Note: Descriptions are shown in the official language in which they were submitted.
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Title: Method of Monitoring the Menstrual Cycle andlor Pregnancy in a
Female
FIELD OF THE INVENTION
The present invention relates to methods of monitoring uterine status
to determine the suitability for a successful pregnancy. More specifically,
the
present invention relates to methods of assessing or monitoring the
menstrual cycle from the pre-ovulatory stage into the luteal phase andlor the
first half of pregnancy by assessing the adhesion of human lymphocytes to
uterine or lymphoid tissues under the influence of gestational hormones.
BACKGROUND OF THE INVENTION
Transient granulated lymphocytes are described in the pregnant uteri
of >20 species'. In women and mice, these cells are Natural Killer (NK)
cells and their activation/maturation depends upon uterine decidualization
rather than presence of conceptuses.2~3 The life history and functions of
uterine NK (uNK) cells are more fully known in rodents than women. In
women, uNK cells are most frequent in first trimester, representing over
70% of the nucleated bone marrow-derived cells in decidual cell
suspensions. Human data also suggest that uNK cells are distinctive,
tissue based cells. Most circulating human NK cells are CD16+ CD56dim;
uNK cells are CD16- t;D56bright 2,4. The minor circulating CD56b~'9"t subset
preferentially expresses (95%) very high levels of L-selectin5, a molecule
central to initiation of extravasation. Fewer CD56d'm circulating cells (24%)
express L-selection and at a much lower surFace density5. The two major
NK cells functions, target cell lysis and cytokine production, may be
displayed separately or dually by single cellso. Human ul\IK cells display
both functions in vitro-9 but their in vivo functions are undefined. Many
current studies of human uNK function address interactions with
trophoblast'°-'2. Ofiher recent reports indicate human uNK cells
express
angiogenic factors including Ang-2'3, an antagonist to endothelial cell TIE-2
and thus, a vessel destabilizing molecule and IVKC~S, a potent endothelial
cell mitogen'4.
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In vivo studies of marine uNK cell functians advanced rapidly after
availability of strains genetically deficient in NK cells'5° 's.
Histological
studies established that NK cell deficient mice da not differentiate uNK
cells"~'$. In NK-deficient strains with unrelated genetic changes, a common
uterine phenotype was developed by 48 hr after implantation. The
anomalies were endothelial cell hypertrophy and damage in mesometrially-
positioned uterine vessels, lack of uterine arteriole wall and lumen changes
indicative of pregnancy and hypocellularity of decidua. Absence of lytic NK
cell function does not explain these results, thus, a cytokine deficiency
hypothesis was pursued. Interferon-gamma (IFN-~y) is the prototypic
cytokine product of NK cells. IFN-y is an induced molecule known to
regulate expression of >1000 genes, many of which are expressed by
vascular and decidual tissues'9~ 2°. IF1V-y is expressed in human and
marine
uteri during normaP gestatian2'-23 but many authors regard IFN-y as
detrimental to pregnancy2a, 2s. In an experimental series, it was found (i)
IFN-
y peaks in mouse mesometrial uterus at gestation day (gd) 10 at 10
IUlimplantation site and (ii) only 10% of this comes from non uNK cells2s.
Transplantation showed that higher levels of uNK-cell derived IFN-'y are
essential for pregnancy-induced modification of the spiral arteries and
integrity of decidua while the lower level, non lymphocyte-derived IFN-y is
adequate for maturing uNK cells and limiting their numbers. Daily
recombinant muIFN-y (100-1000 IUI6 days) in alymphoid mice promoted full
uterine artery modification and normalized deciduasz'. Tumor necrosis
factor-alpha, another NK and uNK cell product, lacked these effectsZS, Zy
Mechanisms that transform endometrium to decidua are endocrine-
related. In humans, decidualization begins 7-14 days after the surge in
luteinizing hormone (LH) (LH+7-14) and continues if pregnancy occurs2s-so.
Early decidual development appears important for implantation and most
human pregnancy wastage occurs in this peri-implantation interval. Human
uNK cells begin to increase in number about LH+3, encircling arteries and
uterine glands. Stromal cell changes that cuff the spiral arteries (Streeter's
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columns) are seen at LH+8. By LH+11 to +13, very large numbers of uNK
cells are found throughout the stroma accounting of 30-40% of all cells4.
Gap junction-like contacts are found between some human uNK cells and
early decidua, that appear essential to the continued differentiation of both
cell types3~, suggesting that normal uNK cell numbers and levels of function
contribute to human implantation success.
Reproductive cycles of mice differ to human by virtual absence of a
luteal phase. Decidualization and uNK cell activation are initiated by
implantation, thus, uNK cell deficits do not influence mouse embryo
implantation~'~ 22. UNK cells proliferate rapidly within decidua2~ 3 but
recently it
was established that self-renewing uNK progenitor cells do not reside
there. Uterine segments from normal mice were grafted by end-to-end
anastomosis into uNK cell deficient or normal recipients who were then
mated. uNK cells were generated only when hosts had NK cell
progenitors3s,sa. In pregnant mammals, thymus and marrow involute during
pregnancy3~~ ~ while spleen and lymph nodes (LN) become hypercellular3'~
38. By transplanting to NK cell deficient mice with established pregnancies,
it
was found that gestation induces acute uNK cell recruitment from spleen
but not from marrow or thymus. uNK cells appear to be recruited to decidua
basalis and then move into the mesometrial triangle, the entry portal for
nerves and vessels supplying the uterus and developing feto-placenta
unltS3~39,40. In decidua basalis of normal mice at mid gestation, 7% of uNK
cells are within vessels homologous to spiral arteries, another 30% are in
walls of these vessels and the remaining cells, as resolved in paraffin
embedded sections, are associated with other tissues4~.
In normal mice, uNK cells are a major source of inducible nitric oxide
synthase22, an IFN-y regulated enzyme producing the powerful vasodilator
nitric oxide (NO). In uNK cell deficient mice, expression of this enzyme i s
induced in trophoblast but at very !ow levels2z that cannot dilate the spiral
arteries (Kiso and Croy, unpublished vascular casting data). Ineffective
dilation of spiral arteries is a hallmark of the human gestational
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complication hypertensionlpre-eclampsia'~2~ '~. Despite extensive study of
this syndrome, its frequency remains constant and there is no consensus
on underlying causes'~° ~. Systemic endothelial cell damage underlies
clinical symptoms and may be mediated by dysregulated blood cytokine
balance4s, 4'. Some authors suggest immunological contributions48-so but
assessment of changes in frequency or functianal activities of uNK cells is
just beginning5'-ss_ Women achieving pregnancy by assisted reproductive
technology (ART) are reported at higher risks for pre-eclampsia than women
carrying naturally conceived conceptusess4~s°. Thus, inappropriate
uterine
recruitment of human NK cells may contribute to two health-related
problems: implantation failure through lack of decidual maintenance and
predisposition in pregnant women to preeclampsia. Therefore, it is critically
important to define the molecules contributing to the movement of human
uNK cells and their progenitors into the uterus and to the specification of
their intrauterine locations. Subnormal uNK cell frequencies are reported in
women with recurrent spontaneous abortions', suggesting an additional
obstetrical group that may benefit from the proposed studies.
Movement of leukocytes from vessels into tissue has been
extensively characterized in non-reproductive organs and many techniques
have been validated for such works2-ss. Lymphocytes constitutively express
the tethering molecule L-selectin, which interacts with Peripheral LN
Addressin (PNAd) and Mucosal Vascular Addressin-1 (MAdCAM-1)
expressed by the microvillous surface of endothelium in LN and Peyers
Patches (PP). Avidity of these interactions is modulated by physiological
responses including cytokines, inflammation and fever which trigger rolling
for egress of non activated lymphocytes from vessels5° ss~s8. Firmer
adhesion
and trans-endothelial migration involve integrins, particularly x4(37, which
uses MAdCAM-1. Recruitment of activated cells requires only the latter
mechanism and down regulation of L-selectin is paired with upregulation of
oc4f37 as naive cells begin to roll and dock. Iln the presence of cytokines,
Vascular Cell Adhesion Molecule 1 (VCAM-1) is induced on endothelium
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and utilized by lymphocytes~9. The f32 integrin, Leukocyte Function
Associated Antigen-1, (LFA-1) interacting with its ligands intercellular
Adhesion IVlolecules (iCAfVI)-1 and --2 also mediates firm adhesion but is
not modified by fever ranges similar fio those seen at human ovulation68.
In view of the foregoing, there is a need in the art to define the
molecules contributing to the movement of human uterine NK cells and their
progenitors into the uterus in order to determine if the uterine environment i
s
amenable to sustaining a pregnancy.
SUMMARY OF THE INVENTIOiV
Uterine natural killer (NK) cells are required to maintain the integrity of
the decidualized uterine stroma and for initiating decidual artery
instability.
Failure to recruit adequate uterine NK cells to the human uterus may lead to
a collapsing decidua that would limit implantation success or contribute to
gestational hypertension with or without pre-eclampsia.
The inventors have determined that uterine NK cells do not self-
renew in the uterus but rather are recruited to the uterus from the periphery.
It is expected that cyclic endocrine changes in the late menstrual cycle and
early pregnancy in women, open molecular gates in uterine endothelium
that promote movement of NK cells and their precursors into the uterus.
Further, the inventors submit that defects in appropriate NK cell trafficking
compromise establishment of pregnancy andlor lead to patients being
classified as infertile or pre-eclamptic.
The inventors have also shown that human lymphocytes show
increased adhesion to uterine or lymphoid tissue from a pregnant or
gestational-hormone-treated mouse compared to uterine or lymphoid
tissue from a non-pregnant, non-gestational~-hormone-treated mouse or
other non-lymphoid or non-uterine tissue. This adhesion of lymphocytes
was found to peak on the day of luteinizing hormone (l_H) surge, which
occurs about mid-way through the menstrual cycle, in the peri-ovulatory
period.
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Accordingly, the present invention provides a method of monitoring a
menstrual cycle andlor pregnancy in a female comprising detecting the
adhesion of lymphocytes from the female with uterine or lymphoid tissue
from a pregnant animal or an animal that has been treated with gestational
hormones, such as luteinizing hormone, chorionic gonadotropin, estrogen
andlor progesterone. The invention also includes the identification of
lymphocyte subsets involved in the adhesion and to determining the effect of
pituitary and ovarian hormones on the interactions between lymphocytes
and uterine or lymphoid endothelium. The invention further relates to the
determination of the effect of controlled ovarian hyperstimulation (CON) with
fertility drugs on the interaction between the lymphocytes and the uterine or
lymphoid endothelium.
The present invention further relates to kits for performing the method
of the invention.
Other features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however, that the detailed description and the specific examples while
indicating preferred embodiments of the invention are given by way of
illustration only, since various changes and modifications within the spirit
and scope of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
Figure 1 shows photomicrographs of grafted uterine segments at day
10 of gestation. Boxed regions in A, C, E are enlarged in B, D and F. A&B)
Lower and higher power images of a CD1 graft site in a CD1 recipient
containing a normally developed implantation site including the
mesometrial aggregation of lymphocytes (MLAp), decidua basalis (DB) and
placenta (p1). B) show numerous, mature uNK cells (open
arrowheads).C&D) Lower and higher power images of a SLID graft
segment in a tgEe26 recipient revealing development of a uterine stroma-
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derived deciduoma (DC). Image D is representative of the serial sections of
the deciduomatae which contained no uNK cells. E) UNK (arrowheads)
cells established within the decidual basalis by spleen cells from gestation
day (gd 5) donors were mature granulated cells. F) Spleen cells from non-
pregnant donors did not generate uNK cells in the 10 day assay protocol.
The blood vessel (asterisk) is an unmodified decidual spiral artery. MT,
mesometrial triangle, DB, decidua basalis, p1 placenta. A F stained with
PAS. Bars in A&C represent 400 p,mm; in B, 40 p.mm and in D-F, 25 p.mm.
Figure 2 is a schematic showing time course studies of human
peripheral blood lymphocytes (PBL) bound per high endothelial venule
(HEV) of peripheral lymph nodes (PLN; top panel), of Peyer's patches (PP;
middle panel), and endothelial cells of small blood vessels of pancreas
(bottom panel) over pregnancy and postpartum periods of the mouse tissue
donor. The depicted figures represent data from one experiment using the
same PBL and same tissue donors. Similar outcomes were obtained in
replicate (n=3) experiments that examined the full gestational timecourse
with peak adherence detected between gd 6 and 8. Datapoints represent
the mean number ~SD of lymphocytes boundlvessel and are based on
triplicate counting of 300-500 HEV or small vessels.
* indicates a significantly different mean (p<0.05) to that in the same tissue
from virgin females (v) as analyzed by ANOVA (SAS 6.12, SAS Institute, Cary,
NC). Each function-blocking mAb treatment lowered adhesion compared to
that seen in untreated samples (p<0.001 using Student's t test).
Figure 3 shows histograms illustrating the influences of 17-beta
estradiol andlor progesterone treatment of ovariectomized B5 mice on
adhesiveness of high endothelial venules (HEV) of peripheral lymph nodes
(PLN; A and C), Peyer's Patches (PP; B and D) and Pancreas (E and F).
Assays were conducted using human PBL indicator cells with PLN and
mouse TK-1 lymphoma cells with PP. Cryostat sections were prepared from
tissues collected from mice treated with 17-[3 estradiol at 100nglday
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(E2low), 1 p.g/day (E2high), progesterone (1 mglday, P4), combined steroids
(E2IP4), and combined steroids plus induction of deciduoma
(E2/P4+deciduoma). Tissues from virgin and Ovx B6 mice were used for
controls. Data are the mean ~SD and are representative of the data from
multiple experiments for Panels A and B (n=3) and for 2 mice per group in
Panels C, D, E and F.
+ = significantly different to the virgin or oil placebo treatment groups
(p<0.05)
* = significantly different level of adhesion to the same tissue compared with
untreated cells (p<0.001)
Figure 4 shows histograms illustrating the influences of 17-beta
estradiol andlor progesterone treatment of ovariectomized B6 mice on
adhesiveness of uterine tissue for CD56~r'9"t human lymphocytes. Assays
were conducted using human PBL pre-labeled with anti-CD56 mAb (NKG1,
Coulter Immunology, diluted 1:100) followed by rabbit anti-mouse Ig-
rhodamine isothiocynate (RITC) Ab as indicator cells and were scored by
fluorescence microscopic examination. Cryostat sections were prepared
from tissues collected from mice treated with 17-~3 estradiol at 100 nglday
(E2low), 1 p.glday (E2high), progesterone (1 mg/day, P4), combined steroids
(E2IP4), and combined steroids plus induction of deciduoma
(E2/P4+deciduoma). Uteri from virgin and Ovx B6 mice were used for
controls. The histograms show means ~ SD and are representative of the
data from at least 3 mice per group.
+ = significantly different to the virgin and oil placebo treatment groups
(p<0.05)
* = significantly different level of adhesion to the same tissue compared with
cells not treated with a function-blocking mAb (p<0.001 )
Figure 5 shows histograms comparing uterine adhesiveness at
gestation day (gd) 6 to that of uteri from ovariectomized mice treated with
placebo (oil) or with therapeutic levels of 17-beta estradiol (E2high) for
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CD56bn9nc human lymphocytes (means ~SD for 10 high power fields; HPF).
CD56 br'9nt cells were <2% of the starting population as analyzed by flow
cytometry (not shown). The % enrichment in this subset, recognized as
CD56bright by microscopy, through binding to uterine tissue is indicated
numerically below each treatment group.
* = significantly reduced adhesion of CD56+ cells (p<0.001) treated with
antibodies DREG-56 ( L-selectin} or HP2/1 (a.~ integrin) as compared to
untreated groups.
Figure 6 shows histological images of uterine tissue. Row A depicts
low power histological images of uterus and implantation sites in B6 mice
stained with H & E, to provide orientation. The mesometrial triangle (M),
where development of the mesometrial lymphoid aggregate of pregnancy
(MLAp) occurs, is uppermost in all panels. The antimesometrial (AM) side of
the uterus is at the bottom of all images. At gestation day (gd) 3,
decidualization has yet to commence. At gd 6, maternal decidual tissue (D)
fills the uterus, with the embryo at the primitive streak stage occupying the
embryonic crypt (EC). By gd 10, there is a fully developed implantation site.
The microdomains of the MLAp and decidua basalis (DB) are maternal in
origin while that of the placental trophoblast (P) is fetally-derived.
Antimesometrial decidua has regressed as the 'Fetus (F) has developed and
grown. Rows B and C show low and high power images, respectively, of
toluidine blue stained human lymphocytes (some marked by arrows)
adhering to thick cryostat sections of mouse uterus at the stages indicated
above the images. Binding to decidualized uteri occurred only in the DB.
Lymphocyte clustering was prominent at gd 10. Lymphocytes binding to
non-decidualized uteri were dispersed as single cells (70% small, 30%
large) with random distribution in the virgin uterus. The most frequently
bound cells at gd 3 were dispersed, typical small lymphocytes (85%, black
arrowheads) while 15% of bound cells were larger (white arrowheads). As
gestation progressed to gd 6, adhesion became restricted to DB, the
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proportion of large cells declined to 1 %, numbers of adherent cells
increased and some appeared as stable clusters of up to 30 cells. The
clusters were much larger at gd 10 than at gd 6, but the proportion of large
adhered cells declined (3%). Row B Bar = 150 p.m, Row C Bar = 40 p.m.
Figure 7 shows histograms illustrating the adhesiveness of
splenocytes for HEV in PLN is altered by ovarian steroid hormones (Panel
A) and by pregnancy to levels seen in fever range hyperthermia (Panel B).
Splenic lymphocytes were collected from ovariectomized mice receiving the
treatments outlined in Fig. 1, from pregnant mice or from virgin mice and
tested for adhesion to PLN from virgin mice. Some of the splenocytes from
virgin mice were incubated at 37°C while others were incubated at
40°C,
prior to use in the adhesion assay. In Panel A, bars represent the mean ~
SD for bound lymphocytes from a normal virgin B6 mouse and for the
ovariectomized mice in each treatment group.
Open bars represent the mean numbers of adhering lymphocytes after
treatment with the mAb MEL-14 which blocks the function of mouse L-
selectin. All blocking was statistically significant (p<0.001 ) using
Student's t-
test. Panel B presents the comparisons between the gains in adhesion
induced by mild temperature elevation in splenocytes from virgin mice and
the peak gains induced by pregnancy (gd 8 and gd 9). The three test groups
were each statistically difFerent to the virgin group (p<0.05) but did not
differ
from each other. Blocking with MEL-14 significantly reduced adhesion
(p<0.001,) when pairs of untreated and treated lymphocytes were
compared.
+ = significantly different between groups (p<0.05 by ANOVA)
* = significantly different from untreated group (p<0.001, Student's t test)
Figure 8 is a bar graph showing data on the role of the L-selectin
molecule in mediating adhesion between murine splenocytes and murine
LN. Splenocytes were collected from both virgin and pregnant (day 6-8 of
gestation) B6 mice and from virgin and pregnant (day 6-8 of gestation) L-
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selectin-~- mice. The cells were labeled with either CMFDA-green or CMAC-
blue tracking dyes, mixed in a 1:1 ratio and overlaid onto firozen sections of
LN from virgin (1St four bars) or pregnant (last flour bars) B6 mice. Adherent
green and blue cells were counted under fluorescence microscopy from 50
hp fields per experiment. The experiment was repeated using reversed
filuors to label cells. Here, the results of both experiments are shown. The
black bars represent the number of adherent B6 splenocytes ~ SD and the
white bars show adhesion of L-selectin-~- splenocytes ~ SD. Adhesion of L-
selectin-~- and B6 splenocytes was significantly different (p<0.001, ANOVA).
Within groups, adhesion of splenocytes to tissue from pregnant animals as
compared to tissue from virgin animals was significantly higher (p<0.001,
ANOVA) and adhesion of splenocytes from pregnant animals was
significantly higher than that of virgin animals (p<0.001, ANOVA).
Figure 9 is a bar graph illustrating the dose response of human
lymphocytes to adhesion to LN from a pregnant mouse. Varying
concentrations of cells (10' to 106 ceIIs/100 p,1, as shown on the lower axis)
were applied to frozen sections of LN, then after fixing and staining, the
numbers of adherent cells were counted. The histogram shows the mean
number of ceIIs/HEV ~ SD of 200 HEV counted. A dose response was
demonstrated and from this data, it was determined that 2.5 x 106 was the
minimum number of cells required to detect a significant difference from
adhesion of control cells.
Figure 10 is a bar graph depicting the effects of the type of anti-
coagulant used in blood collection on the subsequent ability of human
lymphocytes to adhere to LN from pregnant mice. Four samples of blood
were collected into sterile tubes containing ACD, heparin, sodium citrate or
EDTA from each of four subjects. Lymphocytes were isolated, washed thrice
and used immediately in an adhesion assay. After fixing and staining,
adherent lymphocytes were counted from each sample. The bars represent
the mean number of lymphocytes counted per HEV from 200 HEV ~SD.
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Figure 11 is a bar graph summarizing the data collected on
temperature effects on the ability of human lymphocytes to adhere to mouse
LN. Four samples of blood were collected from 4. volunteer subjects, and
incubated for 4 h either at 37°C, at 20°C, at 4°C or at
4°C, then warmed to
37°C. After the incubation period, lymphocytes were isolated and used
in an
adhesion assay to LN from a pregnant mouse. Individual bars represent the
mean number of adherent lymphocytes per HEVtSD of 200 HEV counted. *
indicates significant difference from freshly isolated cells.
Figure 12 shows histograms illustrating adhesion of human PBL taken
over the course of a normal menstrual cycle to mouse tissues. Lymphocytes
were collected from 7 normally cycling, informed and consenting adult
women over the course of one menstrual cycle. In Panel A, it is shown that
the adhesiveness of human peripheral lymphocytes (PBL) for HEV in PLN
from virgin mice is not altered during the menstrual cycle but does respond
to hormonal changes when contacted with LN from pregnant animals
(Panel B) or with PP from both virgin (Panel C) and pregnant animals (Panel
D). Black bars represent the mean ~- SD of bound Iymphocytes/HEV. Open
bars represent the mean numbers of adhering lymphocytes after treatment
with an antibody to L-selectin. All blocking was statistically significant
(p<0.05) using Student's t-test. * denotes significant decline in adhesion as
compared to day 12 (peri-ovulation) of the menstrual cycle (p<0.05).
Figure 13 shows bar graphs illustrating the adhesion of human PBL
taken at 3 timepoints in the menstrual cycle to LN and uterine tissue from
mice at day 8 of gestation. Six informed, consenting female volunteers of
legal and reproductive age were recruited to monitor their cycle by basal
temperature and use of an Lhl detection kit to pinpoint the LH surge and
donate blood at cycle day 8, day of LH surge and at day 20. Serum samples
were also obtained and used to measure concentration of E2, P4 and LH.
Lymphocytes were isolated and used in an adhesion assay. Two hundred
HEV were counted on LN sections and 50 HP (high power) fields were
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counted on uterine sections. In Panel A, the adhesion of lymphocytes from 3
timepoints of the menstrual cycle to LN are shown. Black bars represent the
mean number of celIs/HEV ~ SD. Open bars show adhesion when the
lymphocytes were pre-treated with anti-L-selectin. In Panel B, samples of
the same lymphocytes were applied to uterine tissue. Black bars represent
mean number of adhered ceIIs/HP field ~ SD. Open bars show mean
number of adhered ceIIs/HP field ~ SD when lymphocytes are pre-treated
with anti-L-selectin.
DETAILED DESCRIPTION OF THE INVENTION
~i~ Method of Monitorin~g~ the Menstrual Cycle andlor Pregnancy in a
Female
Prior to the present study, it was unclear whether the immediate
precursors of uNK (pre-uNK) cells self-renew within uterus or are recruited
from the periphery. A series of experiments in which uterine horns from wild-
type mice were transplanted to NK-cell deficient mice demonstrated that
precursors of uterine NK cells do not reside within the uterus, but re-
populate. Subsequent investigations of engraftment of lymphocytes isolated
from various hematopoietic tissues of wild-type mice into NK cell deficient
mice established that the majority of NK cells which populate the pregnant
uterus were recruited from the spleen. Splenic cells lacking the chemokine
receptors, CCR2 and CCRS were recruited equally to the wild-type spleen,
indicating that alternative chemokine receptors are used by NK cells to
home to the uterus (Fig. 1 ).
Lymphocyte recruitment into tissues requires adhesive interactions
with vascular endothelium. This was assessed by examining adhesion of
human lymphocytes to frozen mouse tissue sections under shear. The
Stamper-Woodruff assay of cell adhesion to frozen tissue sections takes
advantage of the fact that human lymphocytes bind to marine adhesion
molecules'°. Human lymphocytes were prepared as indicator cells from
single blood donor buffy coats purchased from the American Red Cross.
Consistent results have been obtained with 20 donors although no
CA 02446871 2003-10-27
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information is available regarding sex or possible menstrual cycle stage of
these donors. For uterine tissue sections, pregnancy induced a dynamic
increase in L-selectin and alpha4 integrin-dependent adhesion of CD56~~'9nt
NK cells that became localized to the developing decidua basalis (DB). Most
of the binding was blocked by monoclonal antibody (MAb) against L-
selectin. These studies are the first to demonstrate dynamic changes in
endothelial adhesion associated with pregnancy. These data support a
model in which the dramatic increases in human and murine uNK cells
during decidualization result from vascular recruitment of precursor cells
from secondary lymphoid organs.
The same lymphocyte preparations were assessed for adhesion to
murine Peyer's Patches, pancreas and uterine tissue sections. Dynamic
changes similar to those in lymph nodes were found in Peyer's Patches.
Importantly, no pregnancy-associated changes occurred in pancreas,
showing pregnancy regulates leukocyte endothelial interactions in selected
tissue micro-environments.
Adhesion was infrequent to non-pregnant uterus, (between
endometrium and myometrium mesometrially and antimesometrially) but
high numbers of human cells localized to decidua basalis of pregnant uteri.
A further remarkable finding was that 10-15% of cells adhering to pregnant
uteri were very large lymphocytes, non-adherent to virgin uterus, LN, PP or
pancreas. The inventors predicted these would be CD56bright NK cells. Buffy
coat cells were prelabelled with anti-CD56 to identify NK cells and then
applied to mouse tissues. No CD56b~'gnt cells adhered to non-pregnant
uterus, but both large and SmaII CD56bright lymphocytes specifically localized
to decidual basalis (gd 6 and 10) in high numbers. Significantly, numerous
clusters of large CD56bright were noted in the gd 6 uteri. The number of
bound lymphocytes/cluster was markedly increased on gd 10 uteri. These
clusters of adherent cells were not observed in uteri from hormone-treated
mice (see below) or in peripheral tissues of any of the mice investigated.
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These innovative and novel results clearly suggest that both small
lymphoid precursors and mature uNK cells circulate in human blood and
move to the uterus, due to changes in uterine endothelium. This agrees
with recent work which mapped endothelial cell expression of adhesion
molecules across murine implantation sites and showed a unique, VCAM-1
rich, microdomain in decidua basalis that excluded all lymphocytes except
mouse uNK CeIIS.39~ 40
The effect of estrogen and progesterone, gestational hormones
associated with the menstrual cycle and with early gestationai success, on
ability of endothelial cells andlor lymphocytes to interact functionally was
investigated in experiments where the mouse providing the lymphoid tissue
source is treated with the hormone. Marine splenocytes, human
lymphocytes and a4 integrin+, L-selectin- TK-1 cells were used in these
assays. It was found that estrogen or progesterone alone or in combination
were equivalent to pregnancy in promoting lymphocyte-L-selectin-
dependent and a4 integrin-dependent adhesion to endothelium under
shear flow conditions. Endothelium was not universally altered in the
estrogen-treated females as there was no increase in adhesion of blood
lymphocytes, or of TK-1 cells to pancreas endothelium.
Estrogen also altered endothelium cell-lymphocyte interactions in
assays using uterine tissues. The percentage of CD55br'gnt cells in the
starting human lymphocyte preparation was 2-3%. By interaction with
uterine tissue (non-pregnant) alone, the percentage of CD56nr~~nt cells was
greatly enriched (2.5 fold) to10%. There was further enrichment to 70% if the
uterine tissue was from a mouse receiving hormone therapy and this
enrichment was not different to that seen for a pregnant uterus. Thus,
estrogen induced changes in the uterine endothelium were proven to
promote interactions specifically with human CD56bright cells, the cell subset
that normally homes to the uterus in pregnancy.
In a similar series of experiments, it was determined that
pragesterone, independent of estrogen, can modify lymphocyte-endothelial
CA 02446871 2003-10-27
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cell interactions. Thus, human lymphocytes were shown to have enhanced
L-selectin-dependent binding (above that measured in tissues from non-
pregnant or ovariectomized placebo treated females) to peripheral lymph
node endothelium from a mouse receiving progesterone replacement
therapy. Also, the a4 integrin-expressing TK1 cell line was shown to have
enhanced, progesterone-dependent, and oc4 integrin-dependent, binding to
endothelium in Pet'er's Patches. /adhesion of human CD56bri9~t cells to
uterine gestational tissues was also promoted in ovariectomized,
progesterone-treated mice.
The dual treatment of the ovariectomized mice with estrogen and
progesterone did not enhance the interactions of human GD56brigh~ cells with
uterine stroma from the treated mice beyond those seen with treatment
using only single steroid therapy. The results with mice receiving dual
treatment with estrogen and progesterone as well as induction of uterine
decidua indicate that the enhanced interactions between human CD56br'gr,c
cells and uterine stroma from the treated mice in the presence of estrogen
and/or progesterone occur independently of whether or not the uterus has
undergone decidualization of its stromal cells.
The clusters of large CD56bri9nt cells that adhered to pregnant uteri at
gd 6 and later, were not seen in experiments involving the adherence of
lymphocytes to uteri from hormone-treated mice, nor in experiments
involving adherence of lymphocytes to peripheral lymph tissues.
Further results have been obtained using murine lymphocytes
isolated from the spleen to replace human blood lymphocytes. Mouse
uterine tissue, lymph node, intestinal Pet'er's Patches and pancreas have
been used as the adhesion substrate. Seven different types of mice have
been used in these adhesion experiments. These mice were 1) normal
cycling virgin young adult females; 2) naturally mated, gestationally timed
primiparous females; 3) ovariectomized virgin adult females treated with a
placebo; 4) ovariectomized virgin adult females receiving estrogen
replacement therapy; 5) ovariectomized virgin adult females receiving
CA 02446871 2003-10-27
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progesterone replacement therapy; 6) ovariectomized virgin adult females
receiving combined estrogen plus progesterone replacement therapy; and
7) ovariectomized virgin adult females receiving combined estrogen plus
progesterone replacement therapy plus induction of uterine decidua. Using
lymph nodes from only non-pregnant mice as a substrate, it was shown that
splenocytes collected from pregnant mice or hormone treated mice were
more adhesive to non-pregnant lymph node endothelium than the
splenocytes isolated from non pregnant mice (Figure 7). Further, the
lymphocytes from mid pregnancy (gd 10) were more adhesive than those
from the first trimester (gd 6). Antibody blocking of the Peripheral Node
Addressin (PNAd) receptor reduced adhesion, confirming involvement of the
ligand for this receptor, L-selectin, as a key molecule in the process. The
level of adhesiveness of the lymphocytes from pregnant mice and hormone-
treated mice was the same as those seen in fever range hyperthermia.
Thus, the elevated adhesion to blood vessel endothelium (a requisite for
moving a cell from the circulation and into a tissue) is a combined result of
the effect of pregnancy on both organ specific endothelium and on the
lymphocytes themselves. This interaction was also shown to involve oc4
integrin-mediated pathways.
Therefore, it has been shown that pregnancy, estrogen replacement
therapy and progesterone replacement therapy each promote functional
interactions between lymphocytes and endothelium of the uterus and the
lymphoid organs. Endothelium from other sites, such as pancreas is not
altered. This predicts that the controlled ovarian stimulation used for oocyte
collection for women undertaking embryo transfer could significantly modify
or disrupt the movement of NK cells into the uterus. It has also been shown
that there are separate actions of pregnancy or hormone replacement that
independently modify the endothelium or the lymphocyte population.
Further, it has been confirmed that C~56bright human blood lymphocytes (the
human uterine phenotype) are enriched when interacting with uterine
endothelial cells from non pregnant uteri and very strongly enriched when
CA 02446871 2003-10-27
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interacting with uterine endothelial cells from pregnant uteri. It has also
been shown that the CD56bright CeIIS form clusters on uteri from pregnant
mice, but not on uteri from hormone-treated mice or in peripheral tissues.
The formation of clusters of lymphocytes on uteri tissue from a pregnant
animal may provide a significant means to monitor the luteal phase of a
menstrual cycle andlor a pregnancy in a female. Finally it has also been
shown that a technical assay for studying the interaction between
lymphocytes and endothelium from uterine and/or lymph nodes is valid for
many blood donors and that the assay gives consistent results when
applied to studies of gestational lymphocytes and endothelium.
In experiments using peripheral blood lymphocytes (P13L) from
healthy, female volunteers of reproductive age in a time-based study of the
effects of the menstrual cycle on the adhesive properties of PBL to adhesion
molecules expressed on vascular endothelium from a mouse, the present
inventors have found that, under normal conditions, adhesion peaks on the
day of luteinizing hormone (LH) surge, which occurs about mid-way through
the menstrual cycle, in the peri-ovulatory period. This confirms that the
assay of the invention may be used to monitor a menstrual cycle andlor
pregnancy in a female in order to assess the female for optimum conditions
for sustaining a pregnancy.
Accordingly, the present invention provides a method of monitoring a
menstrual cycle and/or pregnancy in a female by detecting the adhesion of
lymphocytes from the female with uterine or lymphoid tissue from a
pregnant animal or from a non-pregnant animal that has been treated with
gestational hormones. In particular, the method can be used to determine
whether a uterine environment is conducive for sustaining a pregnancy and
is recognized by the immune system of a woman trying to conceive. This
recognition will indicate that her lymphocytes will home appropriately to her
uterus, optimizing the uterine environment for her embryo. The greater the
adhesion of the lymphocytes, in particular the C~56br'ght ~K cell subset, the
better the uterine environment is to sustaining a pregnancy. Also the greater
CA 02446871 2003-10-27
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the number of clusters of C~56br'9"t IVK cells adhered to uterine tissue from
a pregnant animal, the better the uterine environment is to sustaining a
pregnancy. The method will have particular utility in women having trouble
conceiving, women experiencing habitual miscarriages and women
undergoing in-vitro fertilization (fVF). in such cases the assay of the
invention can be used to determine if the problems in conceiving or
maintaining a pregnancy are related to problems in lymphocyte trafficking to
the uterine environment. The method of the invention can also be used to
monitor an early pregnancy wherein the greater the adhesion the greater the
chance of sustaining the pregnancy. Accordingly, in an embodiment, the
method of the invention is used to monitor changes in the ability of a
lymphocyte from a female to home or adhere to a uterus, the changes being
induced by the menstrual cycle and/or a pregnancy in the female.
In a further embodiment, the method or assay of the invention
comprises:
(a) obtaining lymphocytes from the female; and
(b) contacting the lymphocytes with uterine or lymphoid tissue
from a pregnant animal or from a non-pregnant animal that
has been treated with gestational hormones; and
(c) detecting the adhesion of the lymphocytes with the uterine or
lymphoid tissue.
The female can be any female animal wherein one desires to
monitor an estrous or menstrual cycle or pregnancy. The female l s
preferably a human female.
The lymphocytes can be obtained from any sample from the female
and are preferably obtained from blood or fractions thereof. The
lymphocytes used in the assay are preferably peripheral blood leukocytes
that may be tagged to identify the natural killer cell subset. Most
preferably,
the human lymphocytes are CD56b~'9"t natural killer cells.
The term "uterine or lymphoid tissue°' includes sections or
homogenates of the tissue, or adhesion molecules derived from these
CA 02446871 2003-10-27
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tissues or tissue homogenates, or cells transfected with adhesion
molecules derived from these tissues or tissue homogenates. The uterine
or lymphoid tissue can be from any animal and is preferably from a mouse,
rat, golden hamster, guinea pig, rabbit, human or other species in which
decidual tissues develops in the pregnant or pseudopregnant uterus. The
uterine tissue is preferably from the decidua basalis. The lymphoid tissue
can be from any lymphoid tissue and is preferably from the lymph node or
Peyer's Patches. fn the assay, histological sections, homogenates of the
tissue or molecules derived from the tissue, either adhered to a substrate or
expressed in a cell line, may be used. The tissue, homogenates,
molecules or cells may be placed on or adhered to a coverslip or microtitre
plate to which the lymphocytes can be directly applied. Examples of
adhesion molecules that may be used in the method of the invention
include cell adhesion molecules and integrins. The expression of adhesion
molecules in cells can be carried out using well known recombinant DNA
technology (suitable methods for transforming and transfecting host cells
can be found in Sambrook et al. Molecular Cloning: A Laboratory Manual,
2nd Edition, Cold Spring hlarbor Laboratory press (1989), and other
laboratory textbooks). It is preferred for the adhesion molecules to be
expressed on the surface of such cells. An example of a cell that is suitable
for the expression of such molecules is Bosco's cell line.
The term "gestational hormones" refers to any hormone associated
with gestation including iuteinizing hormone, chorionic gonadotropin,
estrogen andlor progesterone. More than one gestational hormone may be
used to treat the animal. Preferably, the gestational hormone is estrogen or
progesterone, or a mixture thereof.
The term "treated with gestational hormones" as used herein means
to administer an effective amount of one or more gestational hormones to
the animal. In this context, an "effective amount" is an amount sufficient to
induce decidualization in the animal. The use of gestational hormones to
induce decidualization in rodent models is welt known in the art.83
CA 02446871 2003-10-27
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In embodiments of the invention, greater adhesion of the
lymphocytes, in particular the CD56br'9nt NK subset, indicates a better
uterine
environment for sustaining a pregnancy. The term "greater adhesion of
lymphocytes" means the adhesion of lymphocytes from the female is
greater with lymphoid or uterine tissues from a pregnant animal, or a non-
pregnant animal that has been treated with gestational hormones, than with
a control. A suitable control may be, for example, the adhesion of
lymphocytes from the female with uterine or lymphoid tissues from a non-
gestational-hormone-treated, non-pregnant animal or a suitable non-
lymphoid or non-uterine tissue, for example, endothelium from the
pancreas. Other suitable controls include: 1) the adhesion of lymphocytes
from a non-pregnant, non-gestational-hormone-treated animal, or from a
male, to uterine or lymphoid tissues from a non-gestational-hormone-
treated, non-pregnant animal or a suitable non-lymphoid or non-uterine
tissue, for example, endothelium from the pancreas; and 2) the adhesion of
lymphocytes from a non-pregnant, non-gestational-hormone-treated animal,
or from a male, to uterine or lymphoid tissues from a gestational-hormone-
treated or a pregnant animal
The term "detection" as used herein refers to any qualitative or
quantitative determination or assessment of lymphocyte adhesion to target
tissues. In order to detect adhesion of the lymphocytes with the uterine or
lymphoid tissue any known method can be used. For example, lymphocytes
may be enumerated by microscopic observation as described in Example 1,
either by staining with, for example, toluidine blue, or using a fluorescent
label. Automated scoring based on differential spectroscopy or colorimetric
measurement of stained lymphocytes may also be used.
In another embodiment of the invention, the method or assay of the
invention comprises:
(a) obtaining lymphocytes from the female; and
(b) contacting the lymphocytes with uterine tissue from a pregnant
animal; and
CA 02446871 2003-10-27
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(c) detecting the number andior size of clusters of lymphocytes
adhered to the uterine tissue.
In embodiments of the invention, a greater number and/or total size of
clusters of lymphocytes from the female, in particular the G1056br'gnt NK
subset, adhered to decidualized uterine tissue from a pregnant animal
indicates a better uterine environment for sustaining a pregnancy. The term
"greater number and/or total size of clusters of lymphocytes" means the
number andlor total size of clusters of lymphocytes from the female adhered
to decidualized uterine tissue from a pregnant animal is greater than the
number of clusters of lymphocytes from the female adhered to a control, for
example to a non-pregnant uterus. As used herein the term "cluster(s)"
refers to a group of more than 5 lymphocytes adhered in a single location, in
contact with at least 1 other lymphocyte. Individual clusters may be as large
as several hundred cells in contact.
The invention also includes the identification of lymphocyte subsets
involved in the adhesion and to the determination of the effect of pituitary
or
ovarian hormones on the interactions between lymphocytes and uterine or
lymphoid endothelium. The invention further relates to the determination of
the effect of controlled ovarian hyperstimulation (CON) with fertility drugs
on
the interaction between the lymphocytes and the uterine or lymphoid
endothelium.
ii Kits
The development of the method of the invention allows the
preparation of kits for use in monitoring the menstrual cycle and/or a
pregnancy of a female. The kits would comprise the reagents suitable for
carrying out the methods of the invention, packaged into suitable containers
and providing the necessary instructions for use.
Accordingly, the present invention includes a kit for monitoring a
menstrual cycle and/or pregnancy of a female comprising uterine or
lymphoid tissue, wherein the tissue is from a pregnant animal or a non
pregnant animal that has been treated with gestational hormones.
CA 02446871 2003-10-27
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Preferably the tissues are mounted on a solid support. For example,
tissues may be adhered to a coverslip or a microtitre plate.
The kits may also include reagents to separate lymphocytes from
blood and/or reagents, for example antibodies, for tagging or separating the
desired lymphocyte subset, for example CD56~"9"t natural killer cells, from
the blood or the lymphocytes.
The kits may also include reagents to perForm a control. In an
embodiment of the invention, the reagents to perform a control comprise
uterine or lymphoid tissues from a non-gestationai-hormone-treated, non-
pregnant anima! or a suitable non-lymphoid or non-uterine tissue, for
example, endothelium from the pancreas. In another embodiment of the
invention, the reagents for performing a control comprise lymphocytes from
a non-pregnant, non-gestational-hormone-treated female or a male.
With particular regard to assay systems packaged in °'kit°'
form, it is
preferred that assay components be packaged in separate containers, with
each container including a sufficient quantity of reagent for at least one
assay to be conductede A preferred kit is typically provided as an enclosure
(package) comprising one or more containers for the within-described
reagents.
The reagents as described herein may be provided in solution, as a
liquid dispersion or as a substantially dry powder, e.g., in lyophilized form.
Usually, the reagents are packaged under an inert atmosphere.
Printed instructions providing guidance in the use of the packaged
reagents) may also be included, in various preferred embodiments. The
terra '°instructions" or "instructions for use'° typically
includes a tangible
expression describing the reagent concentration or at feast one assay
method parameter, such as the relative amounts of reagent and sample to
be admixed, maintenance time periods for reagent/sample admixtures,
temperature, buffer conditions, and the like.
The following non-limiting examples are illustrative of the present
invention:
CA 02446871 2003-10-27
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EXAMPLES
Example 1: Identification of the source of NK cells in the preginant uterus
Goals of Example 1 were i) to determine if uterus self renews uNK
cells and ii) to examine peripheral lymphoid tissues as sources for pre-
uNK.
Materials and Methods
Animals
Immunocompetent mice (randombred CD1, Charles River
Laboratories, St. Constant, PQ), C5?BIl6J (BC) and C57B1!6x129lJ F1 (F1,
Jackson Laboratories, Bar Harbor, ME)) were housed under standard
husbandry in the Central Animal Facility, University of Guelph.
Immunodeficient mice, (randombred ICR- scidlscid (SCID; NK+,T-,B-,
Taconic, Germantown, N.Y), tgEe26 (H?~'b; NK-,T-,B+) and RAG-2~~-fyc ~- (H-2b;
NK-,T,B-) were housed in the University of Guelph's barrier-husbandry
facility. Both tgEe26 or RAG-2~~-f~c~- lack uNK cells and are referred to as
uNK cell deficient." Mice ablated for CCR2 (I-i-2'~b) and CCRS (H-2wb)'1,72
were bred at University of Texas, Austin, TX and shipped to Guelph.
Females over 8 wks of age were used, unless stated and, if bred, were
mated to males of the same strain. Gd 0 was the morning of copulation
plug detection. Euthanasia was by C02 followed by cervical dislocation.
Uterine segment transplantation
Uterine segments (10 or 5 mm), trimmed of mesentery and vessels,
were grafted from virgin donors to virgin recipients in an orthotopic manner
that preserved both cranial-caudal and mesometrial-antimesometrial
orientations. For autotransplantation (n=2), CD1 females were
anaesthetized (0.35 ml of xylaxine (20mglml) and ketamine (100 mglml))
and the donor horn was reanastomosed with simple interrupted 8-0 Vicryl
(Polysorb~, Norwalk, CT) sutures. For all other grafting, donors (CD1 or
SCID) were euthanised while recipients (tg~e26 or GD1) were
anaesthetized as above. Recipient horns were cut at their midpoint and
donor tissue was inserted and anastomosed. No vascular anastomosis
CA 02446871 2003-10-27
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was attempted. The abdomen was closed surgically. After 7 days,
recipients were paired for breeding and euthanised at gd 10.
Thymic engraftment
Thymuses were dissected from non-pregnant or pregnant (gd 3, 5)
adult or neonatal (48 hr) B6 mice and grafted under the renal capsule of
anaesthetized gd 0 RAG-2-~-lyc ~-.
Adoptive transfer of ~M, LN or splenocytes
Bone marrow (BM) and spleen cell (SC) donors were non-pregnant
or pregnant SLID mice while lymph node (LN) donors were B6 (gd 3, 5 or
7). As pregnancy changes cellularity of these organs, one donor was used
per recipient, pooling donors if several mated recipients were available on
the same day. Uteri from all gd 3 donors were flushed to confirm pregnancy
by detection of pre-implantation blastocysts. BM was flushed from femurs
and tibias of each donor. Microscope-aided dissection was used to harvest
peripheral (P) LN (9 superficial and 3 pelvic LNJdonor) that were pooled and
the mesenteric (M) LN chain. LN and spleens were dissociated
mechanically. PBS (400 ~.ml withlwithout cells) was infused via tail veins
into gd 0 tg~e26 or RAG-2-I lyc-I recipients who were sacrificed on their gd
10.
Morphornetric analyses
Abdominal contents were examined grossly, then uteri were
dissected, fixed in Bouin's solution, processed routinely for paraffin
embedding, serially sectioned at 7 ~.mm (transversely for normal uteri and
longitudinally for surgically manipulated uteri) and stained with
Haematoxylin and Eosin (H&E) for routine histopathology or Periodic Acid
Schiff's (PAS) for uNK cell enumeration. Eleven central tissue sections from
each implantation site were scored as previously described.'$ ~ne
mm2lsection was analysed in each mesometrial microdomain, decidual
basalis (DB) and the mesometrial aggregation of lymphocytes (MLAp).
Circular smooth muscle was used as the boundary between these. When
the MLAp was absent or rudimentary, mesometrial triangle (MT) rather than
CA 02446871 2003-10-27
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MLAp is the term used to describe the scored region. For longitudinally
sectioned uteri, those containing a conceptus Were scored as above. Uteri
containing a deciduoma were scored in two independent 1 mm2 areas of 10
of the serial sections separated by 42p.mm to avoid duplicate counting of
uNK cells which can reach 40p.mm by gd 10. Means and standard
deviations of uNK cefls/mm2, p values and Student-Newman-Keuls test for
ANOVA were conducted using PC-SAS 6.12 for lNindows (SAS Institute Inc.,
NC).
Results
Assessment of uNK cells in uterine segment transplants
Feasibility of orthotopic uterine grafting was assessed in
autologously grafted CD1 mice using grafts of 10mm (n=3) and 5mm (n=4).
The longer grafts showed gross full-length necrosis while the shorter grafts
were viable. All recipients of the 5mm grafts were pregnant with three grafts
containing implantation sites and the fourth a deciduoma (Table 1). Typical
uNK cells with 16-45 pm in diameter and containing 9-25 PAS reactive
granules/cell were found in these decidualized grafts (Fig. 1A&B). Thus,
orthotopically-grafted mouse uterus supports uNK cell differentiation. Next,
5mm uterine segments were grafted from uNK cell competent CD1 or SCID
donors into NK/uNK cell deficient tgEe26 recipients (n=7). Two females
mated but were not pregnant at euthanasia, despite grossly and
histologically normal grafts. Five recipients were pregnant and each grafted
segment contained a large deciduoma indicative of viable, hormonally-
responsive tissue. By serial section analysis, neither the deciduomatae in
donor tissues nor implantation sites in host tissues contained uNK cells
(Fig. 1 C&D). Thus, the donor segments did not contain self-renewing
prolpre-uNK cells that could differentiate in situ or migrate to adjacent
implantation sites.
Development of uNK cells from Thymus, BM, LN and SC
At gd10 in normal and SC1D mice, range in uNK cell frequency is 27-
53 cellslmm2 in DB and 72-129 ceIIsImm2 in MLAp (Table 2). UNK cell
CA 02446871 2003-10-27
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sizes in B6 mice ranged from 11-20 p.m (average 14.3~2.9) and in SCID
mice 11-27 p,m (mean 15.9~4.0). All uNK cells were granulated and
contained 8-35 (mean 18.0~8.2) and 5-30 granules/cells (mean 19.4~9.0)
in B6 and SCID mice, respectively. lJterine segment transplantation
suggested that migration of pre-uNK cells accounts for filling of these
microdomains. Peripheral lymphoid tissues were assessed for pre-uNK
cells by grafting to mated, uNK cell deficient mice. Thymic engraftment
generated limited numbers of uNK cells at gd 10 (Table 2). There were no
statistical differences in reconstitution of DB or MT by thymuses of different
ages or from different donor pregnancy states (p>0.05). BM from non-
pregnant or 3 early times of pregnancy also gave low level uNK cell
reconstitution in all recipients (Table 2). No significant differences were
found in uNK cells/mm2 in DB or MT between the BM donor groups (p>0.05).
MLN failed to reconstitute uNK cells while implantation sites in recipients of
PLN showed MLAp development. Both MLAp and DB of PLN grafted mice
contained mature uNK cells. Implantation sites in uNK cell deficient mice
receiving SC from pregnant donors also showed histological development
of MLAp and high levels of uNK cells in both MLAp and DB. However, if the
SC donors were not pregnant, uNK cell reconstitution was much lower
(p<0.001) in both microenvironments. As shown in Fig. 1(E&F), levels of
engraftment resulting from inoculation of SC from pregnant donors was
sufficient to modify the decidual spiral arteries. In sharp contrast, host
arterial vasculopathy persisted in recipients of SC from non-pregnant
donors. For all SC donors, uNK cells were present at higher frequencies in
the MLAp than in DB (p<0.01), a typical gd 10 pattern in normal mice.
Morphological assessment of graft-derived uNK cells showed that uNK
cells derived from thymus, BM, LN and SC were similar in size (14.5~4.0,
15.6~4.5, 18.7~7.5 and 13.9~4.7 p.m, respectively) and in numbers of
granule/cell (12.8~5.8, 15.6~5.5, 20.5~11.9, 17.8~7.7, respectively). These
morphology were identical to gd 10 uNK cells in unmanipulated B6 and
SCID mice, implying equivalent maturity.
CA 02446871 2003-10-27
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To test the role of specific chemokines expressed by the pregnant
uterus on pre-uNK cell recruitment, mated RAG-2-I-lyc-I- females were
infused with SC from pregnant (gd 3 or 5) CCR2-J- or CCRS-/- mice. High
levels of uNK cells were found in all recipients that did not differ
numerically
or morphologically from uNK cells in gd-matched, F1 controls (Table 2).
These data indicate that the chemokines MIP-1 oc, MCP-1 and RANTES are
not essential for uterine recruitment of pre-uNK cells.
Discussion
This is the first comprehensive study, in any species, to address the
source of the immediate precursors of uNK cells in a pregnant adult.
Availability of NK/uNK cell deficient mice that reliably carried pregnancies
was central to the study's success. Following transplantation of uterine
segments from NK+ mice into NK-IuNK- mice, no uNK cells were found in
decidual tissue within the grafts or at any of the implantation sites in host
tissue. The latter observation excluded migration of prolpre-uNK cells from
the graft segments into host tissue and established that mouse uterus
does not contain self-renewing prolpre-uNK cells. Uterine and oviductal
grafts, used in published studies, may have scarred and died due to
problems of excessive length, inadequate perfusion andlor immune
rejection. The choice of an immune deficient host eliminated host versus
graft rejection. Early graft versus host disease was not a problem as
allografts from CD1 were as equally viable and hormone responsive as T
cell deficient SCID allografts. Duration of the transplantation experiments
was shorter (17 days) than mouse gestation (19-20 days), permitting the
conclusion that uterine recruitment likely occurs during gestations.
Previous grafting of mated, immunocompetent mice with virgin uterine
tissue in sealed diffusion chambers showed that uterus has some pre-uNK
cells with a 12 day maximum survival time.'4
To explain the dramatic rise in human uNK cells during
decidualization some authors suggest that uNK cells self-renew in the
uterus,75 while others suggest precursor trafficking from BM ~6 The present
CA 02446871 2003-10-27
- 29 -
studies indicate that both ideas maybe incorrect. In adoptive transfers, BM
was identified as only a minor pre-uNK cell source. Despite the known and
observed involution of BM (Table 2), there was no loss in progenitors able to
populate uterus with uNK cells, during the first trimester of pregnancy.
Because pregnancy alters lymphocyte frequency in organs, transferred cells
always included all of the nucleated cells recovered from a defined tissue
on the specified gd. This design approximates the physiological situation
better than transfer of fixed cell numbers. Levels of uNK cell generation
from thymus were also low and independent of donor age or pregnancy
status. However, the present results show for the first time that thymus
retains its capacity for NK cell generation into adulthood.
Nat all LN had transplantable pre-uNK. MLN did not hypertrophy and
lacked pre-uNK. Cell yields from other LN or LN chains were too low to
assess each as a source of pre-uNK cells; thus, PLN were pooled.
Hypertrophy was anticipated in the pelvic LN draining the uterus" but was
not measurable in the cell pool. Transplantable pre-uNK were present in
PLN. However, the cellular composition of the pooled LN is probably not
homogeneous because development of some PLN (i.e. cervical, included in
the pool) is regulated in a manner analogous to MLN deveioprnent.'$
Further study would be required to precisely define the PLN-containing
transplantable pre-uNK.
Spleen contained pre-uNK cells that were mobilized for relocation to
the uterus by pregnancy. In comparison to non-pregnant donors, spleen
cell numbers doubled in pregnant donors while numbers of uNK cell
progeny increased ~-47x (Table 2). This suggests that numerical
alterations are not the sole pregnancy-induced changes in SC accounting
for uNK cell reconstitution. The developmental stages of hematopoietic
cells which move into the uterus are not yet known. Because uNK cells
differentiating from thymus, BM, LN and SC are identical morphologically
and morphometrically and match those in gd 10 unmanipulated, genetically
normal mice, the cells which moved into the uterus from these tissues were
CA 02446871 2003-10-27
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probably at relatively similar stages of differentiation. Alternatively, uNK
cells
may differentiate rapidly and cells at various prolpre-uNK stages may have
had sufficient time to complete differentiation under the experimental
conditions described herein. The heterogeneity in size of human
lymphocytes adhering to marine uterus suggests that circulating cells at
more than one stage of differentiationlactivation may have uterine homing
potential. Lack of CCR2 or CCRS did not reduce pre-NK cell homing from
spleen to uterus despite high levels of CC chemokine expression in
pregnant human and mouse uteri'9~$° suggesting that these chemokines
target other cell types, andlor that there is a redundancy in uterine
chemokines adequate to recruit pre-uNK cells through other receptors.
Exam~lale 2: Cell adherence to marine tissue sections under shear and
effect of hormones of this adherence
Goals of Example 2 were i) to determine whether human peripheral
blood lymphocytes (PBL) recognize and bind to adhesion molecules
expressed on the vascular endothelium of the mouse uterus and lymphoid
tissue and ii) to determine whether pregnancy l pregnancy hormones
altered this recognition.
Materials and Methods
Mice and tissue dissections
C57B116J (Jackson Laboratory, Bar Harbor, IUIE) and Balb/c (laconic,
Germantown, NY) mice, aged 7-8 wk were studied. Some females were
used for timed coatings with the morning of the copulation plug designated
gd 0. Other females (n=34) were surgically ovariectomized under Avertin
anaesthesia (24). Females were rested 6 days and then received daily
subcutaneous injections of either hormone dissolved in sesame oil or
sesame oil alone between 9.00-11.00 a.m. as indicated in Fig. 2.8~-~ Some
of the Ovx hormone-treated mice also had 10 ~.! sterile sesame oil injected
into a uterine horn to induce decidualization 48 h prior to euthanasia. Ovx
mice were euthanized 24 hours after receiving their last injection and
CA 02446871 2003-10-27
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vaginal smears were collected and stained with Wright-Giemsa to confirm
treatment success. Non-pregnant (NP) controls were virgin females who
had never been paired with mates. Aff procedures were performed under
appropriate animal utilization protocols.
The following samples were collected from each mouse and
embedded in ~CT (Miles Laboratories) for cryosectioning: i) a pool of 10-12
PLN from subcutaneous and intermuscular sites; ii) a pool of 10-12
intestinal PP; iii) the entire pancreas; and iv) the uterus. For some
experiments, the median iliac lymph nodes that drain the pelvic organs,
including the uterus, were also dissected. Non-decidualized uteri were
trimmed at the ovarian and cervical ends, then incised longitudinally along
one side on an axis midway between the mesometrial and anti-
mesometrial aspects. The mesometrium (the mesentery suspending the
uterus, also known as the broad ligament) was not removed and served as
a positional landmark during analyses. For embedding, samples were
oriented to produce full thickness longitudinal sections that included both
antimesometrial and mesometrial sides. Uteri from gd 3 were flushed for
blastocysts to confirm pregnancy.
Assay of functional cell adhesion under shear to frozen tissue sections
The cell suspensions evaluated for adhesive interactions with
endothelium were human peripheral blood lymphocytes (PBL) isolated from
random (female and male) normal donor bully coat leukocyte concentrates
(American Red Cross, Rochester, N~;85=~ and TK-1 cells (an alpha
integrin"'g", L-selecting°'" mouse T lymphoma cell line).85 In selected
experiments, human PBL were cultured 5 hr at 37°C or 40°C before
being
applied to tissue sections, as previously described'2. 1n all assays, some
cell aliquots were incubated with function blocking mAb specific for human
L-selectin (DREG-56, American Type Cell Collection ~ATCC], Mantissas, e/A,
10 p,glml) or human alpha4 integrin (HP2/1, Coulter Immunology, Hialeah,
FL, 10 ~.glml). To identify adherent human NK cells, PBL aliquots were pre-
CA 02446871 2003-10-27
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labeled with anti-CD56 mAb (NKG1, Coulter Immunology, diluted 1:100)
followed by rabbit anti-mouse Ig-rhodamine isothiocynate (RITC) Ab as
described previously (10;29). Control aliquots were treated with the
secondary reagent alone. Lymphocytes were overlaid on 12 p,m
cryosections of marine tissues and rotated (112 rpm for PBL and
splenocytes, 95 rpm for TK-1 cells) at 4° C for 30 min. In some
instances,
tissue cryosections were pre-treated with MECA-367 a mAb to MAdCAM-1
(ATCC, 1:4 dilution of hybridoma conditioned medium). Following removal of
non adherent cells, giutaraldehyde-fixed specimens were stained with 0.5%
toluidine blue and adhesion was scored on 300 -500 HEV/PLN or PP
specimen or equivalent numbers of small vessels in pancreatic tissue.
When CD56-pre-labeled cells were used, fluorescent cells were quantified
in 10 high power fields (HPF; one HPF = 5 mm2) at 200X magnification
using an Olympus BH2/RFL fluorescence microscope (Olympus Optical,
Tokyo, Japan) (10;29). All enumerations were done thrice.
Results
(a) Pregnancy promotes organ-specific adhesive funcfion in endothelial
cells
To evaluate the effects of pregnancy on endothelial cell interactions in
peripheral organs, adhesion of human PBL to endothelium was assessed
in PLN, PP, and pancreas from virgin, pregnant and postpartum mice (Fig.
3). Onset of pregnancy, prior to blastocyst implantation (gd 3), significantly
elevated adhesion to HEV in PLN above that of PLN from virgin mice
(p<0.05). Peak levels of adhesion were achieved early post implantation (gd
6-8). An equivalent gain in adhesion was demonstrated at gd 6 in LN that
provide pelvic organ drainage (not shown). Adherent cells were uniformly
small in size, 6.8 ~ 0.4 p.m. Once peak levels of adhesion were achieved,
these levels were sustained throughout the remainder of gestation (i.e. to
gd 18 with birth at gd 19). In PLN of mice taken 5 and 9 days postpartum
(times selected to represent completion of the first and second postpartum
CA 02446871 2003-10-27
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estrous cycles), adhesion of PBL dropped to levels found in PLN from virgin
mice. Pregnancy-induced adhesion of PBL to HEV in PLfV was L-
selectin-dependent, as shown using an L-selectin function blocking mAb
(p<0.001, Fig. 3, top panel).
Pregnancy also increased lymphocyte adhesion to HEV in PP.
However, in contrast to LN HEV, adhesion in PP HEV adhesion declined to
baseline during late pregnancy (Fig. 3, middle panel). Adhesion to PP HEV
was blocked by MECA367, a mAb that functionally inhibits both L-selectin
and alpha integrin-mediated binding to MAdCAM-1 (Fig. 3, middle panel). In
sharp contrast to the gains in lymphocyte-endothelial cell adhesion detected
in HEV of LN and PP, adhesion was not altered in the non-differentiated
squamous endothelium lining small vessels of the pancreas at any
gestational time point (Fig. 3, lowest panel). These data suggest that only a
restricted subset of vessels is targeted by pregnancy-driven signals for
lymphocyte egress.
(b) Increased adhesion of lymphocytes t~ HEV is ind~rced by ovarian ster~id
hormones
Receptors for E and P4 are expressed by endothelial cells and may
contribute to the observed pregnancy-induced changes in lymphocyte-
endothelial cell interactions. To address these potential mechanisms, Ovx
mice were studied using hormone replacement strategies. Ovx mice were
pre-treated with physiologic (100 ng)8~ or therapeutic (1 p,g)~ doses of E2
(called low and high), with P4,8'°$2 or with combined low dose E2 plus
P4. In
addition, because decidualization is an important feature of mouse
implantation and not induced by hormone treatment alone, additional
E2~-P4-treated Ovx animals were studied in which decidualization was
induced artificially by injection of sesame oil into a uterine horn.8~~82 All
hormone treatments promoted statistically significant gains in L-selectin-
dependent adhesion of human PBL indicator cells to PLN HEV as
compared to the basal levels of adhesion observed in either placebo-
CA 02446871 2003-10-27
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treated (oil) or virgin control mice (Fig. 3A). The murine TK-1 indicator cell
line (a4a~ integrin"'g", L-selecting°~') was used to identify the
efifects of
hormone treatment on alpha4. integrinlMAdCAM-1-dependent adhesion
events in PP HEV. Ai! hormone conditions significantly elevated TK-1 cell
adhesion to PP HEV through a mechanism that could be blocked by the cx4~3~
integrin-specific DATK-32 mAb (Fig. 3B). Induction of decidua did not
promote adhesion in PLN or PP HEV above that induced by hormone
treatment alone (Fig. 3A, B). The functional levels of adhesive changes
stimulated by E2 treatment of Ovx mice were similar to the peak levels
stimulated by pregnancy in HEV of secondary lymphoid organs (therapeutic
E2 dose compared to gd 10 in Fig, 3C, D). As shown in Fig. 3E and F,
administration of E2 had no effect on adhesion of PBL or TK1 cells to
pancreas.
(c) Increased adhesion of lymphocytes t~ endometrium is promoted by E2
and P4
To assess steroid hormone-mediated effects on adhesiveness of
endometrium for NK cells, CD56-labeled human PBL were applied to uteri
from Uvx mice treated with oil, E2 {low or high dose), P4 or E2~-P4 with or
without decidualization. Adhesion was assessed under mechanical shear.
All hormone treatments promoted similar levels of statistically significant
adhesion compared to adhesion on control uterine tissue (placebo-treated
Ovx or virgin; Fig. 4). The presence of decidualized stroma had no
independent effect. Adhering CD56b"9nt cells were randomly distributed
across all of these uteri as single cells. The adherent CD56b"9"t cells were
heterogeneous in size, some being larger cells {8.01-9.01 ~,m as compared
to 6.8 ~ 0.4~,m). Adhesion was blocked using either DREG56 or HP2/1
mAbs to L-selectin and alpha4 integrin, respectively (Fig. 4).
A high proportion of adherent cells in both naturally pregnant uteri at
gd 6 and in hormone-treated Ovx uteri were CD56brignt {Fig. 5). This
represented a significant enrichment in this NK cell phenotype since less
CA 02446871 2003-10-27
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than 2% of lymphocytes in the original overlays were CD56bright.
Furthermore, numerous clusters of large CD56b'rgnc cells were noted on the
gd 6 uteri but not on hormone-treated uteri. Cells in these clusters could not
be quantified reproducibly and were excluded from the enumeration. The
values presented for pregnant uteri therefore underestimate the actual
numbers of cells bound under pregnancy conditions. Further evaluations
were performed using unlabeled human PBL, stained with toluidine blue for
scoring under fight microscopy. This provided better visualization of
adhering cells and clusters and improved details of anatomic localization.
As shown in Fig. 6 prior to decidualization (virgin and gd 3), adherent cells
were randomly distributed both mesometrially and anti-mesometrially and
bound as single cells. In sharp contrast, using the same cell suspension
within the same experiment, decidualized gd 6 uteri bound some of the
lymphocytes in small clusters. The number of bound lymphocyteslcluster
was markedly increased on gd 10 uteri. Both single cells and clusters
preferentially adhered to DB. In parallel experiments using CD56-pre-
labeled cells, the clusters were shown to be mixtures of both CD56br'9nt and
CD56d'"' cells. These clusters of adherent cells were not observed in uteri
from hormone-treated mice or in peripheral tissues of any of the mice
investigated.
Discussion
The exquisite microdomain compartmentalization of the decidualized
mouse uterus of early pregnancy is due to the transient development of
highly differentiated vessels expressing known vascular adhesion
molecules in non-overlapping patterns.4o,as The biological function of this
compartmentalization is postulated to be recruitment and localization of
specialized, distinct leukocyte subsets. Precursors for the dominant
lymphocyte subset of early pregnancy, the uNK cells, do not self renew in
the uterus but are recruited during the first trimester.$',s8 Transplantable
uNK precursor cells have been found in BM, neonatal and adult thymus,
CA 02446871 2003-10-27
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spleen, LN and liver with PLN and spleens from pregnant donors being the
richest sources.$'~88 Por pre-uNK or uNK cells to leave the circulation and
move into the uterus, interactions with endothelial cells are required. It was
observed that a pregnancy-induced gain in adhesive function of HEV using
human PBL as indicator lymphocytes in adhesion assays conducted under
shear forces and PLN from gd 6 and virgin mice as tissue substrates.8'~$$
Because of the potential biological relevance of this observation to
lymphocyte homing into the pregnant uterus, which appears to be important
for antigen monitoring, for promotion of pregnancy-dependent physiological
changes to the endometrium and its vasculature2' and to lymphoid organ
hypertrophy and involution during pregnancy,3s.37,ss an extended study was
undertaken.
Pregnancy-induced gains in endothelial cell adhesive properties
were found in selected tissues including uterus and secondary lymphoid
tissues (LN, PP). Notably, these changes in adhesion were highly tissue
specific as evidenced by the failure of pancreatic endothelium to respond to
the pregnant state. Consistent with the notion that these changes were
mediated by the ovarian steroids, E2 and P4, it was observed that treatment
of Ovx mice with either steroid fully mimicked the functional gains in
adhesion promoted by pregnancy in secondary lymphoid tissues.
Hormonally-induced changes in adhesion at LN and PP sites were
mediated by L-selectin and a4ø7 integrin respectively, consistent with known
roles of these molecules in trafficking to these secondary lymphoid
tissues.s2,ss,85,$' Thus, it is unlikely that new adhesion pathways are
induced
by pregnancy. More probably, novel regulatory events are being detected.
Since it has been demonstrated that E2 stirnulation of human vascular
endothelial cells (HUVEC) results in increased expression of the adhesion
molecule ICAM-1 (21), and stabilization of mRNA for adhesion molecules
ICAM-1, V CAM, and E-selectin, it is possible that ligands for L-selectin
(PNAd) and a4ø~ (MAdCAM) are similarly influenced by hormonal
CA 02446871 2003-10-27
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stimulation, although that has not been reported to date. VCAM-1 has been
found to be the most dramatically upregulated endothelial cell adhesion
molecule in mouse DB,a6 but PECAM-1 and alpha4 integrin were strongly
upregulated on human uterine myometrial endothelium when cells were
cultured with E2 and P4.9o
Movement of lymphocytes including uNIC cells andlor their precursors
between secondary lymphoid organs into the uterus would involve transit
across two endothelial surfaces, that in a lymphoid tissue and that in the
uterus. Therefore, steroid hormone-mediated effects on uterine promotion
of CD56brignt Cell binding were investigated using Ovx mice in which
hormone levels were controlled. Endometrium from ~vx-placebo treated
mice could not be distinguished functionally from that of virgin females. Both
bound mixtures of CD56br'9ht and CD56d'm cells at low frequency across all of
the endometrial stroma. 1n both types of tissue, occasional larger cells
bound, some of which were CD56bright, as previously reported for virgin and
pregnant uteri.$'°8$ Gain in uterine adhesiveness was induced by all
steroid
hormone treatments. lJnexpectedly, equivalent functional changes were
induced in uterine tissue by both physiofogical$' and pharmacofogical84
doses of E2, by E2 compared to P483 and by E2+P4.~~83 Quite surprisingly,
artificial induction of endometrial strama cell decidualization83 did not
modify
the levels of adhesion beyond that seen following administration of
hormones only. While not wishing to be limited by theory, this may suggest
that maximal functional changes had been induced using single agents and
points to redundancy in the mechanisms used by the pregnant uterus to
promote homing of specific lymphocyte subsets. Blocking studies again
indicated that known adhesion pathways62 were being promoted by the
steroid hormones. Administration of E2 or P4. to mice increases
glycotransferase and siafyltransferase activities in the uterus.9' Notably,
these enzymes are known to modify L-selectin ligands.~2 Alpha
fucotransferase and NAC-glucosamine-6-0-sulfotransferase expression in
CA 02446871 2003-10-27
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the MLAp of gd 6 and 10 B6 mice have also been identified in a cDNA
microarray analysis (unpublished).
Although artificially-induced decidua in normal mice lacks
compartmentalization, these tissues are able to recruit uNK cells.
Lymphocytes adhering to artificially induced decidua were always dispersed
and never found as aggregates. The decidualized, pregnant uterus induced
an aggregatinglclustering behavior in the viable adherent cells that was
microdomain restricted (Fig. 6). This suggests that recruitment is distinct
from localization. Recruitment appears to be hormone mediated while
localization appears to be hormone independent, but requires lymphocyte
recognition of fetal trophoblast. Broadly, these data define limits in the
widely used model of deciduomata induction in rodents and demonstrate
that this model is not fully representative of decidua induced by blastocyst
implantation.
Examale 3
Goals of Example 3 were l) to assess the role of pregnancy andlor
pregnancy hormones on the adhesive properties of mouse splenocytes and
ii) to determine the robe of the adhesion molecule L-selectin in mediating
adhesion.
Materials and Meth~ds
Mice and i~issue dissections
C57BI/6J (Jackson Laboratory, Bar Harbor, ME) and C57B116 mice
genetically altered to lack a functional L-selectin adhesion molecule (L-
selectin-~-), aged 7-8 wk were studied. Some females from both strains of
mice were used for timed matings with the morning of the copulation plug
designated gd 0. Non-pregnant (NP) controls were virgin females who had
never been paired with males. Other C57BIl6J females (n=34) were
surgically ovariectomized under Avertin anaesthesia~4, rested 6 days and
then received daily subcutaneous injections of either hormone dissolved in
sesame oil or sesame oil alone between 9.0U-11.00 a.m. as indicated in
CA 02446871 2003-10-27
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Fig. 2.$'~ Some of the Ovx hormone-treated mice also had 10 p1 sterile
sesame oil injected into a uterine horn to induce decidualization 48 h prior
to euthanasia. Ovx mice were euthanized 24 hours after receiving their last
injection and vaginal smears were collected and stained with Wright-
Giemsa to confirm treatment success. All procedures were performed
under appropriate animal utilization protocols.
Assay of functional cell adhesion under shear f~ frozen fissue secfi~ns
In all assays, some cell aliquats were incubated with function
blocking mAb specific for mouse L-selectin (MEL-14, ATCC, hybridoma
conditioned medium diluted to 1:4), or mouse cc4~7 integrin (~ATK-32,
Coulter Immunology, 10 p,g/rnl). Lymphocytes were overlaid on 12 p.m
cryosections of PLN from virgin mice and rotated at 112 rpm at 4° C for
30
min. In some instances, tissue cryosections were pre-treated with MECA-
367 a mAb to MAdCAM-1 (ATCC, 1:4 dilution of hybridoma conditioned
medium). Following removal of non-adherent cells, glutaraldehyde-fixed
specimens were stained with 0.5% toluidine blue and adhesion was scored
on 300 -500 HEVIPLN. All enumerations were done thrice.
Role of L-selectin in mediating adhesion
In the experiments investigating the role of L-selectin in mediating
adhesion between splenocytes and PLN, splenocytes from C5713L16J mice
and L-selectin-~- mice were pre-labelled with either CeIITracker green
CMFDA (5-chloromethylfluorescein diacetate) or CeIITracker blue CMAC ( 7-
amino-4-chloromethylcoumarin) (Molecular Probes) and mixed in a 1:1 ratio
prior to use in the adhesion assay. To assess the effect of the dye in the
adhesion assay, dyes were reversed in a second experiment and
unlabelled cells were mixed in place of control cells in a third experiment.
Fluorescent cells were quantified in 10 high power fields (HPF; one HPF = 5
mm2) at 200X magnification using an Olympus BH21RFL fluorescence
microscope (Olympus Optical, Tokyo, Japan). All enumerations were done
thrice.
CA 02446871 2003-10-27
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~~'S li ItS
a) Pregnancy and steroid treatment enhances adhesion of splenocytes to
PLlll
Having established that both pregnancy and pregnancy-associated
hormones stimulate endothelial cell adhesion, the functional analysis of
these physiologic mediators was extended to lymphocytes. Splenocytes
from all groups of ~vx mice (Fig. 2) were assessed for L-selectin-dependent
adhesion to HEV of PL~J from virgin mice. As shown in Fig. 7A, all steroid
hormone treatments enhanced L-selectin-dependent adhesion of splenic
lymphocytes under shear compared to levels in the placebo treatment
group. IVloreover, these increases in L-selectin binding function were
comparable to those induced by pregnancy as well as to those induced by
in vitro lymphocyte stimulation using fever-range temperature (Fig. 7B).
Adhesion induced in this assay by fever range hyperthermia was previously
equated to a 4-5 fold increase in lymphocyte homing potential in vivo.85
b) L-selectin has an important, but not solitary role in mediating enhanced
adhesion during pregnancy
In Figure 8, the results of the L-selectin experiment are summarized.
Each experiment gave identical results, regardless of which CeIITracker dye
was used. This demonstrates that the effects of pregnancy are cumulative;
lowest adhesion is observed when splenocytes from a virgin donor are
applied to tissue from a virgin donor. Adhesion to virgin tissue is enhanced
when the donor lymphocytes come from a pregnant animal, further
increased when the tissue source is from a pregnant animal and
maximized when both cells and tissue come from pregnant animals.
Using cells from the C57BL/6J mice, adhesion is consistently higher
on the same tissue samples than cells from the L-selectin-~- mice. Lack of
the L-selectin molecule results in the same pattern of adhesion as is
observed in the B6 mice, with significant increases on pregnant tissue, but
this level of adhesion is approximately 2I3 that of the B6 mine.
CA 02446871 2003-10-27
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~iscussion
Pregnancy and its associated hormones alter not only the vascular
endothelium to enhance cell trafficking but also the ability of lymphocytes
(splenocytes) to recognize and adhere to molecules expressed on vascular
endothelium. This recognition appears to be mediated in part, but not
exclusively, by L-selectin, a molecule highly expressed on the surface of
CD56bri9nt natural killer cells. Previous work also implicates roles for oc4
integrin and LFA-1 in establishing firm adhesion between lymphocytes and
endothelium in the pregnant uterus 39' 4°.
The coordinated effects of pregnancy on endothelium, on
lymphocytes and on their interactions are reminiscent of findings in other
models of normal physiological change such as inflammation and fever.
Amplification of iymphocytelendothelial interactions in specialized hiEV of
selected tissue sites (LN, PP) while sparing non-lymphoid tissues, has
been proposed to focus immune response to these sites, thus preventing
an unproductive exodus to less relevant sites. ~efining the steroid
regulated pathways for lymphocyte recruitment to the uterus has potential
therapeutic importance for promoting lymphocyte localization to uteri of
patients at risk for implantation failure or pre-eclampsia and to non uterine
tissues in patients with other hormone-sensitive diseases such as some
tumours and endometriosis. Such information may also help to explain
inefficiencies in combination therapies that involve steroid hormone
replacement.
Example 4 Effect of hormones on human ~phoc)rtes
Goals of example 4 were to i) determine the effect of the menstrual
cycle on the recognition of human peripheral blood lymphocytes (PBL) of
adhesion molecules expressed on PLN and PP from virgin and pregnant
mice and to ii) correlate the hormones involved in cycle regulation to
adhesion of human PBL to mouse uterine tissue.
CA 02446871 2003-10-27
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Materials and Meth~cIa
Mice and tissue dissections
C57B1/6J (Jackson Laboratory, Bar Harbor, ME) aged 7-8 wk were
used for timed matings with the morning of the copulation plug designated
gd 0. Non pregnant (NP) controls were virgin females who had never been
paired with males. All procedures were performed under approved animal
utilization protocols. A pool of 10-12 PLN from subcutaneous and
intermuscular sites were collected from each mouse and embedded in
OCT (Miles Laboratories) for cryosectioning. Non-decidualized uteri were
trimmed at the ovarian and cervical ends, then incised longitudinally along
one side on an axis midway between the mesometrial and anti-
mesometrial aspects. Implantation sites from pregnant uteri were trimmed
and cut horizontally mid-way through the site and placed cut face down into
biopsy cryomolds and flash frozen.
Human Subjects and Slood Sampling
Male and female subjects of legal age and in good health were
recruited to donate up to 30 ml of blood, using venipuncture into evacuated,
sterile blood collection tubes. All subjects were informed about the risks of
participation in this study and signed approved informed consent forms.
The blood was layered onto an equal volume of Histopaque 1.07a (Sigma)
and centrifuged at 400 x g for 30 min at RT according to manufacturers
instructions. The cells at the interface were collected, washed thrice and
counted.
Preliminary experiments were done to determine the lowest number
of cells possible to detect significant differences in lymphocyte adhesion.
Replicate experiments using 10', 5 x 106, 2.5 x 106, and 106 cells per 100 p.1
from several donors (4) were applied to LN from both virgin and pregnant
animals in a standard adhesion assay. After washing, fixing and staining,
adherent cells were counted.
The effects of anti-coagulants on adhesive properties of isolated PBL
were assessed. Ten ml of blood was collected by venipuncture (40 m I
CA 02446871 2003-10-27
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collected per subject) into sterile evacuated tubes containing either Acid
Citrate Dextrose (ACD), EDTA, Sodium Citrate, or heparin. Lymphocytes
were isolated as described above and applied to sections of LN from
pregnant mice. The sections were assessed for lymphocyte adhesion by
microscopy.
It has been previously demonstrated that fever range temperature
resulted in enhanced adhesion of PBL. To determine the effect of
refrigeration on the adhesive properties of PBL, four samples of blood from
each of four different subjects were incubated at 20°C for 4 h, at
37°C for 4 h,
at 4°C for 4 h or at 4°C for 3.5 h, then warmedback to
37°C for 30 min. Then
lymphocytes were isolated as described above and cells were used in an
adhesion assay. Lymphocytes from freshly drawn blood were used as a
control.
Effects of the menstrual cycle on lymphocyte adhesiveness
Seven women of legal and reproductive age, not using hormonal
birth control and giving informed consent, were recruited to donate blood at
12 timepoints of their menstrual cycle. They were asked to maintain a
record of their temperature throughout one cycle using a basal body
thermometer and they started donating blood at a random timepoint of their
cycle. Twenty five ml of blood were drawn thrice weekly for 4 weeks. White
blood cells were isolated and 2.5 x106 cells were overlaid onto tissue
section of PP and LN from virgin and pregnant (d8) mice. Function-blocking
antibodies to L-selectin or alpha-4 integrin were used and found to
significantly reduce adhesion. Two hundred FiEV were counted per slide
and were scored blind encoded. After the completion of counting, volunteers
submitted their temperature charts and the results were correlated with the
phase of the cycle as indicated by first day of menses and with temperature
shift (indicating that ovulation had occurred). Significant differences were
detected using ANOi/A.
The second group of seven subjects also kept basal body
temperature charts throughout their cycle, but in addition, commenced use
CA 02446871 2003-10-27
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of a commercial LFi detection kit (Ciearplan) on day 11 of their cycle. Blood
was collected by venipuncture on day 8, day of LH surge and day 22 of their
cycle. Serum was collected at each of these timepoints and assayed by
ELISA for concentration of estradiol (E2) (DRG Instruments, Germany),
luteinizing hormone (LH) (Biocheck, lnc. Burlingame, CA) and progesterone
(P4) (Biocheck, Inc. Burlingame, CA).
Results
Dose response
Due to the limited number of PBL available from 25 ml of blood, a
dose response cunre of the number of cells used per tissue section was
done. In Figure 9, the results of 3 independent experiments are shown.
From this, it was determined that the minimum number of cells that could
be used while maintaining the ability to detect a significant increase in
adhesion in comparison to a control was 2.5 x 106 cells.
Effect of anti-coagulants on adhesive properties of lymphocytes
Next, the possible effect of the anti-coagulant used to collect the
blood on the adhesive properties of the isolated lymphocytes was
investigated. Four commonly were used anti-coagulants on 3 subjects
each. The results of those experiments are summarized in Figure 10. The
cells isolated from blood containing acid citrate dextrose (ACD)
demonstrated superior adhesion than those isolated from EDTA or heparin.
The sodium citrate did not interfere with adhesion, but the numbers of
lymphocytes isolated from these samples was consistently lower than with
the other anti-coagulants. Therefore, ACD was selected as the anti
coagulant for future experiments.
Effect of temperature of blood on adhesive properties of lymphocytes
The results of the temperature assay are shown in Figure 11. Freshly
isolated human lymphocytes {first bar) were used as a reference value.
Warming the blood to 37°C results in an increase in adhesive
ability,
significantly higher than the control (freshly isolated lymphocytes), while
storage at room temperature has no effect on adhesion. It was
CA 02446871 2003-10-27
demonstrated that chilling blood significantly reduces the ability of the
isolated human lymphocytes to recognize and bind to ligands expressed on
mouse tissue. Chilled blood, which is then re-warmed to 37°C
demonstrates a rebound in adhesion, but not to levels seen in either the
5 control or the 37°C cells.
Effect of the menstrual cycle orr adhesive properties of lymphocytes
As shown in Figure 12A, there was no significant difference in
lymphocyte adhesion to LN from virgin mice at any stage of the menstrual
cycle. However, we found that lymphocytes taken at the peri-ovulatory period
10 adhered at a significantly higher level than those from the proliferative
or
luteal phase of the menstrual cycle in an L-selectin dependent manner in
LN from a pregnant animal (Fig. 12~). The same pattern of adhesion was
evident in PP from both virgin and pregnant animals (Fig. 12C and D).
Adhesion to PP was shown to be alpha 4 integrin dependent.
15 In Figure 13, the results of the second experiment are shown. Here
blood samples were taken at only 3 timepoints and applied to LN and uteri
from pregnant mice only. In panel A, the same pattern of increased
adhesion to LN is seen in the periovulatory period as was demonstrated in
Fig. 13. Panel B shows that the effect at the LH surge is greater on uterine
20 tissue than was seen on LN.
Discussion
During the latter half of the menstrual cycle, a subset of Natural Killer
(NK) cells expressing the phenotype CD56b«ght9 CD16d'm home to the uterus.
These cells constitute the most abundant uterine lymphocyte population
25 during early to mid-pregnancy. It has been previously shown that in mouse
tissue (uterus, lymph node and Peyer's Patches), pregnancy, as well as
exposure to the pregnancy hormones 17~i-estradiol (E2) or progesterone
(P4), markedly up-regulated both L-selectin and cc4-integrin-dependent
adhesion of human CD56 expressing cells. similarly, adhesion of
30 splenocytes from pregnant or hormone treated animals to LN from virgin
mice was significantly increased over virgin or sham-treated controls. Here,
CA 02446871 2003-10-27
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these studies have been extended using peripheral blood lymphocytes
(PBL) from healthy, female volunteers of reproductive age for a time-based
study of the effects of the menstrual cycle on the adhesive properties of PAL
to mouse tissues. It was found that adhesion of human PAL to pregnant
mouse LN, PP and uterus peaked at mid-cycle, in the peri-ovulatory period.
Peak adhesion was observed on the day of Lhl surge, as determined by a
commercial LH detection method. LH, E2 and P4 levels were confirmed by
ELISA.
The primary differences between the previous experiments and these
experiments is that lymphocytes were immediately prepared and assessed
for adhesion on PLN from virgin or day 7 pregnant mice and on Peyer's
Patches (PP) from the same donors within 3 hours of drawing blood. The
central question for the research program is how to monitor changes in
human lymphocytes modified by pituitary or steroid hormones, in their
interactions with endothelium. It was anticipated that changes which would
promote movement of the lymphocytes from the circulation into the uterus
during pre-decidualization (LH+3-5) and that would be sustained during
early pregnancy. The data confirm that PAL exhibit higher affinity for
adhesion molecules expressed on vascular endothelium in the peri-
ovulation phase, indicating that either peak E2 or LH itself, may trigger NK
cells to traffic to the uterus. The adhesion assay only measures the first 2
steps of the homing process. It has been determined that lymphocytes are
slowed and arrested, not that they exit the blood.
The chemokine receptor repertoire reported for C~56bright cells may
contribute to homing of this lymphocyte subset to discreet regions of the
uterus. The uNK cells belong to the CD16-CD3-C~56br'9"~ subset of NK cells
and express high IeVeIS of L-S2IeCtlr1,5~102,103 This subset also expressed
high levels of CCRS, CCR7, CXCR3 and CXCR4 and low levels of
CX3CR1.1°Z In migration assays, C~16~CD3-CD56b"9nt NK cells were
differentially enriched by responding to RANTED, I-TAC and IP10 with lower
enrichment in response to hiICP-3. In dose response migration assays, the
CA 02446871 2003-10-27
- 4~ -
most dramatic responses were to the CCR7 ligands MIP-3~, and SLC and
to the CXCR3 ligands I-TAC and iP10.
Examples 5 and 6
These examples aim t~ define the normal, hormonally- and gestationally
induced changes in adhesion properties of women's lymphocytes with
endothelial cells that recruit NK cells into the decidualizing uterus for
promotion of successful decidualization, implantation, uterine artery
modification and pregnancy.
Materials and Methods
Human Subjects and Mood Sampling
The study requires 2 groups of women who have given informed
consent and whose ovulation cycles are defined by monitoring of blood
estradiol and LH levels. All donors will be prescreened HIV- and Hepatitis-
free. Example 5 requires serial blood sampling of n = 30-35 patients
across a normal luteal phase of the cycle and into early pregnancy (to day
40). Patients (n=30-35) enrolled in a REI program anticipating transfer of
banked frozen embryos will be recruited. Each woman will give an
additional 20 ml blood at regularly scheduled endocrine monitoring bleeds
(cycle days 10, 12, 14, 16), at the day of transfer and on days 18 and 40
after
transfer, respective times for pregnancy diagnosis endocrinologically and by
ultrasound. Bleeding will be conducted and hormone values measured will
become available, as blind encoded data. Example 6 requires serial blood
sampling during C~H and into early pregnancy (day 40). Patients (n=20)
will again be recruited from a REI program at lJVll~ and monitored serially
as in Example 4. Six or 7 serial collections are anticipated per patient and
each will be encoded and couriered for immediate analysis. Patient
exclusion criteria include lack of 2 good to high quality embryos, and age
over 39. Differences in conception rates in the patient groups are currently
attributed to cryogenic embryo damage. Historical conception rate data will
be available for statistical comparisons.
CA 02446871 2003-10-27
Nliceo
C57B1/6J mice will be purchased from, Jackson Laboratories and
maintained for breeding. Non-pregnant and gd 7 will be used. All
pregnancy time points required in a single experiment must be available on
the same day. Mice will be euthanised, uterus, and LN dissected and
placed in OCT compound for immediate freezing in N2 chilled isopentane
and storage. Adhesion molecule stability is 10 days. On assay days, 12
pm cryostat sections will be cut from selected test tissues and melted onto
glass coverslips.
C~uantitative Frozen Tissue Adhesion Assay under Shear:
Lymphocytes will be prepared by I°listopaque 1.077 (Sigma)
centrifugation and adherent cell depleted using established protocols.~~66,6s
5x106 cells are routinely used per section; 2x106 are adequate. In all
experiments, some lymphocytes will be pre-incubated 30 min at 20°C with
MAb (such as DREG-55, an anti-L-selectin function blocking Ab or T51I22,
an LFA-1 blocking Ab, both from ATCC, anti-hu alpha4 integrin
(Immunotech) anti CD56-I'E (PharMingen), anti-CD3-FITC (Becton
Dickenson) or isotype matched control antibodies before being applied to
tissue sections in a 100 p1 volume of RPMI medium + 10% bovine serum.
In other instances, tissue sections will be pre-incubated 30 min at
4.°C with
antibodies directed against PNAd (MECA-7g), MAdCAM-1 (MECA-367),
VCAM-1 (MK2.7), I-CAM-1 (YN1/1.7) or species appropriate isotype negative
control reagents, prior to initiation of adhesion. These antibodies are on
hand as hybridomas from ATCC. Antibody dilutions will be determined by
prior studies in Dr. Evan's laboratory. In a cold room, adhesion assays are
conducted under shear68~'° and then washed, fixed and stained. 300-500
high endothelial venules are scored 3 times in lymphoid tissues to obtain a
mean binding of Iymphocytes/HEV. For uterine tissue, vessel associated
cells are countered per 10 high power fields and tissue adherent cells/mmz
in different decidual regions as gestationally appropriate. To provide an
internal control for standardization of comparisons between experiments,
CA 02446871 2003-10-27
the murine B cell line 300.19, transfected with and expressing full length
human L-selectin6'~1o6 will be used in each adhesion assay as the positive
control. Some unblocked completed assay slides will be post
immunostained to evaluate IFN-y (antibody from Becton-~ickenson) in
adhering cells.
Estradiol and t_H Assays:
ELISA will be used to quantify estradiol and LH in plasma from
leukophoresis donors and patients.
Statistics: Mean values between paired groups are compared using
Student's T test. ANOVA is used for multiple group comparisons. Statistical
procedures will be optimized to correlate adhesion and endocrine data with
pregnancy outcomes.
Example 5 To quantify chances in l~mphoc~tedendothelial cell
interactions associated with transition from the luteal phase to
pregnancy
Assays will be conducted serially on lymphocytes provided by patient
volunteer recipients of frozen embryos until 5 gd 40 pregnancies have been
monitored. lJterine sections will be non pregnant and a constant pregnancy
day. Inbred mice must be used to maximally reduce variation in the uterine
tissues. This example will characterize the stability or dynamics of
lymphocyte/endothelial cel! interactions within individuals during the late
luteal phase of uterine decidualization and during establishment of
pregnancy.
Example 6 To monitor the effect of C~hl on le~mphoc,rtelendothelial cell
interactions:
Assays will be conducted serially on 9ymphocytes provided by patient
volunteer recipients of fresh embryos following COH until 5 gd 40
pregnancies have been monitored. Adhesion assays will be identical to
those in Example 5 and patients will be run concurrently as available. This
example will reveal the way a standard IVF hormone therapy protocols°'
modifies interactions between lymphocytes and uterine tissue by
CA 02446871 2003-10-27
comparing the results to those from Example ~. The information gained will
be used to to reassess ovulation protocols or' to modify hormone therapy
for other types of patients. The experimental design also permits
correlations of changes in adhesive functions to circulating hormone levels.
While the present invention has been described with reference to
what are presently considered to be the preferred examples, it is to be
understood that the invention is not limited to the disclosed examples. To
the contrary, the invention is intended to cover various modifications and
equivalent arrangements included within the spirit and scope of the
appended claims.
All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as if each
individual publication, patent or patent application was specifically and
individually indicated to be incorporated in its entirety.
CA 02446871 2003-10-27
51-
Tabte 1. Results of uterine segment transplantation
Donor -Host Animal Grafted horn Non-grafted
Identity Norn
number # of fetuses in maternal density of #. of fetuses
graft : host tissues in graft uNK cells at
segments graft sites*
CD1 -->CD1t 1 1 : 3 MLAp$, DBE +++ 0
2 0:0 deciduoma + 7
CD1 -jCD1~ 3 1 : 1 MLAp, DB +++ 4
4 1 : 3 MLAp, DB +++ g
CD1-->tgE26 5 0:0 deciduoma - 6
6 0:0 deciduoma - 1
7 0:1 deciduoma - 3
8 0 : 0 normal uterus - 0
SCID-> tge26 9 0 : 0 deciduoma - 5
0 : 0 normal uterus -
11 0:0 deciduomata - 1
+++ = more than 50 uNK cellslmm2, + = 5 or fewer uNK 2, - =
cellslmm no
5 uNK cells
t autografted, non grafted horn was re-anastomosed
$ MLAp, mesometrial lymphoid aggregate of pregnancy
DB, decidua basalis
~ donor and recipient were different CD1 mice
10
CA 02446871 2003-10-27
-52-
Table 2. Mean density of uNK cellslmm2 on gd 10 in mesometrial tissues of
NKIuNK cell
deficient mice transplanted on gd 0 and controls
Genotype Graft Cells inoculatedDamsl Means
Cells/
mm' SD)
Ireci ient fetusesDB MT
x10'
Confrol
tgE26 PBS _ 3l9 0 0
RAG-2-'-hycPBS - 3/9 0 0
~-
SCID PBS - 218 48.514.3 129.126.0
B6 PBS - 2l4 26.53.3 74.74.8
F1 PBS _ 113 53.38.3 72.15.1
Transplanted _
RAG-2-'~lyc~'-NP B6 adult Na 216 1.62.2* 0.61.3*
thymus ~ ~
RAG-2-'~/ycgd3 B6 thymus n a 2I6 2.51.1 5.22.7
~- * *
RAG-2-'-lyc'-gds B6 thymus na 3I9 9.65.2 7.0-~5.0
* R
RAG-2-'-/yc~-Neonatal B6 na 216 1.70.8 3.71.O
thymus
RAG-2-'~lyc'-NP adult SC1D 0.1 2l6 6.581.3* 1.00.4*
liver
RAG-2-'-lyc'-Gd7 adult SLID0,1 216 0 0
liver
tgE26 NP adult SCID 1.0 319 3.83.3* 2.72.1
BM
tgs26 gd3 SCID BM 0.6 319 5.93.8* 4.94.8*
~ =
tgE26 gds SLID BM 0.5 319 4.71.4 4.30.8
- ~
tgs26 gd7 SCID 8M 0.4 3l9 4.91.8 7.84.3
RAG-2~lrc~ NP B6 MLN 0.5 3I8 1.71.5 1.20.79
RAG-2 Jyc gd3 B6 MLN 0.6 1I3 0 0
RAG-2''/y~c~'gds B6 MLN 0.5 113 0 0
RAG-2~lyc'-gd7 B6 MLN 0.6 1I3 0.20.3 0.90.5
RAG-2~lyc~-NP B6 PLN 1.0-1.2 3/8 6.44.6 14.46.4
RAG-2~/yc~ gd3 B6 PLN 1.2 1/3 15.65.5 34.710.3
_ gd5 B6 PLN 0.5 1I3 3.2 0.8 2.41.4
RAG-2 /yc
RAG-2-~lyc''gd7 B6 PLN 1.5 1I3 14.510.6 0.90.4
tgE26 NP adult SCID 0.76-1.0 3/9 2.91.3*$ 1.21.6r
SC
tgs26 gd3 SCID SC 1.6-2.3 319 16.60.4~$~47.40.9~
tgE26 gds SCID SC 2.0-2.8 319 21.81.7~$n56.710.9~~
tgs26 gd7 SCID SC 2.0-2.8 319 11.25.9*$24.411.2
RAG-2-'lyo-~gd3 CCR2-'-SC 2.0-2.2 214 32.84.7 57.56.7
RAG-2-~'/rc~gds CCR2-'-SC 2.1-2.5 214 32.711.9 54.423.1
RAG-2'/y~c~'gd3 CCRS~'-SC 2.0-2.3 316 49.311.0 70.315.2
~
RAG-2'~lx~ ads CCR5-'-SC 2.3-2.5 316 30.86.2 32.66.3
* significantly different from uNK+controls (p<0.05)
significantly different from uNK cell densities in the MTIMLAp of the members
of the
same treatment group (p<0.01 )
~ spleen cell recipients with significantly different uNK cell numbers than
tgs26 receiving
spleen cells from NP SCID donors (p<0.001)
n spleen cell recipients with significantly different uNK cell numbers from
tgs26 receiving
spleen cells from gd7 SCID donors (p<0.01)
na - not applicable, intact thymus was transplanted
CA 02446871 2003-10-27
-53-
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