By
From the * Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute,
Bethesda, Maryland 20892; and the Division of Hematologic Products, Food and Drug
Administration, Bethesda, Maryland 20892
In the immune system, there is a careful regulation not only of lymphoid development and
proliferation, but also of the fate of activated and proliferating cells. Although the manner in
which these diverse events are coordinated is incompletely understood, cytokines are known to
play major roles. Whereas IL-7 is essential for lymphoid development, IL-2 and IL-4 are vital
for lymphocyte proliferation. The receptors for each of these cytokines contain the common
cytokine receptor chain (
c), and it was previously shown that
c-deficient mice exhibit severely compromised development and responsiveness to IL-2, IL-4, and IL-7. Nevertheless,
these mice exhibit an age-dependent accumulation of splenic CD4+ T cells, the majority of
which have a phenotype typical of memory/activated cells. When
c-deficient mice were
mated to DO11.10 T cell receptor (TCR) transgenic mice, only the T cells bearing endogenous TCRs had this phenotype, suggesting that its acquisition was TCR dependent. Not only
do the CD4+ T cells from
c-deficient mice exhibit an activated phenotype and greatly enhanced incorporation of bromodeoxyuridine but, consistent with the lack of
c-dependent survival signals, they also exhibit an augmented rate of apoptosis. However, because the CD4+
T cells accumulate, it is clear that the rate of proliferation exceeds the rate of cell death. Thus,
surprisingly, although
c-independent signals are sufficient to mediate expansion of CD4+
T cells in these mice,
c-dependent signals are required to regulate the fate of activated CD4+
T cells, underscoring the importance of
c-dependent signals in controlling lymphoid homeostasis.
The common cytokine receptor Mice and Genetic Analysis.
The Flow Cytometric Analysis.
Cells from thymus and spleen were
stained and analyzed on a FACSort® (Becton Dickinson, San Jose,
CA) using Lysis II software (7). For direct staining, the following
conjugated antibodies were purchased from PharMingen (San Diego, CA): anti-CD4 Cy-Chrome (H129.19), anti-CD8 PE (53-6.7),
anti-CD25 PE (7D4), anti-CD44 Cy-Chrome (IM7), anti-CD45R
(B220) PE (RA3-6B2), anti-CD49d FITC (R1-2), anti-CD62L
PE (MEL-14), anti-CD69 PE (H1.2F3), anti-CTLA-4 PE (UC104F10-11), anti-erythroid cells PE (TER-119), anti-Fas biotin
(Jo2), anti-I-Ab FITC (AF6-120.1), anti-I-Ad PE (AMS-32.1),
anti-TCR V Splenic CD4+ T Cell Purification.
Since erythroblastoid cells
and macrophages were increased in the spleens of Cell Survival Assay.
Purified CD4+ T cells were cultured in
RPMI-1640 media supplemented with 10% charcoal-treated fetal
bovine serum (Cocalico Biological, Inc., Reamstown, PA), 2 mM
glutamine, and antibiotics for 8 to 48 h. Cells were harvested and
stained with 5 µg/ml of propidium iodide (PI) (Boehringer Mannheim) and analyzed by flow cytometry.
Annexin V Staining.
After cells were stained with anti-CD4
Cy-Chrome and washed twice with PBS, 1% BSA, cells were
stained with annexin V FITC (R&D Systems) according to the
instructions of the manufacturer. Cells were analyzed on a FACSort®.
TUNEL Method.
The terminal deoxynucleotidyl transferase
(TdT)-mediated dUTP nick end-labeling (TUNEL) method was
performed according to the instructions of the manufacturer (Boehringer Mannheim). Splenocytes were stained with anti-CD4 CyChrome and washed with PBS, 1% BSA. Cells were then fixed
with 70% ethanol for 20 min at 4°C, treated with PBS containing
1% paraformaldehyde and 0.01% Tween-20 for 30 min at 25°C,
washed once with PBS, and incubated with the fluoresceinlabeled TUNEL reaction mix for 60 min at 37°C. As a negative
control, cells were incubated without TdT. Cells were then
washed with PBS, 1% BSA and analyzed on a FACSort®.
Intracellular Staining of Bcl-2.
Bcl-2 levels in CD4+ T cells
were analyzed as previously described (14). In brief, splenocytes
(1 × 106) were permeabilized in PBS, 1% BSA containing 0.04%
saponin (saponin buffer) and incubated with anti-Bcl-2 mAb (clone
3F11, PharMingen, 2 µg/ml final concentration) or purified hamster IgG as a negative control (4°C for 30 min). Cells were
washed twice with saponin buffer, incubated with anti-hamster
IgG cocktail conjugated to FITC (clone G70-204, G94-56, G91140, PharMingen) (4°C for 30 min), washed once with saponin
buffer and once with PBS, 1% BSA, then stained with antiCD62L PE/and anti-CD4 Cy-Chrome, and analyzed on a FACSort®.
In Vivo Bromodeoxyuridine (BrdU) Uptake and BrdU Staining.
8 to 12-wk-old Apparent Rates of Replication and Survival.
Assuming that cells
that have taken up BrdU at 20 h have divided once, the apparent
replication rate/day, r, can be calculated from the equation: percent BrdU uptake/20 h × 24 h/d = 2r/(100 + r). Based on the
data in Fig. 1 D, this yields values for r of 21.43%/d for
Like humans with XSCID, A striking difference between splenic CD4+ T cells from
The accumulation and activation of the CD4+ T cells
appeared to result from a defect in T cells, because the defect was corrected in mice in which Next, we investigated whether the CD62LlowCD69high
phenotype of the Based on the BrdU uptake studies, the
There are at least two possible explanations for why peripheral CD4+ T cells in
In the immune response, there is a careful balance between clonal expansion and clonal deletion (29). Clonal
expansion is believed to be regulated by mitogenic cytokines, especially IL-2. In chain (
c)1 is the genetic defect in X-linked severe combined immunodeficiency (XSCID) (1) and is a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (2). In
humans with XSCID, the lack of functional
c results in
profoundly diminished T cell development and an absence
of natural killer (NK) cells. In contrast, B cell numbers are
normal, although the B cells are nonfunctional with concomitant hypogammaglobulinemia. Mice in which the
c
gene has been targeted by homologous recombination exhibit a related, but somewhat different phenotype (6).
Like humans with XSCID, they have hypoplastic thymuses
and lack NK cells. Unlike humans with XSCID, conventional B cells are greatly diminished in the bone marrow
and the spleens of both young and adult
c-deficient mice,
likely reflecting the key role of IL-7 as a pre-B cell growth
factor in mice, but not in humans (5). In addition, these
mice lacked visible gut-associated lymphoid tissue and peripheral lymph nodes were essentially absent. Although
splenic T cells are diminished in number at 3 wk of age, CD4+ T cells, but not CD8+ T cells, dramatically accumulate in the spleen in an age-dependent manner. Moreover,
the finding of CD4+ T cell infiltrates, particularly in the gut
in association with colitis, suggested that the CD4+ T cells
might be activated and involved in mediating the pathological changes found in these mice. Given that the mice are
compromised in their ability to respond to
c-dependent
cytokines including IL-2, IL-4, and IL-7 (6), this accumulation of CD4+ T cells was unexpected. We now have
performed studies to characterize further these CD4+ T cells
to understand better the role of
c in development of the
immune response in vivo.
c-deficient mice (7) used in
these studies were back-crossed to either C57BL/6 (B6) (H-2b/b) or
BALB/c mice (H-2d/d) (Jackson Laboratory, Bar Harbor, ME) for
three/four generations. Since the
c gene is localized on X chromosome, DO10 TCR transgenic male mice (H-2d/d), specific for
ovalbumin (9), were mated with BALB/c
c+/
heterozygous female mice. These matings yielded four genotypes of male mice
(DO10
c+/Y, DO10+
c+/Y, DO10
c
/Y, and DO10+
c
/Y).
Similarly, AND TCR transgenic male mice (H-2b/b), specific for
cytochrome c (10, 11), were mated with B6
c+/
female mice.
Mice were housed in microisolator cages under pathogen-free conditions. The mice were genotyped by PCR of tail DNA using
the following primer pairs: to detect the wild-type
c gene,
5
-CTTTATTGATAACGATCTATCCCTCACCC-3
and 5
CTCCACTCTGCAGAGTCTATGGAATCC-3
; to detect the
c knockout gene, 5
-GCTGACAGCCGGAACACGGCGG-3
and 5
-GTGCAATCCATCTTGTTCAATGGCCG-3
; to detect
the DO10 transgene, 5
-CAGGAGGGATCCAGTGCCAGC-3
and 5
-TGGCTCTACAGTGAGTTTGGT-3
; to detect the
AND transgene, 5
-GACTTGGAGATTGCCAACCCATATCTAAGT-3
and 5
-TGAGCCGAAGGTGTAGTCGGAGTTTGCATT-3
.
11 FITC (RR8-1), and anti-Qa2a biotin (1-9-9)
(12). KJ1-26 mAb, anti-idiotype for DO10 TCR (13) (provided
by Dr. B.J. Fowlkes), was purified from supernatants of hybridoma cells using protein G columns (Pharmacia, Uppsala, Sweden) and conjugated to FITC.
c-deficient
mice as compared with wild type mice, single cell suspensions
from spleen were treated with ACK lysing buffer to remove red
cells, stained with PE-conjugated anti-CD8 mAb (53-6.7, PharMingen) and PE-conjugated anti-erythroid mAb (TER-119, PharMingen) and anti-CD16/CD32 mAb (Fc
RIII/II receptor, 2.4G2,
PharMingen), washed twice with 1 × PBS containing 1% BSA (PBS, 1% BSA), and applied to T cell enrichment columns (R&D
Systems). The resulting cells from both wild-type and
c-deficient mice were 88-95% pure CD4+ T cells (average 91.8%) as
determined by flow cytometry.
c-deficient or littermate control mice were injected i.p. with 750 µg of BrdU (Sigma) in 250 µl of PBS at time
0 and again at 4 h. Control mice were injected with PBS. 16 h after the second injection, thymocytes and purified splenic CD4+
T cells were fixed with 70% ethanol for 30 min on ice, denatured with 2 N HCl containing 0.5% Triton X-100 for 30 min to produce single-stranded DNA, and neutralized with 0.1 M sodium
tetraborate, pH 9.0 (Sigma). Cells were resuspended in PBS, 1%
BSA containing 0.5% Tween-20, stained with anti-BrdU FITC
(Becton Dickinson) for 30 min at 25°C, washed once with PBS,
1% BSA containing 0.5% Tween-20, resuspended in PBS, 1%
BSA, and analyzed on a FACSort®.
c-deficient mice and 6.32%/d for wild-type mice. Assuming that no
death occurs and that there is no new migration of cells from thymus to periphery (neither assumption is likely to be correct), we
can estimate the number of cells that might exist after 63 d of
growth (from 3-12 wk of age) as 1.3 × 106 × (1.2143)63 = 2.66 × 1011 CD4+ splenic T cells instead of 5.28 × 107 cells for
c-deficient mice and 9.1 × 106 × (1.0632)63 = 4.31 × 108 cells instead
of 1.67 × 107 for wild-type mice. Although not accurate, these
calculations suggest that survival rates for the cells differ between
the wild-type and
c-deficient mice. Again, assuming no new migration from the thymus to periphery and a constant replication
rate, the survival rate/day can be estimated as ~87% for
c-deficient mice and 95% for wild-type mice, suggesting that cells from
the
c-deficient mice had an increased rate of cell death (see below).
Fig. 1.
Activated CD4+ T cells accumulate in an age-dependent
fashion in c-deficient mice and exhibit increased BrdU uptake. (A) The number of splenic CD4+ T cells in
c-deficient (closed diamonds) and wildtype (open squares) mice was calculated as the product of the number of
splenocytes and the percent of CD4+ T cells, based on staining with antiCD4 Cy-Chrome and analysis on a FACSort® (Becton Dickinson) using
Lysis II software. Shown are mean ± SEM (n = 4-9 at each timepoint).
(B) Size of CD4+ T cells. Splenocytes from 6-wk-old mice were stained
with anti-CD4 Cy-Chrome and PI, and cell size of viable cells was assessed by forward light scatter (FSC); data are shown on a linear scale. (C)
Activation markers expressed on CD4+ T cells. Splenocytes were stained
with anti-CD4 Cy-Chrome and either PE conjugated anti-CD62L (L-selectin), anti-CD69, or anti-CD25. Data were gated on CD4+ cells and displayed on a log scale. The data shown are representative of six experiments. (D) BrdU uptake of thymocytes and splenic CD4+ T cells. Mice
were injected twice with BrdU (see Material and Methods). 16 h after the
second injection, purified splenic CD4+ T cells and thymocytes were
stained with anti-BrdU FITC according to the Becton Dickinson protocol and analyzed on a FACSort®. Histograms show BrdU staining (log
scale); the numbers above the gate represent the mean percentage of
BrdU-positive cells from four experiments. When mice were injected
with PBS instead of BrdU, less than 0.1% of thymocytes and splenic
CD4+ T cells were stained with anti-BrdU FITC (data not shown).
[View Larger Version of this Image (23K GIF file)]
c-deficient mice have hypoplastic thymi and lack NK cells (5). However, peripheral T cells are generally not found in humans with XSCID
(4), whereas peripheral CD4+ T cells are found in the
cdeficient mice (5). Although these cells are diminished in
numbers in
c-deficient mice at 3 wk of age (6, 7), in most
mice there is a dramatic age-dependent accumulation of
these cells (7) (Fig. 1 A), even though the thymus remains
small (7). Analysis using a series of different TCR V
-specific mAbs (V
2, V
3, V
4, V
5.1,5.2, V
6, V
8.1,8.2, V
10, and V
11) indicated that the CD4+ T cell accumulation was polyclonal (unpublished data). Interestingly, the
splenic CD4+ T cells in
c-deficient mice were increased
in size (Fig. 1 B). The large size of the cells, coupled with
the finding of CD4+ T cell infiltrates, particularly in the
gut in association with colitis (5, 7), suggested that the
CD4+ T cells might be activated and involved in mediating
the pathological changes found in these mice. Therefore,
we investigated the basis for the CD4+ T cell expansion.
c-deficient and wild type mice was that the cells from the
c-deficient mice exhibited a CD62LlowCD69high phenotype typical of memory/activated cells, whereas those from wild-type mice exhibited a CD62LhighCD69low phenotype
typical of naive cells (Fig. 1 C) (15). Interestingly, CD4+
T cells in mice deficient in IL-2 (16, 17), IL-2R
(18), and
IL-2R
(19) also exhibited activated phenotypes. Expression of CD49d (VLA-4
chain) and CD44 was slightly increased in
c-deficient CD4+ T cells (data not shown),
whereas no significant difference was seen in expression of
IL-2R
(CD25) (Fig. 1 C), or in CD40L, CD28, CTLA-4,
or in Fas (data not shown). The lack of increase in these activation markers suggested that these cells were partially
rather than fully activated and that
c-dependent signals are
required for completing T cell activation. In contrast with
the different phenotypes in splenic T cells, CD4+CD8
thymocytes from
c-deficient mice and wild type mice expressed similar levels of CD62L, CD69, and Qa-2a (data
not shown).
c expression was selectively reconstituted in T cells (transgenic mice in which
c is under control of the CD3
enhancer and promoter)
but not in mice in which
c expression was selectively reconstituted in B cells (transgenic mice in which
c is under
control of the mb-1 promoter and Eµ heavy chain enhancer) (data not shown). A similar activated phenotype in
Jak3-deficient mice was corrected when these mice were
reconstituted with Jak3 under control of the Lck promoter
(19a), a finding consistent with the similar phenotypes in
mice and humans deficient in either
c or Jak3 and with
the conclusion that Jak3 is the major mediator of
c-dependent signals (4, 5).
c-deficient splenic CD4+ T cells reflected active proliferation and found that bromodeoxyuridine (BrdU) uptake in these cells was greater than in
wild-type mice (Fig. 1 D, left). In contrast,
c-deficient thymocytes exhibited lower BrdU uptake than wild-type thymocytes (Fig. 1 D, right). Thus, there was more active cell
division in the spleens than in the thymi of adult
c-deficient mice, correlating with the activated phenotype of the
c-deficient splenocytes. It is important to recognize that
because these mice were not subjected to thymectomy, some splenic BrdU uptake could have occurred in thymocytes before CD4+ T cell migration to the periphery;
however, because the thymi in
c-deficient mice are very
small (0.5-5% of littermate controls) and the BrdU uptake
in thymocytes in these mice was lower than in wild-type
mice, it is likely that the majority of BrdU uptake occurred
in the periphery.
c-deficient mice
had an apparent replication rate much greater than wildtype mice (21.43%/d for
c-deficient mice and 6.32%/d for
wild-type mice; see Materials and Methods). Such an increased rate for the
c-deficient mice should have yielded a
much greater accumulation of cells than actually seen (Fig.
1 A), suggesting that CD4+ T cells from the
c-deficient
mice had an increased rate of cell death (see Materials and
Methods). Indeed, when cultured without stimulation, purified splenic CD4+ T cells from
c-deficient mice exhibited a higher level of death than cells from wild-type mice,
with the greatest difference occurring in the first 8 h (Fig.
2 A). Increased apoptosis was confirmed both by TUNEL
analysis (data not shown) and by staining with annexin V
(Fig. 2 B), which allows the detection of apoptotic cells at an early stage because of its selective affinity for phospholipids, especially phosphatidylserine (PS), which is exposed
on the cell surface in apoptotic cells, while normally PS is
restricted to the inner cell membrane (20). The rapid
increase in death seen at 8 h in
c-deficient mice (Fig. 2 A)
presumably is accounted for by the large number of cells
that stained with annexin V at time 0 (Fig. 2 B), suggesting
that splenic CD4+ T cells of
c-deficient mice may be
primed to die by apoptosis in vivo. Bcl-2 levels correlate
with the survival of lymphoid cells (23, 24) and are induced
by
c-dependent cytokines that protect against apoptosis
(25). Therefore, it was interesting that
c-deficient
splenic CD4+ T cells expressed very low levels of Bcl-2
(Fig. 2 C) (including both CD62Lhigh and CD62Llow subpopulations; data not shown), suggesting that
c is required for maintaining the normal levels of Bcl-2 expression in
both activated and naive peripheral CD4+ T cells.
Fig. 2.
Augmented cell death and diminished Bcl-2 expression in
c-deficient CD4+ T cells. (A) Spontaneous cell death in vitro. Purified
splenic CD4+ T cells from 6-wk-old mice were cultured in RPMI medium containing 10% charcoal-treated fetal bovine serum (Cocalico Biological, Inc.) for 8-48 h, harvested, and cell survival determined by staining with 5 µg/ml of PI, followed by analysis using a FACSort®. Data for
each mouse were analyzed in duplicate. Shown are the means ± SD of
three mice in each group. At time 0, approximately 98% of cells from
wild-type and 93% of cells from
c-deficient mice were PI negative. (B)
Annexin V binding to CD4+ T cells. Splenocytes from 6-wk-old mice
were stained with anti-CD4 Cy-Chrome and then with annexin V FITC.
Annexin V binding (log scale) is shown for CD4+ PI
cells. (C) Bcl-2 expression in splenic CD4+ T cells was analyzed approximately as previously described (14). The histograms show profiles for staining with anti-Bcl-2
(closed histograms) and control IgG (open histograms) in wild-type and
c-deficient mice (log scale).
[View Larger Version of this Image (22K GIF file)]
c-deficient mice were already activated without exogenous stimulation. In the first model,
the activated phenotype does not require TCR stimulation
(TCR-independent activation model); instead,
c-dependent signals are required for keeping mature CD4+ T cells
in a naive stage and, that without such signals, these cells
are activated nonspecifically. In the second model, peripheral CD4+ T cells in
c-deficient mice respond to self- or
environmental antigens (TCR-dependent activation model).
We investigated these possibilities using mice expressing
MHC class II-restricted transgenic TCRs specific for ovalbumin (DO10 mice) or cytochrome c (AND mice). In these experiments, the mice were not exposed to the specific antigens.
In DO10 male mice deficient in
c (DO10+
c
/Y mice),
far fewer splenocytes were found than in DO10
c
/Y
mice (6.4 × 106 versus 79 × 106 cells/spleen) (Fig. 3 A).
Analogous differences were also seen between AND+
c
/Y
and AND
c
/Y mice (2.7 × 106 versus 50 × 106 cells/
spleen). These data suggested that endogenous TCR expression might be important in regulating CD4+ T cell accumulation. Using the DO10+
c
/Y mice, we compared
the relative frequency of memory (CD62Llow) and naive
(CD62Lhigh) phenotypes in splenic CD4+ T cells expressing
transgenic (KJ1-26+) versus endogenous (KJ1-26
) TCRs.
Approximately 70% of CD4+ T cells from DO10+
c
/Y
mice express the transgenic TCR (Fig. 3 B, fourth histogram), analogous to DO10+
c+/Y mice (second histogram).
Surprisingly, although relatively high numbers of KJ126+CD4+ T cells in DO10+
c
/Y mice were CD62Lhigh
(naive phenotype), similar to the finding for DO10+
c+/Y
mice (84.9% versus 84.6%) (Fig. 3 C, bottom), relatively few of the endogenous KJ1-26
CD4+ T cells in DO10+
c
/Y
mice were CD62Lhigh (10%), similar to the finding for
DO10
c
/Y mice (8.3%) (Fig. 3 C, top). Thus, the CD62Llow
phenotype correlated with the presence of endogenous
TCRs. Analogous results were found in AND TCR transgenic
c-deficient (AND+
c
/Y) mice. In AND+
c
/Y mice,
93% of transgenic TCR+ (V
11+) CD4+ T cells were
CD62Lhigh, similar to AND+
c+/Y mice (94%). These results support the TCR-dependent activation model and
suggested that TCR(s) expressed on expanding peripheral CD4+ T cells in
c-deficient mice are specific for self-antigen or environmental antigens. The finding that over 80%
of adult
c-deficient mice (C57BL/6 background) develop
an inflammatory bowel disease (data not shown) is consistent with the possibility of self-reactive CD4+ T cells in
these mice.
Fig. 3.
Activation of splenic
CD4+ T cells in c-deficient mice
is TCR dependent.
c-deficient
mice were back-crossed to
BALB/c mice (H-2d/d, Jackson
Laboratory) for three generations. Since the
c gene is located
on the X chromosome, DO10
TCR transgenic male mice (H-2d/d)
were mated with BALB/c
c+/
heterozygous female mice (H-2d/d).
These matings yielded four
genotypes (assessed by PCR of
tail DNA) of male mice
(DO10
c+/Y, DO10+
c+/Y,
DO10
c
/Y, and DO10+
c
/Y)
(A). In (A), also shown are the
total number and CD4/CD8
staining of splenocytes from
5-wk-old mice. As previously
reported (32), DO10+ male
mice expressing wild-type
c
(DO10+
c+/Y mice) exhibit an
increased CD4/CD8 ratio as
compared with DO10
c+/Y mice
(first two panels), but consistent
with the increased CD4/CD8 ratios previously observed in
c-deficient mice (6, 7), DO10+
c
/Y mice exhibited an
even greater CD4/CD8 ratio
(fourth panel). Results are representative of three separate experiments. (B) Histograms of KJ1-26
mAb (anti-idiotype TCR) staining (gated on CD4+ T cells). (C)
Splenocytes were stained by
three color flow cytometric analysis for expression of KJ1-26,
CD62L, and CD4. Data were
gated on KJ1-26
CD4+ (endogenous TCR) (top histogram)
or KJ1-26+CD4+ (transgenic
TCR) (bottom histogram) T cells.
The histograms show profiles
for CD62L expression (log
scale); the percent of CD62Lhigh
cells is indicated.
[View Larger Version of this Image (38K GIF file)]
c-deficient mice, it was therefore
surprising that although splenic T cells exhibit diminished
in vitro responsiveness to mitogens (5), there was TCRdependent activation and a marked expansion of CD4+
T cells in vivo. In addition to this in vivo proliferation, we also detected an augmented rate of apoptosis in
c-deficient
CD4+ T cells, a finding consistent with the known action
of
c-dependent cytokines as survival factors (25); nevertheless, it was evident that the overall rate of death was
inadequate for the degree of proliferation. Although the
CD4+ T cells were activated, they expressed only low levels of CD25, CTLA-4, and Fas. Therefore, it is conceivable
that
c-dependent signals are required to achieve induction
of pathways that are required to negatively regulate immune responses. Thus,
c-dependent signals are essential
components of a carefully controlled homeostatic mechanism that regulates the fate of activated cells.
Address correspondence to Warren J. Leonard, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, Building 10, Room 7N244, 9000 Rockville Pike, Bethesda, MD 20892.
Received for publication 6 August 1996
H. Nakajima was supported in part by Japan Society for the Promotion of Science and The Naito Foundation.We thank D.Y. Loh for DO10 mice and the sequences of primers for detecting the DO10 transgene, S.M. Hedrick for AND mice, L. Berg for the sequences of primers for detecting the AND transgene, T. Tran for assistance with purification and labeling of KJ1-26 mAb, M. Murakami for valuable discussions, and R. Germain for critical comments.
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