g., decreasing
or downregulating a particular response or activity). Accordingly,
in one embodiment of the method of the invention, production of a
Th2-associated cytokine by the cell is stimulated by contacting the
cell with a stimulatory agent that stimulates c-Maf expression
and/or activity. In another embodiment of the method of the
invention, production of a Th2-associated cytokine by the cell is
inhibited by contacting the cell with a inhibitory agent that
inhibits c-Maf expression and/or activity.
[0031] As demonstrated in the Examples, although c-Maf is
responsible for the tissue specificity of IL-4 gene expression,
c-Maf acts synergistically with one or more additional
transcription factors to activate IL-4 gene transcription. In
particular, c-Maf acts synergistically with an NF-AT protein to
stimulate IL-4 gene expression. Moreover, NF-AT proteins and other
members of the AP-1/CREB/ATF family of transcription factors have
been demonstrated to be involved in regulating expression of both
Th1- and Th2-associated cytokine genes. Accordingly, the method of
the invention for modulating Th2-associated cytokine production by
a cell can further comprise contacting the cell with a second agent
that modulates (i.e., stimulates or inhibits) the expression or
activity of a second transcription factor that contributes to
regulating the expression of a Th1- or Th2-associated cytokine gene
(discussed further below).
[0032] A. Stimulatory Agents
[0033] According to the method of the invention, to stimulate
Th2-associated cytokine production by a cell, the cell is contacted
with a stimulatory agent that stimulates expression and/or activity
of a transcription factor (e.g., c-Maf) that regulates expression
of a Th2-associated cytokine gene. Th2-associated cytokine
production can be stimulated in cell types that do not normally
express such cytokines, such as Th1 cells, B cells or non-lymphoid
cells. Furthermore, Th2-associated cytokine production can be
stimulated in helper precursor cells (Thp) to promote their
differentiation along the Th2 pathway instead of the Th1
pathway.
[0034] A preferred stimulatory agent is a nucleic acid molecule
encoding a maf family protein, wherein the nucleic acid molecule is
introduced into the cell in a form suitable for expression of the
maf family protein in the cell. For example, a c-Maf cDNA is cloned
into a recombinant expression vector and the vector is transfected
into the cell. As demonstrated in Example 3, ectopic expression of
a c-maf recombinant expression vector in Th1 cells, B cells or
non-lymphoid cells leads to activation of the IL-4 promoter.
Additionally, under appropriate conditions (discussed in further
detail below), transcription of the endogenous IL-4 gene is
stimulated, leading to IL-4 production by cells that do not
normally express this cytokine (see Example 4).
[0035] To express a maf family protein in a cell, typically a maf
family cDNA is first introduced into a recombinant expression
vector using standard molecular biology techniques. A maf family
cDNA can be obtained, for example, by amplification using the
polymerase chain reaction (PCR) or by screening an appropriate cDNA
library. The nucleotide sequences of maf family cDNAs are known in
the art and can be used for the design of PCR primers that allow
for amplification of a cDNA by standard PCR methods or for the
design of a hybridization probe that can be used to screen a cDNA
library using standard hybridization methods. Preferably, the maf
family cDNA is that of the c-maf proto-oncogene. The nucleotide and
predicted amino acid sequences of a mammalian (mouse) c-maf cDNA
are disclosed in Kurscher C. and Morgan, J. I. (1995) Mol. Cell.
Biol. 15:246-254 and deposited in the GenBank database at accession
number S74567. This mammalian c-maf is highly homologous to the
avian v-maf sequence (disclosed in Nishizawa, M. K. et al. (1989)
Proc. Natl. Acad. Sci. USA 86:7711-7715 and GenBank accession
numbers D28598 and D28596), indicating that c-maf is well conserved
among species. c-maf cDNAs from other mammalian species, including
humans, can be isolated using standard molecular biology techniques
(e.g., PCR or cDNA library screening) and primers or probes
designed based upon the mouse or avian sequences. Human partial
cDNA sequences homologous to the mouse c-maf cDNA are also
deposited in the GenBank database at accession numbers H24189 and
N75504. The sequences of other maf family members are also known in
the art, for example MafB (Kataoka, K. et al. (1994) Mol. Cell
Biol. 14:7581-91; GenBank accession number D28600), MafG (Kataoka
et al. (1994) Mol. Cell Bio. 14:7581-91; GenBank accession numbers
D28601 and D28602), MafF (GenBank accession number D16184) and MafK
(Igarashi, K. et al. (1995) J. Biol. Chem. 270:7615-7624; GenBank
accession numbers D16187 and D42124).
[0036] Following isolation or amplification of a maf family cDNA,
the DNA fragment is introduced into an expression vector. As used
herein, the term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA segments may be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression
of genes to which they are operatively linked. Such vectors are
referred to herein as "recombinant expression vectors" or simply
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" may be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0037] The recombinant expression vectors of the invention comprise
a nucleic acid in a form suitable for expression of the nucleic
acid in a host cell, which means that the recombinant expression
vectors include one or more regulatory sequences, selected on the
basis of the host cells to be used for expression and the level of
expression desired, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to includes promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell, those which direct expression of the nucleotide
sequence only in certain host cells (e.g., tissue-specific
regulatory sequences) or those which direct expression of the
nucleotide sequence only under certain conditions (e.g., inducible
regulatory sequences). It will be appreciated by those skilled in
the art that the design of the expression vector may depend on such
factors as the choice of the host cell to be transformed, the level
of expression of protein desired, etc. When used in mammalian
cells, the expression vector's control functions are often provided
by viral regulatory elements. For example, commonly used promoters
are derived from polyoma virus, adenovirus, cytomegalovirus and
Simian Virus 40. Non-limiting examples of mammalian expression
vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987), EMBO J. 6:187-195). A variety of mammalian
expression vectors carrying different regulatory sequences are
commercially available. For constitutive expression of the nucleic
acid in a mammalian host cell, a preferred regulatory element is
the cytomegalovirus promoter/enhancer. Moreover, inducible
regulatory systems for use in mammalian cells are known in the art,
for example systems in which gene expression is regulated by heavy
metal ions (see e.g., Mayo et al. (1982) Cell 29:99-108; Brinster
et al. (1982) Nature 296:39-42; Searle et al. (1985) Mol. Cell.
Biol. 5:1480-1489), heat shock (see e.g., Nouer et al. (1991) in
Heat Shock Response, e.d. Nouer, L., CRC, Boca Raton, Fla.,
pp167-220), hormones (see e.g., Lee et al. (1981) Nature
294:228-232; Hynes et al. (1981) Proc. Natl. Acad. Sci. USA
78:2038-2042; Klock et al. (1987) Nature 329:734-736; Israel &
Kaufman (1989) Nucl. Acids Res. 17:2589-2604; and PCT Publication
No. WO 93/23431), FK506-related molecules (see e.g., PCT
Publication No. WO 94/18317) or tetracyclines (Gossen, M. and
Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen,
M. et al. (1995) Science 268:1766-1769; PCT Publication No. WO
94/29442; and PCT Publication No. WO 96/01313). Still further, many
tissue-specific regulatory sequences are known in the art,
including the albumin promoter (liver-specific; Pinkert et al.
(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame
and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters
of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733)
and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen
and Baltimore (1983) Cell 33:741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc.
Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters
(Edlund et al. (1985) Science 230:912-916) and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0038] Vector DNA can be introduced into mammalian cells via
conventional transfection techniques. As used herein, the various
forms of the term "transfection" are intended to refer to a variety
of art-recognized techniques for introducing foreign nucleic acid
(e.g., DNA) into mammalian host cells, including calcium phosphate
co-precipitation, DEAE-dextran-mediated transfection, lipofection,
or electroporation. Suitable methods for transfecting host cells
can be found in Sambrook et al. (Molecular Cloning: A Laboratory
Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)),
and other laboratory manuals.
[0039] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker may be introduced into a host cell on a separate vector from
that encoding a maf family protein or, more preferably, on the same
vector. Cells stably transfected with the introduced nucleic acid
can be identified by drug selection (e.g., cells that have
incorporated the selectable marker gene will survive, while the
other cells die).
[0040] Another form of a stimulatory agent for stimulating
expression of a Th2-associated cytokine in a cell is a chemical
compound that stimulates the expression or activity of an
endogenous maf family protein in the cell. Such compounds can be
identified using screening assays that select for compounds that
stimulate the expression or activity of a maf family protein.
Examples of suitable screening assays are described in further
detail in subsection V below.
[0041] In addition to use of an agent that stimulates the
expression or activity of a maf family protein, the stimulatory
methods of the invention can involve the use of a second agent that
stimulates the expression or activity of a second transcription
factor that contributes to regulating the expression of a Th1- or
Th2-associated cytokine gene. In Example 4, it is shown that
stimulation of the expression of endogenous IL-4 in M12 B lymphoma
cells required the introduction into the cells of both a c-Maf
expression vector and an NF-AT expression vector, thereby
demonstrating that c-Maf and NF-AT act synergistically to activate
IL-4 transcription, with c-maf responsible for the
tissue-specificity of expression. While the skilled artisan will
appreciate that certain cells may express sufficient amounts of
endogenous NF-AT such that use of a second agent that stimulates
the expression or activity of NF-AT is unnecessary, in certain
situations and with certain cell types it may be necessary to
stimulate both c-Maf and NF-AT to achieve IL-4 production.
[0042] Accordingly, in one embodiment, the stimulatory methods of
the invention involve the use of a first agent that stimulates the
expression or activity of c-Maf and a second agent that stimulates
the expression or activity of an NF-AT protein. A preferred second
agent for stimulating NF-AT activity in a cell is a recombinant
expression encoding an NF-AT, wherein the recombinant expression
vector is introduced into the cell and NF-AT is expressed in the
cell. NF-AT-encoding expression vectors can be prepared and
introduced into cells as described above for c-Maf expression
vectors. The nucleotide sequences of NF-AT cDNAs are known in the
art and can be used for the design of PCR primers that allow for
amplification of a cDNA by standard PCR methods or for the design
of a hybridization probe that can be used to screen a cDNA library
using standard hybridization methods. Four NF-AT family members
have been identified (see e.g., Emmel, E. A. et al. (1989) Science
246:1617-1620; Flanagan, W. M. et al. (1991) Nature 352:803-807;
Jain, J. et al. (1993) Nature 365:352-355; McCaffrey, P. G. et al.
(1993) Science 262:750-754; Rao, A. (1994) Immunol. Today
15:274-281; Northrop, J. P. et al. (1994) Nature 369:497).
Preferably, the NF-AT cDNA is that of NF-ATp. The nucleotide and
predicted amino acid sequences of a mammalian NF-ATp cDNA are
disclosed in McCaffrey, P. G. et al. (1993) Science 262:750-754.
The nucleotide and predicted amino acid sequences of a mammalian
NF-ATc cDNA are disclosed in Northrop, J. P. et al. (1994) Nature
369:497 and deposited in the GenBank database at accession number
U08015. The nucleotide and predicted amino acid sequences of
mammalian NF-AT3 and NF-AT4 cDNAs are disclosed in Hoey, T. et al.
(1995) Immunity 2:461-472.
[0043] Alternative to use of an NF-AT cDNA to stimulate the
activity of NF-AT in a cell, one or more chemical compounds that
stimulate NF-AT activity in a cell can be used as a second agent in
a stimulatory method of the invention. Compounds that stimulate
NF-AT activity in cells are known in the art (for a review see Rao,
A. (1994) Immunol. Today 15:274-281). For example, stimulation of
certain cells with the phorbol ester phorbol myristate acetate
(PMA) and a calcium ionophore (e.g., ionomycin) results in
translocation of NF-ATs to the cell nucleus (see e.g., Flanagan, W.
M. et al. (1991) Nature 352:803-807; Jain, J. et al. (1993) Nature
365:352-355). Additionally, stimulation of T cells through the T
cell receptor (TcR), for example with an anti-CD3 antibody, results
in activation of NF-AT in the T cells.
[0044] In addition to NF-AT proteins, AP-1 family members,
including c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, have been
shown to be involved in regulating the expression of both Th1- and
Th2-associated cytokine genes (e.g., IL-2 and IL-4) (see e.g., Rao,
A. et al. (1994) Immunol. Today 15:274-281; Jain, J. et al. (1993)
Nature 365:352-355; Boise, L. H. et al. (1993) Mol. Cell. Biol.
13:1911-1919; Rooney, J. et al. (1995) Immunity 2:545-553; Rooney,
J. et al. (1995) Mol. Cell. Biol. 15:6299-6310). Although these
factors are not responsible for the Th1/Th2 specificity of
expression of the cytokine genes, and these factors do not appear
to synergize with c-Maf in regulating IL-4 gene expression (see the
Examples), AP-1 family members have been shown to increase IL-4
expression in Th2 cells (see e.g., Rooney, J. et al. (1995)
Immunity 2:545-553). Accordingly, in certain circumstances it may
be beneficial, in addition to stimulating c-Maf activity (and
possibly NF-AT activity), also to stimulate the activity of an AP-1
protein. Accordingly, in one embodiment, the stimulatory methods of
the invention involve the use of a first agent that stimulates the
expression or activity of c-Maf and a second agent that stimulates
the expression or activity of an AP-1 protein. In another
embodiment, the invention involves the use of a first agent that
stimulates the expression or activity of c-Maf, a second agent that
stimulates the expression or activity of an NF-AT protein and a
third agent that stimulates the expression or activity of an AP-1
protein.
[0045] A preferred agent for stimulating AP-1 activity in a cell is
a recombinant expression encoding an AP-1 protein, wherein the
recombinant expression vector is introduced into the cell and the
AP-1 protein is expressed in the cell. AP-1-encoding expression
vectors can be prepared and introduced into cells as described
above for c-Maf expression vectors. The nucleotide sequences of
AP-1 cDNAs are known in the art. For example, the nucleotide and
predicted amino acid sequences of human c-fos are disclosed in van
Straaten, F. et al. (1983) Proc. Natl. Acad. Sci. USA 80:3183-3187.
The nucleotide and predicted amino acid sequences of human c-jun
are disclosed in Bohmann, D. et al. (1987) Science 238:1386-1392.
The nucleotide and predicted amino acid sequences of human jun-B
and jun-D are disclosed in Nomura, N. et al. (1990) Nucl. Acids
Res. 18:3047-3048. The nucleotide and predicted amino acid
sequences of human fra-1 and fra-2 are disclosed in Matsui, M. et
al. (1990) Oncogene 5:249-255. These sequences can be used for the
design of PCR primers that allow for amplification of a cDNA by
standard PCR methods or for the design of a hybridization probe
that can be used to screen a cDNA library using standard
hybridization methods. Alternatively, one or more chemical
compounds that stimulate AP-1 activity in a cell can be used as
additional agents in a stimulatory method of the invention.
Compounds that stimulate AP-1 activity in cells are known in the
art, including PMA/calcium ionophore (e.g., ionomycin) and anti-CD3
antibodies.
[0046] B. Inhibitory Agents According to the method of the
invention, to inhibit Th2-associated cytokine production by a cell,
the cell is contacted with an inhibitory agent that inhibits
expression and/or activity of a transcription factor (e.g., c-Maf)
that regulates expression of a Th2-associated cytokine gene.
Th2-associated cytokine production can be inhibited in, for
example, Th2 cells or in helper precursor cells (Thp) to promote
their differentiation along the Th1 pathway instead of the Th2
pathway. Inhibitory agents of the invention can be, for example,
intracellular binding molecules that act to inhibit the expression
or activity of the transcription factor. As used herein, the term
"intracellular binding molecule" is intended to include molecules
that act intracellularly to inhibit the expression or activity of a
protein by binding to the protein or to a nucleic acid (e.g., an
mRNA molecule) that encodes the protein. Examples of intracellular
binding molecules, described in further detail below, include
antisense nucleic acids, intracellular antibodies and dominant
negative inhibitors.
[0047] In one embodiment, an inhibitory agent of the invention is
an antisense nucleic acid molecule that is complementary to a gene
encoding a maf family protein, or to a portion of said gene, or a
recombinant expression vector encoding said antisense nucleic acid
molecule. The use of antisense nucleic acids to downregulate the
expression of a particular protein in a cell is well known in the
art (see e.g., Weintraub, H. et al., Antisense RNA as a molecular
tool for genetic analysis, Reviews--Trends in Genetics, Vol. 1(1)
1986; Askari, F. K. and McDonnell, W. M. (1996) N. Eng. J. Med.
334:316-318; Bennett, M. R. and Schwartz, S. M. (1995) Circulation
92:1981-1993; Mercola, D. and Cohen, J. S. (1995) Cancer Gene Ther.
2:47-59; Rossi, J. J. (1995) Br. Med. Bull. 51:217-225; Wagner, R.
W. (1994) Nature 372:333-335). An antisense nucleic acid molecule
comprises a nucleotide sequence that is complementary to the coding
strand of another nucleic acid molecule (e.g., an mRNA sequence)
and accordingly is capable of hydrogen bonding to the coding strand
of the other nucleic acid molecule. Antisense sequences
complementary to a sequence of an mRNA can be complementary to a
sequence found in the coding region of the mRNA, the 5' or 3'
untranslated region of the mRNA or a region bridging the coding
region and an untranslated region (e.g., at the junction of the 5'
untranslated region and the coding region). Furthermore, an
antisense nucleic acid can be complementary in sequence to a
regulatory region of the gene encoding the mRNA, for instance a
transcription initiation sequence or regulatory element.
Preferably, an antisense nucleic acid is designed so as to be
complementary to a region preceding or spanning the initiation
codon on the coding strand or in the 3' untranslated region of an
mRNA. An antisense nucleic acid for inhibiting the expression of a
Maf family protein in a cell can be designed based upon the
nucleotide sequence encoding the Maf family protein, constructed
according to the rules of Watson and Crick base pairing.
[0048] An antisense nucleic acid can exist in a variety of
different forms. For example, the antisense nucleic acid can be an
oligonucleotide that is complementary to only a portion of a maf
family gene. An antisense oligonucleotides can be constructed using
chemical synthesis procedures known in the art. An antisense
oligonucleotide can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g. phosphorothioate derivatives and
acridine substituted nucleotides can be used. To inhibit Maf
protein expression in cells in culture, one or more antisense
oligonucleotides can be added to cells in culture media, typically
at 200 .mu.g oligonucleotide/ml.
[0049] Alternatively, an antisense nucleic acid can be produced
biologically using an expression vector into which a nucleic acid
has been subcloned in an antisense orientation (i.e., nucleic acid
transcribed from the inserted nucleic acid will be of an antisense
orientation to a target nucleic acid of interest). Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen which direct the expression of
the antisense RNA molecule in a cell of interest, for instance
promoters and/or enhancers or other regulatory sequences can be
chosen which direct constitutive, tissue specific or inducible
expression of antisense RNA. The antisense expression vector is
prepared as described above for recombinant expression vectors,
except that the cDNA (or portion thereof) is cloned into the vector
in the antisense orientation. The antisense expression vector can
be in the form of, for example, a recombinant plasmid, phagemid or
attenuated virus. The antisense expression vector is introduced
into cells using a standard transfection technique, as described
above for recombinant expression vectors.
[0050] In another embodiment, an antisense nucleic acid for use as
an inhibitory agent is a ribozyme. Ribozymes are catalytic RNA
molecules with ribonuclease activity which are capable of cleaving
a single-stranded nucleic acid, such as an mRNA, to which they have
a complementary region (for reviews on ribozymes see e.g., Ohkawa,
J. et al. (1995) J. Biochem. 118:251-258; Sigurdsson, S. T. and
Eckstein, F. (1995) Trends Biotechnol. 13:286-289; Rossi, J. J.
(1995) Trends Biotechnol. 13:301-306; Kiehntopf, M. et al. (1995)
J. Mol. Med. 73:65-71). A ribozyme having specificity for a maf
family mRNA can be designed based upon the nucleotide sequence of
the maf family, preferably c-maf. For example, a derivative of a
Tetrahymena L-19 IVS RNA can be constructed in which the base
sequence of the active site is complementary to the base sequence
to be cleaved in a c-maf mRNA. See for example U.S. Pat. Nos.
4,987,071 and 5,116,742, both by Cech et al. Alternatively, c-maf
mRNA can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See for example
Bartel, D. and Szostak, J. W. (1993) Science 261: 1411-1418.
[0051] Another type of inhibitory agent that can be used to inhibit
the expression and/or activity of a Maf protein in a cell is an
intracellular antibody specific for the Maf protein. The use of
intracellular antibodies to inhibit protein function in a cell is
known in the art (see e.g., Carlson, J. R. (1988) Mol. Cell. Biol.
8:2638-2646; Biocca, S. et al. (1990) EMBO J. 9:101-108; Werge, T.
M. et al. (1990) FEBS Letters 274:193-198; Carlson, J. R. (1993)
Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al.
(1993) Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al.
(1994) Bio/Technology 12:396-399; Chen, S-Y. et al. (1994) Human
Gene Therapy 5:595-601; Duan, L et al. (1994) Proc. Natl. Acad.
Sci. USA 91:5075-5079; Chen, S-Y. et al. (1994) Proc. Natl. Acad.
Sci. USA 91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem.
269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res.
Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.
14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad.
Sci. USA 92:3137-3141; PCT Publication No. WO 94/02610 by Marasco
et al.; and PCT Publication No. WO 95/03832 by Duan et al.).
[0052] To inhibit protein activity using an intracellular antibody,
a recombinant expression vector is prepared which encodes the
antibody chains in a form such that, upon introduction of the
vector into a cell, the antibody chains are expressed as a
functional antibody in an intracellular compartment of the cell.
For inhibition of transcription factor activity according to the
inhibitory methods of the invention, preferably an intracellular
antibody that specifically binds the transcription factor is
expressed within the nucleus of the cell. Nuclear expression of an
intracellular antibody can be accomplished by removing from the
antibody light and heavy chain genes those nucleotide sequences
that encode the N-terminal hydrophobic leader sequences and adding
nucleotide sequences encoding a nuclear localization signal at
either the N- or C-terminus of the light and heavy chain genes (see
e.g., Biocca, S. et al. (1990) EMBO J. 9:101-108; Mhashilkar, A. M.
et al. (1995) EMBO J. 14:1542-1551). A preferred nuclear
localization signal to be used for nuclear targeting of the
intracellular antibody chains is the nuclear localization signal of
SV40 Large T antigen (see Biocca, S. et al. (1990) EMBO J.
9:101-108; Mhashilkar, A. M. et al. (1995) EMBO J.
14:1542-1551).
[0053] To prepare an intracellular antibody expression vector,
antibody light and heavy chain cDNAs encoding antibody chains
specific for the target protein of interest, e.g., a Maf family
protein, are isolated, typically from a hybridoma that secretes a
monoclonal antibody specific for the maf protein. Preparation of
antisera against Maf family proteins has been described in the art
(see e.g., Fujiwara, K. T. et al. (1993) Oncogene 8:2371-2380;
Kataoka, K. et al. (1993) J. Virol. 67:2133-2141; Kerppola, T. K.
and Curran, T. (1994) Oncogene 9:675-684; Igarashi, K et al. (1995)
Proc. Natl. Acad. Sci. USA 92:7445-7449). Anti-Maf protein
antibodies can be prepared by immunizing a suitable subject, (e.g.,
rabbit, goat, mouse or other mammal) with a Maf protein immunogen.
An appropriate immunogenic preparation can contain, for examples,
recombinantly expressed Maf protein or a chemically synthesized Maf
peptide. The preparation can further include an adjuvant, such as
Freund's complete or incomplete adjuvant, or similar
immunostimulatory agent. Antibody-producing cells can be obtained
from the subject and used to prepare monoclonal antibodies by
standard techniques, such as the hybridoma technique originally
described by Kohler and Milstein (1975, Nature 256:495-497) (see
also, Brown et al. (1981) J. Immunol 127:539-46; Brown et al.
(1980) J. Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31;
and Yeh et al. (1982) Int. J. Cancer 29:269-75). The technology for
producing monoclonal antibody hybridomas is well known (see
generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension
In Biological Analyses, Plenum Publishing Corp., New York, N.Y.
(1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L.
Gefter et al. (1977) Somatic Cell Genet., 3:231-36). Briefly, an
immortal cell line (typically a myeloma) is fused to lymphocytes
(typically splenocytes) from a mammal immunized with a maf protein
immunogen as described above, and the culture supernatants of the
resulting hybridoma cells are screened to identify a hybridoma
producing a monoclonal antibody that binds specifically to the Maf
protein. Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-Maf protein monoclonal antibody (see,
e.g., G. Galfre et al. (1977) Nature 266:550-52; Gefter et al.
Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited
supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the
ordinary skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines may be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from the American Type Culture Collection (ATCC),
Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are
fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
that specifically binds the maf protein are identified by screening
the hybridoma culture supernatants for such antibodies, e.g., using
a standard ELISA assay.
[0054] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-maf antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with a maf
protein or peptide to thereby isolate immunoglobulin library
members that bind specifically to a Maf protein. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurJaP.TM. Phage Display
Kit, Catalog No. 240612). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening
antibody display library can be found in, for example, Ladner et
al. U.S. Pat. No. 5,223,409; Kang et al. International Publication
No. WO 92/18619; Dower et al. International Publication No. WO
91/17271; Winter et al. International Publication WO 92/20791;
Markland et al. International Publication No. WO 92/15679;
Breitling et al. International Publication WO 93/01288; McCafferty
et al. International Publication No. WO 92/01047; Garrard et al.
International Publication No. WO 92/09690; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; and
McCafferty et al. Nature (1990) 348:552-554.
[0055] Once a monoclonal antibody specific for the Maf protein has
been identified (e.g., either a hybridoma-derived monoclonal
antibody or a recombinant antibody from a combinatorial library),
DNAs encoding the light and heavy chains of the monoclonal antibody
are isolated by standard molecular biology techniques. For
hybridoma derived antibodies, light and heavy chain cDNAs can be
obtained, for example, by PCR amplification or cDNA library
screening. For recombinant antibodies, such as from a phage display
library, cDNA encoding the light and heavy chains can be recovered
from the display package (e.g., phage) isolated during the library
screening process. Nucleotide sequences of antibody light and heavy
chain genes from which PCR primers or cDNA library probes can be
prepared are known in the art. For example, many such sequences are
disclosed in Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242 and in the "Vbase"
human germline sequence database.
[0056] Once obtained, the antibody light and heavy chain sequences
are cloned into a recombinant expression vector using standard
methods. As discussed above, the sequences encoding the hydrophobic
leaders of the light and heavy chains are removed and sequences
encoding a nuclear localization signal (e.g., from SV40 Large T
antigen) are linked in-frame to sequences encoding either the
amino- or carboxy terminus of both the light and heavy chains. The
expression vector can encode an intracellular antibody in one of
several different forms. For example, in one embodiment, the vector
encodes full-length antibody light and heavy chains such that a
full-length antibody is expressed intracellularly. In another
embodiment, the vector encodes a full-length light chain but only
the VH/CH1 region of the heavy chain such that a Fab fragment is
expressed intracellularly. In the most preferred embodiment, the
vector encodes a single chain antibody (scFv) wherein the variable
regions of the light and heavy chains are linked by a flexible
peptide linker (e.g., (Gly.sub.4Ser).sub.3) and expressed as a
single chain molecule. To inhibit transcription factor activity in
a cell, the expression vector encoding the transcription
factor-specific intracellular antibody is introduced into the cell
by standard transfection methods, as discussed hereinbefore.
[0057] Yet another form of an inhibitory agent of the invention is
an inhibitory form of a Maf protein, also referred to herein as a
dominant negative inhibitor. The maf family of proteins are known
to homodimerize and to heterodimerize with other AP-1 family
members, such as Fos and Jun (see e.g., Kerppola, T. K. and Curran,
T. (1994) Oncogene 9:675-684; Kataoka, K. et al. (1994) Mol. Cell.
Biol. 14:700-712). One means to inhibit the activity of
transcription factors that form dimers is through the use of a
dominant negative inhibitor that has the ability to dimerize with
functional transcription factors but that lacks the ability to
activate transcription (see e.g., Petrak, D. et al. (1994) J.
Immunol. 153:2046-2051). By dimerizing with functional
transcription factors, such dominant negative inhibitors can
inhibit their functional activity. This process may occur naturally
as a means to regulate gene expression. For example, there are a
number of "small" maf proteins, such as mafK, mafF, mafG and p18,
which lack the amino terminal two thirds of c-Maf that contains the
transactivating domain (Fujiwara, K. T. et al. (1993) Oncogene
8:2371-2380; Igarashi, K. et al. (1995) J. Biol. Chem.
270:7615-7624; Andrews, N. C. et al. (1993) Proc. Natl. Acad. Sci.
USA 90:11488-11492; Kataoka, K. et al. (1995) Mol. Cell. Biol.
15:2180-2190). Homodimers of the small maf proteins act as negative
regulators of transcription (Igarashi, K. et al. (1994) Nature
367:568-572) and three of the small maf proteins (MafK, MafF and
MafG) have been shown to competitively inhibit transactivation
mediated by the v-Maf oncoprotein (Kataoka, K. et al. (1996)
Oncogene 12:53-62). Additionally, MafB has been identified as an
interaction partner of Ets-1 and shown to inhibit Ets-1-mediated
transactivation of the transferrin receptor and to inhibit
erythroid differentation (Sieweke, M. H. et al. (1996) Cell
85:49-60).
[0058] Accordingly, an inhibitory agent of the invention can be a
form of a Maf protein that has the ability to dimerize with c-Maf
but that lacks the ability to activate transcription. This dominant
negative form of a Maf protein may be, for example, a small Maf
protein (e.g., MafK, MafF, MafG) that naturally lacks a
transactivation domain, MafB or a mutated form of c-Maf in which
the transactivation domain has been removed. Such dominant negative
Maf proteins can be expressed in cells using a recombinant
expression vector encoding the Maf protein, which is introduced
into the cell by standard transfection methods. To express a mutant
form of c-Maf lacking a transactivation domain, nucleotide
sequences encoding the amino terminal transactivation domain of
c-Maf are removed from the c-maf cDNA by standard recombinant DNA
techniques. Preferably, at least amino acids 1-122 are removed.
More preferably, at least amino acids 1-187, or amino acids 1-257,
are removed. Nucleotide sequences encoding the basic-leucine zipper
region are maintained. The truncated cDNA is inserted into a
recombinant expression vector, which is then introduced into a cell
to allow for expression of the truncated c-maf, lacking a
transactivation domain, in the cell.
[0059] Yet another type of inhibitory agent that can be used to
inhibit the expression and/or activity of a maf protein in a cell
is chemical compound that inhibits the expression or activity of an
endogenous maf family protein in the cell. Such compounds can be
identified using screening assays that select for compounds that
inhibit the expression or activity of a maf family protein.
Examples of suitable screening assays are described in further
detail in subsection V below.
[0060] As discussed above with regard to stimulatory agents, the
inhibitory methods of the invention can involve the use of one or
more additional inhibitory agents that inhibit the expression or
activity of one or more additional transcription factors that
contributes to regulating the expression of a Th1- or
Th2-associated cytokine gene. For example, in one embodiment, the
inhibitory method of the invention comprises contacting a cell with
a first agent that inhibits the expression or activity a maf family
protein and a second agent that inhibits the expression or activity
of an NF-AT protein. In another embodiment, the inhibitory method
of the invention comprises contacting a cell with a first agent
that inhibits the expression or activity a maf family protein and a
second agent that inhibits the expression or activity of an AP-1
protein. In yet another embodiment, the inhibitory method of the
invention comprises contacting a cell with a first agent that
inhibits the expression or activity a maf family protein, a second
agent that inhibits the expression or activity of an NF-AT protein
and a third agent that inhibits the expression or activity of an
AP-1 protein. Examples of types of inhibitory agents for inhibiting
NF-AT or AP-1 proteins include antisense nucleic acids,
intracellular antibodies, dominant negative inhibitors and chemical
compounds that inhibit the endogenous proteins, as described above.
Regarding the latter, it is known in the art that the nuclear
translocation of NF-ATp is inhibited by the immunosuppressive drugs
cyclosporin A and FK506 (see e.g., Rao, A. (1994) Immunol. Today
15:274-281; Rao, A. (1995) J. Leukoc. Biol. 57:536-542).
Accordingly, in one embodiment of the inhibitory method, an
immunosuppressive drug such as cyclosporin A or FK506 (or other
related drug that inhibits the calcineurin pathway) is used in
combination with an agent that inhibits the expression or activity
of c-Maf.
[0061] The method of the invention for modulating production of
Th2-associated cytokines by a cell can be practiced either in vitro
or in vivo (the latter is discussed further in the following
subsection). For practicing the method in vitro, cells can be
obtained from a subject by standard methods and incubated (i.e.,
cultured) in vitro with a stimulatory or inhibitory agent of the
invention to stimulate or inhibit, respectively, the production of
Th2-associated cytokines. For example, peripheral blood mononuclear
cells (PBMCs) can be obtained from a subject and isolated by
density gradient centrifugation, e.g., with Ficoll/Hypaque.
Specific cell populations can be depleted or enriched using
standard methods. For example, monocytes/macrophages can be
isolated by adherence on plastic. T cells or B cells can be
enriched or depleted, for example, by positive and/or negative
selection using antibodies to T cell or B cell surface markers, for
example by incubating cells with a specific primary monoclonal
antibody (mAb), followed by isolation of cells that bind the mAb
using magnetic beads coated with a secondary antibody that binds
the primary mAb. Peripheral blood or bone marrow derived
hematopoietic stem cells can be isolated by similar techniques
using stem cell-specific mAbs (e.g., anti-CD34 mAbs). Specific cell
populations can also be isolated by fluoresence activated cell
sorting according to standard methods. Monoclonal antibodies to
cell-specific surface markers known in the art and many are
commercially available.
[0062] When a stimulatory agent is used in vitro, resulting in
stimulation of the production of Th2-associated cytokines, in
particular IL-4, the cytokine(s) can be recovered from the culture
supernatant for further use. For example, the culture supernatant,
or a purified fraction thereof, can be applied to T cells in
culture to influence the development of Th1 or Th2 cells in vitro.
Alternatively, the culture supernatant, or a purified fraction
thereof, can be administered to a subject to influence the
development of Th1 vs. Th2 responses in the subject.
[0063] Moreover, cells treated in vitro with either a stimulatory
or inhibitory agent can be administered to a subject to influence
the development of a Th1 vs. Th2 response in the subject.
Accordingly, in another embodiment, the method of the invention for
modulating the production of Th2-associated cytokines by a cell
further comprises administering the cell to a subject to thereby
modulate development of Th1 or Th2 cells in a subject. Preferred
cell types for ex vivo modification and readministration include T
cells, B cells and hematopoietic stem cells. For administration to
a subject, it may be preferable to first remove residual agents in
the culture from the cells before administering them to the
subject. This can be done for example by a Ficoll/Hypaque gradient
centrifugation of the cells. For further discussion of ex vivo
genetic modification of cells followed by readministration to a
subject, see also U.S. Pat. No. 5,399,346 by W. F. Anderson et
al.
II. Methods for Modulating Development of Th1 or Th2 Cells in a
Subject
[0064] Another aspect of the invention pertains to a method for
modulating development of Th1 or Th2 cells in a subject. The term
"subject" is intended to include living organisms in which an
immune response can be elicited. Preferred subjects are mammals.
Examples of subjects include humans, monkeys, dogs, cats, mice,
rats, cows, horses, goats and sheep. As discussed above, one way to
modulate Th1/Th2 ratios in a subject is to treat cells (e.g., T
cells, B cells or hematopoietic stem cells) ex vivo with one or
more modulatory agents of the invention, such that production of a
Th2-associated cytokine by the cells is modulated, followed by
administration of the cells to the subject. In another embodiment,
Th1/Th2 ratios are modulated in a subject by administering to the
subject an agent that modulates the activity of a transcription
factor that regulates expression of a Th2-associated cytokine gene
such that development of Th1 or Th2 cells in the subject is
modulated. Preferably, the transcription factor is a maf family
protein and most preferably a c-Maf protein. Preferably, the
Th2-associated cytokine is IL-4. Development of a Th2 response in
the subject can be promoted by administration of one or more
stimulatory agents of the invention, whereas development of a Th1
response in the subject can be promoted by administration of one or
more inhibitory agents of the invention. As discussed above, in
certain situations it may be desirable, in addition to modulating
the activity of a maf family protein, to also modulate the activity
of other transcription factors involved in regulating Th1- or
Th2-associated cytokine genes. Preferably, the activity of an NF-AT
protein(s) is also modulated. Additionally or alternatively, the
activity of an AP-1 protein(s) is also modulated.
[0065] For stimulatory or inhibitory agents that comprise nucleic
acids (including recombinant expression vectors encoding
transcription factors, antisense RNA, intracellular antibodies or
dominant negative inhibitors), the agents can be introduced into
cells of the subject using methods known in the art for introducing
nucleic acid (e.g., DNA) into cells in vivo. Examples of such
methods include:
[0066] Direct Injection: Naked DNA can be introduced into cells in
vivo by directly injecting the DNA into the cells (see e.g., Acsadi
et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science
247:1465-1468). For example, a delivery apparatus (e.g., a "gene
gun") for injecting DNA into cells in vivo can be used. Such an
apparatus is commercially available (e.g., from BioRad).
[0067] Receptor-Mediated DNA Uptake: Naked DNA can also be
introduced into cells in vivo by complexing the DNA to a cation,
such as polylysine, which is coupled to a ligand for a cell-surface
receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol.
Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967;
and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to
the receptor facilitates uptake of the DNA by receptor-mediated
endocytosis. A DNA-ligand complex linked to adenovirus capsids
which naturally disrupt endosomes, thereby releasing material into
the cytoplasm can be used to avoid degradation of the complex by
intracellular lysosomes (see for example Curiel et al. (1991) Proc.
Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.
Acad. Sci. USA 90:2122-2126).
[0068] Retroviruses: Defective retroviruses are well characterized
for use in gene transfer for gene therapy purposes (for a review
see Miller, A. D. (1990) Blood 76:271). A recombinant retrovirus
can be constructed having a nucleotide sequences of interest
incorporated into the retroviral genome. Additionally, portions of
the retroviral genome can be removed to render the retrovirus
replication defective. The replication defective retrovirus is then
packaged into virions which can be used to infect a target cell
through the use of a helper virus by standard techniques. Protocols
for producing recombinant retroviruses and for infecting cells in
vitro or in vivo with such viruses can be found in Current
Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene
Publishing Associates, (1989), Sections 9.10-9.14 and other
standard laboratory manuals. Examples of suitable retroviruses
include pLJ, pZIP, pWE and pEM which are well known to those
skilled in the art. Examples of suitable packaging virus lines
include .psi.Crip, .psi.Cre, .psi..psi.2 and .psi.Am. Retroviruses
have been used to introduce a variety of genes into many different
cell types, including epithelial cells, endothelial cells,
lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro
and/or in vivo (see for example Eglitis, et al. (1985) Science
230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA
85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA
85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA
87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA
88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA
88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van
Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644;
Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992)
Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J.
Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.
4,980,286; PCT Application WO 89/07136; PCT Application WO
89/02468; PCT Application WO 89/05345; and PCT Application WO
92/07573). Retroviral vectors require target cell division in order
for the retroviral genome (and foreign nucleic acid inserted into
it) to be integrated into the host genome to stably introduce
nucleic acid into the cell. Thus, it may be necessary to stimulate
replication of the target cell.
[0069] Adenoviruses: The genome of an adenovirus can be manipulated
such that it encodes and expresses a gene product of interest but
is inactivated in terms of its ability to replicate in a normal
lytic viral life cycle. See for example Berkner et al. (1988)
BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434;
and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral
vectors derived from the adenovirus strain Ad type 5 d1324 or other
strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to
those skilled in the art. Recombinant adenoviruses are advantageous
in that they do not require dividing cells to be effective gene
delivery vehicles and can be used to infect a wide variety of cell
types, including airway epithelium (Rosenfeld et al. (1992) cited
supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl.
Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)
Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin
et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).
Additionally, introduced adenoviral DNA (and foreign DNA contained
therein) is not integrated into the genome of a host cell but
remains episomal, thereby avoiding potential problems that can
occur as a result of insertional mutagenesis in situations where
introduced DNA becomes integrated into the host genome (e.g.,
retroviral DNA). Moreover, the carrying capacity of the adenoviral
genome for foreign DNA is large (up to 8 kilobases) relative to
other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand
and Graham (1986) J. Virol. 57:267). Most replication-defective
adenoviral vectors currently in use are deleted for all or parts of
the viral E1 and E3 genes but retain as much as 80% of the
adenoviral genetic material.
[0070] Adeno-Associated Viruses: Adeno-associated virus (AAV) is a
naturally occurring defective virus that requires another virus,
such as an adenovirus or a herpes virus, as a helper virus for
efficient replication and a productive life cycle. (For a review
see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)
158:97-129). It is also one of the few viruses that may integrate
its DNA into non-dividing cells, and exhibits a high frequency of
stable integration (see for example Flotte et al. (1992) Am. J.
Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J.
Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol.
62:1963-1973). Vectors containing as little as 300 base pairs of
AAV can be packaged and can integrate. Space for exogenous DNA is
limited to about 4.5 kb. An AAV vector such as that described in
Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to
introduce DNA into cells. A variety of nucleic acids have been
introduced into different cell types using AAV vectors (see for
example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA
81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;
Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.
(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol.
Chem. 268:3781-3790).
[0071] The efficacy of a particular expression vector system and
method of introducing nucleic acid into a cell can be assessed by
standard approaches routinely used in the art. For example, DNA
introduced into a cell can be detected by a filter hybridization
technique (e.g., Southern blotting) and RNA produced by
transcription of introduced DNA can be detected, for example, by
Northern blotting, RNase protection or reverse
transcriptase-polymerase chain reaction (RT-PCR). The gene product
can be detected by an appropriate assay, for example by
immunological detection of a produced protein, such as with a
specific antibody, or by a functional assay to detect a functional
activity of the gene product, such as an enzymatic assay.
[0072] A modulatory agent, such as a chemical compound that
stimulates or inhibits endogenous c-Maf activity, can be
administered to a subject as a pharmaceutical composition. Such
compositions typically comprise the modulatory agent and a
pharmaceutically acceptable carrier. As used herein the term
"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 is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0073] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. For
example, 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.
[0074] 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 polyetheylene 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 manitol, 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.
[0075] Sterile injectable solutions can be prepared by
incorporating the active compound 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.
[0076] 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, or corn starch; a lubricant such as
magnesium stearate or Sterotes; 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.
[0077] 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 may be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0078] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of individuals.
III. Applications of the Methods of the Invention
[0079] Identification of the transcription factor that controls the
production of IL-4, and hence continued formation of Th2 cells,
allows for selective manipulation of T cell subsets in a variety of
clinical situations using the modulatory methods of the invention.
The stimulatory methods of the invention (i.e., methods that use a
stimulatory agent) result in production of Th2-associated
cytokines, with concomitant promotion of a Th2 response and
downregulation of a Th1 response. In contrast, the inhibitory
methods of the invention (i.e., methods that use an inhibitory
agent) inhibit the production of Th2-associated cytokines, with
concomitant downregulation of a Th2 response and promotion of a Th1
response. Thus, to treat a disease condition wherein a Th2 response
is beneficial, a stimulatory method of the invention is selected
such that Th2 responses are promoted while downregulating Th1
responses. Alternatively, to treat a disease condition wherein a
Th1 response is beneficial, an inhibitory method of the invention
is selected such that Th2 responses are down-regulated while
promoting Th1 responses. Application of the methods of the
invention to the treatment of disease conditions may result in cure
of the condition, a decrease in the type or number of symptoms
associated with the condition, either in the long term or short
term (i.e., amelioration of the condition) or simply a transient
beneficial effect to the subject.
[0080] Numerous disease conditions associated with a predominant
Th1 or Th2-type response have been identified and could benefit
from modulation of the type of response mounted in the individual
suffering from the disease condition. Application of the
immunomodulatory methods of the invention to such diseases is
described in further detail below.
[0081] A. Allergies
[0082] Allergies are mediated through IgE antibodies whose
production is regulated by the activity of Th2 cells and the
cytokines produced thereby. In allergic reactions, IL-4 is produced
by Th2 cells, which further stimulates production of IgE antibodies
and activation of cells that mediate allergic reactions, i.e., mast
cells and basophils. IL-4 also plays an important role in
eosinophil mediated inflammatory reactions. Accordingly, the
inhibitory methods of the invention can be used to inhibit the
production of Th2-associated cytokines, and in particular IL-4, in
allergic patients as a means to downregulate production of
pathogenic IgE antibodies. An inhibitory agent may be directly
administered to the subject or cells (e.g., Thp cells or Th2 cells)
may be obtained from the subject, contacted with an inhibitory
agent ex vivo, and readministered to the subject. Moreover, in
certain situations it may be beneficial to coadminister to the
subject the allergen together with the inhibitory agent or cells
treated with the inhibitory agent to inhibit (e.g., desensitize)
the allergen-specific response. The treatment may be further
enhanced by administering other Th1-promoting agents, such as the
cytokine IL-12 or antibodies to Th2-associated cytokines (e.g.,
anti-IL-4 antibodies), to the allergic subject in amounts
sufficient to further stimulate a Th1-type response.
[0083] B. Cancer
[0084] The expression of Th2-promoting cytokines has been reported
to be elevated in cancer patients (see e.g., Yamamura, M., et al.
(1993) J. Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc.
Natl. Acad. Sci. USA 89:7708-7712) and malignant disease is often
associated with a shift from Th1 type responses to Th2 type
responses along with a worsening of the course of the disease.
Accordingly, the inhibitory methods of the invention can be used to
inhibit the production of Th2-associated cytokines in cancer
patients, as a means to counteract the Th1 to Th2 shift and thereby
promote an ongoing Th1 response in the patients to ameliorate the
course of the disease. The inhibitory method can involve either
direct administration of an inhibitory agent to a subject with
cancer or ex vivo treatment of cells obtained from the subject
(e.g., Thp or Th2 cells) with an inhibitory agent followed by
readministration of the cells to the subject. The treatment may be
further enhanced by administering other Th1-promoting agents, such
as the cytokine IL-12 or antibodies to Th2-associated cytokines
(e.g., anti-IL-4 antibodies), to the recipient in amounts
sufficient to further stimulate a Th1-type response.
[0085] C. Infectious Diseases
[0086] The expression of Th2-promoting cytokines also has been
reported to increase during a variety of infectious diseases,
including HIV infection, tuberculosis, leishmaniasis,
schistosomiasis, filarial nematode infection and intestinal
nematode infection (see e.g.; Shearer, G. M. and Clerici, M. (1992)
Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer, G. M. (1993)
Immunology Today 14:107-111; Fauci, A. S. (1988) Science
239:617-623; Locksley, R. M. and Scott, P. (1992)
Immunoparasitology Today 1:A58-A61; Pearce, E. J., et al. (1991) J.
Exp. Med. 173:159-166; Grzych, J-M., et al. (1991) J. Immunol.
141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol.
148:3264-3270; Bancroft, A. J., et al. (1993) J. Immunol.
150:1395-1402; Pearlman, E., et al. (1993) Infect. Immun.
61:1105-1112; Else, K. J., et al. (1994) J. Exp. Med. 179:347-351)
and such infectious diseases are also associated with a Th1 to Th2
shift in the immune response. Accordingly, the inhibitory methods
of the invention can be used to inhibit the production of
Th2-associated cytokines in subjects with infectious diseases, as a
means to counteract the Th1 to Th2 shift and thereby promote an
ongoing Th1 response in the patients to ameliorate the course of
the infection. The inhibitory method can involve either direct
administration of an inhibitory agent to a subject with an
infectious disease or ex vivo treatment of cells obtained from the
subject (e.g., Thp or Th2 cells) with an inhibitory agent followed
by readministration of the cells to the subject. The treatment may
be further enhanced by administering other Th1-promoting agents,
such as the cytokine IL-12 or antibodies to Th2-associated
cytokines (e.g., anti-IL-4 antibodies), to the recipient in amounts
sufficient to further stimulate a Th1-type response.
[0087] D. Autoimmune Diseases
[0088] The stimulatory methods of the invention can be used
therapeutically in the treatment of autoimmune diseases that are
associated with a Th2-type dysfunction. Many autoimmune disorders
are the result of inappropriate activation of T cells that are
reactive against self tissue and that promote the production of
cytokines and autoantibodies involved in the pathology of the
diseases. Modulation of T helper-type responses can have an effect
on the course of the autoimmune disease. For example, in
experimental allergic encephalomyelitis (EAE), stimulation of a
Th2-type response by administration of IL-4 at the time of the
induction of the disease diminishes the intensity of the autoimmune
disease (Paul, W. E., et al. (1994) Cell 76:241-251). Furthermore,
recovery of the animals from the disease has been shown to be
associated with an increase in a Th2-type response as evidenced by
an increase of Th2-specific cytokines (Koury, S. J., et al. (1992)
J. Exp. Med. 176:1355-1364). Moreover, T cells that can suppress
EAE secrete Th2-specific cytokines (Chen, C., et al. (1994)
Immunity 1:147-154). Since stimulation of a Th2-type response in
EAE has a protective effect against the disease, stimulation of a
Th2 response in subjects with multiple sclerosis (for which EAE is
a model) is likely to be beneficial therapeutically.
[0089] Similarly, stimulation of a Th2-type response in type I
diabetes in mice provides a protective effect against the disease.
Indeed, treatment of NOD mice with IL-4 (which promotes a Th2
response) prevents or delays onset of type I diabetes that normally
develops in these mice (Rapoport, M. J., et al. (1993) J. Exp. Med.
178:87-99). Thus, stimulation of a Th2 response in a subject
suffering from or susceptible to diabetes may ameliorate the
effects of the disease or inhibit the onset of the disease.
[0090] Yet another autoimmune disease in which stimulation of a
Th2-type response may be beneficial is rheumatoid arthritis (RA).
Studies have shown that patients with rheumatoid arthritis have
predominantly Th1 cells in synovial tissue (Simon, A. K., et al.,
(1994) Proc. Natl. Acad. Sci. USA 91:8562-8566). By stimulating a
Th2 response in a subject with RA, the detrimental Th1 response can
be concomitantly downmodulated to thereby ameliorate the effects of
the disease.
[0091] Accordingly, the stimulatory methods of the invention can be
used to stimulate production of Th2-associated cytokines in
subjects suffering from, or susceptible to, an autoimmune disease
in which a Th2-type response is beneficial to the course of the
disease. The stimulatory method can involve either direct
administration of a stimulatory agent to the subject or ex vivo
treatment of cells obtained from the subject (e.g., Thp, Th1 cells,
B cells, non-lymphoid cells) with a stimulatory agent followed by
readministration of the cells to the subject. The treatment may be
further enhanced by administering other Th2-promoting agents, such
as IL-4 itself or antibodies to Th1-associated cytokines, to the
subject in amounts sufficient to further stimulate a Th2-type
response.
[0092] In contrast to the autoimmune diseases described above in
which a Th2 response is desirable, other autoimmune diseases may be
ameliorated by a Th1-type response. Such diseases can be treated
using an inhibitory agent of the invention (as described above for
cancer and infectious diseases). The treatment may be further
enhanced by administrating a Th1-promoting cytokine (e.g.,
IFN-.gamma.) to the subject in amounts sufficient to further
stimulate a Th1-type response.
[0093] The efficacy of agents for treating autoimmune diseases can
be tested in the above described animal models of human diseases
(e.g., EAE as a model of multiple sclerosis and the NOD mice as a
model for diabetes) or other well characterized animal models of
human autoimmune diseases. Such animal models include the
mrl/lpr/lpr mouse as a model for lupus erythematosus, murine
collagen-induced arthritis as a model for rheumatoid arthritis, and
murine experimental myasthenia gravis (see Paul ed., Fundamental
Immunology, Raven Press, New York, 1989, pp. 840-856). A modulatory
(i.e., stimulatory or inhibitory) agent of the invention is
administered to test animals and the course of the disease in the
test animals is then monitored by the standard methods for the
particular model being used. Effectiveness of the modulatory agent
is evidenced by amelioration of the disease condition in animals
treated with the agent as compared to untreated animals (or animals
treated with a control agent).
[0094] Non-limiting examples of autoimmune diseases and disorders
having an autoimmune component that may be treated according to the
invention include diabetes mellitus, arthritis (including
rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), psoriasis, Sjogren's Syndrome, including
keratoconjunctivitis sicca secondary to Sjogren's Syndrome,
alopecia greata, allergic responses due to arthropod bite
reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis
posterior, and interstitial lung fibrosis.
[0095] E. Transplantation
[0096] While graft rejection or graft acceptance may not be
attributable exclusively to the action of a particular T cell
subset (i.e., Th1 or Th2 cells) in the graft recipient (for a
discussion see Dallman, M. J. (1995) Curr. Opin. Immunol.
7:632-638), numerous studies have implicated a predominant Th2
response in prolonged graft survival or a predominant Th2 response
in graft rejection. For example, graft acceptance has been
associated with production of a Th2 cytokine pattern and/or graft
rejection has been associated with production of a Th1 cytokine
pattern (see e.g., Takeuchi, T. et al. (1992) Transplantation
53:1281-1291; Tzakis, A. G. et al. (1994) J. Pediatr. Surg.
29:754-756; Thai, N. L. et al. (1995) Transplantation 59:274-281).
Additionally, adoptive transfer of cells having a Th2 cytokine
phenotype prolongs skin graft survival (Maeda, H. et al. (1994)
Int. Immunol. 6:855-862) and reduces graft-versus-host disease
(Fowler, D. H. et al. (1994) Blood 84:3540-3549; Fowler, D. H. et
al. (1994) Prog. Clin. Biol. Res. 389:533-540). Still further,
administration of IL-4, which promotes Th2 differentiation,
prolongs cardiac allograft survival (Levy, A. E. and Alexander, J.
W. (1995) Transplantation 60:405-406), whereas administration of
IL-12 in combination with anti-IL-10 antibodies, which promotes Th1
differentiation, enhances skin allograft rejection (Gorczynski, R.
M. et al. (1995) Transplantation 60:1337-1341).
[0097] Accordingly, the stimulatory methods of the invention can be
used to stimulate production of Th2-associated cytokines in
transplant recipients to prolong survival of the graft. The
stimulatory methods can be used both in solid organ transplantation
and in bone marrow transplantation (e.g., to inhibit
graft-versus-host disease). The stimulatory method can involve
either direct administration of a stimulatory agent to the
transplant recipient or ex vivo treatment of cells obtained from
the subject (e.g., Thp, Th1 cells, B cells, non-lymphoid cells)
with a stimulatory agent followed by readministration of the cells
to the subject. The treatment may be further enhanced by
administering other Th2-promoting agents, such as IL-4 itself or
antibodies to Th1-associated cytokines, to the recipient in amounts
sufficient to further stimulate a Th2-type response.
[0098] In addition to the foregoing disease situations, the
modulatory methods of the invention also are useful for other
purposes. For example, the stimulatory methods of the invention
(i.e., methods using a stimulatory agent) can be used to stimulate
production of Th2-promoting cytokines (e.g., IL-4) in vitro for
commercial production of these cytokines (e.g., cells can be
contacted with the stimulatory agent in vitro to stimulate IL-4
production and the IL-4 can be recovered from the culture
supernatant, further purified if necessary, and packaged for
commercial use).
[0099] Furthermore, the modulatory methods of the invention can be
applied to vaccinations to promote either a Th1 or a Th2 response
to an antigen of interest in a subject. That is, the agents of the
invention can serve as adjuvants to direct an immune response to a
vaccine either to a Th1 response or a Th2 response. For example, to
stimulate an antibody response to an antigen of interest (i.e., for
vaccination purposes), the antigen and a stimulatory agent of the
invention can be coadministered to a subject to promote a Th2
response to the antigen in the subject, since Th2 responses provide
efficient B cell help and promote IgG1 production. Alternatively,
to promote a cellular immune response to an antigen of interest,
the antigen and an inhibitory agent of the invention can be
coadministered to a subject to promote a Th1 response to the
antigen in a subject, since Th1 responses favor the development of
cell-mediated immune responses (e.g., delayed hypersensitivity
responses). The antigen of interest and the modulatory agent can be
formulated together into a single pharmaceutical composition or in
separate compositions. In a preferred embodiment, the antigen of
interest and the modulatory agent are administered simultaneously
to the subject. Alternatively, in certain situations it may be
desirable to administer the antigen first and then the modulatory
agent or vice versa (for example, in the case of an antigen that
naturally evokes a Th1 response, it may be beneficial to first
administer the antigen alone to stimulate a Th1 response and then
administer a stimulatory agent, alone or together with a boost of
antigen, to shift the immune response to a Th2 response).
IV. Compositions for Modulating Th2-Associated Cytokine
Production
[0100] Another aspect of the invention pertains to compositions
that can be used to modulate Th2-associated cytokine production by
a cell or Th1/Th2 development in a subject in accordance with the
methods of the invention. The invention provides recombinant
expression vectors comprising a nucleotide sequence encoding a maf
family protein operatively linked to regulatory sequences that
direct expression of the maf family protein specifically in certain
cell types. In a preferred embodiment, the regulatory sequences
direct expression of the maf family protein specifically in
lymphoid cells (e.g., T cells or B cells). In one embodiment, the
lymphoid cells are T cells. T cell specific regulatory elements are
known in the art, such as the promoter regulatory region of T cell
receptor genes (see e.g., Winoto and Baltimore (1989) EMBO J.
8:729-733; Leiden, J. M. (1994) Annu. Rev. Immunol. 11:539-570;
Hettman, T. and Cohen, A. (1994) Mol. Immunol. 31:315-322; Redondo,
J. M. et al. (1991) Mol. Cell. Biol. 11:5671-5680). Other examples
of T cell specific regulatory elements are those derived from the
CD3 gene (see e.g., Clevers, H. et al. (1988) Proc. Natl. Acad.
Sci. USA 85:8623-8627; Clevers, H. C. et al. (1988) Proc. Natl.
Acad. Sci. USA 85:8156-8160; Georgopoulos, K. et al. (1988) EMBO J.
7:2401-2407), the CD4 gene (see e.g., Sawada, S. and Littman, D. R.
(1991) Mol. Cell. Biol. 11:5506-5515; Salmon, P. et al. (1993)
Proc. Natl. Acad. Sci. USA 90:7739-7743; Hanna, Z. et al. (1994)
Mol. Cell. Biol. 14:1084-1094; see also GenBank accession numbers
U01066 and S68043 for human CD4 promoter sequences) and the CD2
gene (see e.g., Zhumabekov, T. et al. (1995) J. Immunol. Methods
185:133-140). A DNA fragment comprising one or more T cell specific
regulatory elements, such as a promoter and enhancer of a T cell
receptor gene, can be obtained by standard molecular biology
methods, such as by PCR using oligonucleotide primers corresponding
to the 5' and 3' ends of the desired region and genomic DNA from T
cells as the template. Once the DNA fragment comprising T cell
specific regulatory elements is obtained, it can be operatively
linked to a cDNA encoding a maf protein (e.g., the two DNA
fragments can be ligated together such that the regulatory elements
are located 5' of the maf sequences) and introduced into vector,
such as a plasmid vector, using standard molecular biology
techniques.
[0101] In another embodiment, the lymphoid cells are B cells (i.e.,
within the recombinant expression vector the nucleotide sequences
encoding a maf family protein are operatively linked to regulatory
sequences that direct expression of the maf family specifically in
B cells). B cell specific regulatory elements are known in the art,
such as the promoter regulatory region of immunoglobulin genes (see
e.g., Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore
(1983) Cell 33:741-748). Other examples of B cell specific
regulatory elements are those derived from the CD20 (B1) gene (see
e.g., Thevenin, C. et al. (1993) J. Biol. Chem. 268:5949-5956;
Rieckmann, P. et al. (1991) J. Immunol. 147:3994-3999), the Fc
epsilon RIIa gene (see e.g., Suter, U. et al. (1989) J. Immunol.
143:3087-3092) and major histocompatibility class II genes (see
e.g, Glimcher, L. H. and Kara, C. J. (1992) Annu. Rev. Immunol.
10:13-49; Benoist, C. and Mathis, D. (1990) Annu. Rev. Immunol.
8:681-715). A DNA fragment comprising B cell specific regulatory
elements, such as a promoter and enhancer of an immunoglobulin
gene, can be obtained by standard molecular biology methods, such
as by PCR using oligonucleotide primers corresponding to the 5' and
3' ends of the desired region and genomic DNA from B cells as the
template. Once the DNA fragment comprising B cell specific
regulatory elements is obtained, it can be operatively linked to a
cDNA encoding a maf protein (e.g. the two DNA fragments can be
ligated together such that the regulatory elements are located 5'
of the maf sequences) and introduced into vector, such as a plasmid
vector, using standard molecular biology techniques.
[0102] In yet another embodiment, the invention provides
recombinant expression vectors comprising a nucleotide sequence
encoding a maf family protein operatively linked to regulatory
sequences that direct expression of the maf family protein
specifically in hematopoietic stem cells. Hematopoietic stem cell
specific regulatory elements are known in the art. Preferably
regulatory elements derived from the CD34 gene are used (see e.g.,
Satterthwaite, A. B. et al. (1992) Genomics 12:788-794; Burn, T. C.
et al. (1992) Blood 80:3051-3059).
[0103] Another aspect of the invention pertains to recombinant host
cells that express a maf family protein. Such host cells can be
used to produce a Th2-associated cytokine (e.g., IL-4). Such host
cells also can be administered to a subject to produce a
Th2-associated cytokine in the subject as a means to manipulate
Th1:Th2 ratios in the subject. The terms "host cell" and
"recombinant host cell" are used interchangeably herein to refer to
a cell into which a recombinant expression vector has been
introduced. It is understood that such terms refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but as long as these
progeny cells retain the recombinant expression vector, these
progeny are still intended to be included within the scope of the
term "host cell" as used herein.
[0104] In one embodiment, the invention provides a host lymphoic
cell into which a recombinant expression vector encoding a maf
family protein has been introduced. The host lymphoid cell can be a
T cell or a B cell. A host T cell of the invention can be, for
example a T cell clone that is cultured in vitro (such as those
described in the Examples) or, alternatively, a normal T cell that
is isolated from a subject (e.g., a peripheral blood T cell or a
splenic T cell). Standard methods for preparing and culturing T
cell clones in vitro, or isolating T cells (e.g., from peripheral
blood) are known in the art, for example through the use of mAbs
that bind T cell specific cell surface markers (e.g., CD3) or
surface markers for specific subsets of T cells (e.g., CD4 or CD8).
The recombinant expression vector can be introduced into the T cell
by one of a variety of known transfection methods for introducing
DNA into mammalian cells, including calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection,
lipofection, or electroporation. Suitable methods for transforming
or transfecting host cells can be found in Sambrook et al.
(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Laboratory press (1989)), and other laboratory manuals.
[0105] In another embodiment, the host lymphoid cell of the
invention is a host B cell into which a recombinant expression
vector encoding a maf family protein has been introduced. The B
cell can be, for example a B lymphoma cell that is cultured in
vitro (such as M12 cells as described in the Examples) or,
alternatively, a normal B cell that is isolated from a subject
(e.g., a peripheral blood B cell or a splenic B cell). Various B
lymphoma cell lines are available in the art and standard methods
for culturing such cells in vitro are known. Additionally, standard
methods for isolating normal B cells (e.g., from peripheral blood)
are known in the art, for example through the use of mAbs that bind
B cell specific cell surface markers (e.g., membrane
immunoglobulin, B7-1, CD20). The recombinant expression vector can
be introduced into the B cell by standard methods, as described
above for T cells.
[0106] In yet another embodiment, the invention provides a host
hematopoietic stem cell into which a recombinant expression vector
encoding a maf family protein has been introduced. Hematopoietic
stem cells can be isolated from a subject (e.g., from peripheral
blood or bone marrow of the subject) using standard methods known
in the art for isolating such stem cells, for example through the
use of mAbs that bind hematopoietic stem cell specific cell surface
markers, preferably CD34 (for further descriptions of isolation of
stem cells, see e.g., Wagner, J. E. et al. (1995) Blood 86:512-523;
Murray, L. et al. (1995) Blood 85:368-378; Bernardi, A. C. et al.
(1995) Science 267:104-108; Bernstein, I. D. et al. (1994) Blood
Cells 20:15-24; Angelini, A. et al. (1993) Int. J. Artif. Organs 16
Suppl. 5:13-18; Kato, K. and Radburch, A. (1993) Cytometry
14:384-392; Lebkowski, J. S. et al. (1992) Transplantation
53:1011-1019; Lebkowski, J. et al. (1993) J. Hematother.
2:339-342). The recombinant expression vector can be introduced
into the hematopoietic stem cell by standard methods, as described
above for T cells.
V. Screening Assays
[0107] Another aspect of the invention pertains to screening assays
for identifying compounds that modulate the activity of a
transcription factor that regulates expression of a Th2-associated
cytokine gene. In various embodiments, these screening assays can
identify, for example, compounds that modulate the expression or
functional activity of the transcription factor, proteins that
interact with the transcription factor, as well as compounds that
modulate these protein-protein interactions, and compounds that
modulate the interaction of the transcription factor with a MARE
within a Th2-associated cytokine gene.
[0108] In a preferred embodiment, the invention provides a method
comprising:
[0109] a) preparing an indicator cell, wherein said indicator cell
contains: [0110] i) a recombinant expression vector encoding a
transcription factor that regulates expression of a Th2-associated
cytokine gene; and [0111] ii) a vector comprising regulatory
sequences of the Th2-associated cytokine gene operatively linked a
reporter gene;
[0112] b) contacting the indicator cell with a test compound;
[0113] c) determining the level of expression of the reporter gene
in the indicator cell in the presence of the test compound;
[0114] d) comparing the level of expression of the reporter gene in
the indicator cell in the presence of the test compound with the
level of expression of the reporter gene in the indicator cell in
the absence of the test compound; and
[0115] e) identifying a compound that modulates the activity of a
transcription factor that regulates expression of a Th2-associated
cytokine gene.
[0116] Preferably, the transcription factor is a member of the maf
family and most preferably a c-Maf protein. Recombinant expression
vectors that can be used for expression of a c-Maf protein in an
indicator cell are known in the art (see discussions above and also
the Examples). In one embodiment, within the expression vector the
c-Maf coding sequences are operatively linked to regulatory
sequences that allow for constitutive expression of c-Maf in the
indicator cell (e.g., viral regulatory sequences, such as a
cytomegalovirus promoter/enhancer, can be used). Use of a
recombinant expression vector that allows for constitutive
expression of c-Maf in the indicator cell is preferred for
identification of compounds that enhance or inhibit the activity of
c-Maf. In an alternative embodiment, within the expression vector
the c-Maf coding sequences are operatively linked to regulatory
sequences of the c-maf gene (i.e., the promoter regulatory region
derived from the endogenous c-maf gene). Use of a recombinant
expression vector in which c-Maf protein expression is controlled
by c-maf regulatory sequences is preferred for identification of
compounds that enhance or inhibit the transcriptional expression of
c-Maf.
[0117] Preferably, the Th2-associated cytokine is interleukin-4. It
has previously shown that Th2-specific, inducible IL-4 expression
can be directed by as little as 157 bp of the proximal IL-4
promoter in Th2 cells (Hodge, M. et al. (1995) J. Immunol.
154:6397-6405). Accordingly, in one embodiment, the method utilizes
a reporter gene construct containing this region of the proximal
IL-4 promoter, most preferably nucleotides -157 to +58 (relative to
the start site of transcription at +1) of the IL-4 promoter.
Alternatively, stronger reporter gene expression can be achieved
using a longer portion of the IL-4 upstream regulatory region, such
as about 3 kb of upstream regulatory sequences. Suitable reporter
gene constructs are described in Todd, M. et al. (1993) J. Exp.
Med. 177:1663-1674. See also the Examples for descriptions of IL-4
reporter gene constructs.
[0118] A variety of reporter genes are known in the art and are
suitable for use in the screening assays of the invention. Examples
of suitable reporter genes include those which encode
chloramphenicol acetyltransferase, beta-galactosidase, alkaline
phosphatase or luciferase. Standard methods for measuring the
activity of these gene products are known in the art.
[0119] A variety of cell types are suitable for use as an indicator
cell in the screening assay. Preferably a cell line is used which
does not normally express c-Maf, such as a B cell (e.g., the M12 B
lymphoma cell line) or a Th1 cell clone (e.g., AE7 cells).
Nonlymphoid cell lines can also be used as indicator cells, such as
the HepG2 hepatoma cell line.
[0120] In one embodiment, the level of expression of the reporter
gene in the indicator cell in the presence of the test compound is
higher than the level of expression of the reporter gene in the
indicator cell in the absence of the test compound and the test
compound is identified as a compound that stimulates the expression
or activity of the transcription factor. In another embodiment, the
level of expression of the reporter gene in the indicator cell in
the presence of the test compound is lower than the level of
expression of the reporter gene in the indicator cell in the
absence of the test compound and the test compound is identified as
a compound that inhibits the expression or activity of the
transcription factor.
[0121] Alternative to the use of a reporter gene construct,
compounds that modulate the expression or activity of c-Maf can be
identified by using other "read-outs." For example, an indicator
cell can be transfected with a c-Maf expression vector, incubated
in the presence and in the absence of a test compound, and
Th2-associated cytokine production can be assessed by detecting
cytokine mRNA (e.g., IL-4 mRNA) in the indicator cell or cytokine
secretion (i.e., IL-4 secretion) into the culture supernatant.
Standard methods for detecting cytokine mRNA, such as reverse
transcription-polymerase chain reaction (RT-PCR) are known in the
art. Standard methods for detecting cytokine protein in culture
supernatants, such as enzyme linked immunosorbent assays (ELISA)
are also known in the art. For further descriptions of methods for
detecting cytokine mRNA and/or protein, see also the Examples.
[0122] In another embodiment, the invention provides a screening
assay for identifying proteins in Th2 cells that interact with
c-Maf These assays can be designed based on the two-hybrid assay
system (also referred to as an interaction trap assay) known in the
art (see e.g., Field U.S. Pat. No. 5,283,173; Zervos et al. (1993)
Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and
Iwabuchi et al. (1993) Oncogene 8:1693-1696). The two-hybrid assay
is generally used for identifying proteins that interact with a
particular target protein. The assay employs gene fusions to
identify proteins capable of interacting to reconstitute a
functional transcriptional activator. The transcriptional activator
consists of a DNA-binding domain and a transcriptional activation
domain, wherein both domains are required to activate transcription
of genes downstream from a target sequence (such as an upstream
activator sequence (UAS) for GAL4). DNA sequences encoding a target
"bait" protein are fused to either of these domains and a library
of DNA sequences is fused to the other domain. "Fish" fusion
proteins (generated from the fusion library) capable of binding to
the target-fusion protein (e.g., a target GAL4-fusion "bait") will
generally bring the two domains (DNA-binding domain and
transcriptional activation domain) into close enough proximity to
activate the transcription of a reporter gene inserted downstream
from the target sequence. Thus, the "fish" proteins can be
identified by their ability to reconstitute a functional
transcriptional activator (e.g., a functional GAL4
transactivator).
[0123] This general two-hybrid system can be applied to the
identification of proteins in Th2 cells that interact with c-Maf by
construction of a target c-Maf fusion protein (e.g., a c-Maf/GAL4
binding domain fusion as the "bait") and a cDNA library of "fish"
fusion proteins (e.g., a cDNA/GAL4 activation domain library),
wherein the cDNA library is prepared from mRNA of Th2 cells, and
introducing these constructs into a host cell that also contains a
reporter gene construct linked to a regulatory sequence responsive
to c-Maf (e.g., a MARE sequence, for example a region of the IL-4
promoter, as discussed above). cDNAs encoding proteins from Th2
cells that interact with c-Maf can be identified based upon
transactivation of the reporter gene construct. Accordingly, the
invention provides a method for identifying a protein in a Th2 cell
that interacts with c-Maf comprising:
[0124] a) providing a two hybrid assay including a host cell which
contains [0125] i) a reporter gene operably linked to a
transcriptional regulatory sequence; [0126] ii) a first chimeric
gene which encodes a first fusion protein, said first fusion
protein including c-Maf; [0127] iii) a library of second chimeric
genes which encodes second fusion proteins, the second fusion
proteins including proteins derived from Th2 cells;
[0128] wherein expression of the reporter gene is sensitive to
interactions between the first fusion protein, the second fusion
protein and the transcriptional regulatory sequence;
[0129] b) determining the level of expression of the reporter gene
in the host cell; and
[0130] c) identifying a protein in a Th2 cell that interacts with
c-Maf.
[0131] Alternatively, a "single-hybrid" assay, such as that
described in Sieweke, M. H. et al. (1996) Cell 85:49-60, can be
used to identify proteins from Th2 cells that interact with c-Maf.
This assay is a modification of the two-hybrid system discussed
above. In this system, the "bait" is a transcription factor from
which the transactivation domain has been removed (e.g., c-Maf from
which the amino-terminal transactivation domain has been removed)
and the "fish" is a non-fusion cDNA library (e.g., a cDNA library
prepared from Th2 cells). These constructs are introduced into host
cells (e.g., yeast cells) that also contains a reporter gene
construct linked to a regulatory sequence responsive to the
transcription factor (e.g., a MARE sequence, for example a region
of the IL-4 promoter, responsive to c-Maf). cDNAs encoding proteins
from Th2 cells that interact with c-Maf can be identified based
upon transactivation of the reporter gene construct.
[0132] In yet another embodiment, the invention provides a
screening assay for identifying compounds that modulate the
interaction of c-Maf with a MARE in an IL-4 gene regulatory region.
Assays are known in the art that detect the interaction of a DNA
binding protein with a target DNA sequence (e.g., electrophoretic
mobility shift assays, DNAse I footprinting assays and the like;
for further descriptions see the Examples). By performing such
assays in the presence and absence of test compounds, these assays
can be used to identify compounds that modulate (e.g., inhibit or
enhance) the interaction of the DNA binding protein with its target
DNA sequence. Accordingly, the invention provides a method for
identifying a compound that modulates the interaction of a c-Maf
protein with a maf response element (MARE) of an IL-4 gene
regulatory region, comprising:
[0133] a) providing a c-Maf protein and a DNA fragment comprising a
MARE of an IL-4 gene regulatory region;
[0134] b) incubating the c-Maf protein and DNA fragment in the
presence of a test compound;
[0135] c) determining the amount of binding of the c-Maf protein to
the DNA fragment in the presence of the test compound;
[0136] d) comparing the amount of binding of the c-Maf protein to
the DNA fragment in the presence of the test compound with the
amount of binding of the c-Maf protein to the DNA fragment in the
absence of the test compound; and
[0137] e) identifying a compound that modulates the interaction of
a c-Maf protein with a MARE of an IL-4 gene regulatory region.
[0138] In one embodiment, the amount of binding of the c-Maf
protein to the DNA fragment in the presence of the test compound is
greater than the amount of binding of the c-Maf protein to the DNA
fragment in the absence of the test compound, in which case the
test compound is identified as a compound that enhances binding of
c-Maf to the MARE. In another embodiment, the amount of binding of
the c-Maf protein to the DNA fragment in the presence of the test
compound is less than the amount of binding of the c-Maf protein to
the DNA fragment in the absence of the test compound, in which case
the test compound is identified as a compound that inhibits binding
of c-Maf to the MARE.
[0139] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by reference.
Nucleotide and amino acid sequences deposited in public databases
as referred to herein are also hereby incorporated by
reference.
EXAMPLE 1
Cytokine Specificity is Due to a Positive Transacting Factor and
Not to a Repressor
[0140] Tissue specificity can be achieved through the action of
repressor or silencer proteins. Thus it was possible that the IL-2
and IL-4 genes were actively repressed in Th2 and Th1 cells
respectively. To test for the existence of repressor proteins,
somatic cell fusions were performed between a Th1(D1.1) and a Th2
(D10) clone of differing MHC Class I haplotypes. The Th1 clone D1.1
(K.sup.d) and the Th2 clone D10 (K.sup.k) were fused according to
the "suspension cell fusion" procedure (Lane, R. D. et al. (1986)
Methods Enzymol. 121:183-192). After fusion, the cells were allowed
to recover for 8 hours and then double-stained using PE-conjugated
anti-K.sup.k and FITC-conjugated anti-K.sup.d antibodies
(Pharmingen, La Jolla, Calif.). Cells were then sorted on the basis
of size to distinguish unfused cells from hetero and homokaryons
and by fluorescence to identify single-positive and double-positive
cells. As indicated in the schematic of this approach shown in FIG.
1A, three populations were sorted for: large PE-positive cells
(D1.1.times.D1.1), large FITC-positive cells (D10.times.D10), and
large PE and FITC positive cells (D1.1.times.D10). Cells expressing
both MHC class I K.sup.b and K.sup.k markers were heterokaryons
while cells expressing only K.sup.b or K.sup.k represented
homokaryons and served as controls.
[0141] The three populations were then stimulated in culture with
antibodies to CD3 to activate cytokine gene expression and RNA
prepared for RT-PCR and Northern blot analysis. Approximately
5.times.10.sup.5 cells were obtained for each population.
Routinely, 5-10% of the cells had undergone fusion. Each of these
three populations was then split in half, one half transferred to
pre-rinsed anti-CD3 coated plates, the remaining half to uncoated
plates. After four hours, the cells were harvested, and poly(A+)
RNA isolated using the Micro-FastTrack.TM. kit (Stratagene, La
Jolla, Calif.). cDNA was made using the SuperScript kit (Gibco/BRL,
Bethesda, Md.), and used for PCR analysis using commercially
available primers specific for murine IL-2, IL-4 and .beta.-actin
according to the manufacturer's instructions (Stratagene, La Jolla,
Calif.). PCR reactions included 0.5 .mu.Ci .alpha.:.sup.32P-dCTP
(3000 Ci/mmol, NEN Dupont). PCR products were ethanol precipitated,
separated by nondenaturing PAGE and dried and visualized by
autoradiography.
[0142] The results of the RT-PCT analysis of cytokine mRNA
expression are shown in FIG. 1B. The Th1 and Th2 clones and the Th
homokaryons transcribed only IL-2 (Th1) or IL-4 (Th2) respectively,
while the Th1/Th2 heterokaryons produced both cytokines. In
contrast, the existence of repressor protein(s) should have
resulted in the extinction of both cytokines in the heterokaryons.
From these experiments, it was concluded that cytokine specificity
in Th1 vs. Th2 cells was mediated by Th-specific positive
transacting factors rather than by selective silencer proteins.
EXAMPLE 2
Isolation of a Th2-Specific c-maf Gene from a cDNA Library Prepared
from an Anti-CD3 Activated Th2 Clone
[0143] In the course of screening a cDNA library prepared from an
anti-CD3 activated Th2 clone, D10, for NF-AT-interacting proteins
by the yeast two-hybrid system (for descriptions of this system,
see e.g., Field U.S. Pat. No. 5,283,173; Zervos et al. (1993) Cell
72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;
Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al.
(1993) Oncogene 8:1693-1696), multiple cDNAs were isolated, all of
which were extremely weak interactors. All cDNAs obtained in this
screen were next evaluated for Th-specific expression by Northern
blot analysis using a panel of Th1 and Th2 clones. One such cDNA,
which was repeatedly isolated (60 of 140) detected transcripts only
in RNA prepared from Th2 clones (D10, CDC35) and not from either
Th1 clones (AR5, OS6, D1) or from a B cell lymphoma, M12, as
illustrated in the Northern blot analysis depicted in FIG. 2A.
Further, the levels of transcripts detected in D10 Th2 cells were
substantially increased upon activation by ligation of the T cell
receptor with anti-CD3 antibody. No induction of the transcript
detected by this cDNA clone occurred in Th1 clones upon anti-CD3
treatment. A control probe, GAPDH, demonstrated approximately equal
loading of RNA in all lanes. Thus, the expression of this cDNA
clone in the lymphoid lineage appeared to be Th2-specific and
sensitive to signals transmitted through the T cell receptor. For
these Northern blots, total RNA was prepared by using Trizol
(GIBCO/BRL) according to manufacturer's instructions. 10 .mu.g of
total RNA from each sample was fractionated on a formaldehyde
agarose gel and transferred to a nylon membrane. A 300 bp DraI
fragment derived from the 3' untranslated region of the isolated
clone was labeled with .alpha.-.sup.32P-dCTP using Random Primed
DNA Labeling Kit (Boehringer Mannheim, Indianapolis, Ind.).
Hybridization was performed using QuikHyb (Stratagene, La Jolla,
Calif.) according to manufacturer's instructions.
[0144] To determine whether the expression of this gene was
tissue-specific and regulated during the course of normal Th cell
development, the following experiment was performed. Naive spleen
cells (Th precursor (Thp) cells) were driven along a Th1 or Th2
pathway by treatment with anti-CD3 in the presence of cytokines and
anti-cytokine antibodies (IFN.gamma. and anti-IL-4 for Th1, IL-4
and anti-IFNg for Th2). Splenic cell suspensions were prepared from
6-8 week-old Balb/c mice, cultured in RPMI 1640 supplemented with
10% FCS at a density of 10.sup.6 cells/ml, and stimulated with
plate bound anti-CD3 antibody in the presence of 5 .mu.g/ml of
anti-IL4 antibody (11B11) for the Th1 lineage, or 5 .mu.g/ml of
anti-IFN.gamma. antibody (XMG-1) for the Th2 lineage. 24 hours
after stimulation, 50 U/ml IL2 was added to all cultures, and 500
U/ml IL4 (Genzyme) was added to Th2 cultures. 7 days after the
primary stimulation, all cells were harvested, washed and
restimulated with plate bound anti-CD3 antibody. Northern blot
analysis of differentiating cells harvested at various time points
after stimulation in a primary (day 0-8) and secondary (0-20 hours)
response was performed, using the methodology described above, and
identification of differentiating Thp cells as Th1 or Th2 was
determined by analyzing culture supernatants by ELISA for IL-10 and
IFN.gamma.. ELISA for cytokine quantitation was performed as
follows. All anti-cytokine antibodies were purchased from
Pharmingen. ELISA was performed according to Pharmingen's
instructions with the exception that Avidin-Alkaline Phosphatase
(Sigma) at 1:500 dilution in PBS/BSA was used in place of
avidin-peroxidase. P-nitrophenyl phosphate (GIBCO BRL) at 4 mg/ml
in substrate buffer (10% diethanolamine, 0.5 mM MgCl.sub.2, 0.02%
sodium azide, pH 9.8) was used as substrate.
[0145] In two independent experiments, representative results of
which are shown in FIG. 2B, this analysis revealed low level or
undetectable expression of this cDNA in naive spleen cells at
baseline at day 0. In cultures differentiating along a Th2 pathway,
substantial induction of transcripts occurred by day 8 in a primary
stimulation and by 20 hr in a secondary stimulation. In contrast,
no induction occurred in cells being driven along a Th1 pathway. A
control probe (GAPDH) showed approximately equal loading of RNA in
all lanes. The low level of transcripts present in cells being
driven along a Th1 pathway likely reflects the presence of residual
Th2 cells since complete skewing does not occur in this in vitro
differentiation system.
[0146] Together, these experiments revealed that the isolated cDNA
is selectively expressed in Th2 clones, where it is induced upon T
cell activation, and that it is absent from Th1 clones and a B
lymphoma. Further, this gene is induced in normal Thp when they are
driven towards the Th2 lineage, but is not induced during Th1
development.
[0147] The cDNA obtained from the yeast two-hybrid screen was used
as a probe to isolate a full-length cDNA from a D10 Th2 cell cDNA
library by standard hybridization methods. A 4.3 kb cDNA clone was
isolated from the Th2 cell library and sequenced by standard
methods. Sequence analysis revealed that this Th2-specific gene
corresponded in sequence to the c-maf proto-oncogene.
EXAMPLE 3
Ectopic Expression of c-Maf in Th1 and B Cells Results in
Activation of the IL-4 Promoter
[0148] The identification of the isolated cDNA described in Example
2 as a member of the AP-1/CREB/ATF gene family, together with its
selective expression in Th2 cells raised the possibility that c-Maf
controlled the tissue-specific transcription of the IL-4 gene.
Additionally, the presence of transcripts encoding c-maf correlated
well with IL-4 expression in Th2 cells and in three of four
transformed mast cell lines examined. To test whether c-Maf could
transactivate the IL-4 promoter, cotransfection experiments were
performed.
[0149] Th1 clones and the B lymphoma M12.4.C3 (M12) neither express
c-maf nor transcribe the IL-4 gene. If c-Maf is the transcription
factor critical for controlling IL-4 gene expression, then forced
expression in these cells should permit IL-4 gene expression. To
test this, the full-length (4.3 kb) c-maf cDNA clone was inserted
into the SalI site of the pMex-NeoI mammalian expression vector,
which utilizes the CMV enhancer to drive expression of the inserted
sequence. The c-Maf expression vector was then cotransfected with
an IL-4 promoter reporter construct into the Th1 clone AE7 and the
B lymphoma M12. The generation of the wild type IL4 CAT reporter
construct, containing an IL4 promoter fragment from -157 to +68
operatively linked to a chloramphenicol acetyltransferase gene is
described in Hodge, M. et al. (1995) J. Immunol. 154:6397-6405. The
Th1 clone was cultured in RPMI 1640 supplemented with 10% FCS and
10% Con-A stimulated rat splenocyte supernatant, and maintained by
bi-weekly stimulation with appropriate antigen and APCs. M12 cells
were cultured in RPMI 1640 supplemented with 10% FCS.
[0150] The Th1 clone AE7 or M12 B lymphoma cells were transiently
transfected by preincubating 0.4 ml of cells, containing
2.times.10.sup.7 cells/ml AE7 or 3.times.10.sup.6 cells/ml M12
cells in serum-free RPMI 1640 with 20 .mu.g (AE7) or 5 .mu.g (M12)
of each plasmid for 10 minutes at room temperature. The samples
were then electroporated using a BIO-RAD Gene Pulser (BIO-RAD,
Richmond, Calif.) set at 975 .mu.F, 280 V, and immediately placed
on ice for 10 minutes. The transfected cells were allowed to
recover overnight in complete media and stimulated with plate bound
anti-CD3 antibody ({Pharmingen, San Diego, Calif.} 1 .mu.g/ml in
1.times.PBS overnight at 4.degree. C.) or with 50 ng/ml PMA (Sigma,
St. Louis, Mo.) and 1 .mu.M Ionomycin (Calbiochem Corp., La Jolla,
Calif.). for 24 hours. Cell lysate was prepared by freeze-thaw
lysis in 0.25 M Tris-Cl, pH 7.8. Equal amounts of protein (between
5-20 .mu.g) were used for CAT assays. CAT assays were performed as
described in Todd, M. et al. (1993) J. Exp. Med. 177:1663-1674.
[0151] It has previously shown that Th2-specific, inducible IL-4
expression can be directed by as little as 157 bp of the proximal
IL-4 promoter in Th2 cells (Hodge, M. et al. (1995) J. Immunol.
154:6397-6405). In cotransfection experiments, the results of which
are summarized in FIG. 3A, it is demonstrated that ectopic
expression of c-Maf in the Th1 clone AE7 results in substantial
activity of the IL-4 promoter reporter after stimulation through
the T cell receptor. The fold induction observed was approximately
5 fold over that observed with the control empty vector alone.
Although expression of a reporter construct containing proximal
(-157 to +58) IL-4 promoter sequences in the subclone of AE7 cells
utilized here has not been previously observed, it has been
demonstrated that small amounts of IL-4 mRNA can be detected by
RT-PCR in other subclones of AE7. To more rigorously test the
ability of c-Maf to transactivate the IL-4 promoter in a non-IL-4
producing cell, the same experiment was performed in the B lymphoma
cell line, M12. Normal B cells and B lymphoma cells do not produce
IL-4. Representative results of the cotransfection experiments are
depicted in FIG. 3B and a summary of three independent experiments
is shown below in Table 1. TABLE-US-00001 TABLE 1 CAT Activity
(fold induction) Plasmids PMA/iono. Exp. I* Exp. II Exp. III
pMEX-NeoI/pREP4 - 1 1 1 + 7.6 1 1.4 pMEX-Maf/pREP4 - 95 5 18.6 +
186 7 37 pMEX-c-Fos/pREP4 - 2.7 1 0.8 + 7.6 1.2 1 pMEX-JunD/pREP4 -
ND** 0.9 0.5 + ND 1.4 1.9 pMEX-NeoI/pREP4-NFATp - 14.2 1.6 0.3 +
41.2 3.5 0.3 pMEX-Maf/pREP4-NFATp - 136 54 26.3 + 138 100 54.7
pMEX-c-Fos/pREP4-NFATp - 7.4 1.6 3 + 15.4 1.9 6.1 *In experiment I,
20 mg of cell lysate was incubated for 2 hours. In experiments II
and III, only 5 mg of cell lysate was incubated for 1 hour in order
to reveal synergy between c-Maf and NFATp **ND = not done
[0152] The results in M12 B lymphoma cells confirmed the findings
in the Th1 clone. Ectopic expression of c-Maf resulted in
substantial activity of the IL-4 promoter in M12 cells, either
unstimulated or stimulated with PMA/Ca++ ionophore. The fold
induction observed when compared to transfection of a control
vector averaged approximately 50 in unstimulated M12 cells.
Stimulation of M12 cells with PMA/Ca++ ionophore, which should
result in translocation of NF-ATs to the nucleus and induction of
other AP-1 family members (Flanagan, W. M. (1991) Nature
352:803-807; Jain, J. et al. (1993) Nature 365:352-355), increased
the basal activity of the IL-4 promoter, but a marked induction in
promoter activity by c-Maf was still present (average of
approximately 25 fold). C-Maf did not transactivate a control
reporter driven by NF-AT multimers, demonstrating the specificity
of transactivation.
[0153] As a control for the specificity of c-Maf as opposed to
other AP-1 family members, the c-Fos and c-Jun proteins were also
overexpressed in M12 cells utilizing murine full-length cDNAs
encoding c-Fos and JunD in the mammalian expression vector of
pMEX-NeoI together with the IL-4 reporter plasmid. No IL-4 promoter
activity could be achieved by overexpression of either of these two
AP-1 family members in M12 cells. Thus, c-Maf has a unique ability
to drive IL-4 gene transcription in M12 B cells. Further, forced
expression of c-Maf in the hepatoma cell line HepG2 also resulted
in IL-4 promoter transactivation. These experiments demonstrate
that the provision of c-Maf to c-Maf negative Th1 or B cells, or to
non-lymphoid cells (e.g., a hepatoma cell line), permits the cells
to transactivate the IL-4 promoter.
[0154] NF-AT proteins have been shown to be critically important in
the regulation of both the IL-4 and IL-2 cytokines. NF-ATp was the
first member of this family to be isolated (McCaffrey, P. G. et al.
(1993) Science 262:750-754). Both AE7 and M12 cells have endogenous
NF-ATp protein, but nevertheless do not transcribe IL-4. Although
NF-ATp could not therefore account for selective IL-4 gene
transcription, it was of interest to test whether overexpression of
NF-ATp in unstimulated or stimulated M12 cells would further
increase the transactivation of the IL-4 promoter by c-maf. M12
cells were cotransfected with the IL-4 reporter construct and
either an NFAPp expression vector (pREP.sub.4-NF-ATp, which also
carries a hygromycin resistance gene) alone or the NFAPp expression
vector together with the c-Maf expression vector. Overexpression of
NF-ATp alone in M12 cells resulted in some modest transactivation
of the IL-4 promoter. This transactivation was markedly increased
by ectopic expression of c-Maf, an increase which was not just
additive but was synergistic (see FIG. 3B and Table 1). In
contrast, c-Fos overexpression did not further increase the modest
transactivation achieved by NF-ATp. These results indicate that
c-maf and NF-ATp interact to achieve maximal induction of the IL-4
promoter, the tissue-specificity being provided by c-maf.
EXAMPLE 4
Ectopic Expression of C-Maf Activates Transcription of the
Endogenous IL-4 Gene in a B Lymphoma
[0155] As demonstrated in Example 3, c-Maf transactivates the IL-4
promoter in transient transfection assays in Th1, B and
non-lymphoid cells. To test whether expression of c-maf in non-IL-4
producing cells can activate the transcription of endogenous IL-4,
the B lymphoma M12 was stably transfected with expression vectors
encoding c-maf, NF-ATp or both, or junD with and without NF-ATp as
a control. For stable transfection, M12 cells were transfected as
described above in Example 3. The transfected cells were allowed to
recover in complete media for 48 hours before the addition of
Neomycin (GIBCO/IBRL, Gaithersburg, Md.) and Hygromycin
(Calbiochem, Corp.) at a concentration of 400 .mu.g/ml of each
antibiotic. The transfected cells were supplemented with fresh
media every other day.
[0156] Stably transfected M12 cells were plated at equal density
supernatants harvested 24 hours later to measure cytokines by
ELISA. ELISAs were performed as described in Example 2. The
results, shown in FIG. 4, demonstrate that in these experiments M12
cells transfected with c-maf, junD or NF-ATp alone did not produce
measurable IL-4 by ELISA. However, M12 cells stably transfected
with both c-maf and NF-ATp did produce detectable, but low level,
IL-4 by ELISA. These results were confirmed by RT-PCR on RNA from
these transfected cells. In contrast, these cells did not produce
detectable IL-2. The requirement for both c-maf and NF-ATp is
consistent with the synergistic effect of these factors in the
transactivation of the IL-4 promoter noted in the transient
transfection experiments in M12 cells. In contrast, transfection of
junD, an AP-1 family member which can increase IL-4 expression in
Th2 cells, alone or together with NF-ATp, did not result in IL-4
production. These results demonstrate the essential and selective
role of c-maf in directing tissue-specific endogenous IL-4
production.
EXAMPLE 5
A Site in the IL-4 Promoter is Footprinted by Extracts from Th2 but
not Th1 Clones
[0157] The experiments described in Examples 3 and 4 demonstrated a
clear functional role for c-maf in controlling tissue-specific
expression of IL-4. Further, c-maf transcripts were expressed in
Th2 but not Th1 cells. However, DNA-protein complexes were not
detected by electrophoretic mobility shift assays (EMSA) when using
nuclear extracts prepared from Th2 cells. To further examine
whether a protein in Th2 nuclear extracts might bind to the MARE,
or nearby sequences, the more sensitive technique of DNAseI
footprinting was used. Two Th2 clones (D10, CDC35) and two Th1
clones (AE7, S53) were activated by ligation of the T cell receptor
with plate-bound anti-CD3 antibody, and nuclear extracts prepared
at time 0 (unstimulated), 2 hours and 6 hours later. DNAseI
footprinting analysis was then performed according to standard
methods using a Klenow end-labeled IL-4 promoter fragment (-157 to
+68). The results are shown in FIG. 5A. Stimulated extracts from
both Th1 and Th2 cells footprinted the two NF-AT sites and the AP-1
site upstream of the distal NF-AT site as described previously
(Rooney, J. et al. (1995) Immunity 2:545-553), consistent with the
demonstrated function of NF-AT and AP-1 proteins in regulating both
the IL-2 and the IL-4 promoters (Rooney, J. et al. (1995) Immunity
2:545-553; Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310).
Furthermore, inspection of the autoradiograph revealed an area of
hypersensitivity on the non-coding strand at residues -28 and -29
when extracts from stimulated Th2 but not stimulated Th1 cells were
used. Unstimulated Th cell extracts did not footprint this region.
The Th2 footprint observed was subtle, but reproducible in two
experiments and is located in a site that has previously been
demonstrated to be critical for IL-4 promoter activation in Th2
cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). A
schematic summary of sites occupied in the IL-4 promoter as
detected by footprint analysis is shown in FIG. 5B. These results
indicate that a site in the proximal IL-4 promoter, previously
shown to be functionally important, is occupied in activated Th2
but not in activated Th1 cells.
EXAMPLE 6
Recombinant c-maf Binds to a MARE Site in the IL-4 Promoter
[0158] The Th2-specific footprint does not contain a c-maf response
element (MARE). However, examination of the proximal IL-4 promoter
revealed a half c-maf binding site (MARE) (residues -42 to -37)
immediately downstream of the proximal NF-AT site (residues -56 to
-51) (shown schematically in FIG. 5B). It has previously been
demonstrated that mutation of this site abolished activity of the
IL-4 promoter in Th2 cells (Hodge, M. et al. (1995) J. Immunol.
154:6397-6405). To determine if c-Maf bound this site, a truncated
c-Maf recombinant protein containing the b-zip domain (amino acids
171-371) was expressed from E. coli, purified on an S-Tag agarose
column and used in electrophoretic mobility shift assays with
radiolabeled MARE oligonucleotide.
[0159] The expression vector for recombinant c-Maf was constructed
by inserting a cDNA fragment encoding a.a. residues 171 to 371 of
c-Maf (disclosed in Kurschner C. and Morgan, J. I. (1995) Mol.
Cell. Biol. 15:246-254) into the NotI site of pET29 (Novagen, Inc.
Madison, Wis.). The truncated c-Maf protein was expressed using T7
polymerase in the BL21(DE3) strain. Cells were induced by the
addition of 1 mM IPTG and incubated at 37.degree. C. for 3 hours.
The induced cells were lysed in 1.times. Bind/Wash buffer (20 mM
Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100) followed by
sonication. The c-Maf protein was then purified from the soluble
fraction by using the S-Tag Purification Kit (Novagen) according to
manufacturer's instructions. Two additional proteins, NF-ATp and
c-Jun, were also used in EMSA assays. The recombinant NF-ATp,
containing the Rel domain of murine NF-ATp, was expressed using an
in vitro transcription/translation vector TP7-NF-ATp, which
contains a cDNA fragment encoding the Rel domain of murine NF-ATp.
The c-Jun expression vector, pGEM-c-Jun, was constructed by
inserting a full-length cDNA of murine c-Jun into the PstI site of
pGEM4.1 .mu.g of each plasmid DNA was transcribed from the T7
promoter and translated in rabbit reticulocyte lysate by using the
TnT Coupled Transcription/Translation Kit (Promega, Madison,
Wis.).
[0160] Electrophoretic mobility shift assays (EMSA) were performed
as follows. 100 ng of double-stranded oligonucleotides were
end-labeled with .gamma.-.sup.32P-dATP (DuPont NEN Research
Product, Wilmington, Del.) using T4 polynucleotide kinase
(Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.). The labeled
ds-oligonucleotides were fractionated on 15-20% polyacrylamide
gels, eluted overnight at 37.degree. C. in 1.times.TE and
precipitated in ethanol. Binding assays were performed at room
temperature for 20 minutes using 0.5 .mu.g of recombinant proteins
or 4 .mu.l of in vitro translated products, 500 ng poly(dI-dC), and
20,000 cpm of probe in a 15 .mu.l volume of 20 mM HEPES (pH 7.9),
100 mM KCl, 5% glycerol, 1 mM EDTA, 5 mM DTT, 0.1% NP-40, and 0.5
mg/ml BSA. The samples were then fractionated in 4% non-denaturing
polyacrylamide gel containing 0.5.times. TBE at room temperature.
Oligonucleotides derived from the murine IL4 promoter used in EMSA
were:
-59 to -27: 5'-CTCATTTTCCCTTGGTTTCAGCAACTTTAACTC-3' (SEQ ID NO:
1);
-79 to -60: 5'-ATAAAATTTTCCAATGTAAA-3' (SEQ ID NO: 2); and
-88 to -61: 5'-TGGTGTAATAAAATTTTCCAATGTAAA-3' (SEQ ID NO: 3).
The sequence of the MARE oligonucleotide used in EMSA was:
5'-GGAATTGCTGACTCAGCATTACT-3'(SEQ ID NO: 4).
All oligonucleotides were annealed with their respective
reverse-complementary strands to form double-stranded
oligonucleotides.
[0161] The results of EMSA with recombinant c-Maf are shown in FIG.
6. The recombinant c-Maf protein bound well to both a consensus
MARE oligonucleotide and to a 33 bp oligonucleotide containing the
NF-AT site and MARE present in the IL-4 promoter. Binding was
specifically competed by unlabeled homologous but not control
probe. Further, c-Maf did not bind to an oligonucleotide containing
only the NF-AT target sequence to which recombinant NF-ATp bound
well. The ability of c-Maf to bind to the IL-4 promoter probe was
specific since in vitro translated c-Jun protein did not bind to
this oligonucleotide. The c-Jun protein was functional since it
could bind to the consensus MARE which contains a core TRE site.
These results indicate that c-Maf, but not another AP-1 family
member (c-Jun), can bind to the MARE site within the proximal IL-4
promoter.
[0162] NF-AT proteins interact cooperatively with AP-1 family
member proteins to form higher mobility complexes on IL-2 and IL-4
promoter DNA on EMSA (Jain, J. (1993) Nature 365:353-355; Rooney,
J. et al. (1995) Immunity 2:545-553). That NF-AT proteins might
interact with c-maf was suggested by the functional studies
described in the previous examples. To determine if c-Maf
interacted with NF-AT in the presence of DNA, recombinant NF-ATp
and c-Maf proteins were used separately or together in EMSA with
the 33 bp oligonucleotide containing both the NF-AT and adjacent
MARE sites. The results are shown in FIG. 6. Each protein alone
bound to IL-4 promoter DNA. Recombinant c-Maf plus recombinant
NF-ATp protein produced these complexes and in addition formed a
higher mobility complex. No higher mobility complex was observed
when c-Jun and NF-ATp proteins were used, consistent with the
failure of c-Jun to bind this site. These results indicate that
c-Maf can specifically bind in vitro to a sequence located in the
proximal IL-4 promoter, previously shown to be functionally
critical in Th2 cells, and that, like other AP-1 proteins, c-Maf
can interact in vitro with NF-AT proteins.
EXAMPLE 7
The Ability of c-Maf to Transactivate the IL-4 Promoter Maps to the
MARE and Th-2 Specific Footprint
[0163] An essential region of the IL-4 promoter located immediately
upstream of the TATA element has been characterized by high
resolution mutagenesis (Hodge, M. et al. (1995) J. Immunol.
154:6397-6405). Mutagenesis of this 33 bp region (-59 to -28)
demonstrated multiple sites required for inducible IL-4
transcription in Th2 cells. These sites included an NF-AT target
sequence, the region footprinted by Th2 extracts, and what is now
recognized as a MARE. A series of IL-4 reporter gene constructs
comprising 4 base pair linker-scanning mutants generated across
this region were used to map the target sequence utilized by c-Maf
in vivo in M12 cells. These cells were cotransfected with the c-maf
expression vector and this series of mutant IL-4 promoter
constructs. The results are shown in FIG. 7A. Mutation of the MARE
(muts 3 and 4), or the site defined by the Th2 footprint (mut 2),
abrogated (muts 2 and 4) or partially abrogated (mut 3) the ability
of transfected c-maf to drive IL-4 transcription. A modest effect
in reducing c-maf transactivation was also observed for mutant 8
which disrupts the NF-AT sequence, consistent with the presence in
M12 cells of endogenous NF-ATp and with the synergy between NF-ATp
and c-maf demonstrated in the previous examples. Mutants 6 and 7
had no significant effect while mutant 5 had enhanced
transactivation ability, consistent with previous observations in
Th2 cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). The
transactivation data is consistent with EMSA performed with
recombinant c-Maf protein using as probe an oligonucleotide which
contains this 33 bp region, and this same series of mutant
oligonucleotides as cold competitors. The results of these EMSA
experiments are shown in FIG. 7B. These experiments indicate that
c-Maf specifically binds to and transactivates the MARE in the
proximal IL-4 promoter and that the adjacent Th2-specific element
is intimately involved in both the binding and function of
c-Maf.
Equivalents
[0164] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *