U.S. patent application number 17/190297 was filed with the patent office on 2021-09-09 for compositions and methods of treating a t cell mediated disorder.
The applicant listed for this patent is CASE WESTERN RESERVE UNIVERSITY. Invention is credited to Feng Lin, M. Edward Medof, Michael G. Strainic.
Application Number | 20210275585 17/190297 |
Document ID | / |
Family ID | 1000005608374 |
Filed Date | 2021-09-09 |
United States Patent
Application |
20210275585 |
Kind Code |
A1 |
Medof; M. Edward ; et
al. |
September 9, 2021 |
COMPOSITIONS AND METHODS OF TREATING A T CELL MEDIATED DISORDER
Abstract
A method of generating a CD4.sup.+FoxP3.sup.+ Treg cell, the
method includes administering at least one complement antagonist to
a naive CD4.sup.+ T cell at an amount effective to substantially
inhibits C3a receptor (C3aR) and/or C5a receptor (C5aR) signal
transduction in the CD4.sup.+ T cell, induce TGF-.beta.1 expression
of the CD4.sup.+ T cell, and induce differentiation of the of the
naive CD4.sup.+ T cell into a CD4.sup.+FoxP3.sup.+ Treg cell.
Inventors: |
Medof; M. Edward;
(Cleveland, OH) ; Lin; Feng; (Cleveland, OH)
; Strainic; Michael G.; (Cleveland, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CASE WESTERN RESERVE UNIVERSITY |
Cleveland |
OH |
US |
|
|
Family ID: |
1000005608374 |
Appl. No.: |
17/190297 |
Filed: |
March 2, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15950038 |
Apr 10, 2018 |
10933093 |
|
|
17190297 |
|
|
|
|
15077256 |
Mar 22, 2016 |
9937206 |
|
|
15950038 |
|
|
|
|
13505976 |
May 3, 2012 |
9290736 |
|
|
PCT/US2010/055445 |
Nov 4, 2010 |
|
|
|
15077256 |
|
|
|
|
61258058 |
Nov 4, 2009 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/17 20130101;
C12N 5/0636 20130101; C12N 2501/998 20130101; A61K 2035/122
20130101; A61K 2035/124 20130101; A61K 38/00 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12N 5/0783 20060101 C12N005/0783 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under Grant
No. NS052471 and AI023598 awarded by The National Institutes of
Health. The United States Government has certain rights in the
invention.
Claims
1. A method of treating inflammation in a subject, the method
comprising: administering at least one complement antagonist to a
naive CD4.sup.+ T cell at an amount effective to substantially
inhibits C3a receptor (C3aR) and/or C5a receptor (C5aR) signal
transduction in the CD4.sup.+ T cell, induce TGF-.beta.1 expression
of the CD4.sup.+ T cell, and induce differentiation of the of the
naive CD4.sup.+ T cell into a CD4.sup.+FoxP3.sup.+ Treg cell; and
administering a therapeutically effective amount of the
CD4.sup.+FoxP3.sup.+ Treg cells to the subject.
2. The method of claim 1, wherein the at least one complement
antagonist substantially inhibits interaction of at least one of
C3a or C5a with the C3aR or C5aR of the CD4.sup.+ T cell.
3. The method of claim 2, the at least one complement antagonist
being selected from the group consisting of a small molecule, a
polypeptide, and a polynucleotide.
4. The method of claim 3, the polypeptide comprising an antibody
directed against at least one of C3, C5, C3 convertase, C5
convertase, C3a, C5a, C3aR, or C5aR.
5. The method of claim 2, the step of administering the at least
one complement antagonist further including administering to the
naive CD4.sup.+ T cell a C3aR antagonist and an a C5aR
antagonist.
6. The method of claim 2, the step of administering the at least
one complement antagonist further including administering to the
cell a C3a antagonist and a C5a antagonist.
7. The method of claim 1, the CD4.sup.+FoxP3.sup.+ Treg cells being
administered systemically to the subject being treated.
8. The method of claim 1, wherein the complement antagonist
substantially induces naive CD4.sup.+cell expression of CD25,
CTLA-4, FoxP3, DAF and C5L2, downregulates dendritic cell B7/CD40
and CD4.sup.+ effector cell CD28/CD40 ligand costimulatory molecule
expression, and inhibits dendritic cell C5a/C3a production and
CD4.sup.+ cell C5aR/C3aR signal transduction in the subject.
9. The method of claim 1, further comprising the step of isolating
the naive CD4.sup.+ Tcells from a mammalian subject.
10. The method of claim 1, further comprising the step of culturing
the naive CD4.sup.+ T cells in the presence of dendritic cells.
11. The method of claim 1, further comprising the step of culturing
the naive CD4.sup.+ T cells with agents which promote the expansion
and survival of CD4.sup.+FoxP3.sup.+ Treg cells.
12. The method of claim 11, the one or more agents being selected
from the group consisting of an anti-CD3/28, IL-2, TGF-.beta. and
combinations thereof.
13. A method of treating a T cell mediated disease in a subject,
the method comprising: administering to the subject a
therapeutically effective amount of at least one complement
antagonist and a pharmaceutically acceptable carrier, wherein the
at least one complement antagonist substantially inhibits
interaction of at least one of C3a or C5a with the C3a receptors
(C3aR) and C5a receptors (C5aR) on interacting dendritic cells and
CD4.sup.+T cells in the subject.
14. The method of claim 13, wherein the complement antagonist does
not substantially systemic complement activation.
15. The method of claim 13, wherein the T cell mediated disease is
selected from the group consisting of achlorhydra autoimmune active
chronic hepatitis, acute disseminated encephalomyelitis, acute
hemorrhagic leukoencephalitis, Addison's disease,
agammaglobulinemia, alopecia areata, Alzheimer's disease,
amyotrophic lateral sclerosis, ankylosing spondylitis, anti-gbm/tbm
nephritis, antiphospholipid syndrome, antisynthetase syndrome,
aplastic anemia, arthritis, atopic allergy, atopic dermatitis,
autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune
hepatitis, autoimmune inner ear disease, autoimmune
lymphoproliferative syndrome, autoimmune peripheral neuropathy,
autoimmune polyendocrine syndrome, autoimmune progesterone
dermatitis, autoimmune thrombocytopenia purpura, autoimmune
uveitis, balo disease/balo concentric sclerosis, bechets syndrome,
Berger's disease, Bickerstaff's encephalitis, blau syndrome,
bullous pemphigoid, castleman's disease, chagas disease, chronic
fatigue immune dysfunction syndrome, chronic inflammatory
demyelinating polyneuropathy, chronic lyme disease, chronic
obstructive pulmonary disease, churg-strauss syndrome, cicatricial
pemphigoid, coeliac disease, cogan syndrome, cold agglutinin
disease, cranial arteritis, crest syndrome, Crohns disease,
Cushing's syndrome, Dego's disease, Dercum's disease, dermatitis
herpetiformis, dermatomyositis, diabetes mellitus type 1,
Dressler's syndrome, discoid lupus erythematosus, eczema,
endometriosis, enthesitis-related arthritis, eosinophilic
fasciitis, epidermolysis bullosa acquisita, essential mixed
cryoglobulinemia, evan's syndrome, fibrodysplasia ossificans
progressive, fibromyalgia, fibromyositis, fibrosing aveolitis,
gastritis, gastrointestinal pemphigoid, giant cell arteritis,
glomerulonephritis, Goodpasture's syndrome, Graves' disease,
Guillain-barre syndrome (gbs), Hashimoto's encephalitis,
Hashimoto's thyroiditis, henoch-schonlein purpura, hidradenitis
suppurativa, Hughes syndrome, inflammatory bowel disease (IBD),
idiopathic inflammatory demyelinating diseases, idiopathic
pulmonary fibrosis, idiopathic thrombocytopenic purpura, iga
nephropathy, inflammatory demyelinating polyneuopathy, interstitial
cystitis, irritable bowel syndrome (ibs), Kawasaki's disease,
lichen planus, Lou Gehrig's disease, lupoid hepatitis, lupus
erythematosus, meniere's disease, microscopic polyangiitis, mixed
connective tissue disease, morphea, multiple myeloma, multiple
sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis
optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus
myoclonus syndrome, ord thyroiditis, Parkinson's disease, pars
planitis, pemphigus, pemphigus vulgaris, pernicious anaemia,
polymyalgia rheumatic, polymyositis, primary biliary cirrhosis,
primary sclerosing cholangitis, progressive inflammatory
neuropathy, psoriasis, psoriatic arthritis, raynaud phenomenon,
relapsing polychondritis, Reiter's syndrome, rheumatoid arthritis,
rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome,
Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome,
spondyloarthropathy, sticky blood syndrome, still's disease, stiff
person syndrome, sydenham chorea, sweet syndrome, takayasu's
arteritis, temporal arteritis, transverse myelitis, ulcerative
colitis, undifferentiated connective tissue disease,
undifferentiated spondyloarthropathy, vasculitis, vitiligo,
Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich
syndrome, hypersensitivity reactions of the skin, atherosclerosis,
ischemia-reperfusion injury, myocardial infarction, and
restenosis.
16. The method of claim 13, the at least one complement antagonist
being selected from the group consisting of a small molecule, a
polypeptide, and a polynucleotide.
17. The method of claim 16, the polypeptide comprising an antibody
directed against at least one of C3, C5, C3 convertase, C5
convertase, C3a, C5a, C3aR, or C5aR.
18. The method of claim 13, the step of administering the at least
one complement antagonist further including administering to the
subject an antibody directed against C5aR and an antibody directed
against C3aR.
19. The method of claim 13, the step of administering the at least
one complement antagonist further including administering to the
subject an antibody directed against C5a and an antibody directed
against C3a.
20. The method of claim 13, the complement antagonist and a
pharmaceutically acceptable carrier being administered locally to a
site of T cell mediated disease in the subject.
Description
RELATED APPLICATION
[0001] This application is a Continuation of U.S. patent
application Ser. No. 15/077,256, filed Mar. 22, 2016, now U.S. Pat.
No. 9,937,206, which is a Continuation of U.S. patent application
Ser. No. 13/505,976, filed May 3, 2012, now U.S. Pat. No.
9,290,736, which is a 371 of PCT/US2010/055445, filed Nov. 4, 2010,
which claims priority from U.S. Provisional Application No.
61/258,058, filed Nov. 4, 2009, the subject matter, which is
incorporated herein by reference.
TECHNICAL FIELD
[0003] The present application generally relates to methods for
generating FoxP3.sup.+ cells and also to methods of treating a T
cell mediated disorder.
BACKGROUND
[0004] T cell responses must adequately defend against pathogens
but should terminate once they have eliminated the pathogen that
elicited them. In the absence of this control, lymphoproliferation
would continue unabated and antithetically would destroy the host.
T cell responses are regulated by dendritic cells (DCs) which are
educated by the local factors they sense. It is widely accepted
that toll like receptor (TLR) signaling triggered by pathogen
components educates them to initiate T effector cell responses.
This process, in large part, involves upregulation of their MHC
class II and B7/CD40 costimulatory molecule expression. The absence
of TLR signals in conjunction with locally produced
immunosuppressive cytokines educates DCs to extinguish T cell
responses by producing T regulatory (Treg) cells. Central among
these cells are antigen specific (induced) FoxP3.sup.+ Treg cells.
In conjunction with nuclear expression of the forkhead/winged-helix
family transcription factor (TF) member, FoxP3, these cells surface
express CD25, the a chain of the IL-2 receptor (IL-2R) which
greatly augments its affinity for IL-2, and CTLA-4, a potent
inhibitor of B7 induced CD28 signaling in T effectors needed both
for their proliferation and their survival.
SUMMARY
[0005] This application relates generally to a method of generating
CD4.sup.+FoxP3.sup.+ Treg cells using complement antagonists, and
also to therapeutic methods of treating T cell mediated disorders
in a subject. According to one aspect of the application, a method
is provided for generating CD4.sup.+FoxP3.sup.+ Treg cells. The
method includes administering at least one complement antagonist to
a naive CD4.sup.+ T cell at an amount effective to substantially
inhibit C3a receptor (C3aR) and/or C5a receptor (C5aR) signal
transduction in the CD4.sup.+ T cell, induce TGF-.beta. expression
of the CD4.sup.+ T cell, and induce differentiation of the of the
naive CD4.sup.+ T cell into a CD4.sup.+FoxP3.sup.+ Treg cell.
[0006] In an aspect of the application, the at least one complement
antagonist is selected from the group consisting of a small
molecule, a polypeptide, and a polynucleotide. In some aspects, the
polypeptide includes an antibody directed against at least one of
C3, C5, C3 convertase, C5 convertase, C3a, C5a, C3aR, or C5aR. In
other aspects, the polypeptide can include decay accelerating
factor (DAF) (CD55) that accelerates the decay of C5 convertase and
C3 convertase. In some aspects, the polynucleotide includes a small
interfering RNA directed against a polynucleotide encoding at least
one of C3, C5, C3aR, or C5aR.
[0007] Another aspect of the application relates to a method of
treating a T cell mediated disorder in a subject. The method
includes administering at least one complement antagonist to a
naive CD4.sup.+ T cell at an amount effective to substantially
inhibits C3a receptor (C3aR) and/or C5a receptor (C5aR) signal
transduction in the CD4.sup.+ T cell, induce TGF-.beta. expression
of the CD4.sup.+ T cell, and induce differentiation of the of the
naive CD4.sup.+ T cell into a CD4.sup.+FoxP3.sup.+ Treg cell. A
therapeutically effective amount of the CD4.sup.+FoxP3.sup.+ Treg
cells is then administered to the subject to treat the T cell
mediated disorder.
[0008] In some aspects of the present application, the T cell
mediated disorder is selected from the group consisting of
achlorhydra autoimmune active chronic hepatitis, acute disseminated
encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's
disease, agammaglobulinemia, alopecia areata, Alzheimer's disease,
amyotrophic lateral sclerosis, ankylosing spondylitis, anti-gbm/tbm
nephritis, antiphospholipid syndrome, antisynthetase syndrome,
aplastic anemia, arthritis, atopic allergy, atopic dermatitis,
autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune
hepatitis, autoimmune inner ear disease, autoimmune
lymphoproliferative syndrome, autoimmune peripheral neuropathy,
autoimmune polyendocrine syndrome, autoimmune progesterone
dermatitis, autoimmune thrombocytopenia purpura, autoimmune
uveitis, balo disease/balo concentric sclerosis, Bechets syndrome,
Berger's disease, Bickerstaff's encephalitis, Blau syndrome,
bullous pemphigoid, Castleman's disease, Chagas disease, chronic
fatigue immune dysfunction syndrome, chronic inflammatory
demyelinating polyneuropathy, chronic lyme disease, chronic
obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial
pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin
disease, cranial arteritis, crest syndrome, Crohns disease,
Cushing's syndrome, Dego's disease, Dercum's disease, dermatitis
herpetiformis, dermatomyositis, diabetes mellitus type 1,
Dressler's syndrome, discoid lupus erythematosus, eczema,
endometriosis, enthesitis-related arthritis, eosinophilic
fasciitis, epidermolysis bullosa acquisita, essential mixed
cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans
progressive, fibromyalgia, fibromyositis, fibrosing aveolitis,
gastritis, gastrointestinal pemphigoid, giant cell arteritis,
glomerulonephritis, Goodpasture's syndrome, Graves' disease,
Guillain-barre syndrome (GBS), Hashimoto's encephalitis,
Hashimoto's thyroiditis, henoch-schonlein purpura, hidradenitis
suppurativa, Hughes syndrome, inflammatory bowel disease (IBD),
idiopathic inflammatory demyelinating diseases, idiopathic
pulmonary fibrosis, idiopathic thrombocytopenic purpura, iga
nephropathy, inflammatory demyelinating polyneuopathy, interstitial
cystitis, irritable bowel syndrome (IBS), Kawasaki's disease,
lichen planus, Lou Gehrig's disease, lupoid hepatitis, lupus
erythematosus, meniere's disease, microscopic polyangiitis, mixed
connective tissue disease, morphea, multiple myeloma, multiple
sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis
optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus
myoclonus syndrome, ord thyroiditis, Parkinson's disease, pars
planitis, pemphigus, pemphigus vulgaris, pernicious anaemia,
polymyalgia rheumatic, polymyositis, primary biliary cirrhosis,
primary sclerosing cholangitis, progressive inflammatory
neuropathy, psoriasis, psoriatic arthritis, raynaud phenomenon,
relapsing polychondritis, Reiter's syndrome, rheumatoid arthritis,
rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome,
Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome,
spondyloarthropathy, sticky blood syndrome, still's disease, stiff
person syndrome, sydenham chorea, sweet syndrome, takayasu's
arteritis, temporal arteritis, transverse myelitis, ulcerative
colitis, undifferentiated connective tissue disease,
undifferentiated spondyloarthropathy, vasculitis, vitiligo,
Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich
syndrome, hypersensitivity reactions of the skin, atherosclerosis,
ischemia-reperfusion injury, myocardial infarction, and
restenosis.
[0009] A further aspect of the application relates to a method of
treating inflammation in a subject. The method includes
administering at least one complement antagonist to a naive
CD4.sup.+ T cell at an amount effective to substantially inhibits
C3a receptor (C3aR) and/or C5a receptor (C5aR) signal transduction
in the CD4.sup.+ T cell, induce TGF-.beta. expression of the
CD4.sup.+ T cell, and induce differentiation of the of the naive
CD4.sup.+ T cell into a CD4.sup.+FoxP3.sup.+ Treg cell. A
therapeutically effective amount of the CD4.sup.+FoxP3.sup.+ Treg
cells is then administered to the subject to treat the
inflammation.
[0010] Yet another aspect of the application relates to a method of
treating a T cell mediated disease in a subject. The method
includes administering to the subject a therapeutically effective
amount of at least one complement antagonist and a pharmaceutically
acceptable carrier. The at least one complement antagonist can
substantially inhibit interaction of at least one of C3a or C5a
with the C3a receptor (C3aR) and C5a receptor (C5aR) on interacting
dendritic cells and CD4.sup.+ T cells in the subject. The at least
one complement antagonist advantageously does not substantially
inhibit innate systemic complement activation in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The foregoing and other features of the application will
become apparent to those skilled in the art to which the
application relates upon reading the following description with
reference to the accompanying drawings, in which:
[0012] FIG. 1 illustrates a schematic drawing of dendritic cell
(DC)--CD4.sup.+ cell C3aR/C5aR signal transduction driving Th1/Th7
responses and the absence of these signals driving FoxP3.sup.+ Treg
induction and TGF-.beta./IL-10 expression.
[0013] FIGS. 2(A-G) illustrate plots and graphs showing: A)
1.times.10.sup.6 CD62L.sup.hiCD25.sup.- CD4.sup.+ cells from
FoxP3-GFP knockin mice were incubated with anti-CD3, 5 ng/ml
rhIL-2, and 1.times.10.sup.5 CD11c.sup.+ WT DCs for 3 days together
with 5 ng/ml rhTGF-.beta.1 (Prospecbio), 10 nM C3aR-A/C5aR-A, or 5
.mu.g/ml anti-C3a/C5a mAbs and assayed for CD25 and FoxP3
expression by flow cytometry. B) Sorted 1.times.10.sup.6
FoxP3.sup.+ cells were incubated with increasing amounts of
CellTracker Red labeled CD25.sup.- CD4.sup.+WT cells and 2 .mu.g/ml
anti-CD3+1.times.10.sup.5 CD11c.sup.+ DCs for 4 days and percent
dividers determined. C) 1.times.10.sup.6 CD62L.sup.hiCD25.sup.-
CD4.sup.+ cells from FoxP3-GFP knockin mice were incubated as in
(A) and assayed for IL-6, TGF-.beta.1, and IL-10 expression by
ELISA. D) 1.times.10.sup.6 CD62L.sup.hiCD25.sup.- CD4.sup.+ cells
from WT or C3aR.sup.-/-C5aR.sup.-/- mice were incubated with CD11
c.sup.+ DCs from C3aR.sup.-/-C5aR.sup.-/- mice and 5 ng/ml rhIL-2
for 3 days after which cells were assayed for FoxP3 expression by
flow cytometry. E-G) CD4.sup.+CD25.sup.- cells from WT or
C3aR.sup.-/-C5aR.sup.-/- mice were incubated with anti-CD3 mAb,
IL-2, and DCs.+-.anti-TGF-.beta. mAb, TGF-.beta.R1 inhibitor or
Smad3 inhibitor for 3 days and E) assessed FoxP3.sup.+ Treg numbers
by flow cytometry, and assayed culture supernatants of CD4.sup.+
cells and DCs for F) TGF-.beta.1 and G) IL-10.
[0014] FIGS. 3(A-I) illustrate graphs showing: A) 1.times.10.sup.6
CD62L.sup.hiCD25.sup.- CD4.sup.+ cells from WT or
C3aR.sup.-/-C5aR.sup.-/- mice were incubated with anti-CD3/CD28
Dynabead activation beads, 5 ng/ml rhIL-2, and 1.times.10.sup.5
CD11c.sup.+ WT DCs for 3 days. The CD4.sup.+ cell and DC partners
were sorted, after which the DCs were assayed for B7 family members
(*P<0.01; **P<0.03; ***P=0.07) and the CD4.sup.+ cells were
assayed for B7 coinhibitory counter-receptors by flow. B)
1.times.10.sup.6 CD62L.sup.hiCD25.sup.- CD4.sup.+ cells from WT
mice were incubated with anti-CD3/CD28 Dynabead activation beads, 5
ng/ml rhIL-2, and 1.times.10.sup.5 CD11c.sup.+ WT DCs for 3
days.+-.10 nM C3aR-A/C5aR-A or.+-.5 ng/ml rhTGF-.beta.1. The
CD4.sup.+ cell and DC partners were sorted, after which the cells
were assayed as in (A; *P<0.01 C3aR-A/C5aR-A vs. TGF-.beta.1).
C) 1.times.10.sup.6 CD62L.sup.hiCD25.sup.- CD4.sup.+ cells from WT
or C3aR.sup.-/-C5aR.sup.-/- were incubated with anti-CD3/CD28
Dynabead activation beads, 5 ng/ml rhIL-2, and 1.times.10.sup.5
CD11c.sup.+ WT DCs for 3 days in the presence of blocking
anti-CTLA-4, anti-PD-1, or anti-ICOS-L Abs and percent FoxP3.sup.+
cells assayed by flow cytometry. D) 1.times.10.sup.6
CD62L.sup.hiCD25.sup.- CD4.sup.+ cells from WT, ICOS .sup.-/-, or
PD-1.sup.-/- were incubated with anti-CD3/CD28 Dynabead activation
beads, 5 ng/ml rhIL-2, and 1.times.10.sup.3 CD11c.sup.+ WT or
PD-L1.sup.-/- DCs for 3 days in the presence of blocking
anti-CTLA-4, anti-PD-1, or anti-ICOS-L Abs and percent FoxP3.sup.+
cells assayed by flow cytometry. E) Anti-CD3/CD28 stimulated
CD4.sup.+ cells were treated with TGF-.beta.1 or buffer and
complement mRNA transcripts were assayed by qPCR. F-H)
1.times.10.sup.4 CD62L.sup.hiCD25.sup.- CD4.sup.+ cells from
C57BL/6 mice were incubated with anti-CD3/CD28 Dynabeads and 5
ng/ml rhIL-2 for 3 days in the presence of TGF-.beta.,
TGF-.beta.+IL-6, or IL-6 after which cells were assayed for (F) C3
mRNA expression by qPCR, (G) C3 and C5a generation by ELSIA, and
(H) C3aR and C5aR surface expression by flow cytometry. I) C57BL/6
CD4.sup.+ T cells were incubated for 1 hr with anti-CD3/CD28.+-.100
ng/ml C5a and FoxP3 mRNA was quantitated by qPCR.
[0015] FIGS. 4(A-F) illustrate plots and graphs showing: A-B)
CFSE-labeled OT-II cells were adoptively transferred into WT mice.
Two days later CD4.sup.+CD25.sup.- Daf1.sup.-/- and
C3aR.sup.-/-C5aR.sup.-/- cells from OT-II mice were adoptively
transferred and the mice were immunized with ovalbumin in CFA. A)
Percent CD25.sup.+ cells in draining LN were assessed by flow
cytometry, and (B) CFSE dilution was assessed 5 days later. CD)
CellTracker Red-labeled OT-II cells were adoptively transferred
into WT or C3aR.sup.-/-C5aR.sup.-/- mice. Two days later
CD4.sup.+CD25.sup.- cells from FoxP3-GFP OT-II mice were adoptively
transferred and the mice were immunized with ovalbumin in CFA. C)
Percent FoxP3.sup.+ cells in draining LN were assessed by flow
cytometry, and (D) CellTracker Red dilution was assessed 5 days
later. E) 10.sup.6 CellTracker Red labeled OT-II cells were
transferred into WT mice. Two days later, Tregs generated from
naive CD4.sup.+cells of FoxP3-GFP mice in vitro by incubation with
TGF-.beta.1 or 10 nM C3aR/C5aR antagonists were adoptively
transferred into WT recipients and the mice were immunized with
ovalbumin in CFA. CellTracker Red dilution in the OT-II cells in
the spleen assessed 5 days thereafter. F) Tregs were generated from
naive CD4.sup.+ OT-II or 2D2 cells (MOG35-55 specific) in vitro as
in (E) using 10 nM C3aR-A/C5aR-A. 10.sup.6 CellTracker Red labeled
OT-II cells were co-transferred into WT mice in 32:1, 8:1, 4:1, and
1:1 E:T ratios with 1) the OT-II Tregs labeled with CellTracker
Violet, 2) the 2D2 Tregs labeled with CFSE, or 3) a 50/50 mixture
of both. Two days thereafter, mice were immunized with ovalbumin in
CFA and 5 days later percent CellTracker Red dividers was
determined by flow cytometry.
[0016] FIGS. 5(A-G) illustrate graphs showing, A-C) EAE was induced
in FoxP3-GFP knockin mice and 10 days after disease onset, CD4
cells were isolated from spleens and lymph nodes and sorted on GFP.
4.times.10.sup.6 GFP negative cells were adoptively transferred
into Rag2.sup.-/- mice. The mice were subsequently injected with
1.times.10.sup.6 FoxP3-GFP positive cells or PBS after which A)
weight change, B) clinical scores, and C) percent FoxP3.sup.+ cells
were assayed. D-E) EAE was induced in WT and
C3aR.sup.-/-C5aR.sup.-/- mice and at day 12 post disease induction
draining LN cells were harvested and assayed for D) % FoxP3.sup.+
cells, and E) suppressive capacity as above. F) EAE was induced in
FoxP3-GFP knockin mice and 10 days after induction mice were
treated with buffer control, C3aR-A/C5aR-A, or ex vivo generated
FoxP3.sup.+ Tregs (from FoxP3-GFP mice with C3aR-A/C5aR-A), after
which C) clinical scores were assessed. G) CD25.sup.+ cells from
1.times.10.sup.6 CD25.sup.-CD4.sup.+ Human T cells isolated by flow
cytometry and incubated with soluble anti-CD3, IL-2, and autologous
DCs in the presence and absence of TGF-.beta., C3aR-A/C5aR-A, or
anti-C3a/C5a mAbs for 3 days were incubated with CFSE labeled
autologous naive T cells, anti-CD3, and autologous DCs and percent
dividers determined 3 days thereafter by CFSE dilution.
DETAILED DESCRIPTION
[0017] Methods involving conventional molecular biology techniques
are described herein. Such techniques are generally known in the
art and are described in detail in methodology treatises, such as
Current Protocols in Molecular Biology, ed. Ausubel et al., Greene
Publishing and Wiley-Interscience, New York, 1992 (with periodic
updates). Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the present application pertains.
Commonly understood definitions of molecular biology terms can be
found in, for example, Rieger et al., Glossary of Genetics:
Classical and Molecular, 5th Edition, Springer-Verlag: New York,
1991, and Lewin, Genes V, Oxford University Press: New York, 1994.
The definitions provided herein are to facilitate understanding of
certain terms used frequently herein and are not meant to limit the
scope of the present application.
[0018] In the context of the present application, the term
"polypeptide" refers to an oligopeptide, peptide, or protein
sequence, or to a fragment, portion, or subunit of any of these,
and to naturally occurring or synthetic molecules. The term
"polypeptide" also includes amino acids joined to each other by
peptide bonds or modified peptide bonds, i.e., peptide isosteres,
and may contain any type of modified amino acids. The term
"polypeptide" also includes peptides and polypeptide fragments,
motifs and the like, glycosylated polypeptides, and all "mimetic"
and "peptidomimetic" polypeptide forms.
[0019] As used herein, the term "polynucleotide" refers to
oligonucleotides, nucleotides, or to a fragment of any of these, to
DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin
which may be single-stranded or double-stranded and may represent a
sense or antisense strand, to peptide nucleic acids, or to any
DNA-like or RNA-like material, natural or synthetic in origin,
including, e.g., iRNA, siRNAs, microRNAs, and ribonucleoproteins.
The term also encompasses nucleic acids, i.e., oligonucleotides,
containing known analogues of natural nucleotides, as well as
nucleic acid-like structures with synthetic backbones.
[0020] As used herein, the term "antibody" refers to whole
antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc.), and
includes fragments thereof which are also specifically reactive
with a target polypeptide. Antibodies can be fragmented using
conventional techniques and the fragments screened for utility
and/or interaction with a specific epitope of interest. Thus, the
term includes segments of proteolytically-cleaved or
recombinantly-prepared portions of an antibody molecule that are
capable of selectively reacting with a certain polypeptide.
Non-limiting examples of such proteolytic and/or recombinant
fragments include Fab, F(ab')2, Fab', Fv, and single chain
antibodies (scFv) containing a V[L] and/or V[H] domain joined by a
peptide linker. The scFv's may be covalently or non-covalently
linked to form antibodies having two or more binding sites. The
term "antibody" also includes polyclonal, monoclonal, or other
purified preparations of antibodies, recombinant antibodies,
monovalent antibodies, and multivalent antibodies. Antibodies may
be humanized, and may further include engineered complexes that
comprise antibody-derived binding sites, such as diabodies and
triabodies.
[0021] As used herein, the term "complementary" refers to the
capacity for precise pairing between two nucleobases of a
polynucleotide and its corresponding target molecule. For example,
if a nucleobase at a particular position of a polynucleotide is
capable of hydrogen bonding with a nucleobase at a particular
position of a target polynucleotide (the target nucleic acid being
a DNA or RNA molecule, for example), then the position of hydrogen
bonding between the polynucleotide and the target polynucleotide is
considered to be complementary. A polynucleotide and a target
polynucleotide are complementary to each other when a sufficient
number of complementary positions in each molecule are occupied by
nucleobases, which can hydrogen bond with each other. Thus,
"specifically hybridizable" and "complementary" are terms which can
be used to indicate a sufficient degree of precise pairing or
complementarity over a sufficient number of nucleobases such that
stable and specific binding occurs between a polynucleotide and a
target polynucleotide.
[0022] As used herein, the terms "effective," "effective amount,"
and "therapeutically effective amount" refer to that amount of a
complement antagonist and/or a pharmaceutical composition thereof
that results in amelioration of symptoms or a prolongation of
survival in a subject with a T cell mediated disease or related
disorder. A therapeutically relevant effect relieves to some extent
one or more symptoms of a T cell mediated disease related disorder,
or returns to normal either partially or completely one or more
physiological or biochemical parameters associated with or
causative of a T cell mediated disease or related disorder.
[0023] As used herein, the term "subject" refers to any
warm-blooded organism including, but not limited to, human beings,
rats, mice, dogs, goats, sheep, horses, monkeys, apes, rabbits,
cattle, etc.
[0024] As used herein, the terms "complement polypeptide" or
"complement component" refer to a polypeptide (or a polynucleotide
encoding the polypeptide) of the complement system that functions
in the host defense against infections and in the inflammatory
process. Complement polypeptides constitute target substrates for
the complement antagonists provided herein.
[0025] As used herein, the term "complement antagonist" refers to a
polypeptide, polynucleotide, or small molecule capable of
substantially reducing expression of C3, C5, C3a, C5a, C5aR, and/or
C3aR in CD4.sup.+ T cells or dendritic cells (DCs), substantially
inhibiting C3aR and/or C5aR signal transduction of CD4.sup.+ T
cells, and/or substantially reducing interaction of C3a and C5a
with C3aR and C5aR expressed by interacting dendritic cells (DCs)
and CD4.sup.+ T cells.
[0026] This application generally relates to a method of generating
CD4.sup.+FoxP3.sup.+ Treg cells using complement antagonists and
also to therapeutic preparations for the treatment of a T cell
mediated disorder or condition. It was found that during wild type
dendritic cell (WT DC)-CD4.sup.+ T cell interactions, both cells
locally synthesize the alternative complement pathway (AP)
components C3, factor B, factor D, in conjunction with C5, C3aR and
C5aR, the latter receptors being G protein coupled receptors
(GPCRs). Concurrently with this, both cells downregulate their
surface expression levels of DAF. As a result of DAF inhibition,
C3/C5 convertase assemble at the adjoining DC-CD4.sup.+ cell
surfaces and act on the locally synthesized C3/C5 to generate
C3a/C5a. Bidirectional interaction of these cytokine-like fragments
with upregulated C3aR/C5aR on the interacting DCs and CD4.sup.+
cells then transduces GPCR signals, which are needed both for
CD4.sup.+ cell IL-2 production and DC innate cytokine production,
which evokes CD4.sup.+ cell differentiation into Th1/Th17 effectors
(FIG. 1). The underlying mechanism is that upon C3aR/C5aR
activation by ligation of locally produced C3a and C5a, PI-3 kinase
.gamma. (PI-3K.gamma.) activity is upregulated leading to increased
intracellular AKT phosphorylation and downstream signaling to
NF-kB. Consistent with this, we found that C3aR/C5aR signals are
required for T cell survival not only during activation but also
tonically in the spleen. The immunological significance of these
GPCR signals is that they 1) upregulate DC MHC class II and DC-T
cell costimulatory molecule expression and 2) concurrently sustain
intracellular Bcl-2/Bcl-x2 expression and suppress surface Fas/FasL
expression in T cells.
[0027] It was found that when autocrine/paracrine C3aR/C5aR
signaling between interacting dendritic cells (DCs) and naive
CD4.sup.+ cells (devoid of FoxP3.sup.+ cells) does not occur,
TGF-.beta. is endogenously produced by both cell partners. In both
primed DCs and naive CD4.sup.+ cells, the elicited TGF-.beta.
enters into autocrine signaling loops that suppresses costimulatory
molecule expression and IL-6 production. Auto-inductive TGF-.beta.
signaling prevents upregulation of costimulatory CD28 and CD40
ligand (CD40L) expression and thereby allows FoxP3.sup.+ Treg
induction.
[0028] As shown in the Example, C3aR and/or C5aR signaling of naive
CD4.sup.+ cells can be substantially inhibited and FoxP3.sup.+
Tregs can be induced from the CD4.sup.+ cells by administering to
the CD4.sup.+cells a pharmaceutical composition comprising C3aR
and/or C5aR antagonists (C3aR-A and/or C5aR-A) and/or anti-C3a
and/or anti-C5a monoclonal antibodies. Human induced FoxP3.sup.+
(iFoxP3.sup.+) Tregs produced in this way exert robust suppressive
activity when added to DC, anti-CD3 and CFSE-labeled CD25.sup.-
CD4.sup.+ cell mixtures from the same individual and confer about 4
about 10 fold greater suppression than FoxP3.sup.+ Tregs generated
by exogenously adding TGF-.beta. CD4.sup.+ cells. This shows that
complement antagonists, such as C3, C5, C3C5aR, C3aR, C5a, or C3a
antagonists (e.g., competitive inhibitors, mAbs, interfering RNA)
as well as DAF, used alone, or in combination, that inhibit C3aR
and/or C5aR signaling in the CD4.sup.+ T cell will not only promote
or induce the generation of FoxP3.sup.+ Treg cells, but when
administered to a subject can be used to treat a T cell mediated
diseases and disorders in a subject.
[0029] One aspect of the application, therefore, relates to a
method of generating CD4.sup.+FoxP3.sup.+ Treg cells by
administering at least one complement antagonist to a naive
CD4.sup.+ T cell at an amount effective to substantially inhibit
C3a receptor (C3aR) and/or C5a receptor (C5aR) signal transduction
in the CD4.sup.+ T cell, induce TGF-.beta. expression of the
CD4.sup.+ T cell, and induce differentiation of the of the naive
CD4.sup.+ T cell into a CD4.sup.+FoxP3.sup.+ Treg cell.
[0030] In some aspects of the application, the complement
antagonist can substantially inhibit the interaction of at least
one of C3a or C5a with the C3a receptor (C3aR) and C5a receptor
(C5aR) on the CD4.sup.+ T cells to substantially inhibit C3a
receptor (C3aR) and/or C5a receptor (C5aR) signal transduction in
the CD4.sup.+ T cell. Disabling these interactions results in the
induction of CD4.sup.+FoxP3.sup.+ Treg cells. In other aspects of
the application, an inhibition or reduction in the functioning of a
C3/C5 convertase can prevent cleavage of C5 and C3 into C5a and
C3a, respectively. An inhibition or reduction in the functioning of
C5a and C3a polypeptides can reduce or eliminate the ability of C5a
and C3a to interact with C5aR and C3aR of CD4.sup.+ cells and
substantially inhibit C3a receptor (C3aR) and/or C5a receptor
(C5aR) signal transduction in the CD4.sup.+ T cell. An inhibition
or reduction in the functioning of a C5aR or C3aR may similarly
reduce or eliminate the ability of C5a and C3a to interact C5aR and
C3aR, respectively, and substantially inhibit C3a receptor (C3aR)
and/or C5a receptor (C5aR) signal transduction in the CD4.sup.+ T
cell.
[0031] In other aspects of the application, the at least one
complement antagonist can substantially induces naive CD4.sup.+
cell expression of CD25, CTLA-4, FoxP3, DAF and C5L2, downregulates
dendritic cell B7/CD40 and CD4.sup.+ effector cell CD28/CD40 ligand
costimulatory molecule expression, and inhibits dendritic cell
C5a/C3a production and CD4.sup.+ cell C5aR/C3aR signal transduction
in the subject.
[0032] In some embodiments of the application, the complement
antagonist can include at least one of a C5a antagonist, a C3a
antagonist, a CSaR antagonist, or a C3aR antagonist. FoxP3.sup.+
cells can be generated by blocking C3a or C5a. C3a and C5a can be
blocked during cognate APC-T cell interactions using mAbs directed
toward C5a and C3a. It is also contemplated that more than one
complement antagonist can be administered concurrently to naive
CD4.sup.+ T cells in the presence of cognate dendritic cells in
order to inhibit C3a/C5a production and/or DC-T cell C3aR/C5aR
signal transduction.
[0033] In some embodiments, the at least one complement antagonist
can include various C5aR antagonists known in the art. For example,
C5aR antagonists include those described by Short et al. (1999)
Effects of a new C5a receptor antagonist on C5a- and
endotoxin-induced neutropenia in the rat. British Journal of
Pharmacology, 125:551-554, Woodruff et al. (2003) A Potent C5a
Receptor Antagonist Protects against Disease Pathology in a Rat
Model of Inflammatory Bowel Disease. The Journal of Immunology,
171:5514-5520, Sumichika et al. (2002) Identification of a Potent
and Orally Active Non peptide C5a Receptor Antagonist. The Journal
of Biological Chemistry, 277(51):49403-49407, all of which are
incorporated herein by reference.
[0034] In one embodiment, CSaRantagonist can include the
peptidomimetic C5aR antagonist JPE-1375 (Jerini AG, Germany). C5aR
antagonists can further include small molecules, such as CCX168
(ChemoCentryx, Mountain View, Calif.).
[0035] In other embodiments, the at least one complement antagonist
can include an antibody or antibody fragment directed against a
complement component that can affect or inhibit the formation of
C3a and/or C5a (e.g., DAF, anti-C5 convertase, and anti-C3
convertase) and/or reduce C5a/C3a-c5aR/C3aR interactions (e.g.,
anti-C5a, anti-C3a, anti-C5aR, C3aR antibodies).
[0036] In still other embodiments, the at least one complement
antagonist can include an antibody or antibody fragment directed
against a complement component that can affect or inhibit the
formation of C3a and/or C5a (e.g., anti-Factor B, anti-Factor D,
anti-C5, anti-C3, ant-C5 convertase, and anti-C3 convertase) and/or
reduce C5a/C3a-C5aR/C3aR interactions (e.g., anti-C5a, anti-C3a,
anti-C5aR, and C3aR antibodies). In one example, the antibody or
antibody fragment can be directed against or specifically bind to
an epitope, an antigenic epitope, or an immunogenic epitope of a
C5, C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase.
The term "epitope" as used herein can refer to portions of C5, C3,
C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase having
antigenic or immunogenic activity. An "immunogenic epitope" as used
herein can include a portion of a C5, C3, C3a, C5a, C5aR, C3aR, C5
convertase, and/or C3 convertase that elicits an immune response in
a subject, as determined by any method known in the art. The term
"antigenic epitope" as used herein can include a portion of a
polypeptide to which an antibody can immunospecifically bind as
determined by any method well known in the art.
[0037] Examples of antibodies directed against C5, C3, C3a, C5a,
C5aR, C3aR, C5 convertase, and/or C3 convertase are known in the
art. For example, mouse monoclonal antibodies directed against C3aR
can include those available from Santa Cruz Biotechnology, Inc.
(Santa Cruz, Calif.). Monoclonal anti-human C5aR antibodies can
include those available from Research Diagnostics, Inc. (Flanders,
N.J.). Monoclonal anti-human/anti-mouse C3a antibodies can include
those available from Fitzgerald Industries International, Inc.
(Concord, Me.). Monoclonal anti-human/anti-mouse C5a antibodies can
include those available from R&D Systems, Inc. (Minneapolis,
Minn.).
[0038] In another aspect of the application, the at least one
complement antagonist can include purified polypeptide that is a
dominant negative or competitive inhibitor of C5, C3, C3a, C5a,
C5aR, C3aR, C5 convertase, and/or C3 convertase. As used herein,
"dominant negative" or "competitive inhibitor" refers to variant
forms of a protein that inhibit the activity of the endogenous,
wild type form of the protein (i.e., C5, C3, C3a, C5a, C5aR, C3aR,
C5 convertase, and/or C3 convertase). As a result, the dominant
negative or competitive inhibitor of a protein promotes the "off"
state of protein activity. In the context of the present
application, a dominant negative or competitive inhibitor of C5,
C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase is a
C5, C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase
polypeptide, which has been modified (e.g., by mutation of one or
more amino acid residues, by posttranscriptional modification, by
posttranslational modification) such that the C5, C3, C3a, C5a,
C5aR, C3aR, C5 convertase, and/or C3 convertase inhibits the
activity of the endogenous C5, C3, C3a, C5a, C5aR, C3aR, C5
convertase, and/or C3 convertase.
[0039] In some embodiments, the competitive inhibitor of C5, C3,
C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase can be a
purified polypeptide that has an amino acid sequence, which is
substantially similar (i.e., at least about 75%, about 80%, about
85%, about 90%, about 95% similar) to the wild type C5, C3, C3a,
C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase but with a
loss of function. The purified polypeptide, which is a competitive
inhibitor of C5, C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3
convertase, can be administered to a naive T cell (e.g.,
CD45.sup.hiCD44.sup.low CD4.sup.+ T cell) expressing C5aR and/or
C3aR, to generate a CD4.sup.+FoxP3.sup.+ Treg cell.
[0040] It will be appreciated that antibodies directed to other
complement components used in the formation of C5, C3, C5a, C3a, C5
convertase, and/or C3 convertase can be used in accordance with the
method of the present application to reduce and/or inhibit
interactions C5a and/or C3a with C5aR and C3aR between dendritic
cells and naive CD4.sup.+ T cells. The antibodies can include, for
example, known Factor B, properdin, and Factor D antibodies that
reduce, block, or inhibit the classical and/or alternative pathway
of the complement system.
[0041] In a further aspect of the present application, the at least
one complement antagonist can include RNA interference (RNAi)
polynucleotides to induce knockdown of an mRNA encoding a
complement component. For example, an RNAi polynucleotide can
comprise a siRNA capable of inducing knockdown of an mRNA encoding
a C3, C5, C5aR, or C3aR polypeptide in the CD4.sup.+ T cells or
dendritic cell.
[0042] RNAi constructs comprise double stranded RNA that can
specifically block expression of a target gene. "RNA interference"
or "RNAi" is a term initially applied to a phenomenon observed in
plants and worms where double-stranded RNA (dsRNA) blocks gene
expression in a specific and post-transcriptional manner. Without
being bound by theory, RNAi appears to involve mRNA degradation,
however the biochemical mechanisms are currently an active area of
research. Despite some mystery regarding the mechanism of action,
RNAi provides a useful method of inhibiting gene expression in
vitro or in vivo.
[0043] As used herein, the term "dsRNA" refers to siRNA molecules
or other RNA molecules including a double stranded feature and able
to be processed to siRNA in cells, such as hairpin RNA
moieties.
[0044] The term "loss-of-function," as it refers to genes inhibited
by the subject RNAi method, refers to a diminishment in the level
of expression of a gene when compared to the level in the absence
of RNAi constructs.
[0045] As used herein, the phrase "mediates RNAi" refers to
(indicates) the ability to distinguish which RNAs are to be
degraded by the RNAi process, e.g., degradation occurs in a
sequence-specific manner rather than by a sequence-independent
dsRNA response.
[0046] As used herein, the term "RNAi construct" is a generic term
used throughout the specification to include small interfering RNAs
(siRNAs), hairpin RNAs, and other RNA species, which can be cleaved
in vivo to form siRNAs. RNAi constructs herein also include
expression vectors (also referred to as RNAi expression vectors)
capable of giving rise to transcripts which form dsRNAs or hairpin
RNAs in cells, and/or transcripts which can produce siRNAs in
vivo.
[0047] "RNAi expression vector" (also referred to herein as a
"dsRNA-encoding plasmid") refers to replicable nucleic acid
constructs used to express (transcribe) RNA which produces siRNA
moieties in the cell in which the construct is expressed. Such
vectors include a transcriptional unit comprising an assembly of
(I) genetic element(s) having a regulatory role in gene expression,
for example, promoters, operators, or enhancers, operatively linked
to (2) a "coding" sequence which is transcribed to produce a
double-stranded RNA (two RNA moieties that anneal in the cell to
form an siRNA, or a single hairpin RNA which can be processed to an
siRNA), and (3) appropriate transcription initiation and
termination sequences.
[0048] The choice of promoter and other regulatory elements
generally varies according to the intended host cell. In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of "plasmids" which refer to circular double
stranded DNA loops, which, in their vector form are not bound to
the chromosome. In the present specification, "plasmid" and
"vector" are used interchangeably as the plasmid is the most
commonly used form of vector. However, the application is intended
to include such other forms of expression vectors which serve
equivalent functions and which become known in the art subsequently
hereto.
[0049] The RNAi constructs contain a nucleotide sequence that
hybridizes under physiologic conditions of the cell to the
nucleotide sequence of at least a portion of the mRNA transcript
for the gene to be inhibited (i.e., the "target" gene). The
double-stranded RNA need only be sufficiently similar to natural
RNA that it has the ability to mediate RNAi. Thus, the application
has the advantage of being able to tolerate sequence variations
that might be expected due to genetic mutation, strain polymorphism
or evolutionary divergence. The number of tolerated nucleotide
mismatches between the target sequence and the RNAi construct
sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or
1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center
of the siRNA duplex are most critical and may essentially abolish
cleavage of the target RNA. In contrast, nucleotides at the 3' end
of the siRNA strand that is complementary to the target RNA do not
significantly contribute to specificity of the target
recognition.
[0050] Sequence identity may be optimized by sequence comparison
and alignment algorithms known in the art (see Gribskov and
Devereux, Sequence Analysis Primer, Stockton Press, 1991, and
references cited therein) and calculating the percent difference
between the nucleotide sequences by, for example, the
Smith-Waterman algorithm as implemented in the BESTFIT software
program using default parameters (e.g., University of Wisconsin
Genetic Computing Group). Greater than 90% sequence identity, or
even 100% sequence identity, between the inhibitory RNA and the
portion of the target gene is preferred. Alternatively, the duplex
region of the RNA may be defined functionally as a nucleotide
sequence that is capable of hybridizing with a portion of the
target gene transcript.
[0051] Production of RNAi constructs can be carried out by chemical
synthetic methods or by recombinant nucleic acid techniques.
Endogenous RNA polymerase of the treated cell may mediate
transcription in vivo, or cloned RNA polymerase can be used for
transcription in vitro. The RNAi constructs may include
modifications to either the phosphate-sugar backbone or the
nucleoside, e.g., to reduce susceptibility to cellular nucleases,
improve bioavailability, improve formulation characteristics,
and/or change other pharmacokinetic properties. For example, the
phosphodiester linkages of natural RNA may be modified to include
at least one of a nitrogen or sulfur heteroatom. Modifications in
RNA structure may be tailored to allow specific genetic inhibition
while avoiding a general response to dsRNA Likewise, bases may be
modified to block the activity of adenosine deaminase. The RNAi
construct may be produced enzymatically or by partial/total organic
synthesis, any modified ribonucleotide can be introduced by in
vitro enzymatic or organic synthesis.
[0052] In certain embodiments, the subject RNAi constructs are
"small interfering RNAs" or "siRNAs." These nucleic acids are
around 19-30 nucleotides in length, and even more preferably 21-23
nucleotides in length, e.g., corresponding in length to the
fragments generated by nuclease "dicing" of longer double-stranded
RNAs. The siRNAs are understood to recruit nuclease complexes and
guide the complexes to the target mRNA by pairing to the specific
sequences. As a result, the target mRNA is degraded by the
nucleases in the protein complex. In a particular embodiment, the
21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group.
[0053] The siRNA molecules can be obtained using a number of
techniques known to those of skill in the art. For example, the
siRNA can be chemically synthesized or recombinantly produced using
methods known in the art. For example, short sense and antisense
RNA oligomers can be synthesized and annealed to form
double-stranded RNA structures with 2-nucleotide overhangs at each
end (Caplen, et al. (2001) Proc Natl Acad Sci USA, 98:9742-9747;
Elbashir, et al. (2001) EMBO J, 20:6877-88). These double-stranded
siRNA structures can then be directly introduced to cells, either
by passive uptake or a delivery system of choice, such as described
below.
[0054] In certain embodiments, the siRNA constructs can be
generated by processing of longer double-stranded RNAs, for
example, in the presence of the enzyme dicer. In one embodiment,
the Drosophila in vitro system is used. In this embodiment, dsRNA
is combined with a soluble extract derived from Drosophila embryo,
thereby producing a combination. The combination is maintained
under conditions in which the dsRNA is processed to RNA molecules
of about 21 to about 23 nucleotides.
[0055] The siRNA molecules can be purified using a number of
techniques known to those of skill in the art. For example, gel
electrophoresis can be used to purify siRNAs. Alternatively,
non-denaturing methods, such as non-denaturing column
chromatography, can be used to purify the siRNA. In addition,
chromatography (e.g., size exclusion chromatography), glycerol
gradient centrifugation, affinity purification with antibody can be
used to purify siRNAs.
[0056] Examples of a siRNA molecule directed to an mRNA encoding a
C3a, C5a, C5aR, or C3aR polypeptide are known in the art. For
instance, human C3a, C3aR, and C5a siRNA is available from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Additionally, C5aR
siRNA is available from Qiagen, Inc. (Valencia, Calif.). siRNAs
directed to other complement components, including C3 and C5, are
known in the art.
[0057] In other embodiments, the RNAi construct can be in the form
of a long double-stranded RNA. In certain embodiments, the RNAi
construct is at least 25, 50, 100, 200, 300 or 400 bases. In
certain embodiments, the RNAi construct is 400-800 bases in length.
The double-stranded RNAs are digested intracellularly, e.g., to
produce siRNA sequences in the cell. However, use of long
double-stranded RNAs in vivo is not always practical, presumably
because of deleterious effects, which may be caused by the
sequence-independent dsRNA response. In such embodiments, the use
of local delivery systems and/or agents, which reduce the effects
of interferon or PKR are preferred.
[0058] In certain embodiments, the RNAi construct is in the form of
a hairpin structure (named as hairpin RNA). The hairpin RNAs can be
synthesized exogenously or can be formed by transcribing from RNA
polymerase III promoters in vivo. Examples of making and using such
hairpin RNAs for gene silencing in mammalian cells are described
in, for example, Paddison et al., Genes Dev, 2002, 16:948-58;
McCaffrey et al., Nature, 2002, 418:38-9; McManus et al., RNA,
2002, 8:842-50; Yu et al., Proc Natl Acad Sci USA, 2002,
99:6047-52). Preferably, such hairpin RNAs are engineered in cells
or in an animal to ensure continuous and stable suppression of a
desired gene. It is known in the art that siRNAs can be produced by
processing a hairpin RNA in the cell.
[0059] In yet other embodiments, a plasmid can be used to deliver
the double-stranded RNA, e.g., as a transcriptional product. In
such embodiments, the plasmid is designed to include a "coding
sequence" for each of the sense and antisense strands of the RNAi
construct. The coding sequences can be the same sequence, e.g.,
flanked by inverted promoters, or can be two separate sequences
each under transcriptional control of separate promoters. After the
coding sequence is transcribed, the complementary RNA transcripts
base-pair to form the double-stranded RNA.
[0060] PCT application WO01/77350 describes an exemplary vector for
bi-directional transcription of a transgene to yield both sense and
antisense RNA transcripts of the same transgene in a eukaryotic
cell. Accordingly, in certain embodiments, the present application
provides a recombinant vector having the following unique
characteristics: it comprises a viral replicon having two
overlapping transcription units arranged in an opposing orientation
and flanking a transgene for an RNAi construct of interest, wherein
the two overlapping transcription units yield both sense and
antisense RNA transcripts from the same transgene fragment in a
host cell.
[0061] In some embodiments, a lentiviral vector can be used for the
long-term expression of a siRNA, such as a short-hairpin RNA
(shRNA), to knockdown expression of C5, C3, C5aR, and/or C3aR in
CD4.sup.+ T cells and dendritic cells. Although there have been
some safety concerns about the use of lentiviral vectors for gene
therapy, self-inactivating lentiviral vectors are considered good
candidates for gene therapy as they readily transfect mammalian
cells.
[0062] It will be appreciated that RNAi constructs directed to
other complement components used in the formation of C5, C3, C5a,
C3a, C5 convertase, and/or C3 convertase can be used in accordance
with the method of the present application to reduce and/or inhibit
interactions between C5a and/or C3a with CSaR and C3aR on the
FoxP3.sup.+ Treg cells. The RNAi constructs can include, for
example, known Factor B, properdin, and Factor D siRNA that reduce
expression of Factor B, properdin, and Factor D.
[0063] Moreover, it will be appreciated that other antibodies,
small molecules, and/or peptides that reduce or inhibit the
formation of C5, C3, C5a, C3a, C5 convertase, and/or C3 convertase
and/or that reduce or inhibit interactions C5a and/or C3a with CSaR
and C3aR on naive CD4.sup.+ cells can be used as a complement
antagonist in accordance with the method of the present
application. These other complement antagonists can be administered
to the subject and/or naive CD4.sup.+ T cells at amount effective
to generate CD4.sup.+FoxP3.sup.+ Treg cells. Example of such other
complement antagonists include CSaR antagonists, such as
AcPhe[Orn-Pro-D-cyclohexylalanine-Trp-Arg, prednisolone, and
infliximab (Woodruff et al,. The Journal of Immunology, 2003, 171:
5514-5520), hexapeptide MeFKPdChaWr (March et al., Mol Pharmacol
65:868-879, 2004), PMX53 and PMX205, and
N-[(4-dimethylaminophenyl)methyl]-N-(4-isopropylphenyl)-7-methoxy-1,2,3,4-
-tetrahydronaphthalen-1- carboxamide hydrochloride (W-54011)
(Sumichika et al., J. Biol. Chem., Vol. 277, Issue 51, 49403-49407,
Dec. 20, 2002), and a C3aR antagonist, such as SB 290157 (Ratajczak
et al., Blood, 15 Mar. 2004, Vol. 103, No. 6, pp. 2071-2078).
[0064] The at least one complement antagonist can be administered
to the naive CD4.sup.+ T cells either in vivo or in vitro. In one
embodiment, the naive CD4.sup.+ T cells can be derived or isolated
from a mammalian subject, from a known cell line, or from some
other source. One example of a naive CD4.sup.+ T cells is a
CD45.sup.hiCD44.sup.low CD4.sup.+ T cell located in a mammalian
subject (e.g., human subject). The CD4.sup.+ T cell may be isolated
or, alternatively, associated with any number of identical,
similar, or different cell types. Where the cell comprises a
lymphocyte, for example, the lymphocyte may be associated with a
costimulatory cell, such as an APC or DC. By way of example, naive
CD4.sup.+ T cells (e.g., CD4.sup.+ FoxP3.sup.- T cells) for use in
the present application can be isolated from a peripheral blood
sample taken from a subject.
[0065] Once the naive CD4.sup.+ T cells are isolated, they can then
be cultured in a growth medium with optionally DCs or APCs.
"Cultured" and "maintained in culture" are interchangeably used
when referring to the in vitro cultivation of cells and include the
meaning of expansion or maintenance of a cell population under
conditions known to be optimal for cell growth.
[0066] A complement antagonist that substantially inhibits C3aR
and/or C5aR signaling can be administered to the cultured
CD4.sup.+FoxP3.sup.- T cells to induce differentiation of
CD4.sup.+FoxP3.sup.- T cells into CD4.sup.+FoxP3.sup.+ Treg cells.
In one exemplary embodiment, the CD4.sup.+FoxP3.sup.- T cells can
be cultured for 72 hours in a growth medium comprising a C3a
antagonist, a C5a antagonist, C3aR antagonist, and/or C5aR
antagonist as well as DCs to generate a population of
CD4.sup.+FoxP3.sup.+ Treg cells. The cell culture can be maintained
under culture conditions including suitable temperature, pH,
nutrients, and proper growth factors which favor the in vitro
expansion and survival of CD4.sup.+FoxP3.sup.+ Treg cells.
Additional agents that can be added to the cell culture to promote
the expansion and survival of CD4.sup.+FoxP3.sup.+ Treg cells
include, but are not limited to, anti-CD3/28 stimulating agents
(e.g., anti-CD3/CD28 Dynabead activation beads) TGF-.beta., and
IL-2.
[0067] The CD4.sup.+FoxP3.sup.+ Treg cells produced by methods of
the present application have the immunoregulatory characteristics
of wild type Treg cells. Accordingly, it is further contemplated
that the CD4.sup.+FoxP3.sup.+ Treg cells generated by the methods
of the application can be used to prevent local and systemic organ
and tissue destruction in cell therapies aimed at alleviating T
cell mediated disorders or diseases.
[0068] The term "T cell mediated disease" or "T cell mediated
disorder" refers to diseases and disorders in which an aberrant
immune reaction involves T cell-mediated immune mechanisms, as
opposed to humoral immune mechanisms. T cell mediated diseases
contemplated by the present application also include T cell
mediated autoimmune diseases or disorders. The language "autoimmune
disorder" is intended to include disorders in which the immune
system of a subject reacts to autoantigens, such that significant
tissue or cell destruction occurs in the subject. The term
"autoantigen" is intended to include any antigen of a subject that
is recognized by the immune system of the subject. The terms
"autoantigen" and "self-antigen" are used interchangeably herein.
The term "self" as used herein is intended to mean any component of
a subject and includes molecules, cells, and organs. Autoantigens
may be peptides, nucleic acids, or other biological substances.
[0069] Thus, the methods of the application pertain to treatments
of immune disorders in which tissue destruction is primarily
mediated through activated T cells and immune cells. For example,
the methods of the present application can be used in the treatment
of autoimmune conditions or diseases, such as inflammatory
diseases, including but not limited to achlorhydra autoimmune
active chronic hepatitis, acute disseminated encephalomyelitis,
acute hemorrhagic leukoencephalitis, Addison's disease,
agammaglobulinemia, alopecia areata, Alzheimer's disease,
amyotrophic lateral sclerosis, ankylosing spondylitis, anti-gbm/tbm
nephritis, antiphospholipid syndrome, antisynthetase syndrome,
aplastic anemia, arthritis, atopic allergy, atopic dermatitis,
autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune
hepatitis, autoimmune inner ear disease, autoimmune
lymphoproliferative syndrome, autoimmune peripheral neuropathy,
autoimmune polyendocrine syndrome, autoimmune progesterone
dermatitis, autoimmune thrombocytopenia purpura, autoimmune
uveitis, balo disease/balo concentric sclerosis, Bechets syndrome,
Berger's disease, Bickerstaff's encephalitis, blau syndrome,
bullous pemphigoid, Castleman's disease, Chagas disease, chronic
fatigue immune dysfunction syndrome, chronic inflammatory
demyelinating polyneuropathy, chronic lyme disease, chronic
obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial
pemphigoid, coeliac disease, Cogan syndrome, cold agglutinin
disease, cranial arteritis, crest syndrome, Crohns disease,
Cushing's syndrome, Dego's disease, Dercum's disease, dermatitis
herpetiformis, dermatomyositis, diabetes mellitus type 1,
Dressler's syndrome, discoid lupus erythematosus, eczema,
endometriosis, enthesitis-related arthritis, eosinophilic
fasciitis, epidermolysis bullosa acquisita, essential mixed
cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans
progressive, fibromyalgia, fibromyositis, fibrosing aveolitis,
gastritis, gastrointestinal pemphigoid, giant cell arteritis,
glomerulonephritis, Goodpasture's syndrome, Graves' disease,
Guillain-barre syndrome (gbs), Hashimoto's encephalitis,
Hashimoto's thyroiditis, henoch-schonlein purpura, hidradenitis
suppurativa, Hughes syndrome, inflammatory bowel disease (IBD),
idiopathic inflammatory demyelinating diseases, idiopathic
pulmonary fibrosis, idiopathic thrombocytopenic purpura, iga
nephropathy, inflammatory demyelinating polyneuopathy, interstitial
cystitis, irritable bowel syndrome (ibs), Kawasaki's disease,
lichen planus, Lou Gehrig's disease, lupoid hepatitis, lupus
erythematosus, meniere's disease, microscopic polyangiitis, mixed
connective tissue disease, morphea, multiple myeloma, multiple
sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis
optica, neuromyotonia, occular cicatricial pemphigoid, opsoclonus
myoclonus syndrome, ord thyroiditis, Parkinson's disease, pars
planitis, pemphigus, pemphigus vulgaris, pernicious anaemia,
polymyalgia rheumatic, polymyositis, primary biliary cirrhosis,
primary sclerosing cholangitis, progressive inflammatory
neuropathy, psoriasis, psoriatic arthritis, raynaud phenomenon,
relapsing polychondritis, Reiter's syndrome, rheumatoid arthritis,
rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome,
Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome,
spondyloarthropathy, sticky blood syndrome, still's disease, stiff
person syndrome, sydenham chorea, sweet syndrome, takayasu's
arteritis, temporal arteritis, transverse myelitis, ulcerative
colitis, undifferentiated connective tissue disease,
undifferentiated spondyloarthropathy, vasculitis, vitiligo,
Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich
syndrome as well as hypersensitivity reactions of the skin,
atherosclerosis, ischemia-reperfusion injury, myocardial
infarction, and restenosis. The methods of the present application
can also be used for the prevention or treatment of the acute
rejection of transplanted organs where administration of a
therapeutic described herein, may occur during the acute period
following transplantation or as long-term post transplantation
therapy.
[0070] In a method of treating a T cell mediated disease or
disorder in a subject, a therapeutically effective amount of the
inventive CD4.sup.+FoxP3.sup.+ Treg cells to the subject. The
therapeutically effective amount of CD4.sup.+FoxP3.sup.+ Treg cells
to be administered to a subject can be determined by a practitioner
based upon such factors as the levels of CD4.sup.+FoxP3.sup.+ Treg
cells induction achieved in vitro, the mode of administration,
and/or the particular T cell mediated disease to be treated.
[0071] The CD4.sup.+FoxP3.sup.+ Treg cells may originate from a
subject into which they are implanted (reimplantation) or from
elsewhere (transplantation). In some aspects, a subject is
administered CD4.sup.+FoxP3.sup.+ Treg cells derived from the
subject's own body because the risk of transmission of an infection
such as HIV is eliminated and the risk of triggering an immune
system-mediated rejection reaction is reduced.
[0072] In general, the CD4.sup.+FoxP3.sup.+ Treg cells are
administered (e.g., implanted) into the mammalian subject by
methods well known in the art. The CD4.sup.+FoxP3.sup.+ Treg cells
of the present application may be introduced into the subject by
any suitable route whether that route is enteral or parenteral, for
example, intravenous or intramuscular. In one exemplary embodiment,
CD4.sup.+FoxP3.sup.+ Tregs cells are administered directly to an
area of a T cell mediated disease.
[0073] In another embodiment of the application, the naive
CD4.sup.+ T cell can comprise a naive CD4.sup.+ T cell in the
subject and the complement antagonist can be used to treat a T cell
mediated disease in the subject. The at least one complement
antagonist can be administered to the subject to treat the T cell
mediated disease in the subject using any one or combination of
known techniques.
[0074] In one aspect of the application, the complement antagonist
can be administered directly or locally to a site of T cell
mediated disease in the subject. Local or direct administration of
the complement antagonist into and/or about the periphery of the
disease site is advantageous because the complement antagonist
localizes at the disease site being treated and does not
substantially affect the subject's innate complement system.
[0075] In another aspect of the application, the complement
antagonist can be administered to the subject systemically by, for
example, intravenous, intraarterial, intraperitoneal,
intramuscular, subcutaneous, intrapleural, intrathecal, oral or
nasal route, to treat the T cell mediated disease or related
disorder in the subject. When administered systemically, the
complement antagonist can be targeted to a disease site to ensure
that the complement antagonist does not adversely affect other
normal cells expressing C5aR and/or C3aR, and to potentially
mitigate adverse systemic effects on the subject's complement
system. Several systems have been developed in order to restrict
the delivery of the complement antagonist to the disease site. With
the identification of cells specific receptors and antigens on
mammalian cells, it is possible to actively target the complement
antagonist using ligand or antibody bearing delivery systems.
Alternatively, the complement antagonist can be loaded on a high
capacity drug carriers, such as liposomes or conjugated to polymer
carriers that are either directly conjugated to targeting
proteins/peptides or derivatised with adapters conjugated to a
targeting moiety.
[0076] Examples of antibodies which can be potentially conjugated
to the complement antagonist to target the complement antagonist to
the T cell mediated disease site include, but are not limited to,
anti-CD20 antibodies (e.g., Rituxan, Bexxar, Zevalin),
anti-Her2/neu antibodies (e.g., Herceptin), anti-CD33 antibodies
(e.g., Mylotarg), anti-CD52 antibodies (e.g., Campath), anti-CD22
antibodies, anti-CD25 antibodies, anti-CTLA-4 antibodies,
anti-EGF-R antibodies (e.g. Erbitux), anti-VEGF antibodies (e.g.
Avastin, VEGF Trap) anti-HLA-DR10.beta. antibodies, anti-MUC1
antibodies, anti-CD40 antibodies (e.g. CP-870,893), anti-Treg cell
antibodies (e.g., MDX-010, CP-675,206), anti-GITR antibodies,
anti-CCL22 antibodies, and the like.
[0077] The complement antagonist, whether administered locally
and/or systemically, can also be provided in a pharmaceutically
acceptable composition. The phrase "pharmaceutically acceptable"
should be understood to mean a material which is not biologically
or otherwise undesirable, i.e., the material may be incorporated
into an antiviral composition and administered to a subject without
causing any undesirable biological effects or interacting in a
deleterious manner with any of the other components of the
composition in which it is contained. When the term
"pharmaceutically acceptable" is used to refer to a pharmaceutical
carrier or excipient, it is implied that the carrier or excipient
has met the required standards of toxicological and manufacturing
testing or that it is included on the Inactive Ingredient Guide
prepared by the U.S. Food and Drug administration. Sterile
phosphate-buffered saline is one example of a pharmaceutically
acceptable carrier. Other suitable carriers are well-known to those
in the art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES,
19th Ed. (1995), and later editions.
[0078] In general, the dosage of the at least one complement
antagonist will vary depending upon such factors as the subject's
age, weight, height, sex, general medical condition and previous
medical history. Typically, it is desirable to provide the subject
with a dosage of the at least one complement antagonist which is in
the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body
weight of patient), although a lower or higher dosage also may be
administered as circumstances dictate. The specific dosage or
amount of complement antagonist administered to a naive CD4.sup.+T
cell (e.g., a human CD45.sup.hiCD44.sup.low CD4.sup.+ T cell,) will
be that amount effective to reduce or inhibit C5a/C3a-C5aR/C3aR
interactions.
[0079] In an example of the method, a therapeutically effective
amount of a pharmaceutical composition comprising a first antibody
directed against C3aR and a second antibody directed against C5aR
can be administered to a naive CD4.sup.+ T cell or a subject having
a T cell mediated disease. The pharmaceutical composition can be
administered to the subject intravenously using, for example, a
hypodermic needle and syringe. Upon administration of the
pharmaceutical composition to the subject, the first and second
antibodies can respectively bind to C3aR and C5aR on at least one T
lymphocyte. Binding of the first and second antibodies can
effectively inhibit or reduce the ability of C3a and C5a to
respectively bind C3aR and C5aR. Consequently, C5a/C3a-C5aR/C3aR
signaling can be reduced or eliminated such that the at least one
naive CD4.sup.+ T cell undergoes differentiation into a
CD.sup.+FoxP3.sup.+ Treg cell.
[0080] The following examples are for the purpose of illustration
only and are not intended to limit the scope of the claims, which
are appended hereto.
EXAMPLE 1
[0081] This Example shows that quiescence of C3aR/C5aR signaling
between DC-CD4.sup.+ cells causes suppression of T cell activation
by simultaneously inducing potent FoxP3.sup.+ iTregs. As a first
test of how DC-CD4.sup.+ cell C3aR/C5aR signal transduction impacts
iTreg induction, we stimulated naive CD4.sup.+ cells from mice in
which the FoxP3 gene promoter is linked to GFP (FoxP3-GFP mice)
with anti-CD3, IL-2 and WT DCs without TGF-.beta.1 in the presence
of C3aR/C5aR pharmaceutical antagonists (C3aR-A/C5aR-A),
anti-C3a/C5a mAbs, or controls. Both treatments induced FoxP3.sup.+
cells (FIG. 2A). The sorted GFP cells failed to produce IL-2
following PMA and ionomycin treatment and were anergic to
anti-CD3/28 stimulation consistent the properties of iTregs. They
conferred .about.4-fold>suppression in mixtures of ova-primed
DCs and CellTracker Red-labeled OT-II cells than sorted GFP cells
conventionally induced with exogenous TGF-.beta.1 (FIG. 2B).
Stimulation of naive C3aR.sup.-/-C5aR.sup.-/- or
C3.sup.-/-C5.sup.-/- CD4.sup.+ cells with anti-CD3, DCs and IL-2
similarly resulted in the induction of FoxP3.sup.+ cells with
potent suppressor function (FIG. 2B). Addition of C5a blocked iTreg
induction and suppressor function when naive C3.sup.-/-C5.sup.-/-
CD4.sup.+ cells were stimulated (FIG. 2B).
[0082] Analyses of day 3 supernatants of mixtures that contained
C3aR/C5aR antagonized FoxP3-GFP CD4.sup.+ cells or
C3aR.sup.-/-C5aR.sup.-/- CD4.sup.+ cells showed abundant
TGF-.beta.1 as well as IL-10, in contrast to little of either
cytokine but IL-6 in WT CD4.sup.+ cell containing mixtures (FIG.
2C). Culturing of flow sorted DCs and CD4.sup.+ cells showed that
both cytokines were being produced by both partners, more IL-10 by
the CD4.sup.+ cells and more TGF-.beta.1 by the DCs. In contrast to
stimulating naive WT cells with anti-CD3/28 plus IL-2 without DCs
which generated IL-6, identical stimulation of naive
C3aR.sup.-/-C5aR.sup.-/- cells generated TGF-.beta.1/IL-10, albeit
12-/20-fold less than when WT DCs were present. Inclusion of
C3aR.sup.-/-C5aR.sup.-/- DCs [which do not produce C3a/C5a]
increased TGF-.beta.1/IL-10 production and iTreg numbers about
2-fold more than inclusion of WT DCs (FIG. 2D), indicating that
while absent C3aR/C5aR transduction in stimulated naive CD4.sup.+
cells is sufficient to induce TGF-.beta.1/IL-10, absent C3aR/C5aR
transduction in interacting DCs markedly augments TGF-.beta.1/IL-10
production by both partners.
[0083] To establish whether the endogenous TGF-.beta.1 that is
produced in the absence of C3aR/C5aR signaling promotes iTreg
induction, we incubated naive C3aR.sup.-/-C5aR.sup.-/- CD4.sup.+
cells with anti-CD3, IL-2, and DCs in the presence and absence of
an anti-TGF-.beta.1 blocking mAb. The presence of the mAb abrogated
FoxP3.sup.+ cell induction (FIG. 2E). To establish whether the
endogenous TGF-.beta.1 enters into an autocrine signaling loop that
amplifies endogenous TGF-.beta.1 production, we added specific
antagonists of TGF-.beta.R1, or Smad3, a requisite partner of Smad4
and transcription factor (TF) essential for TGF-.beta.1 gene
transcription. Both agents markedly reduced TGF-.beta.1 (FIG. 2F)
and IL-10 production in both the CD4.sup.+ cells and DCs (FIG. 2G)
and essentially abrogated FoxP3.sup.+ cell induction (FIG. 2E),
indicating that IL-10 production and FoxP3.sup.+ cell induction are
dependent on auto-inductive TGF-.beta.1 signaling.
[0084] Comparison of the phenotypes of WT DCs that derived from the
co cultures with C3aR.sup.-/-C5aR.sup.-/- or WT CD4.sup.+ cells
(without added TGF-.beta.1) showed that DCs incubated with
stimulated C3aR.sup.-/-C5aR.sup.-/- CD4.sup.+ cells (FIG. 3A)
expressed 11-fold lower levels of costimulatory B7-1, 10-fold lower
costimulatory B7-2, and 2.5-fold lower levels of CD40, while only
small changes (increases) in coinhibitory PD-L1 (P=0.07) and ICOS-L
(P=0.013) were apparent. Similarly FoxP3.sup.+ cells deriving from
the C3aR.sup.-/-C5aR.sup.-/- CD4.sup.+ cells expressed markedly
decreased levels of costimulatory CD28 and CD40 ligand (CD40L),
similar levels of coinhibitory PD-1 and ICOS, and increased levels
of stably surface expressed CTLA-4 (FIG. 3A). Thus, there is a
direct correlation between the absence of C3aR/C5aR signal
transduction and the downregulation of co-stimulatory molecule
expression on DCs and CD4.sup.+ cells [consistent with our earlier
results] and the upregulation of immunosuppressive CTLA-4 on
CD4.sup.+ cells. Comparison of iTregs conventionally generated with
exogenous TGF-.beta.1 to those generated by C3aR/C3aR antagonism
showed 3-6-fold higher costimulatory molecule expression (FIG. 3B)
more closely resembling the phenotype of Th17 effector cells.
[0085] To determine the significance of the changes in surface
phenotypes of DCs and CD4.sup.+ cells in the absence of C3aR/C5aR
signals on the induction of FoxP3.sup.+ T cells derived from the
naive C3aR.sup.-/-C5aR.sup.-/- responder CD4.sup.+ cells, we added
anti-PD-Ll or anti-ICOS-L mAb to the co-cultures. The immunological
blockade in both cases caused marked inhibition of both
TGF-.beta./IL-10 production and FoxP3.sup.+ cell induction (FIG.
3C). Comparable results were obtained when we substituted
PD-L1.sup.-/- DCs for WT DCs or naive PD-1.sup.-/- or LCOS
CD4.sup.+ cells for naive WT CD4.sup.+ cells (FIG. 3D). Interfering
with CTLA-4/CD80OCD86 interactions also inhibited iTreg induction
(FIG. 3C). Collectively, these findings that pertain in the absence
of C3aR/C5aR signaling are consistent with reports that the
interactions of PD-1/PD-L1, ICOS/ICOS-L as well as CTLA-4/CD80-CD86
all play roles in amplifying TGF-.beta.1/IL-10 production and
inducing iTregs
[0086] For C3aR/C5aR signaling to be absent, local complement
production by interacting DCs and CD4.sup.+ cells must not occur or
be suppressed. The above findings connecting absent C3aR/C5aR
signaling with iTreg commitment, together our previous findings
connecting potentiated C3aR/C5aR signaling with Th1 and Th17
commitment suggested that the divergent effects of TGF-.beta.1 vs
IL-6 on naive CD4.sup.+ cell differentiation might be
mechanistically linked to opposing effects on local complement
production by CD4.sup.+ cells. As a first test of this, we
incubated anti-CD3/CD28 stimulated CD4.sup.+ cells in the presence
and absence of TGF-.beta.1. The added TGF-.beta.1 abolished
transcription of all the complement genes connected with C3aR/C5aR
signaling (FIG. 3E) and induced FoxP3.sup.+ cells (FIGS. 2A, D, E).
To test how IL-6 affects CD4.sup.+ cell complement production, we
added IL-6 to naive CD4.sup.+ cells. In contrast to TGF-.beta.1,
IL-6 (or IL-6 in combination with TGF-.beta.1) upregulated
complement mRNA transcription (FIG. 3F). Consistent with the IL-6
induction of naive CD4.sup.+ cell complement gene transcription,
IL-6 (as well as IL-6 plus TGF-.beta.1) evoked DC-CD4.sup.+ cell
C3a/C5a production (FIG. 3G) and upregulated C3aR/C5aR expression
(FIG. 3H), whereas TGF-.beta.1 alone did neither. In accordance
with the absence of C3a/C5a production and consequent C3aR/C5aR
signaling being required for iTreg induction, adding IL-6 or C5a to
anti-CD3/28 stimulated naive CD4.sup.+ cells (FIG. 3I) inhibited
FoxP3 mRNA transcription. These data thus argue that DC control of
naive CD4.sup.+ cell C3aR/C5aR signaling is one switch through
which TGF-.beta.1 alone vs IL-6 (or IL-6 plus TGF-.beta.1 in
combination) bias between iTreg vs Th1/Th17 effector cell
commitment.
[0087] To establish whether the above findings apply in vivo, we
used four systems: 1) In the first, we tested whether C3aR/C5aR
antagonism in CD4.sup.+ cells, in recipients, or both is(are)
involved in iTreg induction. We adoptively transferred
(unfractionated) OT-II cells or C3aR.sup.-/-C5aR.sup.-/- OT-II
cells iv into WT or C3aRC5aR recipients, two days after which we
immunized recipient mice with ovalbumin (ova) in CFA. Five days
thereafter, more ova specific FoxP3.sup.+ OT-II cells (identified
by anti-OT-II TCR mAbs) were present in the spleen when either the
recipient or OT-II cells were C3aR/C5aR deficient and most
(2.5-fold more) when both were C3aR/C5aR deficient. The production
of TGF-.beta.1 and IL-10 by cultured spleen cells of the
C3aR.sup.-/-C5aR.sup.-/- to C3aR.sup.-/-C5aR.sup.-/- and WT to WT
donor-recipient combinations showed the same pattern. To document
that absent C3aR/C5aR signaling in naive CD4.sup.+ cells is
essential, we adoptively transferred CFSE-labeled naive
(CD25.sup.-) OT-II cells as a source of effectors into WT
recipients. Two days later, at the time of ova immunization, we
co-administered CellTracker Red naive (CD25.sup.-) Daf1.sup.-/-
OT-II cells (in which DC C3aR/C5aR signaling is potentiated) or
CellTracker Red naive (CD25.sup.-) C3aR.sup.-/-C5aR.sup.-/- OT-II
cells (in which C3aR/C5aR signaling is precluded) as a source of
iTregs. Five days later CellTracker Red labeled
C3aR.sup.-/-C5aR.sup.-/- OT-II cells but not CellTracker Red
labeled Daft1.sup.-/- OT-II cells were CD25.sup.+ (FIG. 4A). The
CellTracker Red labeled C3aR.sup.-/-C5aR.sup.-/- OT-II cells
suppressed proliferation of the CFSE labeled responder OT-II cells
9-fold more efficiently than the CellTracker Red labeled
Daf1.sup.-/- OT-II cells (FIG. 4B). A repeat experiment utilizing
WT mice in place of Daft1.sup.-/- yielded the same results. To
document that absent C3aR/C5aR signaling in recipients is
essential, we adoptively transferred CellTracker Violet labeled
CD25.sup.- OT-II cells as a source of T effectors to WT or
C3aR.sup.-/-C5aR.sup.-/- recipients. Two days later, at the time of
immunization, we adoptively transferred CD25.sup.- OT-II FoxP3-GFP
cells. Five days later, 18% of splenic CD4.sup.+ cells were
FoxP3.sup.+ cells in C3aR.sup.-/-C5aR.sup.-/- recipients as
compared to 10% in WTs (FIG. 4C). Additionally, 10-fold greater
suppression of CellTracker Violet dilution occurred in the
C3aR.sup.-/-C5aR.sup.-/- recipient than in the WT recipient (FIG.
4D).
[0088] In the second system, we compared the in vivo suppressive
activity of iTregs generated by C3aR/C5aR antagonism (no added
TGF-.beta.1) to that of iTregs conventionally generated (with added
TGF-.beta.1). We adoptively transferred 10.sup.6 CellTracker
Red-labeled naive (CD25.sup.-) OT-II cells to WT mice. Two days
later, we immunized the mice with ova in CFA and at the same time
transferred equal numbers of sorted green (FoxP3.sup.+) cells
derived from stimulated naive CD25.sup.-FoxP3-GFP CD4.sup.+ cells
alternatively treated with C3aR-A/C5aR-A or with TGF-.beta.1.
Cell-Tracker Red dilution of the responder OT-II cells in lymph
nodes 5 days later showed that sorted (green) FoxP3.sup.+ cells
generated with C3aR-A/C5aR-A exerted >2 fold more suppression
than those generated with exogenous TGF-.beta.1 (FIG. 4E).
[0089] We designed a third model to compare the suppressive
function of antigen specific vs antigen non-specific iTregs
generated by C3aR/C5aR antagonism. We transferred 1.times.10.sup.6
CD25.sup.- OT-II cells to WT recipients as a source of responders.
At the same time, we co transferred increasing numbers of iTregs
generated by C3aR-A/C5aR-A treatment of either 1) CellTracker
Violet labeled naive CD25.sup.- OT-II (specific for ova) CD4.sup.+
cells, or 2) nonrelated CFSE labeled naive CD25.sup.-2D2 (specific
for MOG.sub.35-55) CD4.sup.+ cells. One day thereafter, we
immunized both recipients with ova in CFA. Flow cytometry of spleen
cells five days later gating on CFSE and CellTracker Violet (FIG.
4F) showed that ova specific CD25.sup.+ OT-II cells (generated by
anti-CD3, IL-2 and C3aR/C5aR antagonism) exerted robust suppression
(reduced dividers to 11% at an injection ratio of 4:1) whereas the
TCR nonspecific CD25.sup.+ (FoxP3.sup.+) 2D2 iTregs generated the
same way exerted less suppression (reduced dividers to 65%). While
2D2 cells express a clonal TCR for MOG.sub.35-55, they were
activated ex vivo with anti-CD3. Consequently, while they exerted
less suppressive activity than OT-II cells which express a clonal
TCR for ova.sub.323-339, their suppressive activity was similar to
FoxP3.sup.+ Tregs induced from polyclonal naive
(CD25.sup.-)CD4.sup.+ cells (see FIG. 4A). To study whether iTregs
generated by C3aR/C5aR antagonism maintained their phenotype and
suppressive capacity, iTregs were induced for 3 days with
C3aR-A/C5aR-A as before and cells were sorted for FoxP3.sup.+
expression. Following an additional 10 days of incubation with
anti-CD3/28 plus IL-2, >95% remained FoxP3.sup.+ and resorted
cells retained their full suppressive activity.
[0090] In the fourth system, we evaluated how efficacy of iTreg
induction by C3aR/C5aR antagonism for ameliorating autoimmunity
using the EAE model of multiple sclerosis. We first performed
studies with Rag-2.sup.4 recipients devoid of nTregs and T effector
cells to obviate any influence of 1) endogenous nTregs and/or
iTregs and 2) any possibility that FoxP3.sup.+ cells themselves are
not being measured. To prepare donor T effector and iTregs, we
immunized FoxP3-GFP mice with MOG.sub.35-55 in CFA. After
establishment of disease (clinical score>2), we isolated
CD4.sup.+ cells from lymph nodes and the spleen and removed green
cells (endogenous nTregs and iTregs) by flow sorting. We then
isolated CD25.sup.- cells from the nongreen cell population. We
treated half of these cells with C3aR-A/C5aR-A to produce green
iTregs. In one set of Rag-2.sup.-/- recipients (5 mice), we
administered 1.times.10.sup.6 cells from the other half of the
nongreen cells (containing T effector cells) by themselves. In a
second set of the Rag-2.sup.-/- recipients (5 mice), we
administered an identical aliquot (1.times.10.sup.6) of the same
effector cell population together with 1.times.10.sup.6 of the
rested C3aR-A/C5aR-A induced green iTregs. Monitoring the animals
thereafter showed that while the Rag-2.sup.-/- recipients that
received untreated nongreen CD4.sup.+ cells containing T effectors
by themselves showed progressive weakness and weight loss, the
Rag-2.sup.-/- mice that additionally received C3aR-A/C5aR-A induced
green iTregs showed markedly less disease which gradually declined
and their weights recovered (FIG. 5AB). At day 7 (FIG. 5C) post
adoptive transfer, green cells were detectable in lymph nodes and
spinal cords of these mice but not in those that received the
untreated nongreen population by itself. Identical results were
obtained in a repeat experiment in which we pre-immunized the
Rag-2.sup.-/- recipient mice with MOG.sub.35-55in CFA.
[0091] Armed with the above control data, we next examined how
DC-CD4.sup.+ cell C3aR/C5aR signaling is interconnected with iTregs
and EAE disease severity in immune sufficient mice. We induced EAE
in WT and in C3aR.sup.-/-C5aR.sup.-/- mice. Ten days later, less
IFN-.gamma. and more TGF-.beta.1/IL-10 and more FoxP3.sup.+ cells
(FIG. 5D) were present in lymph nodes of C3aR.sup.-/-C5aR.sup.-/-
mice than in WTs. Splenic CD4.sup.+ cells from
C3aR.sup.-/-C5aR.sup.-/- mice (isolated by CD4.sup.+ negative
selection followed by sorting on CD4.sup.+CD25.sup.+ cells)
contained 2-fold more FoxP3.sup.+ cells and conferred
5-fold>suppression than those from WTs (FIG. 5E). In a second
experiment, we established EAE in FoxP3-GFP mice and treated sick
mice (scores >2) with adoptively transferred FoxP3.sup.+ (green)
cells generated ex vivo by incubating naive (CD25.sup.-) CD4.sup.+
cells from FoxP3-GFP mice with C3aR-A/C5aR-A. Following adoptive
transfer of 1.times.10.sup.6 green FoxP3.sup.+ cells, clinical
scores decreased (FIG. 5F) and weights increased. At days 7 and 14
post adoptive transfer, green cells were detectable in lymph nodes
and spinal cords.
[0092] To test whether C3aR/C5aR antagonism is more effective than
exogenous TGF-.beta.1 in preparing human iTregs, we incubated human
CD45RA.sup.+CD25.sup.-CD4.sup.+ cells with anti-CD3, IL-2 and
C3aR-A/C5aR-A for 3 days and quantified FoxP3.sup.+ cells. After
verifying that CD25.sup.hi cells were >95% FoxP3.sup.+ using
multiple anti-FoxP3 Abs and that PMA plus ionomycin treatment of
the sorted CD25.sup.+ cells produced no IL-2 and that the sorted
cells were anergic compared to identically treated naive CD4.sup.+
cells, we added an equal number of sorted CD25.sup.+ cells (that
stained FoxP3.sup.+ in a parallel aliquot) prepared this way or
prepared with exogenous TGF-.beta.1 to mixtures of DCs, anti-CD3,
and CFSE-labeled CD25.sup.-CD4.sup.+ cells from the same
individual. The CD25.sup.+ (FoxP3.sup.+) cells prepared by
C3aR/C5aR antagonism exerted robust suppression (FIG. 5G) whereas
those prepared with TGF-.beta.1 had little effect. As found with
mouse cells (FIG. 3B), phenotyping of the iTregs prepared with
TGF-.beta.1 showed costimulatory molecules, while those prepared by
C3aR/C5aR antagonism did not.
[0093] The above data provide several new insights concerning how T
cell responses are controlled: 1) They demonstrate that absent
C3aR/C5aR signal transduction in naive CD4.sup.+ cells during their
interaction with DC partners that do not produce C3a/C5a leads to
the induction of iTregs. Taken together our findings show that DC
control of this GPCR signaling in naive CD4.sup.+ cells serves as
one switch which regulates whether tolerogenic or effector T cell
responses are mounted in response to peptide bearing DCs. 2) They
provide a molecular mechanism for the findings that TGF-.beta.1
biases toward iTreg lineage commitment, whereas IL-6 or IL-6 plus
TGF-.beta.1 bias toward Th1 and Th17 lineage commitment. 3) They
explain the source of TGF-.beta.1 that evokes iTreg induction and
that of IL-10 which is involved in Treg immunosuppressive function.
4) They demonstrate that while iTregs that arise in the absence of
C3aR/C5aR signaling do not express costimulatory molecules, iTregs
induced by exogenously added TGF-.beta.1 do, and potentially
explain one mechanism of their more robust suppressive function and
greater stability. 5) They provide more information on the
functional effects of coupling of B7 family co-inhibitory molecules
to their CD4.sup.+cell counter-receptors. 6) They open a previously
unrecognized avenue by which iTregs can be induced ex vivo as well
as in vivo.
Materials and Methods
Reagents and antibodies
[0094] Murine C5a was from Cell Sciences, Inc (Canton, Me.). Mouse
C3a and C5a mAbs were from R&D Systems (Minneapolis, Minn.).
Mouse IL-2, IL-10, IFN-.gamma., human IL-2, IL-10, IFN-.gamma. and
human TGF-.beta.1 were from Prospec Bio (Rehovot, Isreal).
Antibodies against mouse B7-1, B7-2, C5aR, PD-1, ICOSL, ICOS,
PD-L1, and CTLA-4 were from BD Biosciences (San Diego, Calif.).
Anti-CD40L mAb was from BioExpress (West Lebanon, N.H.). Anti-C3aR
was purchased from Santa Cruz Biotech (Santa Cruz, Calif.). Mouse
and Human anti-FoxP3 mAbs and Treg staining kits were purchased
from eBiosciences (San Diego, Calif.). CFSE, CellTracker Red, and
CellTracker Violet were purchased from Invitrogen (San Diego,
Calif.) and used per the manufacturer's instructions.
Animals
[0095] C57BL/6, OT-II (specific for OVA.sub.323-339 plus
I-A.sup.b), Rag2.sup.-/-, C3.sup.-/-, and C5 deficient mice were
from Jackson labs (Bar Harbor, Me.). C3.sup.-/- mice and
C3aR.sup.-/- and C5aR.sup.-/- were gifts of Dr. Michael Carroll and
Dr. Craig Gerard (Harvard Medical School and Childrens Hospital,
Boston, Mass.). C5.sup.-/-C3.sup.-/- mice were generated by
crossing C5 deficient B10.2 mice with C57BL/6 congenic C3' mice.
C.5.sup.+/+C3.sup.+/+ littermates used as controls displayed
comparable results to the studies with C57BL/6 mice as controls.
All studies were approved by the Case Western Reserve University
Institutional Animal Care and Use Center (IACUC).
RNA Purification, cDNA Synthesis, and qPCR
[0096] Cells were purified for 5 min at 20.degree. C. using Trizol
(Invitrogen, Carlsbad, Calif.) according to the manufacturer. When
C3aR and C5aR mRNAs were analyzed, preparations were treated with
DNase I (standard protocol) to remove genomic DNA. cDNAs were
synthesized by incubating 20 .mu.l of mRNAs in Sprint PowerScript
Single Shots (Clontech, Mountain View, Calif.). Ten .sub.ill of
diluted cDNA were mixed with 2 .mu.l of primer and 10 .mu.l SYBR
green master mix (Applied Biosystems, Foster City, Calif.) and
assayed in triplicate on an ABI prism 7000 cycler. In all assays
fold increases are relative to each basal level and standardized to
Actin.
Murine DCs and T Cell Isolations
[0097] CD4.sup.+ T cells were isolated from spleens and lymph nodes
using the CD4.sup.+ negative selection cocktail from Miltenyi
(Bergisch Gladbach, Germany) per the manufacturer's instructions.
CD11c.sup.+ cells were isolated from spleens using the positive
selection cocktail from Miltenyi per the manufacturer's
instructions. Both were purified using the Automacs Pro.
Immunizations and Flow Cytometry
[0098] Mice were immunized s.c. with OVA.sub.323-339 or
MOG.sub.35-55 peptide as described. All antibodies unless otherwise
noted were purchased from BD Biosciences (San Diego, Calif.),
stained cells analyzed on a Becton-Dickinson LSR I or II.
Anti-CD3 and anti-CD28 Stimulations
[0099] Cells were stimulated three different ways: 1) 1 .mu.g/ml
anti-CD3 and/or anti-CD28 (BD Biosciences, San Diego, Calif.) in
complete RPMI 1640 +10% FBS; 2) 1 .mu.g/ml anti-CD3 and CD11c.sup.+
DCs in complete RPMI 1640; or 3) with 1:1 ratio of CD3/CD28 coated
Dynabeads (Invitrogen) per the manufacturer's instructions.
FoxP3.sup.+ Staining Assays
[0100] For both Human and Murine Treg staining assays, the relevant
FoxP3.sup.+ T regulatory staining kits (236A/E7 for Human and
FJK-16s for mouse) were purchased from eBiosciences and used per
the manufacturer's instructions.
Murine Treg Suppression Assays
[0101] CFSE-labeled CD4.sup.+CD25.sup.- T cells (1.times.10.sup.6)
sorted by FACS were stimulated with 1.times.10.sup.5 autologous
CD11c.sup.+ DCs and 1 .mu.g/mL anti-CD3 mAb (BD Biosciences) alone
or with various numbers of suppressor cells. The cells were
cultured for 3 days in 96-well flat-bottom plates and CFSE dilution
was analyzed by FACS.
ELISAs
[0102] For C5a, C3a, IL-2, IL-10, and IL-6, capture and
biotinylated detection Ab pairs were purchased from BD Biosciences.
For TGF-.beta.1, the human Ab pair (cross reacts with mouse) was
purchased from eBiosciences. 96-well Costar 3590 plates (Corning,
N.Y.) were coated with 100 .mu.l of 2 .mu.g/ml capture Ab (either
C5a, C3a, IL-2, IL-10, IL-6 or TGF-.beta.1) overnight at 4.degree.
C. Following washing, 100 .mu.l of culture supernatants (acid
activated in the case of TGF-.beta.1) were added to the plates and
again were incubated overnight at 4.degree. C. Well were washed,
then 100 of 2 .mu.g/ml biotinylated detection Ab were added and
plates were incubated at RT for 4 hr. The plates were then washed
and streptavidin-conjugated HRP was added to each well for 30 min.
Wells were then washed 7 times and 60 .mu.l of TMB substrate
(Pierce, Rockford, Ill.) was added to the plates. Reactions were
terminated using 1N H.sub.2SO.sub.4 and plates were read using a
SpectraMax M2 fluorimeter.
Induction and Evaluation of EAE
[0103] Animals were injected sc with 100 .mu.g of MOG.sub.33-35 in
CFA containing 400 .mu.g of mycobacterium tuberculosis H37RA
(Difco, Detroit, Mich.). Upon immunization and two days later, 200
ng of pertussis toxin (List Biological Labs Inc., Campbell, Calif.)
was injected ip. Mice were weighed and scored for neurological
deficits daily: 0=no disease; 1=decreased tail tone or slightly
clumsy gait; 2=tail atony; 3=limb weakness; 4=limb paralysis;
5=moribund state.
Adoptive transfer of T cells
[0104] 10 days after priming 8-12 wk old female mice with MOG35-55
in CFA containing H37RA, 4.times.10.sup.6 washed
CD4.sup.+CD25.sup.- cells were administered iv via tail vein into
WT or Rag2.sup.--- recipients. Clinical scores and weights were
monitored as above.
Human Treg Isolation
[0105] The acquisition of blood products was approved according to
the policies of the University Hospitals Cleveland Case Medical
Center in accordance with the Declaration of Helsinki. Peripheral
blood mononuclear cells (PBMCs) were obtained from 5 to 10 mL blood
from healthy donors. Written informed consent was obtained from all
donors in accordance with the Declaration of Helsinki. PBMCs were
prepared over Histopaq gradient centrifugation (Sigma Aldrich, St
Louis, Mo.) and CD4 as well as blood DCs were purified as follows.
For FACS, CD4.sup.+ cells were enriched over the AutoMACS Pro
Separator by positive selection with human CD4 microbeads (Miltenyi
Biotec, Auburn, Calif.) and DCs were isolated by positive selection
using the Blood Dendritic Cell Isolation Kit II (Miltenyi Biotec,
Auburn, Calif.). The cells were labeled with CD4 FITC, CD25 PE,
CD45RA PE-Cy5.5 (all Invitrogen, Carlsbad, Calif.) and CD127 Alexa
Fluor 647 (BD Biosciences, San Jose, Calif.). The FACSAria flow
cytometer was used to sort Tregs by gating on the top 2%
CD25.sup.hi and non-Tregs by gating on CD4.sup.+CD25.sup.-
CD127.sup.+CD45RA.sup.+ cells.
Human Treg Assays
[0106] CFSE-labeled CD4.sup.+CD25.sup.-- T cells (1.times.10.sup.6)
sorted by FACS were stimulated with 1.times.10.sup.5 autologous DCs
and 1 .mu.g/mL anti-CD3 mAb (BD Biosciences) alone or with various
numbers of suppressor cells. The cells were cultured for 3 days in
96-well flat-bottom plates and CFSE dilution was analyzed by
FACS.
EXAMPLE 2
Increased Numbers of CD4.sup.+CD.sup.25 Foxp3.sup.+ Treg Cells are
Generated by Blocking C5aR
[0107] In view of our previous findings that during APC:T cell
interactions, increased levels of C5a is locally generated from
Daf1.sup.-/- APCs, we next examined whether C5aR signaling is
integrally involved in FoxP3.sup.+ Treg cell generation. We set up
another MLR by mixing WT C57BL/6 peritoneal macrophages (H-2.sup.b)
and WT Balb/C CD4.sup.+ T cells (H-2d) as we did before. In
different wells, we added 200 nM of a potent peptidomimetic C5aR
antagonist JPE-1375, or the same volume of PBS. At day 12, we
quantified the CD4.sup.+CD25.sup.+Foxp3.sup.+ Treg cell numbers by
flow cytometry. These assays showed that blocking C5aR activity
with C5aR antagonist increases FoxP3.sup.+Treg generation by
.about.2 fold. These results suggest that C5aR signaling either in
APCs and/or T cells inhibits Treg cell generation.
Increased Numbers of CD4.sup.+CD25.sup.+ FoxP3.sup.+ GFP Treg Cells
are Generated from CD4.sup.+FoxP3.sup.+ GFP-Cells by Blocking C5a
or C3a
[0108] In the experiments discussed above, it was not possible to
distinguish whether C5aR signaling modulates de novo Treg cell
differentiation or existing Treg cell expansion because subgroups
of CD4.sup.+ T cells were not separated. To address this issue, we
flow sorted CD4.sup.+ Foxp3 GFP- T cells (H-2.sup.b) from foxp3 GFP
knockin mice (kindly provided by Dr. VK Krochroo, Harvard
University) and mixed them with WT Balb/C peritoneal macrophages
(H-2.sup.d) to set up a MLR. To block C3a or C5a activity, we added
neutralizing anti mouse C3a or C5a mAbs or isotye IgGs in each
well. 7 days later, we quantified the
CD4.sup.+CD25.sup.+FoxP3.sup.+ Treg cell numbers by flow cytometry.
Blocking C3a or C5a during cognate APC:T cell interactions
increases Foxp3GFP.sup.+ Treg generation about 2 fold from the
foxp3 GFP-precursors. These results indicate that locally generated
C5a and C3a inhibits Treg cell differentiation from naive
CD4.sup.+foxp3-precursors.
Increased Numbers of CD4.sup.+CD25.sup.+ Treg Cells are Expanded by
C3aR.sup.-/-C5aR.sup.-/- APCs
[0109] To examine whether C3aR/C5aR signaling in APCs regulates the
expansion of existing Treg cells , we next flow sorted
CD4.sup.+CD25.sup.+ Treg cells from Balb/C mice (H-2.sup.d) and
mixed them with WT or C5a12.sup.-/-C3aR.sup.-/- bone marrow-derived
dendritic cells (BM-DCs) (H-2.sup.b) together with 1000 U/ml IL-2
to set up a MLR. After 3 days of incubation, we counted total live
cell numbers by trypan blue staining and assessed the percentages
of CD4.sup.+CD25.sup.+FoxP3.sup.+ Treg cells in the populations.
These assays showed that C3aR.sup.-/-C5aR.sup.-/- DCs expanded--two
fold larger numbers of the sorted Treg cells than WT DCs,
suggesting that C3aR/C5aR signaling in APCs inhibits the expansion
of existing Treg cells.
IL-6 Productions is reduced in C3aR.sup.-/-C5aR.sup.-/- Dendritic
Cells
[0110] The above two experiments using sorted CD4.sup.+Foxp3.sup.-
precursors and CD4.sup.+CD25.sup.+ Treg cells indicate that
C3aR/C5aR signaling inhibits both Treg cell differentiation and
expansion. We next measured levels of IL-6 produced by resting WT
and C3aR.sup.-/-C5aR.sup.--- BM-DCs by ELISA. We isolated bone
marrow cells from WT and C3aR.sup.-/-C5aR.sup.-/- mice and
incubated them with GM-CSF and IL-4 to generate BM-DCs following
the standard protocol as described before. We collected culture
supernatants at day 6 for IL-6 ELISA. These assays showed that
C3aR.sup.-/-C5aR.sup.-/- BM-DCs produce about 3 fold less amounts
of IL-6 than WT BM-DCs. These data, together with previous reports
on the critical role of IL-6 in Treg cell differentiation and
maintenance, suggest that IL-6 could be mechanistically linked to
the above observations in which C3aR/C5aR signaling in APCs
inhibits Treg cell differentiation/expansion. Increased numbers of
CD4.sup.+CD25.sup.+Foxp3.sup.+ Treg cells are generated from
CD4.sup.+CD25- T cells deficient of C3aR and C5aR.
[0111] The above experiments address potential impacts of C3aR/C5aR
signaling in APCs on Treg cell differentiation/expansion. As
indicated above, since both APCs and T cells express C3aR and C5aR,
both types of cells could be modulated by the locally generated C3a
and C5a. We next tested whether the absence of C3aR/C5aR signaling
in T cells favors FoxP3.sup.+ Treg cell generation. We isolated
CD4.sup.+CD25.sup.- T cells from WT and C3aR.sup.-/-C5aR.sup.-/-
mice by flow sorting and incubated them with plate-bound anti CD3
mAb (5 .mu.g/ml) together with 25 ng/ml TGF-.beta.. We assessed
numbers of CD4.sup.+CD25.sup.+Foxp3.sup.+ Treg cells 3 days later
and the assays showed that the generation of
CD4.sup.+CD25.sup.+Foxp3.sup.+ Treg cells from C3aR and C5aR
deficient CD4.sup.+CD25.sup.- T cells was more than 2 fold larger
than that from the WT T precursors (39.4% vs. 14.9%). These results
indicate that in addition to its effects on APCs, C3aR/C5aR
signaling in T cells directly inhibits Treg cell generation from
their CD4.sup.+CD25.sup.- precursors.
Increased Numbers of CD4.sup.+CD25.sup.+FoxP3.sup.+ Treg Cells are
Expanded from Existing CD4.sup.+CD25.sup.+ Treg Cells Deficient of
C3aR and C5aR
[0112] To study whether C3aR/C5aR signaling in T cells will have
direct impact on the expansion of existing Treg cells, we flow
sorted CD4.sup.+CD25.sup.+ Treg cells from WT and C5a12.sup.-/-
C3a12.sup.-/- mice, then activated them with plate coated anti CD3
mAb as described above together with 2000 U/ml IL-2. After 3 days
of culture, we counted total live cell numbers by trypan blue
staining and assessed CD4.sup.+CD25.sup.+FoxP3.sup.+ Treg cell
percentages by flow cytometry. These assays showed that there were
3.5.times.10.sup.5 total FoxP3.sup.+ Treg cells expanded from
1.times.10.sup.5 WT Treg cells and 6.3.times.10.sup.5 FoxP3.sup.+
Treg cells expanded from the same numbers of
C3aR.sup.-/-C5aR.sup.-/- Treg cells. These results indicate that
C3aR/C5aR signaling in T cells directly suppresses existing Treg
cell expansion.
CTLA-4 Levels are Increased in C3aR.sup.-/-C5aR.sup.-/- Treg
Cells
[0113] As described (Background), previous studies indicated that
CTLA-4 is critical for Treg cell function. To investigate the
mechanism underlying increased suppressive activity of
C3aR.sup.-/-C5aR.sup.-/- Treg cells in the above experiments, we
next flow sorted WT and C3aR C5aR
[0114] Treg cells then compared total CTLA-4 levels between them by
staining both the cell surface and intracellular CTLA-4 with a PE
conjugated anti CTLA-4 mAb (BD Biosciences, Calif.). Because
previous studies showed that Treg cells exhibit significantly
augmented suppressive activity after activation, we stimulated the
WT and C5aR.sup.-/-C3aR.sup.-/- Treg cells by anti CD3/CD28 beads
plus 1000 U/ml of IL-2 for 18 hr, then assessed the CTLA-4 levels
again. These assays showed that WT Treg cells(MFI 6125 v.s. 6809).
After activation, both types of Treg cells markedly upregulate
their CTLA-4 expression (.about.10 fold), while
C5aR.sup.-/-C3aR.sup.-/- Treg cells have significantly higher
levels of total CTLA-4 than WT Treg cells (MFI 79660 v. 58004),
suggesting that C3aR/C5aR signaling in Treg cells could inhibit
their CTLA-4 expression thereby suppressing their immunoregulatory
function.
cAMP Levels are Augmented in C3aR.sup.-/-C5aR.sup.-/- CD4.sup.+ T
Cells and Reduced in WT CD4.sup.+ T Cells Treated with C3a/C5a
[0115] Previous work by others has shown that cAMP induces CTLA-4
expression in CD4.sup.+ T cells. The underlying mechanism could be
that cAMP binds to and activates protein kinase A (PKA), which in
turn phosphorylates downstream transcription factor cAMP response
element binding protein (CREB) that binds to the cAMP-responsive
element (CRE) in the CTLA-4 promoter region and therefore enhancing
CTLA-4 transcription. We have analyzed the promoter region (-1302
bp) of mouse CTLA-4 gene using the software package Genomatix
(www.genomatix.de) and found that there are three CRE sites in the
CTLA-4 promoter region In view of these results, to explore the
mechanism underlying increased levels of CTLA-4 in
C3aR.sup.-/-c5aR.sup.-/- Treg cells, we compared cAMP levels
between flow sorted WT and C3aR.sup.-/-C5aR.sup.-/- Treg cells
using a cAMP-GLO kit (Promega, MI) after stimulation by forskolin,
an adenylyl cyclase activator. These assays showed that
C3aR.sup.-/-C5aR.sup.-/- Treg cells possess higher levels of cAMP
than WT Treg cells, suggesting that C3aR/C5aR signal in Treg cells
might inhibit cAMP production thereby suppressing CTLA-4
expression.
[0116] While this application has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the application encompassed by the appended claims. All
patents, publications and references cited in the foregoing
specification are herein incorporated by reference in their
entirety.
* * * * *