Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF PRODUCING A THYMIC MICROENVIRONMENT THAT SUPPORTS
THE DEVELOPMENT OF DENDRITIC CELLS
This application claims priority from U.S.
Provisional Application No. 60/031,445, filed
October 10, 1996, the entire contents of which is
incorporated herein by reference.
TECHNICAL FIELD
The present invention relates, in general, to
thymic microenvironment and, in particular, to a method
of producing a thymic microenvironment in vitro that
supports the development of dendritic cells from -
hematopoietic pregenitor cells. The invention further
relates to a method of treating congenital and acquired
IS immunodeficiencies, including malignant and autoimmune
diseases and traditional T cell immunodeficiency
diseases such as occurs in AIDS.
BACKGROUND
Dendritic cells are antigen presenting cells (APC)
distributed widely in lymphoid and non-lymphoid tissues -
(Steinman et al, Advances in Experimental Medicine &
Biology 329:1-9 (1993); Steinman, Experimental
Hematology 24:859-62 (1996); Young et al, Stem Cells
14:376-87 (1996); Steinman et al, Immunological Reviews
156:2S-37 (1997)). Several different subsets of
dendritic cells have been demonstrated in peripheral
blood, skin, lymphoid organs and thymus (Caux et al,
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Blood Cell Biochemistry 7:263-301 (1996); Caux et al,
J. Exp. Med. 184:695-706 (1996); Caux et al, J.
Immunol. 155:5427-5435 (1995); Chu et al, Br. J.
Cancer. Suppl 23:S4-10 (1994)). Dendritic cells posses
a distinct morphologic appearance, express high levels
of MHC class I and II and have a potent ability to
process antigens and activate T cells (Wettendorff et
al, Adv. Exp. Med. Biol. 378:371-374 (1995); Wu et al,
J. Exp. Med. 184:903-11 (1996); Shortman et al, Ann.
Rev. Immunol. 14 :29-47 (1996) ; Res et al, J. Exp. Med.
185:191-51 (1997); Mommaas et al, Eur. J. Immunol.
25:520-5 (1995); Mackensen et al, Blood 86:2699-2707
(1995); Caux et al, J. Exp. Med. 180:1841-7 (1994)).
They also appear to play a critical role in mounting
effective immune responses against microorganisms,
neoplasms, and transplanted organs and may play a vital
role in the induction of tolerance (Steinman et al,
Advances in Experimental Medicine & Biology 329:1-9
(l993), Steinman, Ann. Rev. Immunol. 9:271-96 (1991)).
Dendritic cells developing within the thymus
appear to be biologically distinct from extrathymic
dendritic cells (Shorman et al, Ciba Found. Symp. -
204:130-41 (1997); Shortman et al, Adv. Exp. Med. Biol.
378:21-9 (1995); Saunders et al, J. Exp. Med. 184:2185-
96 (1996); Ardavin et al, Nature 362:761-3 (1993);
Ardavin et al, Immunology Letters 38:19-25 (1993);
Ardavin et al, Eur. J. Immunol. 22:859-62 (1992); Suss
et al, J. Exp. Med. 183:1789-96 (1996); Winkel et al,
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Immunology Letters 40:93 -9 (1994)). Bone marrow,
peripheral blood and umbilical cord blood (UCB)
hematopoietic progenitors cultured with GM-CSF, TNF-a,
and other cytokines in vitro generate mixed colonies
containing both monocytes and dendritic cells which
typically express primarily myeloid cell markers (Caux
et al, Blood Cell Biochemistry 7:263-301 (1996); Caux
et al, J. Exp. Med. 184:695-706 (1996); Caux et al, J.
Immunol. 155:5427-5435 (1995); Caux et al, Nature
360:258-61 (1992); Rosenzwajg et al, Blood 87:535-44
(1996); Szabolcs et al, Blood 87:4520-30 (1996); Young
et al, J. Exp. Med. 182:1111-1119 (1995)). In
contrast, thymic dendritic cells express molecules
normally considered as markers of lymphoid cells
(Steinman et al, Immunological Reviews 156:25-37
(1997); Shortman et al, Adv. Exp. Med. Biol. 378:21-9
(1995); Saunders et al, J. Exp. Med. 184:2185-96
(l996); Ardavin et al, Nature 362:761-3 (1993); Ardavin
et al, Immunology Letters 38:19-25 (l993); Ardavin et
al, Eur. J. Immunol. 22:859-62 (1992); Winkel et al,
Immunology Letters 40:93-9 (1994); Li et al, Exp.
Hematol 23:21-5 (1995); Maraskovsky et al, J. Exp. Med. -
184:1953-62 (1996); Sotzik et al, J. Immunol. 152:3370-
7 (1994); Ardavin et al, Immunology Today 18:350-61
(1997); Lafontaine et al, Cellular Immunology 142:238-
251 (l992)). In addition, thymic dendritic cell
progenitors have been reported to generate both
lymphoid cells and thymic dendritic cells (Saunders et
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al, J. Exp. Med. 184:2185-96 (1996); Ardavin et al,
Nature 362:761-3 (1993); Li et al, Exp. Hematol 23:21-5
(1995); Shortman et al, Ann. Rev. Immunol. 14:29-47
(1996)). Shortman and his colleagues have shown that
murine "low-CD4" precursors isolated from the thymus
can develop into dendritic cells and lymphoid cells,
but not myeloid cells, following in vivo injection into
the thymus or in vitro culture under certain conditions
(Saunders et al, J. Exp. Med. 184:2185-96 (1996);
Ardavin et al, Nature 362:761-3 (1993); Li et al, Exp.
Hematol 23:21-5 (1995); Shortman et al, Ann. Rev.
Immunol. 14:29-47 (l996)). These observations
indicated that thymic dendritic cells may be more
closely related to lymphoid cells than extrathymic
dendritic cells. The growth and development of
intrathymic dendritic cells may also be governed by
different cytokines than those important in the
development of extrathymic dendritic cells. Saunders
et al demonstrated that murine "low CD4" thymic
precursors developed into thymic type dendritic cells
in vitro with a combination of TNF-a, IL-1f3, IL-3, IL-
7, and SCF (Saunders et al, J. Exp. Med. 184:2185-96
(1996)). In contrast, the generation of dendritic
cells from peripheral blood and bone marrow progenitors
required GM-CSF (Caux et al, Blood Cell Biochemistry
7:263-301 (1996); Caux et al, Nature 360:258-61 (1992);
Reid et al, J. Immunol. 149:2681-2688 (1992); Santiago-
Schwarz et al, J. Leukoc. Biol. 52:274-81 (l992);
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Strunk et al, Blood 87:1292-1302 (1996)). Thymic
dendritic cells may also have different functional
properties than extrathymic dendritic cells. In
particular, thymic dendritic cells are believed to
5 participate in the process of T cell negative selection
and tolerance induction within the thymus (Steinman et
al, Immunological Reviews 156:25-37 (1997); Shortman et
al, Adv. Exp. Med. Biol. 378:21-9 (Z995); Colic et al,
Develop Immunol. 5:37-51 (1996); Tanaka et al, Eur. J.
1o Immunol. 23:2614-21 (1993y; Douek et al, Internat.
Immunol. 8:1413-20 (1996)). Taken together, these
observations indicate that thymic dendritic cells
constitute a subset of dendritic cells with distinct
developmental, immunophenotypic and functional
IS properties.
Currently, most studies evaluating thymic
dendritic cell biology have utilized murine models, in
part because human thymic dendritic cells have been
difficult to isolate and culture efficiently. For
20 example, while Barcena et al demonstrated that human
fetal thymic organ cultures (FTOC) could support the
development of human fetal liver CD34+lin- cells into
monocytoid cells which displayed dendritic cell
morphology (Barcena et al, J. Exp. Med. 180:123-32
25 i1994)), too few putative dendritic cells were
recovered to be fully characterized. In another study,
Res et al demonstrated that individual human
CD34+CD38dim thymocytes could differentiate into both T
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and NK cells in FTOC and develop into dendritic cells
when cultured in vitro with GM-CSF and TNFoc (Res et al,
Blood 87:5196-206 (1996)). However, the role that the
thymus played in mediating the development of dendritic
cells from intrathymic or extrathymic progenitors
remained unclear because extrathymic culture of
CD34+CD38dim cells was required to generate dendritic
cells. Consequently, certain aspects of human thymic
dendritic cell biology remain uncharacterized.
Prior to the present invention, no experimental
systems existed, other than FTOC, that generated thymic
dendritic cells in vitro in a thymic microenvironment.
The present inveniton provides systems that can be used
to generate thymic dendritic cells from hematopoietic
progenitors.
OBJECTS OF THE INVENTION
It is a general object of the invention to provide
a thymic microenvironment in vitro.
It is a specific object of the invention to
provide a thymic microenvironment suitable for use in -
human thymic transplantation and in the generation of
thymic dendritic cells.
It is another object of the invention to provide a
method of treating a congenital or acquired
immunodeficiency disease or disorder.
The foregoing objects are met by the present
invention which provides a method of producing, in
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vitro, a thymic microenvironment and a method of
generating dentritic cells from hematopoietic
progenitor cells using same.
Further objects and advantages of the present
invention will be clear from the description that
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA-F. Generation of CDla+ cells from
CD34~CD38-lin- and CD34+CD38+1in- umbilical cord blood
cells by co-culture with human thymic stromal cells.
CD34+CD38-lin- and CD34~'CD38+ lin- cells were separated
from lin- UCB cells using fluorescence activated cell
sorting (FACS) using the gates shown in Fig. lA. A
post sort analysis of the sorted CD34~'CD38+lin- cells
(gate R1) is shown in Fig. 1B, and of the sorted
CD34+CD38-lin- cells (gate R2) is shown in Fig. 1C.
After 21 day co-culture with mixed thymic stromal cells
(predominantly TE cells with 5-30~ TF), CD34+CD38-lin-
UC8 cells expanded approximately 40-fold and acquired -
cell surface CDla (Fig. 1F). CD34+CD38+lin- UCB cells
also acquired cell surface CDla (Fig. lE), but to a
lesser extent than CD34+CD38-lin- UCB cells co-cultured
with thymic stroma. Data presented are representative
of 5 experiments.
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Figures 2A-2T. Phenotype of CDla+ cells derived
from CD34+CD38-lin- UCB cells cultured on thymic
stromal monolayers for 21 days. Cells were processed
for 2- or 3-color staining with CDla conjugated to FITC
or PE and monoclonal antibodies labeled with
complementary fluorescent molecules (FITC, PE, or Cy)
as indicated. Histograms show fluorescent intensity of
test (thick line) and isotype-matched control (thin
line) antibodies gated on CDla+ cells. Results are
representative of more than 3 experiments for each
antibody tested.
Figures 3A and 3B. CDla+CD14- cells generated in
vitro from CD34'~38'lin- or CD34+CD38+lin- UCB cells on
thymic stromal cell monolayers are good stimulators in
allogeneic mixed lymphocyte reactions (MLR).
Irradiated CDla+CD14- and CDla-CD14+ cells grown on
thymic stromal cell monolayers for 21 days and
separated by FACS were used to stimulate 1.5 x 105
allogeneic monocyte-depleted peripheral blood
mononuclear cells at different responder to stimulator
ratios. Shown is a representative experiment of two
performed. Error bars are mean ~ standard deviation of
triplicate wells in the experiment shown. Shown in
Fig. lA is the thymidine uptake (in counts per minute
less background of CD4+ responder T cells alone)
induced by CDla+CD14- and CDla-CD14~ cells derived from
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CD34+CD38-lin-UCB progenitors. CDla+CD14- cells induce
significantly more proliferation in MLR than CDla-CD14'~
cells (p<0.001 at a11 stimulator: responder ratios).
Fig. 1B shows the thymidine uptake induced by
CDla+CD14- and CDla-CD14+ cells derived from more
mature CD34+CD38+lin- UCB progenitors. CDla'~CD14'
cells generated from CD34+CD38+lin- UCB are also good
stimulators in MLR, but are less potent that those
generated from CD34+CD38-lin- progenitors (p=0.001).
CDla-CD14+ cells generated from CD34+CD38-lin-
progenitors are not potent stimulators in MLR.
Figures 4A-4F. DCs grown on thymic stroma acquire
markers of differentiated DCs in response to TNF-a.
Shown are the phenotypes of CD34+CD38-lin- UCB cells
cultured in thymic stromal monolayers for 3 weeks with
(Figs. 4D-4F) and without (Figs. 4A-4C) stimulation
with 10 nglml TNF-a for 48 hours prior to harvest.
When compared to cells not treated with TNF-a, a higher
percentage of TNF-a treated CD34'~CD38'lin- cells
express markers of DC including CD86, CD80 and CD83.
Data are representative of 2 experiments performed.
Figures 5A-5D. Human thymic epithelial (TE) cells
and thymic fibroblasts (TF) cultured in an artificial
capillary system aggregate into nodules that
recapitulate the human thymic microenvironment. Human
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TE cells and TF from postnatal thymus were cultured in
vitro and mixed at a ratio of 95 TE:S TF in an
artificial capillary system. Over a two to six week
period, the TE and TF aggregated into nodules
5 containing TE cells in an intertwined pattern
encapsulated by fibroblasts (N=10). Shown are low
power (Fig. 5A and Fig. 5B) and high power (Fig. SC and
Fig. 5D) photomicrographs of a representative nodule
stained by indirect immunofluorescence with anti-
10 keratin mAb AE-3 (Fig. 5A and Fig. 5C) and anti-stromal
mAb TE7 (Fig. 5B and Fig. 5D). The arrowheads point to
the fibrous capsule surrounding the nodule and the
arrows point to a rest of thymic epithelium forming a
rosette in sequential sections. Data are
representative of 10 experiments.
Figures 6A and 6B. Umbilical cord blood
progenitor cells differentiate into CDla+ cells with
dendritic morphology in thymic nodules in vitro. Shown
is the reactivity of anti-CDla mAb Nal/34 in sections
of thymic stromal nodules (Fig. 6A) cultured in media
alone or (Fig. 6B) co-cultured with lin(-) umbilical -
cord blood (UCB) cells for 4 weeks. Note that the
thymic stromal nodules co-cultured with UCB cells
contain numerous CDla+ cells with a dendritic
morphology while the thymic stromal nodules cultured in
media alone do not contain any CDla+ cells. Dendritic
processes of a single CDla+ cell in Fig. 6B are
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indicated by arrows. The CDla+ cells were cytoplasmic
CD3-, CD331o and CD83-, which is consistent with the
interpretation that these cells are early dendritic-
lineage cells. Data are representative of 3
experiments.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an in vitxo
method of producing a thymic microenvironment from
cultured thymic cells and to a method of reconstituting
the immune system of a mammal, for example, a mammal
suffering from an immunodeficiency, using same. The
invention also relates to a method of generating thymic
dendritic cells from hematopoietic cells using such a
thymic microenvironment.
IS Thymic epithelial cells suitable for use in the
production of the thymic microenvironment of the
present invention can be cultured from either fresh or
frozen fetal or postnatal thymic tissues. The thymic
epithelial cells can be cultured and isolated as
described, for example, by Singer et al, Human Immunol.
13:161 (l985) and Singer et al, J. Invest. Dermatol.
92:166 (1989). Culture medium can contain any of a
variety of growth factors, such as EGF, FGF, IGF, and
TGF a/13 and insulin, and/or cytokines, such as IL-6,
IL-8 and IFN-y. Cells so derived can be used
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immediately or stored frozen, for example, in a medium
containing a cryoprotectant such as DMSO.
As indicated above, the present invention relates,
in one embodiment, to a method of producing a thymic
microenvironment from thymic stroma, particularly,
human thymic stroma, and to a method of using same to
support the development of primative hematopoietic stem
cells into dendritic cells. In accordance with this
embodiment, thymic stromal cells (thymic fibroblasts
IO and thymic epithelial cells) obtained from human thymus
tissue as decribed above are depleted of T cells, for
example, by adding hydrocortisone to the culture medium
(Singer et ai, Human Immunol. 13:161 (1985)) or other T
cell depleting agent such as deoxyguanosine (Hong,
Clin . Immunol . Immunopathol . 4 0 : 13 6 ( 19 8 6 ) ) , and
extensive washing. Reduction in the number of thymic
fibroblasts relative to thymic epithelial cells can be
effected, for example, by complement-mediated lysis
and/or growth on a feeder layer of irradiated NIH 3T3
fibroblasts.
Monolayers of thymic stromal cultures prepared as
described above can be used directly for dendritic cell
production or the thymic stromal cells can be cultured,
for example, in an artificial capillary system (eg
optionally, with a coating of ProNectinTM F), to
provide 3-dimension cell aggregates or nodules (see
Example 1). In addition to the technique described in
Example 1, various zero gravity culture strategies or
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strategies providing for three-dimensional cell
aggregation with low to no shear stress can also be
used to optimize thymic cell growth. Rotating-wall
vessel (RWV) technology (Schwartz et al, J. Tiss. Cult.
Meth. 14:51 (1992); Tsao et al, The Physiologist 35:549
(I992)) has shown three dimensional growth of many
epithelial cell types including ovarian cancer cells
(Becker et al, J. Cell. Biochem. 51:283 (1993)),
primary salivary gland cells (Lewis et al, J. Cell
Biochem. 51:265 (1993)), normal small intestine
epithelium (Goodwin et al, Proc. Soc. Exp. Biol. Med.
202:181 (1993)), and embryonic kidney cells (Goodwin et
al, J. Cell Biochem 51:301(1993)). Since RWV
technology maintains low shear while simulating
microgravity compared, for example, to impeller-stirred
biorectors (Spaulding et al, J. Cell. Biochem. S1:249
(1993)), this system can be used for in vitro growth of
functional thymic stroma suitable for engrafting.
(Preliminary data suggest that proliferation and tissue
formation in the spinner culture system (impeller-
stirred biorector) may be limited by shear forces as
increased shear forces (>5-7 dynes/cm2) resulted in
decreased aggregation of and cellular destruction on
microcarriers).
Stem cells suitable for coculture with the thymic
stromal cultures described above can be obtained from
human umbilical cord blood, bone marrow and GCSE
mobilized peripheral blood stem cells (Siena et al,
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Exp. Hem. 23:1463 (1995)) (G-PBSCs). Preferred stem
cells are CD34+CD38-lin- or CD34+CD38+lin-. Such cells
can be isolated from the indicated sources using
commercially available lineage depleting antibody
cocktails and art recognized cell sorting techniques
(see Example 1).
The data presented in Example 1 demonstrate that
umbilical cord blood CD34+CD38-lin- and CD34+CD34+lin-
hematopoeitic progenitor cells cocultured on thymic
stromal monolayers in serum free medium develop into
cells with phenotypic, morphologic and functional
characteristics of thymic dendritic cells. As also
described in Example l, thymic nodules produced as
indicated above can support development of dendritic
IS cells from hematopoietic cell progenitors by incubating
the nodules with the progenitor cells under conditions
such that the progenitor cells migrate into and
differentiate in the nodules.
It will be appreciated that the thymic stromal
cells used in the production of a thymic
microenvironment as described herein can be genetically
engineered so as to express various factors (eg
secreted or surface bound factors) such as CD40 ligand
and flt-3-ligand which can increase the yield and
activity of the resulting thymic dendritic cells.
Retroviral vectors can be used to effect the genetic
manipulation, as can a variety of other engineering
techniques. In addition, the thymic stromal cells can
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be selected/designed to express specific MHC molecules.
The thymic stromal cells can be used as packaging
systems for transfer of genetic materials into
developing hematopoietic cells) (Liu et al, Cell 86:367
5 (1996)). Such cells can be used to reconstitute an
immune system that is superior to that of the
recipient, for example, in its ability to defend
against infection (specific MHC molecules to defend
against infection with HIV) or its resistance to
l0 infection (CCRS mutations in preventing infection with
HIV) .
In another embodiment, the present invention
relates to immortalized human thymic epithelial cells
and to a method of producing same. The establishment
15 of immortalized human thymic epithelial stroma and
individual clonal lines derived from such stroma is
useful for several reasons. A readily available and
consistent source of stroma which supports human thymic
dendritic cell generation reduces intra-experimental
variation and improves the logistics of generating
thymic dendritic cells for study. An immortalized
thymic epithelial stromal line can also be extensively
characterized, validated for clinical use and can be
expanded to scale up the generation of thymic dendritic
cells. In addition, identification of.cytokines..
important in the process of thymic deficient cell
generation is aided by the development of such a line.
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In accordance with this embodiment of the present
invention, human thymic epithelial cells in thymus
chunks can be immortalized via retroviral vector gene
transfer of the papilloma virus E6E7 genes (Furukawa et
al, Am. J. Pathol. 148:1763 (I996); LePoole et aI, In
Vitro Cell. & Devel. Biol. Animal 33:42 (1997)) in
order to increase the number of passages these lines
can be propagated. One such line, designated TE750, was
deposited at the American Type Culture Collection,
12301 Parklawn Drive, Rockville, MD 20852, USA, on
October 10, 1997, under the terms of the Budapest
Treaty, and was given Accession No.
In order to demonstrate that cells such as TE750
cells could support the generation of thymic dendritic
cells, they were co-cultured with CD34+CD38-lin- cells
derived from umbilical cord blood. CDla+CD14-HLA DR+
cells were generated indicating that immortalized
thymic epithelial can also support the development of
thymic dendritic cells from primitive progenitors. In
order to demonstrate that these immortalized cells
could support the generation of thymic dendritic cells
through soluble factors, CD34+CD38-lin- cells were -
placed into the insert of Transwell cultures, separated
from the thymic epithelial by a permeable membrane
which precluded cell-cell contact. While the overall
cell number was reduced compared to cells grown in
standard thymic epithelial cell co-cultures, CDla+CD14-
HLA-DR+ cells could be readily identified in the
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Transwell cultures. Similar observations were made
with co-cultures containing primary non-immortalized
thymic epithelial stroma. These findings indicate that
soluble factors produced by both primary and
immortalized thymic epithelial stroma support the
development of thymic dendritic cells from primitive
progenitors. The thymic stroma cells of the invention
can be used as sources of such factors both defined and
undefined. These findings also indicate that cell
surface bound factors are important in amplifying the
generation of thymic dendritic cells. C-kit ligand and
flt-3-ligand are logical candidates to account for this
activity (Siena et al, Exp. Hem. 23:1463 (1995);
Szabolcs et al, J. Immunol. 1S4:5851 (1995)).
In another embodiment, the present invention
relates to a method of treating or preventing an
autoimmune disease. This embodiment of the invention
results from the fact that thymic dendritic cells play
a critical role in the negative selection of
autoreactive T cells. Accordingly, dendritic cells
pulsed with antigens that incite autoimmune disease,
for example, diabetes or multiple sclerosis (or other -
autoimmune disease), can be used in treatment or
prevention protocols.
Taking as an example diabetes, insulin and other
islet cell autoantigens (including GAD65, GAD57 and
ICA69) are expressed in the thymus and the intrathymic
expression of insulin mRNA is regulated by a known
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disease susceptibility locus for Type I diabetes
(Atkinson et al, New Engl. J. Med. 331:1428 (1994);
Pugliese et al, Nat. Genetics 15:293 (1997)). Thymic
expression of these antigens can mediate the negative
selection and tolerance of islet cell reactive T cells.
For tolerance to occur, these antigens must be
expressed at very high levels in any thymic stromal
cell type or they must be expressed by cells
specializing in negative selection (preferably,
dendritic cells). In accordance with the present
embodiment, dendritic cells propagated and pulsed ex
vivo with a diabetes inciting antigen (such as insulin)
can be used to prevent or treat Type I diabetes.
Specifically, dendritic cells expanded from progenitor
cells of preferably a patient's own bone marrow and
pulsed with diabetes inciting antigen (or encoding
sequence) (Khoury et al, J. Exp. Med. 182:357 (1995))
can be used to treat or prevent this disease. Such
cells can be administered intravenously or
intrathymically. A similar approach can be used to
treat other autoimmune diseases. In the case of
multiple sclerosis, for example, dendritic cells
produced as described above can be pulsed with myelin
basic protein.
In a further embodiment, the present invention
relates to a method of producing a cancer vaccine using
dendritic cells prepared as described herein. A
variety of murine studies have demonstrated that
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dendritic cells generated or isolated from the spleen
or bone marrow, when pulsed with tumor antigens in
vitro and inoculated into tumor bearing animals, serve
as extremely effective cancer vaccines (Porgador et al,
J. Exp. Med. 182:255 (1995); Mayordomo et al, Nat. Med.
1:1297 (1995); Boczkowski et al, J. Exp. Med. 184:465
(1996); Alijagic et al, Eur. J. Immunol. 25:3100
(1995); Bernhard et al, Can. Res. 55:l099 (1995); Yang
et ate, Cell. Immunol. 179:84 (1997); Lotze, Ann. Surg.
226:1 (1997); Engleman, Biol. Bone Marrow Transp. 2:115
(1996); Murphy et al, Prostate 29:371 (1996); Tjoa et
al, Prostate 27:63 (l995); Vieweg et al, Surg. ?Oncol.
Clin. N. A. 4:203 (1995); Nair et al, Inter. J. Can.
70:706 (1997)). More recently, several groups, have
initiated clinical trials designed to determine whether
dendritic cell vaccines can be administered safely and
effectively. In most of these trials, the dendritic
cells have either been isolated or generated from
peripheral blood mononuclear cells (Morse et al, Ann.
Surg. 226:6 (1997); Engelman, Biol. Bone Marrow Transp.
2:115 (l996)). Recently, several methods have been
described for improving the yield, safety, and
efficiency of generating dendritic cells from the
peripheral blood including using serum free media,
adding macrophage conditioned media, and collecting
peripheral blood mononuclear cells after treatment with
chemotherapy and/or cytokines (Morse et al, Ann. Surg.
226:6 (1997); Bender et al, J. Immunol. Meth. 196:121
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(1996); Romani et al, J. Immunol. Meth. 196:137 (1996):
Maraskovsky et al, J. Exp. Med. 184:1953 (1996)).
Immature dendritic cells are preferred as the cellular
platform in a dendritic cell vaccine (Morse et al, Ann.
5 Surg. 226:6 (1997); Romani et al, J. Immunol. Meth.
l96:137 (1997)) as immature dendritic cells are better
antigen processors than mature dendritic cells. In
accordance with this embodiment, immature thymic
dendritic cells produced in accordance with the present
10 invention are pulsed with a tumor antigen (eg, MAGE,
CEA, and her-2/neu (Boon et al, J. Exp. Med. l83:725
(1996)), or nucleic acid (RNA or DNA) encoding same
using, for example, standard techniques. The pulsed
cells can be used in vaccination therapies to treat
15 existing tumors or prevent tumor development in
individuals at increased risk (Boon et al, J. Exp. Med.
l83:725 (1996); Hsu et al, Nature Med. 2:52 (1996)).
In yet a further embodiment of the present
invention, the thymic microenvironments produced as
20 described above can be used in thymic transplantation
for treating congenital and acquired immunodeficiencies
including, but not limited to, DiGeorge syndrome, -
Ataxia-Telangiectasia and Nezelof's disease (congenital
immunodeficiencies) and acquired immunodeficiency
syndromes, such as AIDS and cancer after ablative
chemotherapy (Mackall et al, N. Engl. J. Med. 332:143
(1995)). The present thymic microenvironments can be
transplanted into patients by a variety of methods
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21
including implantation into the omentum or readily
accessible muscles including, but not limited to, the
forearm, thigh and calf muscles.
Certain aspects of the present invention are
described in greater detail in the non-limiting
Examples that follow.
EXAMPLE I
l0 Dendritic Cell Development
in Human Thymic Stroma
Experimental Details
Thymic stromal cultures: Thymic epithelial (TE)
cells and thymic fibroblasts (TF) were cultured by an
explant technique and propagated in enriched medium
containing 67~ DMEM (Gibco BRL, Grand Island, NY), 220
F12 (Gibco BRL), 5~ Fetal Clone II serum (HyClone,
Logan, Utah), 0.4 ug/ml hydrocortisone, 5 ug/ml
insulin, 11 ng/ml recombinant human epidermal growth
factor (Collaborative Biomedical, Bedford MA), 0.18 uM
adenine, 10 10 M cholera toxin (ICN Biomedicals,
Aurora, OH), 0.25 ug/ml fungizone and 50 lZg/ml
genatmicin (TE medium) on irradiated NIH 3T3 fibroblast
feeder layers as described (Singer et al, J. Invest.
Dermatol. 92:166-176 (1989), Singer et al, Hum.
Immunol. 13:161-76 (1985)). Human thymus tissue was
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obtained from the Department of Pathology, Duke
University Medical Center, as discarded tissue from
children undergoing corrective cardiovascular surgery.
Thymic stromal cells (thymic fibroblasts and TE cells)
were depleted of T cells by culture in TE medium, and
extensive washing. Cells were either used immediately
or stored frozen in 7.5% DMSO-containing medium prior
to expansion and use for reconstruction of the thymic
stromal microenvironment. Contaminating thymic
fibroblasts were removed from TE cell monolayers by
treatment with 0.02% EDTA in PBS followed by
complement-mediated lysis with mAb 1B10, which binds to
a cell-surface antigen on human fibroblasts (Singer et
al, J. Invest. Dermatol. 92:166-176 (1989)). TE cell
preparations were >95% positive for the keratin marker
AE-3 and negative for CDla, CD7 and CD14. For
coculture with sorted cord blood CD34+CD38- cells, 2.5
x 105 TE cells were plated in 24 well plates on
irradiated NIH 3T3 fibroblast feeder layers, and
irradiated with 2500 cGy once cells became confluent.
Thymic fibroblasts (TF) were obtained by an explant
technique and grown in TE medium without an NIH 3T3
feeder layer. Typically, TF outgrew TE cells and were
~98% pure by the first passage. The TF cultures used
in this study were >98% positive for M38 (procollagen),
<2% positive for AE3 and negative for CDIa, CD7 and
CD14.
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PCT/US97118317
Lineage depletion and stem cel3 isolation by
fluorescence-activated cell sorting (FRCS): Human
umbilical cord blood (UCB) was obtained as discarded
material from the Department of Obstetrics and
Gynecology of Duke University Medical Center. The UCB
used in these studies was collected in sterile bottles
containing an anticoagulant citrate buffer and
processed within 18 hours of collection. The blood was
diluted 1:2 with Dulbecco's phosphate buffered saline
l0 (PBS) and red blood cells were agglutinated at room
temperature using 1~ Hespan (DuPont Pharma, Wilmington,
DE). Non-agglutinated white blood cells were harvested
and residual red cells were hemolysed at 37~C in 0.17 M
NH4C1 containing 10 mM Tris-HC1, pH 7.2 and 200 mM
EDTA. For lineage depletions, the white cell fractions
were brought to 5-8 X107 cells/ml in PBS containing 4%
fetal calf serum (FCS) and were depleted through the
addition of a commercial antibody cocktail and magnetic
colloid as per the manufacturers instructions (CD34+
StemSep enrichment cocktail, StemCell Technologies,
Vancouver, BC). The mixtures of cells, antibodies and
magnetic colloid were cleared of lineage-marked cells
over a column held in a wrap-around magnet. The cells
that passed through the column (lin- cells) were
collected, washed in Dulbecco's modified MEM (DMEM)
with IO~s FCS and stored on ice.
For fluorescence activated cell sorting (FACS),
lin- cells were pelleted and resuspended in 100 ul
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PBS/2~ FCS, incubated with anti-CD34 and anti-CD38 for
20-30 minutes. After 3 washes in PBS/2o FCS, cells
were sorted on a FACStar Plus cell sorter (Becton
Dickinson) and collected in sterile polystyrene tubes
containing 100 FCS. Post sort analysis was performed
on the last 10~ of cells which were collected in
separate tubes and reanalyzed on the flow cytometer.
Coculture of sorted stem cell and thymic stromal
monolayers: Sorted CD34+38-lin-or CD34+CD38+ lin-
cells were added onto irradiated confluent thymic
stromal monolayers at 103 to 104 cells/well and
cultured in 1 mL of serum free media. This media was
made with 80o IMDM (Gibco BRL) 20o BIT 9500 (StemCell
Technologies), 1 mg/ml glutamine, 40 mg/L lipoprotein
(Sigma) and 0.1~ mercaptoethanol. Cells were fed thrice
weekly by carefully removing 0.5 mL of supernatant and
replacing it with fresh media.
Antibody reagents: mAbs to the following antigens
were used for indirect immunofluorescence staining:
P3x63/Ag8 (IgGl, from American Type Culture Collection
(ATCC), Rockville, MD); CDla (Na1/34, from Andrew
McMichael) (McMichael et al, Eur. J. Immunol. 9:205-10
(1979)); CD2 (35.1, ATCC), CD3 (Leu4, Becton Dickinson,
Mountain View, CA), CD4 (Leu3a, ATCC), CD7 (3Ale)
(Haynes et al, Proc. Natl. Acad. Sci. 76:5829-33
(1979)), CD14 (LeuM3) (Dimitriu-Bona et al, J. Immunol.
130:145-52 (1983)), AE3 (keratin from TT Sun)
(Woodcock-Mitchell et al, J. Cell. Biol. 95:580-88
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(1982)), 1B10 (fibroblasts) (Singer et al, J. Invest.
Dermatol. 92:166-176 (1989)), M38 (C-terminal region of
type I procollagen) (McDonal et al, J. Clin. Invest.
78:1237-94 (1986)), and fluorescein-conjugated goat
5 anti-mouse Ig (Kirkegaard & Perry Laboratories,
Gaithersburg, MD). Directly-conjugated antibodies to
the following antigens were also used for multicolor
analyses of cell surface antigens: CD2 (leu5, FITC),
CD3 (leu4, PerCP), CD5 (leul, PE), CD7 (leu9, FITC),
10 CD8 (SK1, FITC} , CDllc (S-HCL-3, PE) , CD14 (MfP9,
FITC), CD16 (B73.1, FITC), CD19 (1eu12, FITC), CD25
(2A3, FITC), CD33 (leuM9, PE), CD34 (HPCA2, FITC, PE
and Cy), CD38 (ieul7, PE), CD56 (1eu19, PE), CD80
(L307.4, PE) HLA-DR (L243, FITC), IgG1 (X40, FITC and
is PE) from Becton Dickinson Immunocytometry Systems
(BDIS, San Jose, CA) ; CDla (T6, PE) , CD4 (T4, PE) , and
CD83 (HBlSa, PE) from Coulter (Hialeah, FL); CD3
(UCHT1, Cy) from Immunotech, Inc. (Westbrook, ME); CDla
(HI149, FITC), CD2 (RPA-2.10, Cy}, CD40 (5C3, FTTC),
20 CD8 6 ( 2331 ( FUN-1 ) , FITC ) , CD95 ( DX2, FITC) , HLA A, B, C
(G46-2.6, FITC) and IgGl (MOPC-21, Cy) from
PharMingen, Inc. (San Diego, CA)~ and CDS (DK25, RPE-
Cy5) and CD13 (F0831, FITC) from Dako Corporation
(Carpenteria, CA).
25 Phenotypic analysis using flow cytometry: For
FACS analysis of cultured cells, cells were gently
resuspended to leave thymic monolayers undisturbed,
pelieted and resuspended in 100 ul of PBS/ 4~ FCS and
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held on ice. Fluorescence-conjugated antibodies were
added directly to the cell suspensions. Following
incubations for 20-30 minutes at 4~C, the cells were
washed 3 times in PBS/4% FCS. Where necessary, the
cells were fixed in 1% formaldehyde in PBS/2% FCS.
Irrelevant isotype-matched mAbs were used as negative
control. Quantitation of the surface staining was
performed on a FACScan and a FACScalibur (Becton-
Dickinson) using a 488 argon laser for fluorescence
excitation. Data was analyzed using CellQuest software
(Becton Dickinson). In a11 experiments, cells stained
with isotype-matched control antibodies were used to
set cursors so that <1% of the cells were considered
positive.
Microscopy: Sorted cells were centrifuged onto
glass slides using a Shandon cytocentrifuge (Shandon
Southern Instrument Co., Sewickley, PA) at 1000 RPM for
3 minutes. Cytospins were air-dried and stained with
Wright Giemsa stain and examined by light microscopy.
For transmission electron microscopy, thymic nodules
and sorted cells were fixed with 2% glutaraldehyde in
150 nM sodium cacodylate buffer plus 2.5 mM CaCl2, pH
7.2, washed, and embedded in 1% agar. After post
fixation for one hour on ice with 2% osmium tetroxide
plus 1% potassium ferrocyanide, blocks were washed with
cacodylate buffer followed by 200 mM sodium acetate, pH
5.2. Samples were stained en bloc for one hour with 1%
uranyl acetate in sodium acetate buffer. After
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dehydration with ethanol, the pellet was infiltrated
with and embedded in EMBED 812 (EM Sciences, Fort
Washington, PA). 90 nm sections were cut on a Reichart
Ultracut E microtome and stained with uranyl acetate,
followed by Sato lead, washed and examined with a
Philips EM300 electron microscope (Philips, Eindhoven,
The Netherlands).
Mixed lymphocyte reactions: Allogeneic responder
PBMCs (1.5 x 105) obtained f=om healthy donors were
l0 cultured in RPMI 1640 supplemented with loo FCS or 10%
human AB serum in 96 well U-bottom tissue culture
plates. Irradiated (3500 rads) sorted CDla+CD14- and
CDla-CD14+ cells were added in graded doses of 1.5x102
(l:1,000) to 1.5x104 (1:10) cells in a total volume of
200 ul. Cell proliferation after 96 hours was
quantified by adding 1 ~ZCi (37kBq) of [methyl3H]
thymidine (NEN-DuPont, Boston, MA) to each well. After
16 hours, the cells were harvested onto filters and
radioactivity was measured in a scintillation counter
with results presented as the mean cpm for triplicate
cultures.
Development of human thymic stromal
microenvironment nodules: Cultured thymic stromal
cells were co-cultured in an artificial capillary sytem
(Cellmax; Cellco, Inc., Germantown, MD) with a coating
of ProNectin'~' F to promote adhesion of stromal cells to
the capillaries. 20-100 x 106 thymic stromal cells
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(95% TE cells by reactivity with anti-keratin antibody
AE-3 and 5~ TF by reactivity with anti-procollagen
antibody M38) were seeded per capillary module with an
extracapillary space of 12 ml. TE medium (Singer et
al, Hum. Immunol. 13:161-76 (1985)) was pumped through
the capillaries at a rate of 10 ml/min. Within 2-6
weeks, 1-2 mm nodules were readily apparent by visual
inspection in the extracapillary space of the capillary
modules. Nodules were harvested by cutting the module
t0 with a sterile pipe cutter. The nodules were separated
from the capillaries by scraping with a sterile rubber
policeman, and washed with DME containing 5o FCS.
Sorted CD34+38-lin- or CD34+CD38+ lin- cells at 103 to
104 cells/well were added onto 24 well plates
containing approximately 10 micronodules/well and
cultured in 1 mL of serum free media.
Results
CD39+ cord blood cells differentiate into CDla+
cells on thymic stromal cell monolayers: In order to
determine whether human thymic stroma could support the
development of DCs from hematopoietic progenitors,
CD34+CD38- lin- (R2 in Fig. lA) and CD34+CD38+lin-
umbilical cord blood cells (R1 in Fig. lA) were
isolated by sterile cell sorting and co-culture with
pre-established irradiated human thymic stromal
monolayers (Herbein et al, Stem Cells (Dayt) 22:187-97
(1994), Terstappen et al, Blood 777:1218-27 (1991)).
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Prior to co-culture, the sorted populations had greater
than 98$ purity (Figure 1B and 1C) and were >98$ CDla-
(Figure 1D). Following co-culture with thymic stromal
monolayers in serum free media for 21 days, CD34+CD38-
cells expanded 43 ~ 17 fold (N=3) and the CD34+CD38+
cells expanded 32 ~ 16 fold (N=3). UCB progenitors
cultured in serum-free media alone did not expand nor
change in morphology. Immunophenotypic analysis of co-
cultured cells revealed the presence of a number of
CDla+CD14-HLA-DR+ cells (Figures 1 and 2) similar to
previous descriptions of human DCs (Caux et al, Blood
Cell Biochemistry 7:263-301 (1996)). The percentage of
CDla+CD14- cells generated from CD34+CD38- cells ranged
from 5-15% (mean 8.2o N=3) and from CD34+CD38+ cells
ranged from 2-100 (mean 4.8~ N=3). The observation
that CDla+CD14- cells could be generated from both the
CD34+CD38-lin- and CD34+CD38+lin-populations suggested
that both of these cell types could develop into DCs in
the thymic stroma monolayers.
Morphology of CD34+ cells expanded in thymic
stroma: In order to confirm that the CDla+ cells grown
in thymic monolayers were DCs, CDla+CD14- cells
generated after 21 days of culture from both CD34+CD38-
lin- and CD34+CD38+lin- umbilical cord cells were
isolated by FACS and examined by light and electron
microscopy. CDla-CD14+ cells were also sorted from
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both cultures to serve as controls. Analysis of the
sorted cells revealed a purity greater than 97$. By
light microscopy, CDla+CD14- cells possessed a DC
morphology with an irregular shape and multiple
5 dendritic processes. Examination of the ultrastructure
by EM showed that CDla+CD14- cells had euchromatic,
lobulated or indented nuclei and a clear cytoplasm with
rough endoplasmic reticulum and well-developed Golgi
apparati. These cells did not contain Birbeck
10 granules. These findings are consistent with these
cells being mature thymic type DCs. In contrast, the
control CDla-CD14+ cells from both precursor types had
the morphologic appearance of macrophages, with
indented nuclei and foamy cytoplasm and no evidence of
15 cytoplasmatic dendritic projections.
Immunophenotype of CDla+ cells expanded in thymic
stroma: In order to better characterize the DCs
generated from UCB progenitors on thymic monolayers,
20 extensive phenotypic evaluations were performed using
multiparameter FACS analysis (Fig. 2). CDIa+ cells
generated on thymic stroma from CD34+CD38-lin- UCB
cells were negative for surface CD3, CD8, CD19, CD25,
CD34, and CD95, expressed CD2, CD4, CDllc, CD13, CD16,
25 CD33, CD38, CD40, CD45, CD49e, CD80, CD83, CD86, MHC
class I and MHC class II.
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Ability of CDla+CD1Q- and CDla-CD14+ cells
generated in thymic stroma to act as antigen presenting
cells: In order to determine whether the putative DCs
generated on thymic stroma were able to activate T
cells, CDla+CD14- and CDla-CD14+ cells were sorted by
FACS and tested in allogeneic MLRs. CDla+CD14- cells
were much more potent stimulators in the MLRs than
CDla-CD14+ cells (Fig. 3). Further, CDla+CD14- cells
generated from CD39+CD38-lin- UCB cells were more
potent stimulators of the MLR on a per cell basis than
the CDla+CD14- cells generated from CD34+CD38+lin-
cells (Fig. 3). This suggests that more primitive
progenitors may not only generate larger numbers of DCs
but that these DCs may be qualitatively different from
DCs generated from more mature progenitors.
Effect of TNF-a on thymic DC: Analysis of CDla
and CD14 expression on CD34+CD38-lin- UCB progenitors
co-cultured with thymic stroma revealed the presence of
several phenotypically distinct populations of cells.
One possible explanation for this observation is that
the co-cultures contained DCs at multiple stages of
development. To test this hypothesis, the co-cultures
were treated for 48 hours with TNF-a (10 ng/ml), a
previously described DC maturation factor (Caux et al,
Blood Cell Biochemistry 7:263-301 (l996), Caux et al,
J. Exp. Med. 184:695-706 (1996), Caux et al, Nature
CA 02268284 1999-04-08
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360:258-61 (1992)). TNF-a treatment induced expression
of CDla, CD83, CD80 and CD86 on large numbers of cells
derived from CD34+CD38-lin- progenitors (Fig. 45). In
addition, most of these cells displayed a dendritic
cell morphology. While TNF-a treatment of co-cultures
established with CD34+CD38+lin- cells caused an
increase in the fraction of cells with mature DC
markers, not a11 cells expressed DC markers and a
significant number of CDla-CD33+ cells were also
observed. This suggested that these cultures may have
contained a significant fraction of non-DC myeloid
cells. This was confirmed by light microscopic
examination that revealed a number of myeloid lineage
cells including neutrophils and macrophages at
different stages of maturation in the CD34+CD38+lin-
co-cultures treated with TNF-a.
Formation of thymic microenvironment nodules from
cultured thymic epithelial cells and thymic
fibroblasts: Since thymic stromal monolayers do not
have the full differentiation capacity of reaggregation
cultures such as that seen with fetal thymic organ
cultures (Barcena et al, J. Exp. Med. l80:123-32
(l994), Res et al, Blood 87:5196-206 (1996), Spits et
al, Blood 85:2654-70 (1995)), and due to the
difficulties of obtaining sufficient human fetal thymus
for studies, a culture system was developed to form
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three-dimensional aggregates of cultured post natal TE
cells and thymic fibroblasts. After 2-6 weeks of co-
culture in an artificial capillary system, human thymic
fibroblasts and TE cells aggregated to form 1-2 mm
nodules with a morphology and phenotype consistent with
a thymic stromal microenvironment devoid of
hematopoietic cells (N=10) (Fig. 5). The nodules
contained TE cells (keratin positive) in a fibroblast
matrix (identified by TE7) that was encapsulated by a
l0 layer of procollagen-positive fibroblasts. By
transmission electron microscopy, the thymic stromal
nodules contained numerous desmosomes and hemi-
desmosomes indicating that the epithelial cells within
the nodules are able to interconnect and form a network
similar to that seen in normal thymus (Haynes et al, J.
Exp. Med. 159:l149-68 (1984), Haynes et al, J. Immunol.
132:2678 (1984)).
TE cells in nodules did not terminally
differentiate as determined by lack of reactivity with
mAbs STE1, STE2 and 11.24 (CD44v9) (Patel et al, J.
Clin. Immunol~. 15:80-92 (1995)), nor did they form
Hassall's bodies. This pattern is similar to that seen
in the thymic stroma of patients with severe combined
immunodeficiency (reviewed in Haynes et al, J. Exp.
Med. 159:1149-68 (1984), Patel et al, Int. Immunol.
6:247-254 (1996) ) .
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CD34+ cord blood cells differentiate in thymic
nodules into CDla+ cells with DC morphology: To test
the functional status of the thymic nodules, an
evaluation was made as to whether umbilical cord blood
hematopoietic cell progenitors migrate into and
differentiate in the nodules in vitro. Lin- UCB cells
were incubated with thymic nodules in a 24-well flat-
bottom plate in serum free medium at 37~C. After 28
days of co-culture with thymic nodules, the nodules
were analyzed for markers of T and NK cells (CDla, CD3,
CD7), progenitor cells (CD33, CD34), myeloid cells
(CD14) and DC (CDla, CD83). Nodules cultured in the
absence of UCB progenitor cells were also analyzed. No
CD3 or CD7 expressing cells were detected in the
nodules by indirect immunofluorescence. However, there
were numerous CDlabright cells with dendritic
morphology in the nodules seeded with lin- UCB cells
(Fig. 6B), but not in the nodules cultured without UCB
cells (Fig. 6A). The CDla bright cells were CD331o and
CD83-. Further, at day 0, lin- cells did not express
CDla, suggesting that CDIa+ cells resulted from the
differentiation of progenitor cells within the nodule
and not from spontaneous expansion of dendritic cells
contaminating the lin- population. Taken together,
these findings indicate that progenitor cells migrated
into the thymic nodules, and that thymic stromal
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nodules were able to support CD34+lin- UCB cell
development into DCs.
EXAMPLE 2
The slow turning lateral vessel (STLV} (Synthecon,
5 Inc., Friendswood, TX) can be used to assess the
utility of low fluid shear forces (typically 0.81
dyn/cm2; Tsao et al, The Physiologist 35:549 (1992)) on
the growth and differentiation of TE cells and thymic
fibroblasts (TF). The STLV, filled with GM (Table 1}
l0 can be inoculated with Cytodex-3 microcarrier beads
(collagen-coated dextran, Pharmacia) at a concentration
of 10 cellslbead. After attachment of either TF or TE
cells to the beads, the vessel can be rotated at
calculated shear forces ranging from 0.51 dyn/cm2 to
15 0.92 dyn/cm2 (Tsao et al, The Physiologist 35:549
(1992); Goodwin et al, Proc. Soc. Exp. Biol. Med.
202:181 (1993)) for 7d. Aliquots of beads can be
removed every 1-2 days and cell number and viability
enumerated by the method of Goodwin et a1, Proc. Soc.
20 Exp. Biol. Med. 202:181 (1993)). Aggregate formation
can be assessed by visual inspection under light
microscopy and scanning electron microscopy. To
determine the status of epithelial cell
differentiation, indirect immunofluorescent studies can
25 be performed with mAbs STE1, STE2, A3D8 and 11.24
(Patel et al, Int. Immunol. 7:277 (1995)) on frozen
sections of microcarriers or by indirect
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immunofluorescence and flow cytometry on cells
liberated from Cytodex-3 beads by collagenase
treatment.
s Table 1
Components of thymic epithelia (TE) feeding medium
Component Final Concentration Supplier
DMEM 67% Gibco
F12 22% Gibco
Fetal Clone II 5% HyClone
Hydrocortisone 0.4 ~g/mt Calbiochem
Cholera toxin 10'' M SchwarzlMann
Insulin 0.135 I.U.lml Sigma
Adenine 0.18 ~M Sigma
Sodium pyruvate 110 ~g/ml Gibco
EGF* 11.2 ng/ml Amgen
Fungizone 247 nglml Gibco
Gentamicin 50 ~g/ml Whitaker
*Recombinant human EGF
io
One of the problems in growing TE cells has been
the overgrowth of fibroblasts which divide more rapidly
than TE cells in two-dimensional tissue culture
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systems. Growth of TF and TE cells may be different in
a low gravity setting than on planar surfaces as has
been demonstrated in Example 1. The problem of
fibroblast overgrowth can be circumvented by purging
the system of TF by complement mediated lysis after
treatment with the fibroblast-specific mAB 1B10
(Singer, J. Invest. Dermatol. 92:l66 (1989)). Thymic
fibroblasts are, however, stimulatory for TE cell
growth. Thus, to avoid overgrowth of fibroblasts but
retain their effects on TE cell proliferation and
tissue architecture, TF and TE cells can be co-cultured
at different ratios(1:1, 1:5 and I:25) on Cytodex-3
microcarrier beads in the STLV to determine the optimal
ratio of TF to TE cells. Cell number, viability and
differentiation status can be assessed as above. TF
cells can be differentiated from TE cells by reactivity
with anti-keratin mAb AE-3 on cytopreps or sections or
by flow cytometry using antibodies to CD104 and CD205.
TE cells express cytoplasmic keratins and CD104 while
TF do not. TF do express surface CD105. To assess
tissue architecture, immunofluorescence can be
performed on frozen sections of beads using mAbs AE3
(to label TE cells), TE7 (to label TF) and STE1 and
STE2 (to assess differentiation status of TE cells)).
Tissue structure can be analyzed by transmission
electron microscopy which can easily differentiate TF
from TE cells, as TE cells (but not TF) contain
tonofilaments and desmosomes.
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EGF, insulin and~IL-6 are growth factors for human
TE cells and combinations of these factors may improve
the growth of TE cells. Le et al, J. Exp. Med.
174:1147 (1991) have proposed that TGF-alpha is a
growth factor for TE cells and that TGF-beta may be
inhibitory for TE cell growth. IL-8 may be an
autocrine growth factor for TE cells or may influence
the expression of surface molecules. To increase the
rate of proliferation and longevity of TE cell
cultures, EFG, FGF, TGF-alpha, TGF-beta, IL-6 and IL-8
can be used alone or in combination to supplement TE
cell and/or fibroblast growth. Not only may these
factors affect growth and differentiation, they may
(like IFN-gamma) affect the expression of adhesion and
MHC molecules involved in T cell development. The
effect of these factors can be determined by indirect
immunofluorescence and flow cytometry on the expression
of surface molecules expressed on TE cells.
To establish that precursor or mature T cells
migrate to the thymic stromal tissues grown in
microgravity, thymic stromal grafts can be implanted
under the renal capsule of SCID mice using techniques
previously reported (Barry et al, J. Exp. Med. 173:167
(1991)). SCID mice can be treated with anti-asialo GM-
1 to abrogate endogenous NK cell activity. The ability
of autologous and allogeneic thymocytes to migrate to
the grafts can be tested by IP injection of 50 x 106
thymocytes. Migration to grafts can be assessed by IF
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using anti-human CD2, CD3, CD4, CD6, CD7 and CD8 mAbs
(all specific to thymocytes at different stages of T
cell development). It can be determined whether thymic
stromal cell cultures grown in microgravity function to
induce thymocyte differentiation in vitro. Immature
populations of autologous thymocytes, both triple
negative (CD3-4-8-, CD7+) and double positive
(CD31~4+8+), can be purified based upon expression of
cell surface antigens by a combination of panning and
fluorescence activated cell sorting (FACS) on the
Becton-Dickinson FACStarPlus, The immature thymocytes
can be injected into autologous stromal tissues (grown
in vitxo in microgravity) using a Narishigi
micromanipulator, and as a control into thymocyte-
depleted chunks of autologous thymus. After in vitro
culture, aliquots of tissues harvested at intervals up
to 4 weeks can be analyzed for T cell differentiation
by LF on frozen sections using mAbs to CD1, CD2, CD3,
CD4, CD6, CD7 and CD8.
A11 documents cited above are hereby incorporated
in their entirety by reference.
One skilled in the art will appreciate from a
reading of this disclosure that various changes in form
and detail can be made without departing from the true
scope of the invention.
