Originally published online as doi:10.1189/jlb.0406252 on December 12, 2006
Published online before print December 12, 2006
(Journal of Leukocyte Biology. 2007;81:757-765.)
© 2007
by Society for Leukocyte Biology
Distinct regulation of autoreactive CD4 T cell expansion by interleukin-4 under conditions of lymphopenia
Natasha J. Hill,
Aleksandr B. Stotland and
Nora E. Sarvetnick1
The Scripps Research Institute, La Jolla, California, USA
1 Correspondence: The Scripps Research Institute, 10550 N. Torrey Pines Road, IMM-23, La Jolla, CA 92037, USA. E-mail: noras{at}scripps.edu
ABSTRACT
IL-4 is protective against Type 1 diabetes in the NOD mouse.
IL-4 promotes T cell survival in vitro, but little is known
about the effect of IL-4 on clonal expansion in vivo. Here,
we show that IL-4 only enhances the expansion of autoreactive
CD4 T cells during lymphopenia and that neither the presence
of islet IL-4 nor IL-4 deficiency affects T cell expansion significantly
under conditions of immunosufficiency. The accumulation of proliferating
cells induced by IL-4 in a lymphopenic host is inhibited incrementally
by increasing the number of bystander cells and is prevented
by cell numbers well below that of unmanipulated NOD mice. The
ability of IL-4 to promote autoreactive CD4 T cell expansion
is therefore sensitive to the degree of host immunodeficiency.
Paradoxically, IL-4 receptor-deficient, autoreactive CD4 T cells
proliferate more extensively than wild-type T cells in immunodeficient
hosts, suggesting that the growth-promoting effect of islet
IL-4 acts indirectly. These results suggest that IL-4-mediated
protection against autoimmunity and diabetes may be outweighed
during immunodeficiency by a pathogenic, IL-4-induced expansion
of autoreactive T cells.
Key Words: cytokine • autoimmunity • diabetes
INTRODUCTION
IL-4 is an effective growth factor for resting [
1
,
2
] and
activated/memory [
3
] T cells in vitro. It is therefore in some
ways paradoxical that IL-4 treatment should also be highly protective
in models of autoimmune diseases such as Type 1 diabetes [
4
]
and multiple sclerosis (MS) [
5
], which depend on a pathogenic
activation and expansion of self-reactive T cells. The expression
of IL-4 in islets prevents diabetes in the NOD mouse and induces
a functional tolerance to islet antigens [
6
]. Islet IL-4 also
prevents lymphocytic choriomeningitis virus (LCMV)-induced diabetes,
and inhibits the acquisition of cytotoxicity by CD8 T cells
following viral infection by blocking the maturation of antigen-presenting
dendritic cells [
7
]. In this model, islet IL-4 promotes a slight
increase in the expansion of CD8 T cells specific for the immunodominant
epitope of LCMV yet is still able to inhibit cytotoxicity. The
growth-promoting effects of IL-4 are therefore not necessarily
linked to the development of autoimmunity. However, expression
of islet IL-4 in immunodeficient NOD
Scid recipients greatly
enhances the expansion of adoptively transferred islet-specific
CD4 T cells and accelerates insulitis [
8
]. The ability of IL-4
to affect T cell expansion in vivo, and the contribution of
this aspect of IL-4 activity in mediating protection against
autoimmunity, are therefore important questions for understanding
how IL-4 may be used therapeutically.
The question of whether IL-4 can promote T cell survival in vivo is controversial. Coinjection of IL-4 with staphylococcal enterotoxin A (SEA) inhibits the deletion of Vß3+ cells that occurs following SEA injection alone [3
], and deprivation of IL-4 and IL-7 enhances the loss of naïve CD4 T cells following thymectomy, suggesting that IL-4 can prevent T cell loss [9
]. IL-4 promotes the homeostatic proliferation and survival of CD8 T cells in lymphoid organ culture, although it is not required for homeostatic proliferation of CD4 or CD8 T cells in vivo [10
]. However, there is also evidence that IL-4 can inhibit T cell survival. IL-4-deficient mice have a twofold increase in thymocyte number by 6 weeks of age compared with wild-type [11
], whereas T cell-specific, transgenic expression of IL-4 reduces thymocyte number dramatically [12
]. In IL-4-deficient mice on the NOD background, the decay of peripheral T cell numbers following thymectomy is lessened, suggesting that IL-4 inhibits T cell survival in these mice [13
].
In animal models of diabetes and MS, IL-4 has been shown to improve protection induced by tolerizing DNA vaccination [14
15
16
]. IL-4 also plays an important role in 
-galactosylceramide-induced protection against diabetes [17
]. Furthermore, glatiramer acetate (copolymer 1) is approved for the clinical treatment of MS, and its effectiveness is thought to depend on the induction of IL-4-producing Th2 cells [18
]. However, there are also concerning reports that IL-4 may, under some circumstances, be involved in autoimmune pathogenesis [19
, 20
]. It is therefore of clinical importance to understand the mechanisms by which IL-4 can protect against autoimmunity and the conditions under which the protective role of IL-4 is compromised.
As IL-4 appears able to both promote and counter T cell survival in vivo, we tested whether the biological effects of this molecule may be dependent on T cell density in the host. We show that the dramatic expansion of autoreactive CD4 T cells induced by islet IL-4 under conditions of severe lymphopenia is inhibited incrementally by the presence of increasing numbers of bystander T cells. Therefore, the ability of IL-4 to promote the expansion of autoreactive CD4 T cells depends on the degree of T cell deficiency. Paradoxically, IL-4 receptor (IL-4R)-deficient, autoreactive CD4 T cells have enhanced proliferation also, suggesting that islet IL-4 may act indirectly to promote proliferation in immunodeficient recipients. These results may explain the unexpected pathogenicity of Th2 cells in immunodeficient hosts [19
] and suggest that the beneficial effects of IL-4 in protecting against autoimmunity may be outweighed by a pathogenic expansion of autoreactive T cells by IL-4 under conditions of immunodeficiency.
MATERIALS AND METHODS
IL-4NOD [
6
] and IL-4NOD
Scid [
8
] mice, which express IL-4 specifically
in their islet ß cells, have been described previously.
BDC2.5NOD mice [
21
] were a kind gift of Drs. Diane Mathis and
Christophe Benoist (Harvard University, Cambridge, MA, USA),
and NODIL-4 knockout (KO) mice were obtained from Jackson Laboratory
(Bar Harbor, ME, USA; Stock 004222). BALB/c-
IL-4ratm1Sz was
obtained from Jackson Laboratory (Stock 003514) and backcrossed
to NOD mice for nine generations using a speed congenic screening
strategy at each generation. N9 mice were NOD-derived at all
markers tested across the genome, except for a congenic interval
of less than 5 cM flanking the IL-4R-targeted mutation on Chr.
7. The mice were then intercrossed to generate homozygous NOD.IL-4RKO
mice. Specific details of the polymorphic markers used for screening
are given in
Supplemental Figure 1. Inheritance of the IL-4R
mutation was tested as described in the Jackson Laboratory PCR
protocol. NOD.IL-4RKO mice were then crossed with BDC2.5.NOD
mice to generate BDC2.5NOD.IL-4RKO mice expressing a single
copy of the BDC2.5 transgene. All strains were maintained under
specific, pathogen-free conditions in the rodent-breeding colony
at The Scripps Research Institute (La Jolla, CA, USA). Live
animal experiments were approved by the Institutional Animal
Care and Use Committee and the Animal Research Committee and
were conducted in accordance with institutional guidelines for
animal care and use.
Adoptive transfer
Adoptive transfer of 20 million CFSE-labeled splenocytes from 5- to 6-week-old BDC2.5NOD or BDC2.5NOD.IL-4RKO donor mice was performed as described previously [8
]. Approximately 20% splenocytes from BDC2.5NOD mice were islet-specific BDC2.5 T cells. Typically, less than 25% BDC2.5 T cells transferred were CD44hi, and therefore the majority of cells was phenotypically naïve. On Day 4 following transfer, single-cell suspensions from recipient pancreatic lymph nodes (panLN) were prepared for analysis by flow cytometry to detect the CFSE+CD4+Vß4+ BDC2.5 population transferred, as described previously [8
]. For annexin staining, cells were gated on the live CFSE+CD4+7-amino-actinomycin– population. Flow cytometry was performed using a FACSCaliber cytometer and CellQuest software or digital LSR II with DiVa and FlowJo software. All average values are shown ± SEM, and an unpaired Student’s t-test (two-tailed) was used where indicated to test statistical significance. Bystander cells were whole splenocytes isolated from 8-week-old NOD mice, and 0, 20, or 100 million cells were injected into Scid recipients at the same time as the CFSE-labeled BDC2.5 splenocytes.
Immunohistochemistry
The pancreas of recipient mice was quick-frozen in OCT medium at Day 4 following transfer, and 4 µm sections from three levels, each level separated by at least 120 µm, were stained with anti-insulin (Dako, Carpinteria, CA, USA; 1/400) and Texas Red-conjugated antiguinea pig secondary antibody (Vector Laboratories, Burlingame, CA, USA; 1/200). 4',6-Diamidino-2-phenylindole (DAPI) nuclear dye was included in the mounting media to visualize nuclei (blue), and the transferred cells were visible as a result of the CFSE label (green). Images were taken using a Bio-Rad (Zeiss) Radiance 2100 rainbow laser-scanning confocal microscope and 40x oil objective lens using Bio-Rad LaserSharp (v3.2) software. ImageJ [National Institutes of Health (NIH), Bethesda, MD, USA] was used for image analysis. The endogenous and CFSE+ infiltration were scored separately for each islet, and islets were scored as having no insulitis, peri-insulitis, insulitis, or severe insulitis, and differences in the distributions were determined using a 
2 test. Sections from a total of two IL-4NOD and two NOD pancreata, from two independent experiments, were examined.
RESULTS
We have shown previously that the presence of islet IL-4 enhances
the expansion of islet-specific, autoreactive CD4 T cells dramatically
when adoptively transferred into lymphopenic (
Scid) recipients
[
8
]. To test our hypothesis that immunodeficiency is important
for the increased expansion of autoreactive T cells by IL-4,
we performed adoptive transfer experiments into islet-IL-4 NOD
mice, which contain their normal complement of lymphocytes.
Islet-reactive CD4 T cells from BDC2.5NOD mice [
21
] were CFSE-labeled
and adoptively transferred into wild-type or islet-IL-4 recipients
on the NOD and NOD
Scid backgrounds in parallel. At Day 4 following
transfer, the number of CFSE+CD4+ BDC2.5 T cells in the panLN
was determined (
Fig. 1A
). Consistent with our results reported
previously, in control, lymphopenic
Scid recipients, islet IL-4
increased recovery of the autoreactive BDC2.5 T cell population
(6.2-fold,
P=0.02,
n=3) [
8
]. However, on the immunocompetent
NOD background, the presence of IL-4 did not increase the recovery
of BDC2.5 cells, and in fact, their number was reduced slightly
in IL-4NOD compared with NOD mice (1.7-fold,
n=3), although
the difference did not reach statistical significance. To test
whether the BDC2.5 T cells in IL-4NOD mice undergo enhanced
expansion but that an increased rate of apoptosis counters accumulation,
we examined the CFSE profile and annexin staining of these cells.
However, no significant difference in the CFSE division profile
(Fig. 1B
and 1C)
or annexin staining
(Fig. 1D)
of the BDC2.5
T cell population was observed between IL-4NOD and NOD recipients.
In IL-4NOD
Scid recipients, the BDC2.5 cells were seen to undergo
increased proliferation and reduced annexin-V staining compared
with NOD
Scid recipients, as reported previously [
8
]. IL-4 therefore
only promotes the expansion of autoreactive CD4 T cells under
conditions of lymphopenia and does not impact autoreactive CD4
T cell expansion significantly in normal NOD mice. We also performed
adoptive transfers using IL-4-deficient NOD recipients and observed
an equivalent number of BDC2.5 T cells to that in wild-type
NOD recipients
(Fig. 1E)
and a comparable CFSE profile
(Fig. 1F)
.
Autoreactive CD4 T cell expansion is therefore unaffected by
IL-4 deficiency and the presence of islet IL-4 in immunocompetent
mice.
Reduced islet infiltration by adoptively transferred, autoreactive CD4 T cells in NOD mice expressing islet IL-4
In NOD
Scid recipients, the increased expansion of autoreactive
CD4 T cells in mice expressing islet IL-4 was associated with
an acceleration of insulitis [
8
]. To test whether the lack
of this expansion in IL-4NOD recipients is associated with protection
against insulitis, we examined the islets of IL-4NOD and NOD
recipients for the presence of transferred BDC2.5 cells. As
shown in
Figure 2A
, CFSE-labeled BDC2.5 splenocytes can be
found in the islets of NOD and IL-4NOD recipients at Day 4 post-transfer.
Insulitis scoring
(Fig. 2B)
was performed for injected (CFSE-positive)
and endogenous (areas of dense nuclear stain) cells and was
found to be significantly less severe in IL-4NOD recipients
(
P<0.05 in both cases). Islet IL-4 therefore protects against
islet infiltration by endogenous and injected BDC2.5 T cells.
This also suggests that the slight reduction in the number of
BDC2.5 T cells in the panLN of IL-4NOD recipients compared with
NOD is unlikely to be a result of increased homing of activated
cells to the pancreas. The number of CFSE-positive cells within
each islet was observed to correlate with the degree of endogenous
infiltration, and there was no difference in the distribution
of insulitis scoring between endogenous and BDC2.5 cells in
either strain. Therefore, in immunosufficient NOD mice, where
endogenous islet inflammation is already present, the degree
of pre-existing inflammation may be the primary determinant
of BDC2.5 T cell islet recruitment. Hence, on the NOD background
islet, IL-4 does not significantly affect expansion in the panLN,
but the extent of islet infiltration is reduced, possibly as
a result of the reduction in endogenous insulitis in this strain.
Cotransfer of bystander cells decreases the recovery of autoreactive CD4 T cells in recipients expressing islet IL-4 and increases their recovery in wild-type mice
The inability of IL-4 to enhance autoreactive T cell expansion
except in lymphopenic
Scid mice suggests that the growth-promoting
activity of IL-4 occurs only in the absence of adequate T cell
numbers. However, homeostatic proliferation in congenitally
T cell-deficient hosts occurs more rapidly than in lymphocyte-depleted,
normal hosts, and one suggestion is that this is driven by the
immunogenic effect of increased levels of foreign antigens [
22
].
The presence of islet IL-4 could potentially be enhancing this
immunogenic effect rather than reflecting T cell deficiency,
or compounding another effect that is specific to congenitally
immunodeficient mice. We therefore wanted to test whether we
could inhibit the growth-promoting effect of IL-4 on BDC2.5
T cells in
Scid recipients by cotransferring increasing numbers
of bystander cells.
We cotransferred 0, 20, or 100 million unlabeled splenocytes from NOD mice, in addition to the 20 million CFSE-labeled BDC2.5 splenocytes, into Scid recipients. Transfer of increasing bystander cell numbers increased the size of the bystander CD4 T cell population in wild-type and islet IL-4-expressing recipients (data not shown). Cotransferring an increasing number of bystander cells into wild-type Scid recipients increased the recovery of BDC2.5 cells in the panLN (Fig. 3
). It therefore appears that the presence of bystander cells increases the percentage of injected cells recovered. However, in contrast, the recovery of BDC2.5 cells from IL-4NODScid recipients was reduced by the cotransfer of bystander cells and decreased incrementally as the number of bystander cells was increased. Hence, there is a clearly contrasting positive and negative correlation between bystander cell number and BDC2.5 recovery in wild-type and islet-IL-4 recipients, respectively, which is evident despite the inherent problem of variability when determining absolute cell counts in adoptively transferred Scid recipients. This suggests that the increasing, growth-promoting effect of IL-4 on autoreactive CD4 T cells under conditions of immunodeficiency outweighs the loss of the positive effects of bystander cells on T cell recovery. These experiments also demonstrate that the ability of IL-4 to promote autoreactive CD4 T cell expansion is critically dependent on the absence of sufficient T cell numbers.
The accumulation of proliferating cells by IL-4 is gradually inhibited by increasing the number of bystander cells
The CFSE dilution profile of autoreactive CD4 T cells transferred
to
Scid mice expressing islet IL-4 shows a pronounced accumulation
of proliferating cells at Day 4 following transfer (
Fig. 1C
and ref. [
8
]). To test whether bystander cells inhibit this
accumulation of dividing cells, we compared the CFSE profile
of BDC2.5 T cells cotransferred into
Scid recipients with titrated
numbers of bystander cells (
Fig. 4A
). Somewhat surprisingly,
the CFSE profile of transferred BDC2.5 T cells in wild-type
Scid recipients was unaffected by the number of bystander cells
present. We quantified the percentage of BDC2.5 T cells, which
have not undergone cell division
(Fig. 4B
and 4C)
and found
that the percent of undivided cells was not different in wild-type
Scid mice, with and without cotransferred bystander cells, and
in NOD mice. The proliferation of BDC2.5 T cells that we observe
in these experiments is therefore unaffected by the degree of
lymphopenia in the recipient. In contrast, the accumulation
of proliferating cells by IL-4 was gradually inhibited by the
transfer of an increasing number of bystander cells, demonstrating
that this accumulation is dependent directly on T cell deficiency.
The CFSE profile of BDC2.5 T cells in IL-4NOD
Scid recipients
cotransferred with 100 million bystander cells was equivalent
to that seen in wild-type recipients. The number of CD4 T cells
present in the panLN of
Scid mice reconstituted with 100 million
cells is still less than 10% of the normal complement of CD4
T cells in full NOD mice in the presence of islet IL-4 or not
(data not shown). The growth-promoting effects of IL-4 are therefore
inhibited by the presence of T cell numbers well below those
of a normal NOD mouse. IL-4 therefore differentially affects
the expansion of autoreactive CD4 T cells according to the degree
of lymphopenia, and under conditions of severe lymphopenia,
IL-4 is able to dramatically promote autoreactive CD4 T cell
expansion.
Gating on CFSE+ cells excludes primarily endogenous T cells in NODScid recipients
To compare the CFSE profiles in NOD mice, it was necessary to
gate on the CFSE-positive cells to distinguish the injected
BDC2.5 T cells from endogenous CD4+Vß4+ T cells, which
are present at high frequency. The same gate was used for NOD
Scid recipients. However, a population of CFSE– cells in NOD
Scid recipients is excluded by this gate, and it is not clear if
these are highly proliferating cells that are being excluded
from the analysis or endogenous cells that can arise in "leaky"
Scid mice. We therefore used an antibody raised specifically
against the BDC2.5 TCR to test whether these cells are injected
or endogenous cells [
23
]. In control, uninjected NOD mice,
the endogenous BDC2.5 T cell population stained by the antibody
was observed to make up 0.8% of the CD4 T cell pool (
Fig. 5A
),
comparable with results reported previously [
23
]. As shown
in
Figure 5B
, the majority of CFSE+CD4+ cells in NOD and
Scid recipients stains positively with the anti-BDC antibody. However,
the CD4+CFSE– cells in
Scid mice, which have been excluded
by the CFSE gate, are largely endogenous, non-BDC2.5 T cells
rather than injected cells that have proliferated extensively
and lost the CFSE signal. Consistent with this, a similar population
can be observed in control, uninjected mice. Therefore, although
some of the highly proliferating BDC2.5 T cells in IL-4NOD
Scid mice are likely to be lost with the CFSE gate, this should not
affect the conclusion that expansion is greater in this strain,
and comparisons between BDC2.5 T cell proliferation in NOD and
Scid recipients are likely to be impacted only mildly.
IL-4R deficiency on BDC2.5 T cells promotes their expansion in immunodeficient NODScid mice
To test whether the growth-promoting effect of IL-4 under conditions
of immunodeficiency is directly a result of signaling through
IL-4R on the BDC2.5 T cells, or an indirect effect via another
cell type, we adoptively transferred CFSE-labeled BDC2.5.IL-4RKO
T cells into NOD
Scid recipients. The recovery of IL-4R-deficient
BDC2.5 T cells from the panLN of
Scid mice at Day 4 following
transfer was not different than that of wild-type BDC2.5 T cells
(
Fig. 6A
). However, the CFSE profile demonstrates that BDC2.5.IL-4RKO
T cells exhibit a much greater accumulation of proliferating
cells in the panLN than wild-type BDC2.5 T cells
(Fig. 6B
and 6C)
.
This suggests that the presence of IL-4R on autoreactive CD4
T cells normally acts to inhibit proliferation in immunodeficient
mice. Therefore, although islet expression of IL-4 promotes
BDC2.5 T cell expansion in the panLN of immunodeficient mice,
when BDC2.5 T cells lack IL-4R, this also results paradoxically
in enhanced panLN proliferation. This suggests that either there
is a dose-dependence of the effect of IL-4 on autoreactive T
cell expansion or that the growth-enhancing effect of islet
IL-4 on autoreactive T cells during immunodeficiency may act
indirectly through another cell type.
We also performed additional controls to test the dependence
of IL-4R deficiency-induced proliferation on the presence of
autoantigen. In the ingLN of NOD
Scid mice, where islet antigen
is not present, proliferation of BDC2.5.IL-4RKO cells was not
increased compared with wild-type BDC2.5 cells
(Fig. 6D)
. This
suggests that, as in IL-4NOD
Scid mice [
8
], the increased proliferation
of BDC2.5.IL-4RKO T cells in panLN is dependent on the presence
of autoantigen. We also transferred BDC2.5 T cells into immunosufficient
NOD recipients in parallel. As an additional control, we used
NOD mice at 3.5 weeks of age as recipients (NODy) as this is
the approximate age at which loss of tolerance to islet autoantigens
in the panLN is thought to be initiated [
24
]. In these mice,
the expansion of BDC2.5 T cells was seen to be reduced compared
with the usual 6- to 7-week-old NOD recipients, suggesting that
it is the presence of large amounts of autoantigen resulting
from T cell-dependent islet damage that is driving the high
degree of proliferation in the panLN of immunosufficient mice.
Figure 6
also shows that BDC2.5 T cells in the ingLN of NODScid mice have undergone one to two rounds of division, and little proliferation is evident in the ingLN of NOD mice. Lymphopenia-induced proliferation of BDC2.5 T cells is therefore evident in the ingLN of NODScid mice. However, the effect of the Scid environment in increasing cell division in panLN BDC2.5 T cells was again less evident than expected. Proliferation of islet-reactive CD4 T cells in the panLN is clearly autoantigen-driven, and particularly, as NOD mice are mildly lymphopenic [25
], this may represent a maximal rate of proliferation in NOD or NODScid mice, except where increased levels of exogenous cytokines are present. Yet, the recovery of injected BDC2.5 T cells from the ingLN of NODScid recipients is reduced even more strikingly compared with NOD recipients than in the panLN, supporting the idea that although proliferation may increase in Scid recipients, the recovery of naïve cells recruited to LN is markedly reduced.
DISCUSSION
These experiments demonstrate that the ability of islet IL-4
to promote the accumulation of proliferating cells is dependent
on conditions of severe immunodeficiency. However, IL-4R-deficient,
autoreactive CD4 T cells were found to proliferate more extensively
in immunodeficient recipients, suggesting that islet IL-4 perhaps
acts indirectly to promote autoreactive T cell expansion. The
opposing effects of IL-4 under immunosufficient and immunodeficient
conditions may therefore reflect the predominance of IL-4 acting
directly or indirectly on T cells. The results also suggest
that the presence of the IL-4Rß chain limits the proliferation
of autoreactive CD4 T cells in immunodeficient hosts. Further
experiments will be required to understand the mechanism that
underlies this, but one possibility is that responsiveness to
another
c cytokine is enhanced in T cells lacking IL-4Rß.
It is important to understand the growth-promoting and homeostatic effects of cytokines on autoreactive T cells in an in vivo context. The NOD mouse was itself recently shown to be mildly lymphopenic [25
], and homeostatic expansion or hypersensitivity to homeostatic cytokines can drive self-reactive T cells to cause autoimmunity [25
, 26
]. The growth-promoting effects of IL-4 in an immunodeficient context are associated with an acceleration of insulitis [8
]. Our results suggest that direct and indirect effects of IL-4, as well as the degree of T cell immunodeficiency, can differentially affect the outcome of IL-4 exposure on autoreactive CD4 T cell expansion. Therefore, although IL-4 is profoundly protective against diabetes under conditions of immunosufficiency and is able to induce tolerance via its effects on APC, our results suggest that in the context of immunodeficiency, the growth-promoting effects of IL-4 on autoreactive T cells may predominate and overcome protection and even accelerate disease.
ACKNOWLEDGEMENTS
This work was supported by NIH Grant DK054063 and American Diabetes
Association Mentor-Based Postdoctoral Fellowship Award #7-05-MN-53.
We thank Cecile King for useful, general discussions, Jared
Purton and members of the Sarvetnick lab for comments about
the manuscript, and the TSRI Flow Cytometry Core Facility.
Received April 5, 2006;
accepted November 16, 2006.
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ABSTRACT
INTRODUCTION
