U.S. patent number RE46,382 [Application Number 14/874,249] was granted by the patent office on 2017-05-02 for methods and compositions for modulating peripheral immune function.
This patent grant is currently assigned to SanBio, Inc.. The grantee listed for this patent is SanBio, Inc.. Invention is credited to Casey C. Case, Mo Dao.
United States Patent |
RE46,382 |
Dao , et al. |
May 2, 2017 |
Methods and compositions for modulating peripheral immune
function
Abstract
Disclosed herein are cell preparations useful for modulating
various peripheral immune functions, methods for making said cell
preparations, and methods for their use.
Inventors: |
Dao; Mo (Huntington Beach,
CA), Case; Casey C. (San Mateo, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
SanBio, Inc. |
Mountain View |
CA |
US |
|
|
Assignee: |
SanBio, Inc. (Mountain View,
CA)
|
Family
ID: |
47139503 |
Appl.
No.: |
14/874,249 |
Filed: |
October 2, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
61516637 |
Apr 6, 2011 |
|
|
|
|
61541248 |
Sep 30, 2011 |
|
|
|
Reissue of: |
13441311 |
Apr 6, 2012 |
8785190 |
Jul 22, 2014 |
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
35/28 (20130101); C12N 5/0639 (20130101); C12N
5/0663 (20130101); A61P 11/06 (20180101); C12N
5/0637 (20130101); C12N 5/0645 (20130101); A61P
17/02 (20180101); A61P 37/06 (20180101); A61P
19/02 (20180101); A61P 17/00 (20180101); A61P
1/04 (20180101); A61P 29/00 (20180101); A61P
37/02 (20180101); A61P 3/10 (20180101); A61P
25/00 (20180101); A61P 11/00 (20180101); A61P
11/08 (20180101); A61P 43/00 (20180101); C12N
5/0663 (20130101); A61K 35/28 (20130101); C12N
5/0639 (20130101); C12N 5/0645 (20130101); C12N
5/0637 (20130101); C12N 15/85 (20130101); C12N
15/85 (20130101); C12N 2501/42 (20130101); C12N
2501/42 (20130101); C12N 2501/2302 (20130101); C12N
2502/1358 (20130101); C12N 2502/1358 (20130101); C12N
2501/2302 (20130101) |
Current International
Class: |
C12N
15/00 (20060101); C12N 5/0784 (20100101); A61K
35/28 (20150101); C12N 5/0786 (20100101); C12N
5/0775 (20100101); C12N 15/85 (20060101); C12N
5/0783 (20100101) |
Field of
Search: |
;435/373,377,455
;436/455 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO 2005/100552 |
|
Oct 2005 |
|
WO |
|
WO 2008/102460 |
|
Aug 2008 |
|
WO |
|
WO 2009/023251 |
|
Feb 2009 |
|
WO |
|
WO 2009/134409 |
|
Nov 2009 |
|
WO |
|
Other References
Medzhitov R. Nat Rev Immunol, Nov. 2001;1(2):135-45 Toll-like
receptors and innate immunity. cited by examiner .
Abdallah, et al., "DLK1/FA1 Regulates the Function of Human Bone
Marrow Mesenchymal Stem Cells by Modulating Gene Expression of
Pro-Inflammatory Cytokines and Immune Response-Related Factors," J.
Biol. Chem. 282:7339-7351 (2007). cited by applicant .
Artavanis-Tsakonas, et al., "Notch Signaling," Science
268(5208):225-232 (1995). cited by applicant .
Bergmann, et al., "Effect of NF-KB Inhibition on TNF-A-Induced
Apoptosis and Downstream Pathways in Cardiomyocytes," J. Mol. Cell.
Cardiol. 33:1223-1232 (2001). cited by applicant .
Campagnoli, et al., "Identification of Mesenchymal Stem/Progenitor
Cells in Human First-Trimester Fetal Blood, Liver, and Bone
Marrow," Blood 98(8):2396-2402 (2001). cited by applicant .
Cheng, et al., "Notch-1 Regulates NF-KappaB Activity in Hemopoietic
Progenitor Cells," J. Immunol. 167:4458-4467 (2001). cited by
applicant .
Chomarat, et al., "IL-6 Switches the Differentiation of Monocytes
From Dendritic Cells to Macrophages," Nature Immunol. 1:510-514
(2000). cited by applicant .
Coppe, et al., "Senescence-Associated Secretory Phenotypes Reveal
Cell-Nonautonomous Functions of Oncogenic RAS and the P53 Tumor
Suppressor," PLoS Biol. 6:2853-2868 (2008). cited by applicant
.
Dezawa, et al., "Sciatic Nerve Regeneration in Rats Induced by
Transplantation of In Vitro Differentiated Bone-Marrow Stromal
Cells," Eur. J. Neurosci. 14(11):1771-1776 (2001). cited by
applicant .
Djouad, et al., "Mesenchymal Stem Cells Inhibit the Differentiation
of Dendritic Cells Through an Interleukin-6-Dependent Mechanism,"
Stem Cells 25(8):2025-2032 (2007). cited by applicant .
Dogusan, et al., "Macrophages and Stromal Cells Phagocytose
Apoptolic Bone Marrow-Derived B Lineage Cells," J. Immunol.
172:4717-4723 (2004). cited by applicant .
Ehebauer, et al., "Notch Signaling Pathway," Sci. STKE
2006(364):cm7 (2006) DOI:10.1126/stke.3642006cm71. cited by
applicant .
Erices, et al., "Mesenchymal Progenitor Cells in Human Umbilical
Cord Blood," Br. J. Haematol. 109(1):235-242 (2000). cited by
applicant .
Ferguson, et al., "MMP-2 and MMP-9 Increase the Neurite-Promoting
Potential of Schwann Cell Basal Laminae and are Upregulated in
Degenerated Nerve," Mol. Cell. Neurosci. 16:157-167 (200). cited by
applicant .
Freund, et al., "Inflammatory Networks During Cellular Senescence:
Causes and Consequences," Trends Mol. Med. 16(5):238-246 (2010).
cited by applicant .
Glavaski-Joksimovic, et al., "Reversal of Dopaminergic Degeneration
in a Parkinsonian Rat Following Micrografting of Human Bone
Marrow-Derived Neural Progenitors," Cell Transplant. 18(7):801-814
(2009). cited by applicant .
Hilton, et al. "Notch Signaling Maintains Bone Marrow Mesenchymal
Progenitors by Suppressing Osteoblast Differentiation," Nature
Medicine 14:306-314 (2008). cited by applicant .
Hou, et al., "Induction of Umbilical Cord Blood Mesenchymal Stem
Cells Into Neuron-Like Cells In Vitro," Int. J. Hematol.
78(3):256-261 (2003). cited by applicant .
Jiang, et al., "Pluripotency of Mesenchymal Stem Cells Derived From
Adult Marrow," Nature 418:41-49 (2002). cited by applicant .
Jin, et al., "Mesenchymal Stem Cellls Cultured Under Hypdxia Escape
From Senescene Via Down-Regulation of P16 and Extracellular Signal
Regulated Kinase," Biochem. Biophys. Res. Commun. 391(3):1471-1476
(2010). cited by applicant .
Kong, et al., "Administration of Bone Marrow Stromal Cells
Ameliorates Experimental Autoimmune Myasthenia gravis by Altering
the Balance of TH1/TH2/TH17/TREG Cell Subsets Through the Secretion
of TGF-B," J. Neuroimmunol. 207(1-2):83-91 (2009). cited by
applicant .
Kriete, et al., "Cell Autonomous Expression of Inflammatory Genes
in Biologically Aged Fibroblasts Associated With Elevated NF-KappaB
Activity," Immunity & Ageing 5:5-12 (2008). cited by applicant
.
Kriete, et al., "Atypical Pathways of NF-KappaB Activation and
Aging," Exp. Gerontology 44:250-255 (2009). cited by applicant
.
Mimura, et al., "Vascular Endothelial Growth Factor Inhibits the
Function of Human Mature Dendritic Cells Mediated by VEGF
Receptor-2," Cancer Immunol Immunother 56:761-770 (2007). cited by
applicant .
Mumm, et al., "Notch Signaling: From the Outside In," Dev. Biol.
228(2):151-165 (2000). cited by applicant .
Orjalo, et al., "Cell Surface-Bound IL-1.alpha. is an Upstream
Regulator of the Senescence-Associated IL-6/IL-8 Cytokine Network,"
PNAS 106(40):17031-17036 (2009). cited by applicant .
Oswald, et al., "NF-KappaB2 is a Putative Target Gene of Activated
Notch-1 Via RBP-J Kappa," Mol. Cell. Biol. 18:2077-2088 (1998).
cited by applicant .
Park, et al., "IL-6 Regulates in Vivo Dendritic Cell
Differentiation Through STAT3 Activation," J. Immunol.
173(6):3844-3854 (2004). cited by applicant .
Park, et al., "Transforming Growth Factor-.beta.1 Regulates the
Fate of Cultured Spinal Cord-Derived Neural Progenitor Cells," Cell
Prolif. 41:248-264 (2008). cited by applicant .
Pittenger, et al., "Multilineage Potential of Adult Human
Mesenchymal Stem Cells," Science 284(5411):143-147 (1999). cited by
applicant .
Prockop, et al., "Marrow Stromal Cells As Stem Cells for
Nonhematopoietic Tissues," Science 276(5309):71-74 (1994). cited by
applicant .
Rodier, et al., "Persistent DNA Damage Signalling Triggers
Senescence-Associated Inflammatory Cytokine Secretion," Nature Cell
Biol. 11:973-979 (2009). cited by applicant .
Sanchez-Abarca, et al., "Oligodendrocytes Use Lactate As a Source
of Energy and as a Precursor of Lipids," Glia 36:321-329 (2001).
cited by applicant .
Scaffidi, et al., "Lamin A-Dependent Misregulation of Adult Stem
Cells Associated With Accelerated Ageing," Nature Cell Biol.
10:452-459 (2008). cited by applicant .
Schober, et al., "Glial Cell Line-Derived Neurotrophic Factor
Rescues Target-Deprived Sympathetic Spinal Cord Neurons But
Requires Transforming Growth Factor-Beta as Cofactor In Vivo," J.
Neurosci. 19:2008-2015 (1999). cited by applicant .
Schober, et al., "GDNF Applied to the MPTP-Lesioned Nigrostriatal
System Requires TGF-Beta for its Neuroprotective Action,"
Neurobiol. Dis. 25:378-391 (2007). cited by applicant .
Schulte-Herbruggen, et al., "Tumor Necrosis Factor-Alpha and
Interleukin-6 Regulate Secretion of Brain-Derived Neurotrophic
Factor in Human Monocytes," J. Neuroimmunol. 160:204-209 (2005).
cited by applicant .
Skihar, et al., "Promoting Oligodendrogenesis and Myelin Repair
Using the Multiple Sclerosis Medication Glatiramer Acetate," PNAS
USA 106:17992-17997 (2009). cited by applicant .
Takahashi, et al., "Vascular Endothelial Growth Factor Inhibits
Maturation of Dendritic Cells Induced by Lipopolysaccharide, But
Not by Proinflammatory Cytokines," Cancer Immunol. Immunother.
53(6):543-550 (2004). cited by applicant .
Tate, et al., "Human Mesenchymal Stromal Cells and Their
Derivative, SB623 Cells, Rescue Neural Cells Via Trophic Support
Following In Vitro Ischemia," Cell Transplant. 19(8):973-984
(2010). cited by applicant .
Van Den Bos, et al., "P21CIP1 Rescues Human Mesenchymal Stem Cells
From Apoptosis Induced by Low-Density Culture," Cell & Tissue
Res. 293:463-470 (1998). cited by applicant .
Van Hall, et al., "Blood Lactate is an Important Energy Source for
the Human Brain," J. Cereb. Blood Flow Metab. 29:1121-1129 (2009).
cited by applicant .
Villoslada, et al., "Immunotherapy for Neurological Diseases,"
Clin. Immunol. 128(3):294-305. cited by applicant .
Wagner, et al., "Replicative Senescence of Mesenchymal Stem Cells:
A Continuous and Organized Process,"PLoS One 3(5):e2213 (2008).
cited by applicant .
Yasuhara, et al., "Notch-Induced Rat and Human Bone Marrow Stromal
Cell Grafts Reduce Ischemic Cell Loss and Ameliorate Behavioral
Deficits in Chronic Stroke Animals," Stem Cell Dev.
18(10):1501-1514 (2009). cited by applicant .
Zhu, et al., "Transforming Growth Factor-B1 Increases Bad
Phosphorylation and Protects Neurons Against Damage," J. Neurosci.
15:3898-3909 (2002). cited by applicant .
Zuo, et al., "Neuronal Matrix Metalloproteinase-2 Degrades and
Inactivates a Neurite-Inhibiting Chondroitin Sulfate Proteoglycan,"
J. Neurosci. 18:5203-5211 (1998). cited by applicant.
|
Primary Examiner: Ponnaluri; Shri
Attorney, Agent or Firm: Levine Bagade Han LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application .Iadd.is a reissue of U.S. Pat. No.
8,785,190, which .Iaddend.claims the benefit the benefit of U.S.
provisional patent applications No. 61/516,637 filed on Apr. 6,
2011 and 61/541,248 filed on Sep. 30, 2011, the disclosures of
which are incorporated herein by reference in their entireties for
all purposes.
Claims
What is claimed is:
1. A method for modulating production of one or more cytokines by a
T-lymphocyte (T-cell) or a monocyte; the method comprising:
contacting the T-cell or monocyte with an effective amount of
.[.SB623.]. .Iadd.MASC-derived immune modulatory .Iaddend.cells;
wherein said contacting results in stimulation of the production of
one or more anti-inflammatory cytokines and/or inhibition of
production of one or more pro-inflammatory cytokines by the T-cell
or the monocyte; further wherein said .[.SB623.].
.Iadd.MASC-derived immune modulatory .Iaddend.cells are obtained by
(a) providing a culture of marrow adherent stromal cells
.Iadd.(MASCs).Iaddend.; (b) contacting the cell culture of step (a)
with a polynucleotide comprising sequences encoding the Notch
intracellular domain (NICD) wherein said polynucleotide dos not
encode a full-length Notch protein; (c) selecting cells that
comprise the polynucleotide of step (b); and (d) further culturing
the selected cells of step (c) in the absence of selection for the
polynucleotide, such that said marrow adherent stromal cells are
induced to form .[.SB623.]. .Iadd.MASC-derived immune modulatory
.Iaddend.cells by expression of the NICD.
2. The method of claim 1, wherein the pro-inflammatory cytokine is
interferon gamma (IFN-.gamma.).
3. The method of claim 1, wherein the pro-inflammatory cytokine is
tumor necrosis factor alpha (TNF-.alpha.).
4. The method of claim 1, wherein the anti-inflammatory cytokine is
interleukin-10 (IL-10).
5. The method of claim 1, wherein the T cell is a helper
T-cell.
6. The method of claim 5, wherein the helper T-cell is T.sub.H1
cell.
7. The method of claim 1, wherein the T-cell is a regulatory
T-cell.
8. The method of claim 7, wherein the regulatory T-cell is a
T.sub.R1 cell.
9. The method of claim 1, wherein said modulation of cytokine
production results in conversion of a T-cell population from one
that is pro-inflammatory to one that is anti-inflammatory.
Description
STATEMENT REGARDING FEDERAL SUPPORT
Not applicable.
FIELD
The disclosure is in the field of immunomodulation (e.g.,
immunosuppression).
BACKGROUND
Peripheral (i.e., non-CNS) immunity in vertebrates is mediated by
two systems: the innate immune system and the adaptive immune
system. The innate immune system provides an early, non-specific
response to injury and/or infection. By contrast, the adaptive
immune system is brought into play later in the process of injury
or infection, and is specific to the invading pathogen. The innate
immune system, being evolutionary more ancient, is active in
plants, invertebrates and vertebrates, while the adaptive immune
system is active in vertebrates only.
As noted above, the innate immune system becomes active immediately
upon infection, at the site of infection, and does not depend on
prior exposure to the infecting pathogen. It thus provides a set of
general defense mechanisms that are not specific to any particular
pathogen. Cellular elements of the innate immune system include
macrophages, dendritic cells, neutrophils and natural killer (NK)
cells. Macromolecular components of the innate immune system
include defensin peptides and the complement system. Additional
elements of innate immunity include physical barriers to infection
(such as the keratinization of the skin, tight junctions between
epithelial cells, stomach acid and the mucus secreted by many
epithelial tissues) and cell-intrinsic responses such as, for
example, phagocytosis (sometimes coupled with lysosomal fusion of
phagocytosed material) and degradation of double-stranded RNA.
Activation of the innate immune system is mediated, in part, by
recognition of pathogen-associated molecules such as, for example,
N-formyl methionine-containing polypeptides, cell wall
peptidoglycans, bacterial flagella, lipopolysaccharides, techoic
acid, and fungal-specific molecules such as mannan, glucan and
chitin. In addition, certain nucleic acid sequences common to
microorganisms (such as unmethylated CpG dinucleotides) can trigger
innate immune responses. Recognition of such pathogen-associated
immunostimulants results in the mounting of an inflammatory
response and phagocytosis of the pathogen by macrophages,
neutrophils and/or dendritic cells.
Certain of the pathogen-associated immunostimulants noted above
occur in repeating patterns called pathogen-associated molecular
patterns (PAMPs), which can be recognized by pattern recognition
receptors on the surfaces of innate immune system cells. These
receptors include soluble members of the complement system and
membrane-bound receptors such as members of the Toll-like receptor
family (TLRs) and the so-called NOD proteins. The membrane-bound
receptors can stimulate phagocytosis and activate programs of gene
expression responsible for various innate and adaptive immune
responses.
Finally, the innate immune system is involved in activating
adaptive immunity, in part by secreting extracellular signaling
molecules which stimulate proliferation and differentiation of
cells of the adaptive immune system, and also by processing and
presenting antigens to cells of the adaptive immune system.
The adaptive immune system, in contrast to the innate immune
system, is not activated immediately upon infection, and generates
specific, long-lived responses to pathogens. Activation of the
adaptive immune system occurs not at the site of injury, but in
lymphoid organs, and depends on presentation of antigens by
components of the innate immune system to activate cells of the
adaptive immune system. The principal cells of the adaptive immune
system are B-lymphocytes (B cells), which synthesize and secrete
antibodies, and T-lymphocytes (T cells).
There are three major classes of T cells: cytotoxic, helper, and
regulatory (or suppressor) T cells. Cytotoxic T cells are able to
kill infected host cells. Helper T cells participate in activation
of macrophages, dendritic cells, B cells and cytotoxic T cells by
secreting cytokines and/or by surface expression of one of a number
of different co-stimulatory molecules. There are two types of
helper T cells: T.sub.H1 cells participate in activation of
macrophages, cytotoxic T cells and B cells to provide immunity to
intracellular pathogens and secrete the macrophage-activating
cytokines interferon gamma (IFN-.gamma.) and tumor necrosis factor
alpha (TNF-.alpha.). T.sub.H1 cells are also capable of stimulating
inflammatory responses. T.sub.H2 cells help activate B cells to
produce antibodies, primarily in response to extracellular
pathogens, and secrete the cytokines interleukin 4 (IL4) and
interleukin 10 (IL10). Development of a naive helper T cell into a
T.sub.H1 cell is stimulated by interleukin 12 (IL12); while
pathogen-induced expression of the Jagged protein by a dendritic
cell will guide a naive helper T cell to develop into a T.sub.H2
cell producing IL4, which stimulates antibody production by B
cells. Regulatory T cells (T.sub.regs) inhibit the function of
cytotoxic T cells, helper T cells and dendritic cells, and are
unique in expressing the Foxp3 transcription factor. Thus, the
interplay between helper T cells and regulatory T cells helps keep
the immune response in balance, with sufficient activity to clear
an invading pathogen without excessive damage to the host.
A class of lymphocytes in the adaptive immune system known as
memory cells retains receptors to a pathogen subsequent to
infection and clearance, enabling the host organism to mount a more
rapid adaptive immunological response to a subsequent encounter
with the same pathogen, and providing the basis for natural or
vaccination-induced immunity to many infections diseases. By
contrast, the innate immune system does not retain such
immunological memory.
Mesenchymal stem cells (MSCs, also known as "marrow stromal cells"
or "marrow adherent stem cells"), that have been transfected with a
plasmid expressing the Notch intracellular domain (NICD), are
useful for the treatment of a number of diseases and disorders of
the central and peripheral nervous systems. See, for example, U.S.
Pat. No. 7,682,825 (Mar. 23, 2010); US Patent Application
Publication No. 2006/0216276 (Sep. 28, 2006); US Patent Application
Publication No. 2010/0034790 (Feb. 11, 2010) US Patent Application
Publication No. 2010/0310523 (Dec. 9, 2010); International Patent
Application Publication No. WO08/102,460 (Aug. 28, 2008); Yasuhara
et al. (2009) Stem Cells and Development 18:1501-1513 and
Glavaski-Joksimovic et al. (2009) Cell Transplantation
18:801-814.
The ability of these cells, known as SB623 cells, to rescue damaged
neural tissue is associated, in part, with their secretion of
various trophic factors and their elaboration of various
extracellular matrix components. See, for example, US Patent
Application Publication No. 2010/0266554 (Oct. 21, 2010) and US
Patent Application Publication No. 2010/0310529 (Dec. 9, 2010).
Current cell transplantation therapies have significant
disadvantages, including, for example, host peripheral
immunological reactions to the transplanted cells. In addition,
inflammation is a hallmark of many neurodegenerative diseases, such
as, for example, Parkinson's disease and multiple sclerosis.
Villoslada et al. (2008) Clin. Immunol. 128:294-305. MSCs have been
reported to attenuate peripheral immune activity through mechanisms
that include blocking production of antigen-presenting cells and
altering the cytokine profile of helper T-cells. Kong et al. (2009)
J. Neuroimmunol. 207:83-91. However, MSCs have limited regenerative
potential, becoming senescent following ex vivo manipulation.
Wagner et al. (2008) PLoS One 3:e2213; Jin et al. (2010) Biochem
Biophys Res Commun. 391:1471-1476. Although senescent cells secrete
a number of cytokines which could be beneficial for tissue
regeneration, the overall senescent cell secretory profile is
pro-inflammatory. Rodier et al. (2009) Nature Cell Biol.
11:973-979; Coppe et al. (2008) PLoS Biol. 6:2853-2868; Freund et
al. (2010) Trends Mol. Med. 16 (5): 238-246.
For these and other reasons, there remains a need for methods and
compositions for cell transplantation that do not provoke host
peripheral immune responses, and/or that reduce inflammatory, and
other immune, responses.
SUMMARY
The inventors have identified, within cultures of MSCs that have
been transfected with sequences encoding a Notch intracellular
domain and their descendants (i.e., SB623 cells), a population of
senescent cells. Although SB623 cells have been shown to be capable
of treating a number of central nervous system disorders, the
present application discloses the surprising ability of SB623 cells
to modulate a number of peripheral immune functions. For example,
SB623 cells can inhibit human T cell proliferation in both
allogeneic and xenogeneic mixed lymphocyte reactions, stimulate
IL-10 production by T-cells, and block the differentiation of
monocytes to dendritic cells. SB623 cells also inhibit maturation
of dendritic cells and, compared to the parental MSCs, SB623 cells
exert a greater inhibitory effect on dendritic cell maturation, as
evidenced by greater reduction in the surface expression of the
co-stimulatory molecule, CD86. SB623 cells can also convert the
cytokine profile of a T-cell population from one that is
pro-inflammatory to one that is anti-inflammatory. These properties
of SB623 cells are additionally surprising and unexpected in light
of studies reporting that senescent cells secrete pro-inflammatory
cytokines. Orjalo et al. (2009) Proc. Natl. Acad. Sci. USA
106:17031-17036.
Accordingly, SB623 cells, and/or their subpopulation of senescent
cells, are useful in a number of therapeutic methods, as
exemplified in the following embodiments.
1. A method for peripheral immunosuppression in a subject, the
method comprising administering in the subject an effective amount
of SB623 cells; wherein said SB623 cells are obtained by (a)
providing a culture of mesenchymal stem cells; (b) contacting the
cell culture of step (a) with a polynucleotide comprising sequences
encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein; (c)
selecting cells that comprise the polynucleotide of step (b); and
(d) further culturing the selected cells of step (c) in the absence
of selection.
2. A method for inhibiting a peripheral inflammatory response in a
subject, the method comprising administering to the subject an
effective amount of SB623 cells; wherein said SB623 cells are
obtained by (a) providing a culture of mesenchymal stem cells; (b)
contacting the cell culture of step (a) with a polynucleotide
comprising sequences encoding a Notch intracellular domain (NICD)
wherein said polynucleotide does not encode a full-length Notch
protein; (c) selecting cells that comprise the polynucleotide of
step (b); and (d) further culturing the selected cells of step (c)
in the absence of selection.
3. The method of embodiment 2, wherein the peripheral inflammatory
response results from an allogeneic transplantation, ischemia or
necrosis.
4. A method for suppressing peripheral T-cell activation in a
subject; the method comprising administering to the subject an
effective amount of SB623 cells; wherein said SB623 cells are
obtained by (a) providing a culture of mesenchymal stem cells; (b)
contacting the cell culture of step (a) with a polynucleotide
comprising sequences encoding a Notch intracellular domain (NICD)
wherein said polynucleotide does not encode a full-length Notch
protein; (c) selecting cells that comprise the polynucleotide of
step (b); and (d) further culturing the selected cells of step (c)
in the absence of selection.
5. The method of embodiment 4, wherein said peripheral T-cell
activation comprises expression of CD69 and/or HLA-DR by the
T-cells.
6. The method of embodiment 4, wherein said peripheral T-cell
activation comprises proliferation of CD4.sup.+ T-cells.
7. A method for suppressing the function of peripheral helper
T-cells in a subject; the method comprising administering to the
subject an effective amount of SB623 cells; wherein said SB623
cells are obtained by (a) providing a culture of mesenchymal stem
cells; (b) contacting the cell culture of step (a) with a
polynucleotide comprising sequences encoding a Notch intracellular
domain (NICD) wherein said polynucleotide does not encode a
full-length Notch protein; (c) selecting cells that comprise the
polynucleotide of step (b); and (d) further culturing the selected
cells of step (c) in the absence of selection.
8. The method of embodiment 7, wherein said peripheral helper
T-cell function is cytokine secretion.
9. The method of embodiment 7, wherein said peripheral helper
T-cell function is associated with the pathology of rheumatoid
arthritis.
10. A method for expanding a population of peripheral regulatory
T-cells (T.sub.regs) in a subject; the method comprising
administering to the subject an effective amount of SB623 cells;
wherein said SB623 cells are obtained by (a) providing a culture of
mesenchymal stem cells; (b) contacting the cell culture of step (a)
with a polynucleotide comprising sequences encoding a Notch
intracellular domain (NICD) wherein said polynucleotide does not
encode a full-length Notch protein; (c) selecting cells that
comprise the polynucleotide of step (b); and (d) further culturing
the selected cells of step (c) in the absence of selection.
11. A method for modulating peripheral production of a cytokine in
a subject; the method comprising administering to the subject an
effective amount of SB623 cells; wherein said SB623 cells are
obtained by (a) providing a culture of mesenchymal stem cells; (b)
contacting the cell culture of step (a) with a polynucleotide
comprising sequences encoding a Notch intracellular domain (NICD)
wherein said polynucleotide does not encode a full-length Notch
protein; (c) selecting cells that comprise the polynucleotide of
step (b); and (d) further culturing the selected cells of step (c)
in the absence of selection.
12. The method of embodiment 11, wherein the cytokine is a
pro-inflammatory cytokine and production of the cytokine is
reduced.
13. The method of embodiment 12, wherein the cytokine is produced
by a T cell.
14. The method of embodiment 13, wherein the cytokine is interferon
gamma (IFN-.gamma.).
15. The method of embodiment 12, wherein the cytokine is produced
by a monocyte.
16. The method of embodiment 15, wherein the cytokine is tumor
necrosis factor-alpha (TNF-.alpha.).
17. The method of embodiment 11, wherein the cytokine is an
anti-inflammatory cytokine and production of the cytokine is
stimulated.
18. The method of embodiment 17, wherein the cytokine is
interleukin-10 (IL-10).
19. The method of embodiment 18, wherein the cytokine is produced
by a T cell or a monocyte.
20. The method of embodiment 19, wherein the T cell is a helper
T-cell.
21. The method of embodiment 20, wherein the helper T-cell is a
T.sub.H1 cell.
22. The method of embodiment 19, wherein the cytokine is produced
by a regulatory T-cell.
23. The method of embodiment 22, wherein the regulatory T-cell is a
T.sub.R1 cell.
24. A method for inhibiting the differentiation of a peripheral
monocyte to a dendritic cell in a subject; the method comprising
administering to the subject an effective amount of SB623 cells;
wherein said SB623 cells are obtained by (a) providing a culture of
mesenchymal stem cells; (b) contacting the cell culture of step (a)
with a polynucleotide comprising sequences encoding a Notch
intracellular domain (NICD) wherein said polynucleotide does not
encode a full-length Notch protein; (c) selecting cells that
comprise the polynucleotide of step (b); and (d) further culturing
the selected cells of step (c) in the absence of selection.
25. A method for inhibiting the maturation of a peripheral
dendritic cell in a subject; the method comprising administering to
the subject an effective amount of SB623 cells; wherein said SB623
cells are obtained by (a) providing a culture of mesenchymal stem
cells; (b) contacting the cell culture of step (a) with a
polynucleotide comprising sequences encoding a Notch intracellular
domain (NICD) wherein said polynucleotide does not encode a
full-length Notch protein; (c) selecting cells that comprise the
polynucleotide of step (b); and (d) further culturing the selected
cells of step (c) in the absence of selection.
26. The method of embodiment 25, wherein maturation comprises an
increase in expression of CD86 by the dendritic cell.
27. A method for treating GVHD in a subject; the method comprising
administering to the subject an effective amount of SB623 cells;
wherein said SB623 cells are obtained by (a) providing a culture of
mesenchymal stem cells; (b) contacting the cell culture of step (a)
with a polynucleotide comprising sequences encoding a Notch
intracellular domain (NICD) wherein said polynucleotide does not
encode a full-length Notch protein; (c) selecting cells that
comprise the polynucleotide of step (b); and (d) further culturing
the selected cells of step (c) in the absence of selection.
28. A method for inhibiting graft rejection in a subject; the
method comprising administering to the subject an effective amount
of SB623 cells; wherein said SB623 cells are obtained by (a)
providing a culture of mesenchymal stem cells; (b) contacting the
cell culture of step (a) with a polynucleotide comprising sequences
encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein; (c)
selecting cells that comprise the polynucleotide of step (b); and
(d) further culturing the selected cells of step (c) in the absence
of selection.
29. A method for treating a peripheral autoimmune disorder in a
subject; the method comprising administering to the subject an
effective amount of SB623 cells; wherein said SB623 cells are
obtained by (a) providing a culture of mesenchymal stem cells; (b)
contacting the cell culture of step (a) with a polynucleotide
comprising sequences encoding a Notch intracellular domain (NICD)
wherein said polynucleotide does not encode a full-length Notch
protein; (c) selecting cells that comprise the polynucleotide of
step (b); and (d) further culturing the selected cells of step (c)
in the absence of selection.
30. The method of embodiment 29, wherein the peripheral autoimmune
disorder is selected from the group consisting of multiple
sclerosis, ulcerative colitis, chronic obstructive pulmonary
disease (COPD), asthma, lupus and Type I diabetes.
31. The method of any of the preceding embodiments, wherein the
subject is an experimental animal.
32. The method of any of embodiments 1-30, wherein the subject is a
human.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows measurements of CFSE dilution, in MSCs and SB623
cells, by flow cytometry.
FIG. 2 shows changes in cell count in cultures of MSCs and SB623
cells, determined by Trypan Blue exclusion after three days of
culture. Each culture was started with one million cells.
FIGS. 3A and 3B show the results of propidium iodide staining of
cultures of MSCs and SB623 cells. FIG. 3A shows representative FACS
data. The peak labeled "M1" represents resting (G0/G1) phase cells.
FIG. 3B shows the fraction of cells in the resting phase of the
cell cycle, for MSCs and SB623 cells, determined by measuring the
area of the M1 peak in FIG. 3A.
FIG. 4 shows measurements of p16Ink4A levels in MSCs and SB623
cells.
FIG. 5 shows levels of certain surface markers in MSCs and SB623
cells.
FIG. 6 shows measurements of CD54 expression in MSCs and SB623
cells.
FIG. 7 shows levels of certain cytokines in MSCs and SB623
cells.
FIG. 8 shows levels of transforming growth factor beta-1
(TGF-.beta.-1) and vascular endothelial growth factor-A (VFGF-A) in
MSCs and SB623 cells.
FIGS. 9A and 9B, show the effect of SB623 cells and MSCs on T-cell
activation in an allogeneic MLR. FIG. 9A shows representative FACS
traces, gating on CFSE and phycoerythrin-labeled anti-CD69, for
control unstimulated human T-cells (upper left panel, indicated by
"-"); human T-cells stimulated by allogeneic PBMCs (upper right
panel, indicated by "MLR"); MLR as before with 10.sup.4 MSCs (lower
left panel, indicated by "MLR+MSC") and MLR as before with 10.sup.4
SB623 cells (lower right panel, indicated by "MLR+SB623"). FIG. 9B
shows quantitation of CD69 expression in MLR cultures. Control,
unstimulated T-cell cultures are represented by "Serum;"
PBMC-stimulated T-cells in a mixed lymphocyte reaction are
represented by "MLR;" a mixed lymphocyte reaction as before in the
presence of mesenchymal stem cells is represented by "MSC;" and a
mixed lymphocyte reaction as before in the presence of SB623 cells
is represented by "SB623." The values for "MSC" and "SB623" are
averages of three cultures, each containing MSCs or SB623 cells
from different donors.
FIGS. 10A and 10B show a comparison of T-cell proliferation rates
in an allogeneic MLR, quantitated by measuring CFSE dilution. FIG.
10A shows representative FACS traces for control unstimulated human
T-cells (upper left panel, indicated by "T cells alone"); human
T-cells stimulated by allogeneic PBMCs (upper right panel,
indicated by "MLR"); MLR as before with 10.sup.4 MSCs (lower left
panel, indicated by "MLR+MSC") and MLR as before with 10.sup.4
SB623 cells (lower right panel, indicated by "MLR+SB623"). FIG. 10B
shows quantitation of CSFE dilution in MLR cultures. Compositions
of the cultures are as indicated in FIG. 10A.
FIG. 11 shows a comparison of HLA-DR expression under different
culture conditions. Control, unstimulated T-cell cultures are
represented by "Serum;" PBMC-stimulated T-cells in a mixed
lymphocyte reaction are represented by "MLR;" a mixed lymphocyte
reaction in the presence of mesenchymal stem cells is represented
by "MSC;" and a mixed lymphocyte reaction in the presence of SB623
cells Is represented by "SB623." The values for "MSC" and "SB623"
are averages of three cultures, each containing MSCs or SB623 cells
from different donors.
FIG. 12 shows effect of SB623 cells and MSCs on T-cell
proliferation in a xenogeneic lymphocyte stimulation reaction.
Proliferation was measured by dilution of PKH26, a cell-permeable
dye. The percentage of CD32.sup.+ T-cells containing PKH26 were
measured for unstimulated T-cells ("T cells alone"); T-cells
co-cultured with glial mix cells ("xeno-MLR"); T-cells co-cultured
with glial mix cells and mesenchymal stem cells ("xeno-MLR+MSC")
and T-cells co-cultured with glial mix cells and SB623 cells
("xeno-MLR+SB623"). Preparations of MSCs and SB623 cells were
obtained from three different donors, as indicated in the
figure.
FIGS. 13A and 13B show assays for regulatory T-cells (T.sub.regs)
in in vitro T-cell cultures, using coexpression of CD4 and CD25 as
a marker for T.sub.regs. FIG. 13A shows representative FACS traces,
measuring CD4 and CD25, for IL-2-stimulated T-cells ("T cells"),
and IL-2-stimulated T-cells co-cultured for seven days with either
mesenchymal stem cells ("T cells+MSCs") or SB623 cells ("T
cells+SB623 cells"). FIG. 13B shows mean CD4/CD25 expression levels
for 5 different matched lots of MSCs and SB623 cells. Note that,
for "Donor 1 PBL" a significant increase in CD4.sup.+CD25.sup.+
cells is observed (p<0.05) in the co-culture with SB623 cells,
compared to the co-culture with MSCs.
FIGS. 14A and 14B show levels of the FoxP3 transcription factor in
T-cells cultured in the presence of IL-2, measured by staining for
intracellular FoxP3 with a PE-conjugated anti-FoxP3 antibody,
followed by flow cytometry analysis. FIG. 14A shows representative
FACS traces for T-cells cultured in the absence of IL-2 (indicated
by "RPMI/10% FBS"), T-cells cultured in 10 ng/ml IL-2 (indicated
"+IL-2"), T-cells cultured in IL-2 as above and co-cultured with
MSCs (indicated "+MSC"), and T-cells cultured in IL-2 as above and
co-cultured with SB623 cells (indicated "+SB623").
FIG. 14B shows the mean percentage of FoxP3-expressing T-cells
after culture in the presence of IL-2 (indicated "T cells alone")
or after co-culture with MSCs ("T cells+MSC") or SB623 cells ("T
cells+SB623") in the presence of IL-2. Co-culture was conducted
with 3 different matched lots of MSCs and SB623 cells.
FIGS. 15A and 15B show results of measurements of intracellular
IL-10 levels in CD4.sup.+ T-cells cultured in the presence of IL-2.
FIG. 15A shows representative FACS traces for T-cells cultured in
the presence of IL-2 ("T cells alone"), T-cells cultured in IL-2 as
above and co-cultured with MSCs (indicated "T cells+MSC"), and
T-cells cultured in IL-2 as above and co-cultured with SB623 cells
(indicated "T cells+SB623"). Alexa 488 fluorescence, indicative of
IL-10 levels, is shown on the abscissa. FIG. 15B shows mean
percentage of IL-10-positive cells in co-cultures of T-cells with
three different matched lots of MSCs ("T cells+MSC") and SB623
cells ("T cells+SB623"), compared T-cells that were not co-cultured
("T cells alone").
FIGS. 16A and 16B, show levels of cytokines in T-cells cultured in
the absence of IL-2 and in the presence of non-maximally-inducing
levels of PMA and ionomycin. In FIG. 16A, levels of
interferon-gamma (IFN-g) are shown in freshly-isolated T-cells
prior to culture ("Fresh cells"), T-cells cultured for seven days
in the absence of other cells ("Culture control"), T-cells
co-cultured with SB623 cells for seven days ("SB623"), and T-cells
co-cultured with MSCs for seven days ("MSC"). In FIG. 16B, levels
of interleukin-10 (IL-10) are shown in freshly isolated T-cells
prior to culture ("Fresh cells"), T-cells cultured for seven days
in the absence of other cells ("Culture control"), T-cells
co-cultured for seven days with SB623 cells ("SB623"), and T-cells
co-cultured for seven days with MSCs ("MSC"). The values for "MSC"
and "SB623" are averages of three cultures, each containing MSCs or
SB623 cells from a different donor.
FIG. 17 shows levels of IL-17 in IL-23-stimulated T-cells. Percent
expression was determined by flow cytometry after staining cells
for IL-17 with a fluorescent antibody. T-cells were cultured with
or without IL-23, as indicated, and alone or in co-culture with
MSCs or SB623 cells, as indicated.
FIGS. 18A and 18B show levels of CD11a and CD14 in monocyte
cultures after 7 days of culture or co-culture. FIG. 18A shows
representative FACS traces of cells stained for CD1A and CD14.
Monocytes contain a population of CD1A.sup.+ CD14.sup.+ dendritic
cell precursors (leftmost panel). When monocytes were cultured in
the presence of IL-4 and GM-CSF for 7 days, this dendritic cell
precursor population is reduced and replaced by a population of
CD1A.sup.+CD14.sup.- dendritic cells (second panel from left). When
monocytes are co-cultured with MSCs (third panel from left) or
SB623 cells (rightmost panel) in the presence of IL-4 and GM-CSF,
the CD1A.sup.+CD14.sup.- dendritic cell population is reduced and
the CD1A.sup.+CD14.sup.+ precursor cell population is increased.
FIG. 18B shows mean expression data for monocytes (leftmost pair of
bars), monocytes cultured in the presence of IL-4 and GM-CSF for 7
days (second pair of bars from left), monocytes co-cultured with
MSCs in the presence of IL-4 and GM-CSF for seven days (third pair
of bars from left) or monocytes co-cultured with SB623 cells in the
presence of IL-4 and GM-CSF for seven days (right-most pair of
bars). Results for the co-culture experiments were obtained from
three different matched lots of MSCs and SB623 cells.
FIG. 19 shows levels of CD86, expressed as mean fluorescent
intensity, in TNF-.alpha.-stimulated monocyte cultures. Cultures
indicated by "Control" contained PBMCs cultured for five days in
the presence of IL-4 and GM-CSF, then for a further 48 hours in
TNF-.alpha.. Cultures indicated as "with CsA" contained PBMCs
cultured for five days in the presence of IL-4 and GM-CSF, then for
a further 48 hours in TNF-.alpha.+1 ug/ml cyclosporine A. Cultures
indicated as "MSC" contained PBMCs cultured for five days in the
presence of IL-4 and GM-CSF, then for a further 48 hours in
TNF-.alpha.+10.sup.4 MSCs. Cultures indicated as "SB623" contained
PBMCs cultured for five days in the presence of IL-4 and GM-CSF,
then for a further 48 hours in TNF-.alpha.+10.sup.4 SB623 cells.
The results for MSCs and SB623 cells are the average of three
experiments, each using a sample from a different donor. Monocyte
donor was the same in all cases. All cultures were started with
10.sup.5 PBMCs.
FIGS. 20A and 20B show measurements of cytokine expression in
monocytes. FIG. 20A shows the percentage of monocytes in culture
that express the inflammatory cytokine TNF-.alpha.. FIG. 20B shows
the percentage of monocytes in culture that express the
anti-inflammatory cytokine IL-10. Monocytes, selected on the basis
of surface expression of CD14, were cultured without supplement
("negative"), with macrophage colony-stimulating factor ("MCSF"),
with granulocyte/macrophage colony-stimulating factor ("GMCSF"),
with MSCs or with SB623 cells. MSCs and SB623 cells were obtained
from three different donors, indicated as D52, D55 and D65 in the
figure.
DETAILED DESCRIPTION
Practice of the present disclosure employs, unless otherwise
indicated, standard methods and conventional techniques in the
fields of cell biology, toxicology, molecular biology,
biochemistry, cell culture, immunology, oncology, recombinant DNA
and related fields as are within the skill of the art. Such
techniques are described in the literature and thereby available to
those of skill in the art. See, for example, Alberts, B. et al.,
"Molecular Biology of the Cell," 5.sup.th edition, Garland Science,
New York, N.Y., 2008; Voet, D. et al. "Fundamentals of
Biochemistry: Life at the Molecular Level," 3.sup.rd edition, John
Wiley & Sons, Hoboken, N.J., 2008; Sambrook, J. et al.,
"Molecular Cloning: A Laboratory Manual," 3.sup.rd edition, Cold
Spring Harbor Laboratory Press, 2001; Ausubel, F. et al., "Culture
Protocols in Molecular Biology," John Wiley & Sons, New York,
1987 and periodic updates; Freshney, R. I., "Culture of Animal
Cells: A Manual of Basic Technique," Fifth Edition, Wiley, New
York, 2005; and the series "Methods in Enzymology," Academic Press,
San Diego, Calif. Standard techniques in immunology are described,
for example, in "Current Protocols in Immunology," (R. Coico,
series editor), Wiley, updated August 2010.
For the purposes of the present disclosure, the term "peripheral"
is used to refer to portions of the body outside of the central
nervous system. These include, for example, the bone marrow,
peripheral circulation and lymphoid organs.
Preparation of SB623 Cells
Mesenchymal stem cells (MSCs) can be obtained by selecting adherent
cells from bone marrow, and can be induced to form SB623 cells by
expression of the Notch intracellular domain (NICD) in the adherent
cells. In one embodiment, a culture of MSCs is contacted with a
polynucleotide comprising sequences encoding a NICD (e.g., by
transfection), followed by enrichment of transfected cells by drug
selection and further culture. See, for example, U.S. Pat. No.
7,682,825 (Mar. 23, 2010); U.S. Patent Application Publication No.
2010/0266554 (Oct. 21, 2010); and WO 2009/023251 (Feb. 19, 2009);
all of which disclosures are incorporated by reference, in their
entireties, for the purposes of describing isolation of mesenchymal
stem cells and conversion of mesenchymal stem cells to SB623 cells
(denoted "neural precursor cells" and "neural regenerating cells"
in those documents). See also Example 1, infra.
In these methods, any polynucleotide encoding a Notch intracellular
domain (e.g., vector) can be used, and any method for the selection
and enrichment of transfected cells can be used. For example, in
certain embodiments, a vector containing sequences encoding a Notch
intracellular domain also contains sequences encoding a drug
resistance marker (e.g. resistance to G418). In these embodiments,
selection is achieved, after transfection of a cell culture with
the vector, by adding a selective agent (e.g., G418) to the cell
culture in an amount sufficient to kill cells that do not comprise
the vector but spare cells that do. Absence of selection entails
removal of said selective agent or reduction of its concentration
to a level that does not kill cells that do not comprise the
vector.
Senescence in SB623 Cells
As described above, SB623 cells are derived from MSCs by expression
of a NICD in cultured MSCs. Because MSCs that have undergone
manipulation in culture often become senescent; the SB623 cells
derived therefrom were tested for senescence.
SB623 cells do not form colonies in soft agar, indicating that they
are not transformed cells. In addition, when SB623 cells were
prelabelled with carboxyfluorescein diacetate succinimidyl ester
(CFSE), a cell-permeable dye that is diluted by cell division, a
sub-population of cells retained high concentrations of CFSE after
5 days of culture (FIG. 1). This slowly-proliferating (or
non-proliferating) sub-population was not observed in MSC cultures.
Certain cells in the SB623 cell population were also observed to
stain intensely for beta-galactosidase (a marker of cell
senescence) and such cells were more plentiful in SB623 cultures
than in MSC cultures. These results are consistent with the
existence of a pool of non-proliferating, senescent cells in the
SB623 cell population.
Cell proliferation was measured by plating one million MSCs or
SB623 cells and, after three days in culture, measuring viable
cells by Trypan Blue exclusion. FIG. 2 shows that a higher number
of viable cells were observed in the MSC cultures, indicating a
lower proliferative index for the SB623 cells. Cell cycle status
was assessed by propidium iodide staining, which revealed a higher
proportion of cells in resting phase (G0/G1) in SB623 cultures
(FIG. 3), providing further support for a reduced rate of
proliferation in SB623 cells.
An additional assessment of senescence was conducted by staining
populations of SB623 cells for expression of the p16Ink4A protein.
p16Ink4A inhibits the progression from the G1 to S phases of the
cell cycle and is expressed in senescent cells. FIG. 4 shows that a
higher percentage of p16Ink4A-expressing cells were detected in
cultures of SB623 cells, compared to MSCs. Moreover, when cells in
SB623 cultures that retained high CFSE levels after 5 days of
culture were tested for p16Ink4A expression, the sub-population of
SB623 cells expressing p16Ink4A coincided with the fraction
containing high CFSE levels. These results, taken together,
indicate the existence of a subpopulation of senescent cells within
SB623 cultures.
Surface Marker and Cytokine Expression
SB623 cells express a number of surface markers in common with
MSCs. These include CD29, CD44, CD73, CD90, CD105 and vascular cell
adhesion molecule-1 (VCAM-1 or CD106). Levels of CD44 and CD73 were
higher, and VCAM-1 levels were lower, in SB623 cells compared to
MSCs. SB623 cells also express intercellular adhesion molecule-1
(ICAM-1 or CD54), which is not normally expressed by MSCs. See
FIGS. 5 and 6. MSCs and SB623 cells do not express the surface
markers CD31, CD34 and CD45.
SB623 cells also secrete a number of cytokines and trophic factors.
The identity of certain of these factors was determined by blocking
protein secretion with Brefeldin A and testing for intracellular
cytokines by antibody staining and flow cytometry. These studies
showed that SB623 cells produce, among other factors, interleukin
1.alpha. (IL-1.alpha.), interleukin-6 (IL-6),
granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular
endothelial growth factor-A (VEGF-A) and transforming growth factor
beta-1 (TGF.beta.-1). See FIGS. 7 and 8. Amounts of IL-6 and GM-CSF
produced by SB623 cells were generally greater than those produced
by MSCs.
Because senescent cells have been reported to synthesize and
secrete certain growth-stimulatory cytokines and trophic factors
(Orjalo et al. (2009) Proc. Natl. Acad. Sci. USA 106: 17031-17036),
the existence of a population of senescent cells within SB623
cultures suggested the utility of SB623 cell transplantation to
support various types of tissue regeneration. However, the
secretory profile of senescent cells has also been reported to be
pro-inflammatory, which, if it were the case for SB623 cells, might
reduce the usefulness of SB623 cells for cell transplantation
therapy.
Surprisingly, and despite the presence of a population of senescent
cells in SB623 cultures, SB623 cells possess a number of
immunosuppressive properties, as disclosed herein. For example,
SB623 cells suppress proliferation and activation of T-cells, after
the cytokine profile of T-cells, block the differentiation of
monocytes to dendritic cells, and are superior to their parental
MSCs at slowing maturation of dendritic cells.
Suppression of T-Cell Activation and T-Cell Proliferation by SB623
Cells
SB623 cells were added to mixed lymphocyte reactions (MLRs)
containing 10.sup.5 CFSE-labeled peripheral blood T-cells and
10.sup.5 peripheral blood mononuclear cells from an unrelated
donor. Levels of CD69, an early marker of T-cell activation, were
measured to examine the ability of SB623 cells to modulate T-cell
activation. In control mixed lymphocyte reactions, surface
expression of CD69 was robustly induced. However, after one day in
the presence of 10.sup.4 SB623 cells, the fraction of CD4.sup.+
T-cells (i.e., helper T-cells) in the MLR expressing surface CD69
was significantly reduced. See Example 4.
After five days in the presence of SB623 cells, dilution of CFSE in
prelabelled CD4.sup.+ T-cells (indicative of cell proliferation)
indicated that proliferation of CD4.sup.+ T-cells in the MLR was
reduced in the presence of SB623 cells. See Example 4. Thus, SB623
cells are capable of suppressing both T-cell proliferation and
T-cell activation.
Additional effects of SB623 cells on T-cell function included
reduction of surface HLA-DR expression (Example 4 herein),
increased production of regulatory T-cells in in vitro cultures of
naive T-cells (Example 6 herein) and alteration of cytokine
secretion (Examples 7 and 8 herein). SB623 cells were also
effective at reducing T-cell proliferation in a xenogenic
lymphocyte activation system. See Example 5 herein.
Inhibition of Dendritic Cell Development by SB623 Cells
Differentiation of monocytes into dendritic cells (a type of
antigen-presenting cell) and further maturation of dendritic cells
can be stimulated in vitro by exposure of monocytes to the
cytokines interleukin-4 (IL-4) and granulocyte/macrophage
colony-stimulating factor (GM-CSF). This differentiation can be
blocked by interleukin-6 (IL-6) or vascular endothelial growth
factor (VEGF), both of which are among the cytokines known to be
secreted by SB623 cells. See, for example, Tate et al. (2010) Cell
Transplant. 19:973-984 and WO 2009/023251.
The inventors show herein that co-culture of monocytes with SB623
cells reduces both the differentiation of monocytes into CD
1a.sup.+ dendritic cells and the maturation of dendritic cells to a
CD86.sup.+ status. See Examples 9 and 10 infra. Because of their
abilities to reduce production of new dendritic cells and inhibit
the function of existing dendritic cells, SB 623 cells can be used
to treat and/or ameliorate graft-versus-host-disease (GVHD)
resulting from activation of T-cells by presentation of peptides by
antigen-presenting cells, such as dendritic cells.
Because of their various immunosuppressive properties as described
herein, SB623 cells can be used in place of other biological and
chemical immunosuppressants (e.g., cyclosporine, tacrolimus,
sirolimus, interferons, mycophenolic acid, fingolimod, myriocin,
azathioprine, mercaptopurine, dactinomycin, mitomycin C, bleomycin,
mithramycin, anthracyclines, methotrexate, FK506,
cyclophosphamides, nitrosoureas, platinum compounds and
glucocorticoids). Moreover, use of immunosuppressive agents is not
required to accompany SB623 allogeneic transplantation in cell
therapy, e.g., for neuroregeneration and treatment of nervous
system disorders.
Progenitor Cells
Progenitor cells, which can be converted to SB623 cells, can be any
type of non-terminally differentiated cell. For example, totipotent
stem cells as disclosed for example, in U.S. Pat. Nos. 5,843,780;
6,200,806 and 7,029,913 can be used as progenitor cells. Totipotent
stem cells can be cultured (e.g., U.S. Pat. Nos. 6,602,711 and
7,005,252) and differentiated into various types of pluripotent
cells (e.g., U.S. Pat. Nos. 6,280,718; 6,613,568 and 6,887,706),
which can also be used as progenitor cells in the practice of the
disclosed methods.
Another exemplary type of progenitor cells are marrow adherent
stromal cells (MASCs), also known as marrow adherent stem cells,
bone marrow stromal cells (BMSCs) and mesenchymal stem cells
(MSCs). Exemplary disclosures of MASCs are provided in U.S. patent
application publication No. 2003/0003090; Prockop (1997) Science
276:71-74 and Jiang (2002) Nature 418:41-49. Methods for the
isolation and purification of MASCs can be found, for example, in
U.S. Pat. No. 5,486,359; Pittenger et al. (1999) Science
284:143-147 and Dezawa et al. (2001) Eur. J. Neurosci.
14:1771-1776. Human MASCs are commercially available (e.g.,
BioWhittaker, Walkersville, Md.) or can be obtained from donors by,
e.g., bone marrow aspiration, followed by selection for adherent
bone marrow cells. See, e.g., WO 2005/100552.
MASCs can also be isolated from umbilical cord blood. See, for
example, Campagnoli et al. (2001) Blood 98:2396-2402; Erices et al.
(2000) Br. J. Haematol. 109:235-242 and Hou et al. (2003) Int. J.
Hematol. 78:256-261.
Conversion of MSCs to SB623 cells has been described, for example,
in U.S. Pat. No. 7,682,825 (Mar. 23, 2010) and WO 2009/023251 (Feb.
19, 2009); both of which disclosures are incorporated by reference,
in their entireties, for the purposes of describing isolation of
mesenchymal stem cells and conversion of mesenchymal stem cells to
SB623 cells (denoted "neural precursor cells" and "neural
regenerating cells" in those documents).
Notch Intracellular Domain
The Notch protein is a transmembrane receptor, found in all
metazoans, that influences cell differentiation through
intracellular signaling. Contact of the Notch extracellular domain
with a Notch ligand (e.g., Delta, Senate, Jagged) results in two
proteolytic cleavages of the Notch protein, the second of which is
catalyzed by a .gamma.-secretase and releases the Notch
intracellular domain (NICD) into the cytoplasm. In the mouse Notch
protein, this cleavage occurs between amino acids gly1743 and
val1744. The NICD translocates to the nucleus, where it acts as a
transcription factor, recruiting additional transcriptional
regulatory proteins (e.g., MAM, histone acetylases) to relieve
transcriptional repression of various target genes (e.g., Hes
1).
Additional details and information regarding Notch signaling are
found, for example in Artavanis-Tsakonas et al. (1995) Science
268:225-232; Mumm and Kopan (2000) Develop. Biol. 228:151-165 and
Ehebauer et al. (2006) Sci. STKE 2006 (364), cm7. [DOI:
10.1126/stke.3642006cm7].
Cell Culture and Transfection
Standard methods for cell culture are known in the art. See, for
example, R. I. Freshney "Culture of Animal Cells: A Manual of Basic
Technique," Fifth Edition, Wiley, New York, 2005.
Methods for introduction of exogenous DNA into cells (i.e.,
transfection) are also well-known in the art. See, for example,
Sambrook et al. "Molecular Cloning: A Laboratory Manual," Third
Edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel et al.,
"Current Protocols in Molecular Biology," John Wiley & Sons,
New York, 1987 and periodic updates.
Autoimmune Disorders and Allergic Reactions
Autoimmune disorders result from an immune response that attacks
normal healthy tissue. Exemplary autoimmune disorders include, but
are not limited to, amyotrophic lateral sclerosis, ankylosing
spondylitis, thrombocytopenic purpura, Hashimoto's thyroiditis,
Guillain Barre syndrome, pernicious anemia, dermatosyositis,
Addison's disease, Type I diabetes, rheumatoid arthritis, systemic
lupus erythematosus ("lupus"), dermatomyositis, Sjogren's syndrome,
multiple sclerosis, Myasthenia gravis, polymyositis, biliary
cirrhosis, psoriasis, reactive arthritis, Grave's disease,
ulcerative colitis, inflammatory bowel disease, vasculitis, Crohn's
disease, and celiac disease--sprue (gluten sensitive
enteropathy).
Allergies result from an immune hypersensitivity to external
substances that would not normally stimulate an immune response.
Common allergens include pollen, mold, pet dander and dust. Certain
foods and drugs can also cause allergic reactions.
The immunosuppressive properties of SB623 cells, as disclosed
herein, make SB623 cells useful for the treatment of autoimmune
disorders and allergies.
Formulations, Kits and Routes of Administration
Therapeutic compositions comprising SB623 cells as disclosed herein
are also provided. Such compositions typically comprise the cells
and a pharmaceutically acceptable carrier.
The therapeutic compositions disclosed herein are useful for, inter
alia, immunomodulation (e.g., reducing immune activation) and
reversing the progression of various immune disorders. Accordingly,
a "therapeutically effective amount" of a composition comprising
SB623 cells can be an amount that prevents or reverses immune
activation. For example, dosage amounts can vary from about 10;
500; 1,000; 2,500; 5,000; 10,000; 20,000; 50,000; 100,000; 500,000;
1,000,000; 5,000,000 to 10,000,000 cells or more; with a frequency
of administration of, e.g., once per day, twice per week, once per
week, twice per month, once per month, depending upon, e.g., body
weight, route of administration, severity of disease, etc.
Supplementary active compounds can also be incorporated into the
compositions. For example, SB623 cells are useful in combination
with other immune modulators such as cyclosporine for treatment of,
e.g., autoimmune disease or to inhibit transplant rejection and/or
GVHD. Accordingly, therapeutic compositions as disclosed herein can
contain both SB623 cells and cyclosporine (or any other
immunosuppressant). When a composition of SB623 cells is used in
combination with another therapeutic agent, one can also refer to
the therapeutically effective dose of the combination, which is the
combined amounts of the SB623 cells and the other agent that result
in immunomodulation, whether administered in combination, serially
or simultaneously. More than one combination of concentrations can
be therapeutically effective.
Various pharmaceutical compositions and techniques for their
preparation and use are known to those of skill in the art in light
of the present disclosure. For a detailed listing of suitable
pharmacological compositions and techniques for their
administration one may refer to texts such as Remington's
Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., "Goodman
and Gilman's The Pharmacological Basis of Therapeutics,"
McGraw-Hill, 2005; University of the Sciences in Philadelphia
(eds.), "Remington: The Science and Practice of Pharmacy,"
Lippincott Williams & Wilkins, 2005; and University of the
Sciences in Philadelphia (eds.), "Remington: The Principles of
Pharmacy Practice," Lippincott Williams & Wilkins, 2008.
The cells described herein may be suspended in a physiologically
compatible carrier for transplantation. As used herein, the term
"physiologically compatible carrier" refers to a carrier that is
compatible with the other ingredients of the formulation and not
deleterious to the recipient thereof. Those of skill in the art are
familiar with physiologically compatible carriers. Examples of
suitable carriers include cell culture medium (e.g., Eagle's
minimal essential medium), phosphate buffered saline, Hank's
balanced salt solution+/-glucose (HBSS), and multiple electrolyte
solutions such as Plasma-Lyte.TM. A (Baxter).
The volume of a SB623 cell suspension administered to a patient
will vary depending on the site of implantation, treatment goal and
number of cells in solution. Typically the amount of cells
administered to a patient will be a therapeutically effective
amount. As used herein, a "therapeutically effective amount" or
"effective amount" refers to the number of transplanted cells which
are required to effect treatment of the particular disorder; i.e.,
to produce a reduction in the amount and/or severity of the
symptoms associated with that disorder. A therapeutically effective
amount further refers to that amount of the composition sufficient
to result in full or partial amelioration of symptoms of the
relevant medical condition, or an increase in rate of treatment,
healing, prevention or amelioration of such condition. For example,
in the case of treatment for graft-versus-host disease,
transplantation of a therapeutically effective amount of SB623
cells typically results in immunosuppression of grafted cells. If
the disorder is graft rejection, for example, a therapeutically
effective amount is that number of SB623 which, when transplanted,
results in sufficient immunosuppression in the host such that a
graft is accepted. Therapeutically effective amounts will vary with
the type of disease or disorder, extensiveness of the disease or
disorder, and size of the organism suffering from the disease or
disorder.
The disclosed therapeutic compositions further include
pharmaceutically acceptable materials, compositions or vehicle,
such as a liquid or solid filler, diluent, excipient, solvent or
encapsulating material, i.e., carriers. These carriers can, for
example, stabilize the SB623 cells and/or facilitate the survival
of the SB623 cells in the body. Each carrier should be "acceptable"
in the sense of being compatible with the other ingredients of the
formulation and not injurious to the subject. Some examples of
materials which can serve as pharmaceutically-acceptable carriers
include: sugars, such as lactose, glucose and sucrose; starches,
such as corn starch and potato starch; cellulose and its
derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients, such as cocoa butter and suppository
waxes; oils, such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and soybean oil; glycols, such as
propylene glycol; polyols, such as glycerin, sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl
laurate; agar; buffering agents, such as magnesium hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, emulsifiers and
lubricants, such as sodium lauryl sulfate and magnesium stearate,
as well as coloring agents, release agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the compositions.
Another aspect of the present disclosure relates to kits for
carrying out the administration of SB623 cells, optionally in
combination with another therapeutic agent, to a subject. In one
embodiment, a kit comprises a composition of SB623 cells,
formulated in a pharmaceutical carrier, optionally containing,
e.g., cyclosporine or another immunosuppressant, formulated as
appropriate, in one or more separate pharmaceutical
preparations.
Exemplary formulations include, but are not limited to, those
suitable for parenteral administration, e.g., intrapulmonary,
intravenous, infra-arterial, intra-ocular, intra-cranial,
sub-meningial, or subcutaneous administration, including
formulations encapsulated in micelles, liposomes or drug-release
capsules (active agents incorporated within a biocompatible coating
designed for slow-release); ingestible formulations; formulations
for topical use, such as eye drops, creams, ointments and gels; and
other formulations such as inhalants, aerosols and sprays. The
dosage of the compositions of the disclosure will vary according to
the extent and severity of the need for treatment, the activity of
the administered composition, the general health of the subject,
and other considerations well known to the skilled artisan.
In additional embodiments, the compositions described herein are
delivered locally. Localized delivery allows for the delivery of
the composition non-systemically, thereby reducing the body burden
of the composition as compared to systemic delivery. Such local
delivery can be achieved, for example, through the use of various
medically implanted devices including, but not limited to, stents
and catheters, or can be achieved by inhalation, phlebotomy,
injection or surgery. Methods for coating, implanting, embedding,
and otherwise attaching desired agents to medical devices such as
stents and catheters are established in the art and contemplated
herein.
EXAMPLES
Example 1
Preparation of MSCs and SB623 Cells
Bone marrow aspirates from adult human donors were obtained from
Lonza Walkersville, Inc. (Walkersville, Md.) and plated in
.alpha.-MEM (Mediatech, Herndon, Va.) supplemented with 10% fetal
bovine serum (Hyclone, Logan, Utah), 2 mM L-glutamine (Invitrogen,
Carlsbad, Calif.) and penicillin/streptomycin (Invitrogen). Cells
were cultured for three days at 37.degree. C. and 5% CO.sub.2, to
obtain a monolayer of adherent cells. Alter removal of non-adherent
cells, culture was continued under the same conditions for two
weeks. During this time, cells were passaged twice, using 0.25%
trypsin/EDTA. A portion of the cells from the second passage were
frozen as MSCs.
The remaining cells from the second passage were plated and
transfected, using Fugene6 (Roche Diagnostics, Indianapolis, Ind.),
with a plasmid containing sequences encoding a Notch intracellular
domain operatively linked to a cytomegalovirus promoter
(pCMV-hNICD1-SV40-Neo.sup.R). This plasmid also contained sequences
encoding resistance to neomycin and G418 under the transcriptional
control of a SV40 promoter. Transfected cells were cultured at
37.degree. C. and 5% CO.sub.2 in the growth medium described in the
previous paragraph, supplemented with 100 .mu.g/ml G418
(Invitrogen, Carlsbad, Calif.). After seven days, G418-resistant
colonies were expanded and the culture was passaged twice. After
the second passage, the cells were collected and frozen as SB623
cells.
MSCs and SB623 cells prepared as described herein were thawed as
required and used for further study.
Example 2
Proliferative Capacity of MSCs and SB623 Cells
To quantify cell proliferation, one million MSCs or SB623 cells
were plated and cultured for three days. Viable cells were counted
by trypan blue exclusion on Day 3. FIG. 2 shows that fewer live
cells were present in the SB623 cultures, compared to the MSC
cultures.
The cell cycle profile of MSC and SB623 cultures was assessed by
propidium iodide staining. Propidium iodide is a DNA-intercalating
dye that stains cells in the resting phase of the cell cycle more
strongly than proliferating cells. After three days of culture, one
million MSCs or SB623 cells were fixed in 70% ethanol overnight at
4.degree. C. After two washes in PBS/2% FBS, cells were incubated
in one ml of staining buffer (50 .mu.g/ml propidium iodide, 50
.mu.g/ml RNAse) (Sigma, St. Louis, Mo.) in PBS/2% FBS for 30 min in
the dark. Acquisition and analysis were done on a FACSCAlibur.TM.
flow cytometer (BD Biosciences) using a CellQuestPro.TM. program
(BD Biosciences, San Jose, Calif.) on the FL-2 linear channel. FIG.
3 shows greater propidium iodide staining of SB623 cells, compared
to MSCs, indicating a higher fraction of cells in the G0/G1 resting
phase of the cell cycle in SB623 cell cultures.
Dilution of the cell-autonomous dye 5-(-6-)carboxyfluorescein
diacetate (CFSE) was used as an additional measure of the kinetics
of proliferation. For this analysis, an equal number of MSCs and
SB623 cells were labeled for 2 min at room temperature with of 5
.mu.M of 5-(-6-)carboxyfluorescein diacetate (Invitrogen, Carlsbad,
Calif.), then cultured for five days. Flow cytometry acquisition
and analysis (for CFSE) were done on a FACSCAlibur.TM. flow
cytometer (BD Biosciences) using the FL-1 log channel. The results,
(FIG. 1) show that SB623 cell cultures contained a population of
cells with high CFSE content, compared to MSCs, indicating the
presence, in SB623 cell cultures, of a population of non-dividing
or slowly-dividing cells.
The levels of intracellular p16Ink4A protein in MSCs and SB623
cells were assessed as follows. Cells were cultured for three days,
then fixed with 4% paraformaldehyde and permeabilized with PBS
containing 0.1% Triton X-100. After two washes in PBS containing 2%
fetal bovine serum (PBS/2% FBS), cell pellets were resuspended in
0.2 ml of PBS/2% FBS and divided into two samples. One cell sample
was stained with phycoerythrin (PE)-conjugated anti-p 16Ink4A
antibody (BD Biosciences, San Jose, Calif.) and the other sample
was incubated with PE-conjugated mouse IgG as an isotype control.
Samples were analyzed by flow cytometry on a FACSCAlibur.TM. flow
cytometer (BD Biosciences) and the data was converted to percentage
of cells in the culture expressing p16Ink4A by gating on cells that
stained positive for p16Ink4A and negative for IgG. FIG. 4 shows
that SB623 cell cultures contain a significantly higher fraction of
cells expressing p16Ink4A.
Example 3
Surface Marker and Cytokine Expression by MSCs and SB623 Cells
For measurements of cell surface markers, MSCs or SB623 cells were
harvested from culture using 0.25% Trypsin/EDTA (Invitrogen,
Carlsbad, Calif.), washed in PBS/2% FBS and resuspended in 1 ml of
PBS/2% FBS. Cells were incubated with fluorochrome conjugated
antibody to CD29, CD31, CD34, CD44, CD45, CD73, CD90 (all from BD
Biosciences, San Jose, Calif.) or CD105 (Invitrogen, Carlsbad,
Calif.) for 15 min on ice. Cells were then washed once with PBS/2%
FBS and acquired on a FACSCalibur.TM. flow cytometer (BD
Biosciences, San Jose, Calif.). The CellQuestPro.TM. software (BD
Biosciences) was used for data analysis. Results were expressed as
dMFI ("delta mean fluorescence intensity"), using IgG as a control;
i.e., MFI for IgG was subtracted from the MFI obtained for a given
surface marker to obtain the dMFI.
The results are shown in FIGS. 5 and 6. FIG. 5 shows that, although
both MSCs and SB623 cells express CD44, CD73 and CD105, SB623 cells
consistently express higher levels of these surface markers. FIG. 6
shows that SB623 cells also express consistently higher levels of
CD54 than do MSCs.
For detection of intracellular cytokines, cells were cultured for
three days and treated with a 1:1,000 dilution of Brefeldin A
(eBioscience, San Diego, Calif., final concentration of 3 ug/ml)
for six hours prior to harvest. Cells ware fixed and permeabilized
as described above for measurement of intracellular pInk4A, and
incubated with fluorochrome-conjugated antibodies to human GM-CSF
(BD), IL-1 alpha (eBioscience, San Diego, Calif.), IL-6 (BD) or
TGF.beta.-1 (R&D Systems, Minneapolis, Minn.) for one hour
followed by two washes with PBS/2% FBS. Data acquisition and
analysis was performed on a BD FACSCalibur.TM. instrument using
CellQuestPro.TM. software.
The results of these analyses, presented in FIG. 7 show roughly
equivalent levels of expression of IL-1.alpha., IL-6 and GM-CSF by
MSCs and SB623 cells; while FIG. 8 shows that comparable levels of
TGF-.beta.-1 and VEGF-A are produced by MSCs and SB623 cells.
Example 4
Allogeneic Mixed Lymphocyte Reaction (Allo-MLR)
Cells for allogeneic mixed lymphocyte reactions were obtained from
10 ml samples of peripheral blood from healthy, unrelated
individuals. To obtain responder T-cells, a RosetteSep T-cell
enrichment kit (Stemcell Technologies, Vancouver, BC, Canada) was
used according to the manufacturer's specifications. Enriched
T-cells (responder cells) were labeled for 2 minutes at room
temperature with 5 uM 5-(-6-)carboxyfluorescein diacetate (CFSE),
obtained from Invitrogen, Carlsbad, Calif. After serum quenching
and three washes in PBS, the labeled responder cells were plated,
in a volume of 0.1 ml of complete lymphocyte medium (RPMI
(Mediatech, Manassas, Va.)+10% FBS (Lonza, Allendale, N.J.)
containing 10.sup.5 cells, in the well of a 96-well U-bottom
plate.
To prepare stimulator cells, peripheral blood buffy coat
mononuclear cells were recovered after Ficoll.TM. density gradient
centrifugation. Red cell lysis buffer (Sigma-Aldrich, St. Louis,
Mo.) was added for 10 min at 37.degree. C.; then the cells were
washed twice with PBS/2% heat-inactivated FBS. The mononuclear
stimulator cells were either added to the well containing responder
cells (10.sup.5 cells in a volume of 0.1 ml) or 10.sup.5 stimulator
cells were mixed with 10.sup.4 SB623 cells or 10.sup.4 MSCs,
centrifuged and the pelleted cells resuspended in a volume of 0.1
ml of complete lymphocyte medium (as described above) which was
then added to a well of CFSE-labeled responder cells prepared as
described above.
Display of CD69 (an early T-cell activation marker) on the surface
of CD4.sup.+ T-cells in Ute culture, two days after initiation of
the reaction, was used as an assay for T-cell activation. For
analysis of CD69 expression, cells were harvested by pipette after
two days, stained with a peridinin chlorophyll protein
(PerCP)-conjugated anti-CD69 antibody (eBioscience, San Diego,
Calif.), and analyzed using a FACSCalibur.TM. flow cytometer
(Becton, Dickinson & Co., San Jose, Calif.), gating on
CD4.sup.+ lymphocytes.
For measurements of T-cell proliferation, cells were harvested
after seven days of culture and stained with a phycoerythrin
(PE)-conjugated anti-CD4 antibody (BD). A BD FACSCalibur flow
cytometer was used for data acquisition.
In a control allo-MLR, the fraction of T-cells within the CD4.sup.+
population, in which expression of surface CD69 had been induced,
was significantly increased after two days (FIGS. 9A and 9B).
The effect of co-culture with MSCs and SB623 cells on T-cell
activation in the MLR was also assessed. In these experiments,
10,000 MSCs or 10,000 SB623 cells were added to the culture at the
start of the MLR. Under these conditions, the increase in surface
CD69-expressing cells that was observed in control cultures after
two days was significantly reduced by co-incubation with MSCs or
SB623 cells (p<0.05; FIGS. 9A and 9B).
As another measure of T-cell activation, the proliferation rate of
CD4.sup.+ T-cells was assayed 7 days after initiation of the MLR.
For these experiments, cells were harvested from the MLR by pipette
and stained with a PE-labeled anti-CD4 antibody.
Flow cytometry was conducted using a Becton-Dickinson
FACSCalibur.TM. apparatus, gating on CD4.sup.+ cells; and dilution
of CSFE was evaluated as an indicator of the proliferation rate of
the CD4.sup.+ responder T-cells. In a control allo-MLR, more than
80% of the CD4.sup.+ responder T-cells had proliferated after seven
days. In the presence of SB623 cells or MSCs, T-cell proliferation
was significantly reduced (i.e., higher levels of CFSE staining
were observed, FIG. 10).
Induction of surface HLA-DR expression is also a measure of T-cell
activation. Both SB623 cells and MSCs reduced the percentage of
HLA-DR-expressing T-cells in the allo-MLR (FIG. 11).
Thus, by a number of different, independent criteria, SB623 cells
suppressed T-cell activation. The ability to block T-cell
activation indicates the usefulness of SB623 cells for
immunosuppression.
Example 5
Xenogeneic Lymphocyte Activation Reaction
The immunosuppressive properties of SB623 cells were also
demonstrated in a xenogenic transplantation model system. Xenogenic
lymphocyte reactions were established using Sprague-Dawley rat
glial mix cells (comprising astrocytes and microglial cells) as
stimulators and human peripheral blood T-cells, labeled with PKH26
according to the manufacturer's instructions (Sigma-Aldrich, St.
Louis, Mo.), as responders. To obtain glial mix cells, postnatal
day 9 rat brains were harvested and triturated prior to treatment
with 0.25% Trypsin for 30 min. Cell suspensions were filtered
through a 70 .mu.M cell strainer and overlaid on Ficoll.TM. prior
to centrifugation. Glial mix cells were cultured in DMEM/F12/10%
FBS/pen-strep for 14 days prior to use in the assay. The xenogeneic
reaction was performed using cell ratios similar to those used in
the allogeneic MLR (100,000 glial mix cells: 100,000 CFSE-labeled
human T-cells; and optionally 10,000 MSCs or SB623 cells) over a
5-day period. PKH26 dilution in human CD3-gated T-cells (which
includes both CD4.sup.+ and CD8.sup.+ T-cells) was assessed by flow
cytometry.
As in the allogeneic MLR, addition of SB623 cells or MSCs to the
xenogeneic system reduced the degree of proliferation of responder
T-cells otherwise observed after stimulation by the glial mix cells
(FIG. 12). Thus, the immunosuppressive properties of MSCs and SB623
cells are not limited to autologous or allogeneic environments.
Example 6
Effect of SB623 Cells on Development of Regulatory T-Cells
Regulatory T-cells (T.sub.regs) are capable of dampening or
suppressing immune responses. Accordingly, the ability of SB623
cells to support the generation of T.sub.regs was investigated. To
this end, enriched T-cells from peripheral blood, purified as
described in Example 2, were cultured in the presence of
interleukin-2 (IL-2), which has been shown to stimulate the
differentiation of naive T-cells into T.sub.regs, and the effect of
co-culture with MSCs or SB623 cells on this process was assessed.
Co-cultures contained a 10:1 ratio of T-cells to SB623 cells or a
10:1 ratio of T-cells to MSCs (10.sup.5 T-cells:10.sup.4 MSCs or
SB623 cells). Co-expression of the surface markers CD4 and CD25,
secretion of the cytokine interleukin-10 (IL-10) and intracellular
production of the transcription factor FoxP3 were used as markers
for T.sub.regs.
For these experiments, human T-cells were enriched from peripheral
blood using a T-cell isolation kit (StemCell Technologies,
Vancouver, Canada) according to the manufacturer's protocol.
Enriched T cells were cultured overnight in RPMI-1640/10%
heat-inactivated FBS/pen/strep prior to use. On Day -1, 10,000 MSCs
or SB623 cells were plated per well in 96-well U-bottom plates. On
Day 0 of the co-culture assay, 100,000 enriched T cells were
transferred to each well of pre-established MSC or SB623 cell
monolayer also containing 10 ng/ml IL-2. As internal controls,
T-cell cultures were also maintained in the absence of MSCs or
SB623 cells. On day 7, cells were stained for surface CD4 (a helper
T-cell marker) and CD25 (the IL-2 receptor alpha chain), and for
intracellular FoxP3.
The results of the assays for the surface markers CD4 and CD25 are
shown in FIG. 13. Co-culture of SB623 cells with IL-2-stimulated
T-cells significantly increased the number of CD4.sup.+CD25.sup.+
T.sub.reg cells (compare left-most and right-most panels of FIG.
13A) and that this stimulation of T.sub.reg development was greater
when the T-cells were co-cultured with SB623 cells than when they
were co-cultured with MSCs (compare center and right panels of FIG.
13A).
Assays for the forkhead box P3 (FoxP3) protein confirmed these
results. FoxP3 is a transcription factor that regulates the
development and function of T.sub.regs. Intracellular FoxP3 was
detected by culturing or co-culturing T-cells for seven days (as
described above), then fixing and permeabilizing cells with
CytoFix/Perm (eBioscience, San Diego, Calif.). PE-conjugated
anti-FoxP3 antibody (clone PCH101, eBioscience, 1:50 dilution) was
used to stain cells for 30 min, and stained cells were analyzed by
flow cytometry gating on lymphocytes based on cell size. The
results, shown in FIG. 14, demonstrate that co-culture with MSCs
and SB623 cells increased FoxP3 expression by T-cells, in the
presence of IL-2, compared to its expression in T-cells that were
not co-cultured.
One mechanism of immunosuppression by T.sub.regs is through
secretion of anti-inflammatory cytokines such as, for example,
interleukin-10 (IL-10). Accordingly, the percentage of T-cells
producing IL-10 in IL-2-containing T-cell cultures, or in
co-cultures with MSCs or SB623s, was determined by staining for
intracellular IL-10 with a fluorochrome-conjugated anti-IL-10
antibody after seven days of culture or co-culture.
Accordingly, after 7 days of culture or co-culture, cells were
treated with a 1:1,000 dilution of Brefeldin A (eBioscience, San
Diego Calif.) (to prevent secretion of extracellular proteins) for
six hours, fixed with 2% paraformaldehyde for 15 min, then
permeabilized with 0.05% (v/v) Triton-X-100 in PBS/2% FBS for 15
min on ice. Alexa 488-conjugated anti-human IL-10 antibody
(eBioscience, San Diego, Calif.) was then added and the cultures
were incubated on ice for 30 min. Wells were washed twice with 2%
fetal bovine serum/0.01% (v/v) Tween 20; cells were acquired by
pipette and analyzed using a FACSCalibur.TM. flow cytometer
(Becton, Dickinson & Co., San Jose, Calif.). Data analysis was
conducted using CellQuestPro.TM. software (Becton, Dickinson &
Co., San Jose, Calif.).
Results of this analysis revealed that T-cells cultured in the
presence of IL-2 did not express intracellular IL-10; while low
levels of IL-10 were produced by CD4.sup.+ T-cells when they were
co-cultured with either SB623 cells or MSCs in the presence of
IL-2, with slightly more IL-10 being produced by T-cells that were
co-cultured with SB623 cells (FIG. 15).
Example 7
Conversion of Pro-Inflammatory to Anti-Inflammatory Cytokine
Profile by SB623 Cells
The effect of co-culture of MSCs and SB623 cells, on the relative
amounts of pro- and anti-inflammatory cytokines produced by
T-cells, was assessed by measuring levels of IL-10 (an
anti-inflammatory cytokine) and interferon-gamma (IFN-.gamma., a
pro-inflammatory cytokine) in T-cells that had been sub-optimally
activated by treatment with phorbol myristate acetate (PMA) and
ionomycin. For these experiments, T-cells were enriched from
peripheral blood and cultured, or co-cultured with MSCs or SB623
cells, as described above (Example 6), except that culture was
conducted in the absence of IL-2. On Day 7, non-activating doses of
25 ng/ml of phorbol 12-myristate 13-acetate (PMA)/0.5 .mu.M
ionomycin (Io) (both from Sigma-Aldrich, St Louis, Mo.) were added
in the presence of 3 .mu.g/ml BrefeldinA (eBioscience, San Diego,
Calif.) and, 6 hours later, cells were harvested and analyzed for
intracellular expression of IL-10 and IFN-gamma. The non-activating
doses of PMA and ionomycin used in these experiments did not induce
T-cell proliferation, but were sufficient to induce cytokine
synthesis by T-cells. IL-10 levels were measured using an Alexa
488-conjugated anti-human IL-10 antibody (eBioscience, San Diego,
Calif.) as described in Example 6. IFN-.gamma. levels were measured
by FACS, using a PE-labeled anti-human IFN-.gamma. antibody
(eBioscience, San Diego, Calif.).
The results of this analysis are shown in FIG. 16. More than 20% of
freshly-isolated T-cells expressed IFN-.gamma., while less than 1%
expressed IL-10, after suboptimal stimulation with PMA/ionomycin
(i.e., of the cells that expressed either IFN-.gamma. or IL-10,
over 95% expressed IFN-.gamma. and less than 5% expressed IL-10).
However, after 7 days' co-culture with either SB623 cells or MSCs,
of the cells expressing either IFN-.gamma. or IL-10, more than 95%
expressed IL-10, while less than 5% expressed IFN-.gamma.. Thus,
co-culture with either MSCs or SB623 cells converted the T-cell
secretome from one that was pro-inflammatory to one that was
anti-inflammatory.
The secretion of the inflammatory cytokine IFN-.gamma. is a
characteristic of the T.sub.H1 subset of helper T-cells; while
IL-10 secretion is characteristic of T.sub.H2 cells and T.sub.reg
cells. Thus, the shift from IFN-.gamma. secretion to IL-10
secretion, observed upon co-culture of naive T-cells with SB623
cells or MSCs, is consistent with conversion of a population rich
in T.sub.H1 cells into one that contains a large amount of T.sub.H2
cells, T.sub.reg cells, or both. This result also indicates that
co-culture with SB623 cells, or MSCs, directed the differentiation
of T-cells from an inflammatory population (characterized by
T.sub.H1 cells) to an more anti-inflammatory population
(characterized by T.sub.H2 cells and/or T.sub.reg cells), in part
through altering cytokine production by the T-cells.
Example 8
Effect of MSC and SB623 Cell Co-Culture on Production of IL-17 by
T-Cells
Two of the cytokines known to be secreted by MSCs and SB623 cells,
TGF.beta.-1 and IL-6 (see Example 3, above) are also known to play
a role in the development of Th17 helper T-cells (i.e., helper T
cells that secrete IL-17). Accordingly, T-cells were cultured in
the presence of IL-23, which is known to stimulate the development
of Th17 helper T-cells, and the effect of co-culture with MSCs or
SB623 cells, on Th17 cell number, was determined.
For these experiments, human T-cells were isolated and cultured as
described in Example 7, above, with the addition of 10 ng/ml of
IL-23 (Peprotech, Rocky Hill, N.J.) to the cultures. After
treatment with Brefeldin A for 6 hours, cells were harvested, fixed
and permeabilized as described in Example 7, stained with a
PE-conjugated anti-IL17 antibody (eBioscience) and analyzed by flow
cytometry. The results indicated that culture of T-cells in the
presence of IL-23 increased the number of IL-17-expressing cells.
In addition, co-culture of T-cells with MSCs or SB623 cells
resulted in a small increase in the number of IL-17-expressing
cells, in both the absence and presence of IL-23. (FIG. 17).
Example 9
Inhibition of the Differentiation of Monocytes into Dendritic Cells
by Co-Culture with SB623 Cells
The normal course of development of monocytes (expressing CD14)
into dendritic cells (which express CD11a) can be recapitulated in
vitro by culturing monocytes in the presence of interleukin-4
(IL-4) and granulocyte/macrophage colony-stimulating factor
(GM-CSF). MSCs, when co-cultured with monocytes in vitro, are able
to block the differentiation of monocytes into dendritic cells, an
effect that is mediated, in part, by secretion of interleukin-6
(IL-6) by MSCs. Chomarat et al. (2000) Nature Immunology 1:510-514;
Djouad et al. (2007) Stem Cells 25:2025-2032. SB623 cells also
secrete IL-6. See U.S. Patent Application Publication No.
2010/0266554 (Oct. 21, 2010). VEGF, which is also secreted by MSCs
and SB623 cells, is also involved in dendritic cell
differentiation. Therefore, the effect of SB623 cells on monocyte
differentiation was investigated.
Peripheral blood was collected from healthy donors and subjected to
density gradient centrifugation using Ficoll-Paque.TM. (GE
Healthcare, Piscataway, N.J.). Mononuclear cells were recovered by
aspirating the buffy coat, resuspended in RPMI/10% fetal bovine
serum and plated. After overnight culture at 37.degree. C., 5%
CO.sub.2, non-adherent cells were washed off and adherent monocytes
were recovered using 0.25% trypsin/2 mM EDTA. Staining with
FITC-conjugated anti-human CD14 antibody (Becton, Dickinson &
Co., San Jose, Calif.) indicated that over 90% of the cells in
these preparation were monocytes).
Monocytes were cultured in RPMI-1640 (Meidatech, Manassas, Va.)
containing 10% fetal bovine serum (Lonza, Allendale, N.J.), 2 mM
L-glutamine, 2 mM L-sodium pyruvate, 100 Units/ml penicillin, 100
ug/ml streptomycin, 40 ng/ml GM-CSF (Peprotech, Rocky Hill, N.J.)
and 20 ng/ml IL-4 (Peprotech, Rocky Hill, N.J.). Co-culture with
SB623 cells (or MSCs, as control) was conducted at a 10:1 ratio of
monocytes to SB6323 cells (or MSCs); i.e., 100,000 monocytes to
10,000 SB623 cells or MSCs. After 7 days of culture (or
co-culture), a portion of the cells were harvested using
trypsin/EDTA (as above) and incubated with PR-conjugated anti-CD14
antibody and FITC-labeled anti-CD1a antibody (both from
eBioscience, San Diego, Calif.). Acquisition and analysis were
performed using a FACSCalibur.TM. cell sorter using
CellQuestPro.TM. software (both from Becton, Dickinson & Co.,
San Jose, Calif.). Another portion of the cultures were observed by
phase-contrast microscopy.
The results of the cell sorting analysis (FIG. 18) indicated a
higher percentage of CD14.sup.+ cells (i.e., a higher fraction of
monocytes) following co-culture of monocytes with SB623 cells or
MSCs. Moreover, the effect was greater when monocytes were cultured
with SB623 cells, compared to co-culture with MSCs. In addition,
fewer CD1a.sup.+dendritic cells were observed in the co-cultures.
These results indicate that SB623 cells (and, to a lesser extent,
MSCs) are able to block the differentiation of monocytes into
dendritic cells.
Microscopic analysis confirmed these observations. In monocyte
cultures, clusters of dendritic cells were readily observed by
microscopy; but in co-cultures with MSCs or SB623 cells, such
clusters were rarely observed.
Example 10
Inhibition of Dendritic Cell Maturation by Co-Culture with SB623
Cells
After differentiating from monocytes, dendritic cells mature into a
cell that expresses the CD86 surface marker. This maturation can be
recapitulated in vitro by culturing dendritic cells in the presence
of tumor necrosis factor-alpha (TNF-.alpha.). IL-6 and VEGF have
been shown to block the maturation of dendritic cells. Park et al.
(2004) J. Immunol. 173:3844-3854; Takahashi et al. (2004) Cancer
Immunol. Immunother 53:543-550. Since SB623 cells secrete both of
these cytokines, the effect of SB623 co-culture on dendritic cell
differentiation was investigated.
To assess the effect of co-culture of SB623 cells on maturation of
dendritic cells, monocytes were obtained from peripheral blood and
differentiated in vitro into dendritic cells, as described in
Example 9. After 5 days of culture, human (TNF-.alpha. (Peprotech,
Rocky Hill, N.J.) was added to the cultures to a final
concentration of 10 ng/ml. In some cultures, SB623 cells or MSCs
were also added at this time. All samples contained 10.sup.5
monocytes and, in co-cultures, 10.sup.4 MSCs or SB623 cells. As a
control, Cyclosporin A, which inhibits maturation of dendritic
cells to a CD86.sup.+ state, was added to TNF-.alpha.-stimulated
cultures to a final concentration of 1 ug/ml. Two days later, cells
were stained with PE-conjugated anti-CD86 antibodies (Becton
Dickinson & Co., San Jose, Calif.), acquired on a FACSCalibur
cell sorter and analyzed using CellQuest Pro.TM. software (both
from Becton, Dickinson & Co., San Jose, Calif.).
The results, shown in FIG. 19, indicate that a significant fraction
of TNF-.alpha.-matured dendritic cells express CD86, and that this
fraction is lowered by treatment with Cyclosporine A, as expected.
Co-culture with SB623 cells and MSCs also lowers the fraction of
CD86.sup.+ cells. Notably, SB623 cells had a stronger inhibitory
effect on dendritic cell maturation, as measured by CD86
expression, than did MSCs.
Example 11
Alteration of the Secretory Profile of Monocytes/Macrophages by
Co-Culture with SB623 Cells
Human peripheral blood monocytes expressing the CD14 cell surface
marker (i.e., macrophage precursors) were obtained from cells of
the buffy coat by magnetic selection, using anti-CD14-coated
magnetic beads (Miltenyi Biotec, Auburn, Calif.). Separate cultures
of the CD14.sup.+ monocytes were exposed to granulocyte/macrophage
colony-stimulating factor (GM-CSF), which converts them to M1
(pro-inflammatory) macrophages; or to macrophage colony-stimulating
factor (M-CSF), which converts them to M2 (anti-inflammatory)
macrophages; or were co-cultured with either SB623 cells or
MSCs.
The percentage of cells expressing tumor necrosis factor-alpha
(TNF-.alpha., a pro-inflammatory cytokine characteristic of M1
macrophages) and interleukin 10 (IL-10, an anti-inflammatory
cytokine characteristic of M2 macrophages) were determined in these
cultures, as follows. Cultures were exposed to 100 ng/ml bacterial
lipolysaccharide (LPS, Sigma, St. Louis, Mo.) for 24 hours. During
the final 6 hours of exposure to LPS, Brefeldin A and monensin
(both from eBioscience San Diego, Calif.; and both used at 1:1,000
dilution) were added to the cultures. Cells were then stained with
either PE-conjugated anti-TNF-.alpha. or FITC-conjugated anti-IL-10
and analyzed by flow cytometry.
The results of these studies, shown in FIG. 20, indicated that
co-culture with MSCs or SB623 cells increased the fraction of
monocytes in the culture that produced anti-inflammatory cytokines.
Co-culture with MSCs or SB623 cells did not increase the percentage
of cells that produced TNF-.alpha., as did exposure to GM-CSF (FIG.
20A). Notably, the percentage of cells expressing the
anti-inflammatory cytokine IL-10 was increased when monocytes were
co-cultured with MSCs, and was increased even further when
monocytes were co-cultured with SB623 cells (FIG. 20B).
* * * * *