Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 8187–8192, July 1998
HD mice: A novel mouse mutant with a specific defect in the
generation of CD4?T cells
VIBHUTI P. DAVE, DAVID ALLMAN, ROBERT KEEFE, RICHARD R. HARDY, AND DIETMAR J. KAPPES*
Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111
Communicated by Robert Palese Perry, Fox Chase Cancer Center, Philadelphia, PA, May 11, 1998 (received for review March 15, 1998)
in mice, which we term HD for ‘‘helper T cell deficient.’’ This
mouse is distinguished by the virtual absence of peripheral T
cells of the CD4?8?major histocompatibility complex (MHC)
class II-restricted T helper subset due to a specific block in
thymic development. The developmental defect is selective for
CD4?8?cells; the maturation of CD4?8?and ?? T cells is
normal. The autosomal recessive mutation underlying the HD
phenotype is unrelated to MHC class II, since it segregates
independently of the MHC class II locus. Moreover, the HD
phenotype is not caused by a defect of the CD4 gene. Bone
marrow transfer experiments demonstrate that the defect is
intrinsic to cells of the hematopoietic lineage, i.e., most likely
to developing thymocytes themselves. The frequency of
CD4?8lowintermediate cells is markedly increased in HD
mice, suggesting that class II-restricted thymocytes are ar-
rested at this stage. This is the first genetic defect of its kind
to be described in the mouse and may prove highly informative
in understanding the molecular pathways underlying lineage
We have identified a spontaneous mutation
Developing ?? T cells mature through three major stages
characterized by differential expression of the coreceptor
molecules CD4 and CD8. Initially, thymocytes express neither
coreceptor (CD4?CD8?: double negative or DN), then up-
regulate both molecules (CD4?CD8?: double positive or DP),
and finally selectively down-modulate CD4 or CD8
(CD4?CD8?or CD4?CD8?: single positive or SP). The vast
that complete ?? T cell antigen receptor (TCR) is first
expressed at the cell surface and thymocytes undergo selection
on the basis of their antigen receptor specificity. Thymocytes
are selected to undergo alternate developmental fates depend-
ing largely on the relative affinities of their clonotypic TCRs
for thymic-selecting ligands. Thymocytes interacting too
strongly with thymic ligands undergo negative selection, i.e.,
suicide by apoptosis, whereas those interacting with interme-
diate affinity undergo positive selection, i.e., maturation to the
Thymocytes that undergo positive selection mature into
either of two cell types, SP CD4?helper T cells or SP CD8?
cytotoxic T cells. The decision to enter one or the other
developmental pathway is referred to as lineage commitment.
Mature cells of the CD4 and CD8 lineages express TCRs
restricted to major histocompatibility complex (MHC) class II
or class I ligands, respectively. Several alternative models have
been proposed to explain how this correlation is achieved: In
the instructive model, TCR specificity directs coreceptor
down-modulation, i.e., when a TCR recognizes a class I ligand,
a specific signal is sent to turn off CD4 expression and vice
versa (1). The stochastic model, on the other hand, postulates
that positive selection leads to random coreceptor down-
modulation; thymocytes that down-modulate the wrong core-
ceptor are unable to respond to subsequent TCR-mediated
stimuli and die (2, 3). In the asymmetric model, only differ-
entiation to the CD8 lineage requires an instructive signal,
whereas development to the CD4 lineage occurs by default (4).
The signal strength model, finally, proposes that lineage com-
mitment is determined by the overall intensity of signaling
through the TCR complex and appropriate coreceptor, such
that stronger and weaker signals promote development to the
CD4 and CD8 lineages, respectively (5).
Recent studies have shown that progression from the DP to
the SP stage is marked by passage through a series of transi-
tional stages characterized by intermediate levels of CD4 and
CD8 expression (4, 6–8). An important transitional stage in
this progression is the CD4?8lowstage, originally identified in
both normal and class II-deficient mice (9, 10). Differentiation
to the CD4?8lowstage indicates that thymocytes have received
a primary positive selection signal (4, 6–8). CD4?8lowcells in
normal mice include progenitors of both the CD4 and CD8
lineages (4, 6–8) and can either give rise directly to CD4?8?
thymocytes or, via two further intermediate stages (CD4low8low
and CD4low8?), to CD4?8?thymocytes (4, 8). There is com-
pelling evidence that the DP to SP transition is a multistep
process requiring repeated or continual stimuli (11, 12). In
particular, it has been proposed that CD4?8lowcells must
receive a distinct secondary selective signal to undergo final
maturation to the SP stage (2).
The differences in signaling processes that mediate alternate
maturation to either CD4 or CD8 lineages have yet to be
clarified. Although there is abundant evidence that CD4 and
CD8 molecules are intimately involved in signal transduction
and influence lineage commitment (13–20), they are not
essential for this process (21, 22). There is some evidence that
additional receptors, specifically homologues of the Drosophila
cell fate regulator Notch, can influence lineage choice of
developing thymocytes (23). The functionally important out-
come of the lineage commitment process is the initiation of
distinct patterns of gene expression consistent with the thy-
mocyte’s specificity toward class I or II MHC. The silencing of
one or the other coreceptor molecule represents the most
obvious example of this divergence in gene expression. In the
case of the CD4 gene, a cis-acting negative regulatory element
has been defined that is required for silencing of CD4 in cells
committed to the CD8 lineage (24). In the case of the CD8?
expression of CD8 only in mature SP CD8?not DP cells (25,
26). The transcription factors that bind to these regulatory
elements remain unknown.
The mutant helper T cell-deficient (HD) mouse that we
describe here is characterized by a striking deficiency in the
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© 1998 by The National Academy of Sciences 0027-8424?98?958187-6$2.00?0
PNAS is available online at http:??www.pnas.org.
Abbreviations: MHC, major histocompatibility complex; DN, double
(CD4?8?or CD4?8?); TCR, T cell antigen receptor; PBL, peripheral
*To whom reprint requests should be addressed. e-mail: dj_kappes@
generation of peripheral CD4?T cells. No mouse model with
a similar phenotype has been described previously, with the
exception of induced mutants lacking expression of CD4 or
MHC class II products (10, 27, 28). We provide evidence that
the HD phenotype is not caused by mutations of the CD4 or
class II loci. Instead, we speculate that HD mice may bear a
defect in a critical signaling pathway initiated either by the
TCR/coreceptor complex or some other receptor expressed on
thymocytes, e.g., a Notch family member or a cytokine recep-
tor. The eventual identification of the specific gene defect in
HD mice should provide significant insights into the mecha-
nisms underlying lineage commitment.
MATERIALS AND METHODS
Animals. The HD mouse arose as a spontaneous mutant in
our animal colony at the Fox Chase Cancer Center. HD mice
are derived from an intercross between CD3?-deficient and
human CD3? transgenic mice which have been described
previously (29). RAG2-deficient mice were provided by F. Alt
(Howard Hughes Medical Institute, Children’s Hospital, Bos-
ton, MA). I-A??/?(MHC class II-deficient) and CD4?/?mice
were obtained, respectively, from Taconic Farms and The
Jackson Laboratory. C57BL/6 and BALB/c mice were ob-
tained from the Fox Chase Cancer Center Laboratory Animal
Facility. Progeny of (HD ? BALB/c) intercrosses were H-2
typed by staining of peripheral blood lymphocytes (PBLs) with
specific antibodies against MHC class I products Kband Kd.
Cell Preparation and Flow Cytometry. PBLs were obtained
by retro-orbital bleeding and purified by density gradient
centrifugation over Lympholyte M (Cedarlane, ON). PBLs
(105) or thymocytes were incubated with the relevant combi-
nations of fluorescently labeled antibodies for 15 min at 4°C
and analyzed using a Becton Dickinson FACStar Plus or
FACScan. Fluorescent-labeled antibodies against CD4, CD8,
??TCR, ??TCR, I-Ab, H-2 Kb, H-2 Kd, CD69, HSA, and
Mel14 were obtained from PharMingen.
Radiation Chimeras. Bone marrow was isolated from the
rear leg bones of a HD mouse and depleted of ??TCR?cells
by fluorescence-activated cell sorting. Five to 10 ? 105T
cell-depleted bone marrow cells in 0.2 ml of RPMI 1640 were
injected i.v. into 10 RAG2-deficient H-2brecipients that had
been sublethally irradiated (900 rads) 24 hr previously. Pe-
ripheral blood and thymus samples were obtained 5 wk after
bone marrow transfer. Seven of 10 animals showed reconsti-
tution of both B and T cell compartments.
HD Mice Lack Peripheral SP CD4?T Cells. We have
identified a cohort of mice in our colony that essentially lack
peripheral CD4?T cells and instead possess elevated numbers
designated as HD mice. Although SP CD4?T cells are lacking
in HD mice, the total number of peripheral ??TCR?cells is
roughly the same as for wild-type (wt) littermates [i.e., in
peripheral blood 24.5 ? 5.4% (n ? 6) for HD and 26.7 ? 7.9%
(n ? 9) for wt littermates]. The HD phenotype is highly
consistent (no animals display intermediate reductions of CD4
T cells) and does not undergo significant variation with age up
to 8 mo (the oldest animals examined). HD mice are healthy
under specific pathogen-free conditions.
The HD Defect Is Due to an Autosomal Recessive Mutation.
To determine whether the HD phenotype has a genetic basis
and, if so, whether it is due to a single dominant or recessive
mutation, we have backcrossed HD mice either to other HD
mice or to wt C57BL/6, animals. All (HD ? HD) progeny bear
the HD phenotype, consistent with a heritable defect, whereas
all (HD ? wt) progeny are normal in phenotype, i.e., possess
mutation is not dominant. Finally, among F2s, i.e., progeny of
an (HD ? wt)F1 intercross, 5 of 24 animals show the HD
phenotype (21%), in close agreement with the expected fre-
quency of 6 of 24 (25%) predicted for a single recessive gene
(Table 1). Interestingly, (HD ? HD) intercrosses consistently
produce small litters of two to four pups. This is also true of
intercrosses between wt males and HD females, but not HD
PBLs from a HD mouse or a normal littermate were stained with
antibodies specific for CD4, CD8, and ??TCR and then analyzed by
HD mice specifically lack peripheral CD4?T lymphocytes.
II products and is genetically unlinked to the class II locus. (a) PBLs
from HD mice, normal littermates, and MHC class II-deficient mice
were stained with antibody specific for the MHC class II I-Abproduct.
(b) F2 progeny from the intercross between HD and BALB/c mice
were stained with antibodies against CD4 and CD8. Data are shown
for one mouse each of the HD phenotype that typed as H-2 b/b, b/d,
HD phenotype is not caused by the absence of MHC class
Table 1. Transmission of HD phenotype
(HD ? HD)*
(HD ? C57BL?6)†F1
*A total of 14 progeny were obtained from four separate matings.
†All 12 progeny are from a single mating.
‡A total of 24 progeny were obtained from three separate matings
between 6 of the 12 F1progeny.
8188 Immunology: Dave et al.Proc. Natl. Acad. Sci. USA 95 (1998)
males and wt females, suggesting that female fertility is im-
paired in HD mice.
Originally, seven HD mice were discovered during routine
screening by PBL analysis of 34 mixed F1 and F2 progeny
component of the TCR/CD3 complex and a transgenic strain
any other crosses between individuals of these two strains,
indicating that the HD phenotype is due to a spontaneous
mutation in one of the parents in this particular mating. In
backcrosses with wt mice, the HD phenotype segregates
independently of both knockout and transgenic loci (data not
The HD Phenotype Is Not Due to a Mutation in MHC Class
II. The HD phenotype is superficially similar to that of
engineered mutants lacking expression of class II MHC.
Hence, it was important to exclude a structural defect in class
HD phenotype. All original HD animals are homozygous for
the H-2bhaplotype and would be expected to express a single
class II product, I-Ab. Indeed, flow cytometric analysis of PBLs
from HD mice shows normal expression of the I-Abmolecule
on peripheral blood B cells and macrophages (Fig. 2a). The
possibility of genetic linkage between the HD phenotype and
the MHC region was directly tested by backcrossing HD mice
to BALB/c mice, which express a different MHC haplotype,
H-2d. If the HD phenotype is unlinked to the MHC, it would
segregate independently of the H-2bhaplotype, i.e., F2mice
would arise bearing the HD phenotype, as defined by the lack
of peripheral SP CD4?T cells, on both heterozygous b/d and
homozygous d/d H-2 backgrounds. In fact, several such prog-
eny were identified (Fig. 2b). Thus, we can exclude mutations
in the coding regions as well as in cis-acting regulatory
elements of class II genes as the cause of the HD phenotype.
HD Mice Are Not CD4 Deficient. To determine whether the
deficit in peripheral CD4?T cells in HD mice reflects inac-
tivation of the CD4 gene, thymocytes were isolated from 6- to
8-wk-old mice previously identified as HD by PBL analysis and
compared with their normal littermates. HD mice displayed
normal thymic cellularity (1.5–2 ? 108). Most significantly,
CD4?CD8?(DP) thymocytes were present at a normal fre-
quency (80–85%), demonstrating that the HD phenotype is
not caused by a null mutation at the CD4 locus (Fig. 3 a and
b). TCR and CD4 levels on DP thymocytes have been shown
to be regulated by the interaction between CD4 and class II
ligands, such that when this interaction is disrupted TCR and
CD4 levels increase (30, 31). In HD mice, both CD4 and TCR
levels on DP thymocytes are normal, indicating that the ability
of CD4 to interact with class II MHC is unimpaired (Fig. 3k:
note the difference in TCR levels between HD mice and class
II?/?mice, in which the interaction with CD4 is abrogated). In
this context, it should be noted that a small but reproducible
fraction of peripheral ??TCR?cells also express cell surface
CD4, mostly in combination with CD8 giving rise to a DP
phenotype (Fig. 4; note the absence of DP cells in the normal
II-deficient controls were stained with antibodies specific for CD4, CD8, ??TCR, and CD69 and analyzed by flow cytometry. Histograms represent
total thymocytes (a–c and k) or thymic subsets, as specified. In e–l, solid and dashed dark lines correspond to HD and wt mice, respectively, whereas
the solid light line in k represents class II?/?mice. In m and n, solid and dashed dark lines correspond to total thymocytes stained either with
anti-Bcl-2 and fluoresceinated goat anti-hamster fluorescein isothiocyanate (G?H–FITC) or only G?H–FITC, respectively, whereas the solid light
lines represent DP thymocytes stained with anti-Bcl-2 and G?H-FITC. Bcl-2highpopulations correspond to SP thymocytes. Thymic subset
designations are defined in d. Gates used to define CD4?8?, CD4?8low, and CD4?8?subsets are shown in a–c. Arrows point out key differences
between HD mice and wt or class II?/?controls as follows: In e and f, arrows identify the mature CD69?population that is present in wt but absent
in HD mice. In h, arrows highlight the 2-fold difference in TCR expression between CD4?8?cells from HD versus wt mice. In k, the arrow highlights
the higher TCR expression levels seen on DP thymocytes from class II?/?but not HD or wt mice.
Thymic development of CD4 lineage cells is abrogated in HD mice. Thymocytes from HD mice, normal littermates, or MHC class
Immunology: Dave et al. Proc. Natl. Acad. Sci. USA 95 (1998)8189
littermate control). In induced mutants lacking expression of
CD4, a distinct population of CD4?CD8?(DN) T cells arises
in the periphery at relatively high frequency (10–15% of total
presumed to belong to the CD4 lineage, although CD4 ex-
pression is blocked due to the induced structural mutation (28,
32). The fact that such cells do not arise in HD mice provides
further confirmation that the HD phenotype is not due to a
defect in CD4. Finally, we have crossed HD and CD4?/?mice
to test for genetic linkage. The fact that F1 progeny are
phenotypically normal, formally demonstrates that the HD
defect cannot lie in the CD4 protein or in cis-acting transcrip-
tional regulatory elements that control its expression (Fig. 4,
Thymic Development of CD4 Cells Is Specifically Abrogated
in HD Mice. Although generation of DP thymocytes was
normal in HD mice, progression to the SP stage was altered in
several respects (Fig. 3): (i) The ratio of SP CD8?to SP CD4?
(CD4?8?? CD4?8low) thymocytes is reversed from 1:3 to 2:1.
(ii) The great majority of SP CD4?thymocytes from HD mice
are actually immature CD4?8lowrather than CD4?8?cells
(Fig. 3 a and b; note that only 1.4% of HD thymocytes fall
within the CD4?8?gate). Furthermore, all SP CD4?thymo-
cytes from HD mice express the lower TCR surface levels that
are characteristic of CD4?8lowcells (4, 8) (Fig. 3 h and i). (iii)
CD4?SP thymocytes from HD mice are uniformly CD69?,
specifically lacking the CD69?subpopulation found in normal
mice (Fig. 3 e and f). The absence of CD69?SP CD4?
thymocytes in HD mice is highly significant, because these
comprise the most mature thymocytes that are ready to
emigrate to the periphery (33). In contrast, CD8?SP thymo-
cytes from HD mice show the typical biphasic CD69 staining
pattern (Fig. 3g), indicative of normal maturation. Develop-
ment of ?? T lineage cells, which diverge from the ?? lineage
at the DN stage, is also unaffected in HD mice, as judged by
the normal frequency with which they arise from the DN
compartment (Fig. 3l). Finally, since it has been shown that
expression of Bcl-2 at the DP stage can cause skewing to the
CD8 lineage (34), we investigated whether Bcl-2 is abnormally
up-regulated in DP thymocytes from HD mice. Intracellular
staining with specific antibody showed that there is no differ-
ence in Bcl-2 expression between DP thymocytes of normal
and HD mice (Fig. 3 m and n).
The HD Defect Is Intrinsic to the Hematopoietic Lineage. In
principle, the HD defect in CD4 development could reflect a
defect in gene expression intrinsic to the developing thymo-
cytes themselves or intrinsic to another thymic cell type that
provides an essential developmental signal. Radiation chime-
ras can be used to dissect the role of radioresistant epithelial
cells from that of radiosensitive hematopoietic cells, including
in CD4?/?mice. PBLs from a HD mouse, a wt littermate, or a
CD4-deficient mouse were stained with antibodies against CD4, CD8,
and ??TCR and analyzed by flow cytometry. Plots are gated to show
CD4:CD8 expression profiles for ??TCR?cells.
HD mice lack the ??TCR?peripheral T cell subset found
Sublethally irradiated RAG2?/?recipients were reconstituted with T
cell-depleted bone marrow from wt (a–d) or HD (e–h) mice. PBLs and
thymocytes were collected after 4 wk and stained with antibodies
against CD4, CD8, HSA, CD69, and L-selectin. Staining profiles are
shown for total PBLs (a and e), total thymocytes (b and f), and SP
CD4?thymocytes (c, d, g, and h). Insets in c, d, g, and h indicate the
position of the most mature SP CD4?subpopulation which is present
in wt but not in HD mice.
The HD defect maps to the hematopoietic compartment.
8190 Immunology: Dave et al.Proc. Natl. Acad. Sci. USA 95 (1998)
thymocytes, macrophages, and dendritic cells. Thus, we iso-
lated bone marrow cells from a HD mouse, depleted mature
T cells by fluorescence-activated cell sorting, and transferred
them into sublethally irradiated RAG2-deficient hosts. Five
weeks later the chimeras were typed for reconstitution of their
peripheral immune system. A total of seven chimeras with
reconstituted immune systems were identified. All of these
bore an abundance of peripheral CD8?T cells but virtually
lacked CD4?cells, thereby recapitulating the original HD
phenotype (Fig. 5e). As would be predicted, analysis of thy-
mocytes from these chimeras confirmed that the specific
defect in CD4 development had also been transferred (Fig. 5
was reversed and that SP CD4?cells were predominantly
replaced by CD4?8locells (Fig. 5f). A more detailed four-color
fluorescence-activated cell sorting analysis of these CD4?8lo
cells was carried out using a panel of antibodies specific for the
differentiation markers CD69, HSA, and L-selectin. In wt mice
fully mature cells that are competent to exit the thymus
comprise a distinct and readily detectable subset of SP CD4?
thymocytes that type as HSA?CD69?L-selectin?(Fig. 5,
populations in insets in c and d) (33). In contrast, in HD
chimeras these mature cells are essentially lacking (Fig. 5 g and
We present the initial phenotypic characterization of a novel
immunodeficient mouse mutant, the HD mouse, which bears
a striking defect in T lymphoid development. We show that
these animals are characterized by the inability to support
thymic development of mature SP CD4?thymocytes and a
virtual absence of peripheral CD4?T cells. Two types of
superficially similar to that of HD mice, i.e., engineered
mutants lacking MHC class II or CD4 (10, 27, 28). We provide
compelling evidence that neither of these genes is defective in
phenotype segregates independently of the MHC locus; (ii)
peripheral B cells and macrophages express normal surface
levels of class II products; and (iii) intrathymic interactions
between CD4 and class II appear unperturbed as evidenced by
normal TCR surface expression levels on DP thymocytes. A
defect in CD4 function or expression is excluded because: (i)
CD4 expression is normal on immature DP and intermediate
CD4?8lowthymocytes; (ii) peripheral T cells committed to the
CD4 lineage fail to arise in HD mice, although these cells can
develop in induced mutant mice in the complete absence of
CD4; and (iii) F1progeny of an intercross between HD and
CD4?/?mice are phenotypically normal. We conclude that the
HD mutation affects expression of a novel gene or, at least, a
gene whose critical role in lineage commitment is unappreci-
Using bone marrow transfer experiments, we demonstrate
that the HD defect is intrinsic to the hematopoietic lineage
rather than the epithelial lineage. Coreconstitution experi-
ments demonstrate that wt thymocytes mature efficiently to
the CD4 lineage, whereas HD-derived thymocytes present in
the same thymus give rise only to SP CD8 thymocytes (V.P.D.
and D.J.K., unpublished work). Hence, the HD defect does not
affect thymocytes that do not themselves carry the HD mu-
tation. It has previously been shown that positive selection and
lineage commitment of CD4?cells requires expression of class
II, and hence the capacity for antigen presentation, only on
cortical epithelial cells and specifically not on cells of hema-
topoietic origin (35). Thus, our data demonstrate that the HD
defect cannot lie in antigen presentation or costimulatory
function provided by epithelial cells. The most likely possibility
is that the defect is intrinsic to developing thymocytes.
We have shown that the lack of peripheral CD4?T cells in
HD mice reflects a specific block in thymic development of the
CD4 lineage. We postulate that the blockade does not affect
positive selection of class II-restricted thymocytes but rather a
transition from the DP to the SP stages is not a simple one-step
process, but rather involves progression through a number of
intermediate stages defined by different relative levels of
surface expression of the CD4 and CD8 molecules (4, 6–8; see
Fig. 6a). The CD4?8lowpopulation may represent a key stage
in this process, since it is the last intermediate common to both
the CD4 and CD8 maturation pathways. CD4?8lowcells have
of CD69 and TCR surface expression (4, 6–8). It has been
proposed that further maturation is not automatic for these
show a striking accumulation of these CD4?8lowcells, as
compared with wt and particularly class II-deficient mice (Fig.
6b). In class II-deficient mice, positive selection of class
II-restricted thymocytes is blocked and CD4?8lowcells should
way to the SP CD8?compartment. HD mice possess 6-fold
more CD4?8lowcells than class II-deficient mice. We postulate
thymocytes that have successfully undergone positive selection
but are blocked in a subsequent maturation stage (Fig. 6b).
However, since CD4?8lowcells can also give rise to SP CD8?
thymocytes, it is alternatively possible that in HD mice these
intermediate cells are all CD8 committed and that develop-
ment of class II-restricted thymocytes is actually arrested
earlier, e.g., at the DP stage. The increased number of
CD4?8lowcells could then reflect enhanced maturation to the
CD8 lineage, as also suggested by the increased number of
mature SP CD8?thymocytes. It has been reported that the
LEC rat strain also bears an unknown defect in generation of
SP CD4?thymocytes (36). The defect is likely to be different,
since LEC rats lack substantial numbers of CD4?8lowinter-
mediate thymocytes. Another difference is that LEC rats
actually bear substantial numbers of peripheral SP CD4?T
cells, particularly in the lymph nodes where they reach almost
normal levels by 12 mo of age (37).
The nature of the genetic defect in HD mice remains
unclear. However, we think that it is unlikely to affect proximal
components of the TCR-mediated signaling pathway, since
induced mutants of such components, i.e., CD3?, p56lck, and
ZAP-70, affect both lineages and, in the case of CD3? and
p56lck, already block the DN to DP transition (38–42). In
addition, we have preliminary data indicating that early events
in TCR/coreceptor-mediated signaling, i.e., phosphorylation
of CD3? and ZAP-70, are normal in HD mice. Rather we
suspect that the defect lies in a different process critical for
reflects a blockade of development at the CD4?8lowstage. (a)
Developmental routes postulated to be followed by class I- (light
arrows) and class II-restricted (dark arrows) thymocytes in wt mice. (b)
Model of HD defect as a blockade in CD4 development at the
Absence of mature SP CD4?thymocytes in HD mice
Immunology: Dave et al.Proc. Natl. Acad. Sci. USA 95 (1998) 8191
lineage commitment. The impairment in female fertility in HD Download full-text
mice, a feature not usually associated with immunodeficiency,
suggests that the effect of the mutation is not lymphoid
In conclusion, we have established that the HD mouse bears
a novel mutation of a gene that is specifically required for CD4
lineage commitment. The defect appears to be intrinsic to
developing thymocytes themselves. Given that lineage com-
mitment remains very poorly understood at the molecular
HD phenotype should provide important new insights into our
understanding of this phenomenon.
We thank D. L. Wiest for critical reading of the manuscript. This
work was supported by National Institutes of Health Grants CA74620
and AI34472, Institutional Grant CA06927 from the National Insti-
tutes of Health, and also by an appropriation from the Commonwealth
1. Robey, E. A., Fowlkes, B. J., Gordon, J. W., Kioussis, D., von
Boehmer, H., Ramsdell, F. & Axel, R. (1991) Cell 64, 99–107.
Chan, S. H., Cosgrove, D., Waltzinger, C., Benoist, C. & Mathis,
D. (1993) Cell 73, 225–236.
Davis, C. B., Killeen, N., Crooks, M. E. C., Raulet, D. & Littman,
D. R. (1993) Cell 73, 237–247.
Suzuki, H., Punt, J. A., Granger, L. G. & Singer, A. (1995)
Immunity 2, 413–425.
Matechak, E. O., Killeen, N., Hedrick, S. M. & Fowlkes, B. J.
(1996) Immunity 4, 337–347.
Kydd, R., Lundberg, K., Vremec, D., Harris, A. W. & Shortman,
K. (1995) J. Immunol. 155, 3806–3814.
Lundberg, K., Heath, W., Ko ¨ntgen, F., Carbone, F. R. & Short-
man, K. (1995) J. Exp. Med. 181, 1643–1651.
Lucas, B. & Germain, R. N. (1996) Immunity 5, 461–477.
Guidos, C. J., Danska, J. S., Fathman, C. G. & Weissman, I. L.
(1990) J. Exp. Med. 172, 835–845.
Cosgrove, D., Gray, D., Dierich, A., Kaufman, J., Lemeur, M.,
Benoist, C. & Mathis, D. (1991) Cell 66, 1051–1066.
Kisielow, P. & Miazek, A. (1995) J. Exp. Med. 181, 1975–1984.
Petrie, H. T., Strasser, A., Harris, A. W., Hugo, P. & Shortman,
K. (1993) J. Immunol. 151, 1273–1279.
Veillette A., Bookman, M. A., Horak, E. M. & Bolen, J. B. (1988)
Cell 55, 301–308.
Ravichandran, K. S. & Burakoff, S. J. (1994) J. Exp. Med. 179,
Killeen, N. & Littman, D. R. (1993) Nature (London) 364,
Fung-Leung, W. P., Louis, M. C., Limmer, A., Ohashi, P. S., Ngo,
K., Chen, L., Kawai, K., Lacy, E., Loh, D. Y. & Mak, T. W. (1993)
Eur. J. Immunol. 23, 2834–2840.
Norment, A. M., Forbush, K. A., Nguyen, N., Malissen, M. &
Perlmutter, R. M. (1997) J. Exp. Med. 185, 121–130.
Crooks, M. E. C. & Littman, D. R. (1994) Immunity 1, 277–285.
Nakayama, K., Nakayama, K., Negishi, I., Kuida, K., Louie,
M. C., Kanagawa, O., Nakauchi, H. & Loh, D. Y. (1994) Science
20. Itano, A., Cado, D., Chan, F. K. M. & Robey, E. (1994) Immunity
Sebzda, E., Choi, M., Fung-Leung, W. P., Mak, T. W. & Ohashi,
P. S. (1997) Immunity 6, 643–653.
Robey, E., Chang, D., Itano, I., Cado, D., Alezander, H., Lans,
D., Weinmaster, G. & Salmon, P. (1996) Cell 87, 483–492.
Sawada, S., Scarborough, J. D., Killeen, N. & Littman, D. R.
(1994) Cell 77, 917–929.
Ellmeier, W., Sunshine, M. J., Losos, K., Hatam, F. & Littman,
D. R. (1997) Immunity 7, 537–547.
Hostert, A., Tolaini, M., Roderick, K., Harker, N., Norton, T. &
Kioussis, D. (1997) Immunity 7, 525–536.
Grusby, M. J., Johnson, R. S., Papaioannou, V. E. & Glimcher,
L. H. (1991) Science 253, 1417–1420.
Rahemtulla, A., Fung-Leung, W. P., Schilham, M. W., Kuendig,
T. M., Samhara, S. R., Narendran, A., Arabian, A., Wakeham, A.,
Paige, C. J., Zinkernagel, R. M., Miller, R. G. & Mak, T. W.
(1991) Nature (London) 353, 180–184.
G., Lafaille, J., de la Hera, A., Tonegawa, S. & Kappes, D. J.
(1997) EMBO J. 16, 1360–1370.
Nakayama, T., June, C. H., Munitz, T. I., Sheard, M., McCarthy,
S. A., Sharrow, S. O., Samelson, L. E. & Singer, A. (1990) Science
McCarthy, S. A., Kruisbeek, A. M., Uppenkamp, I. K., Sharrow,
S. O. & Singer, A. (1988) Nature (London) 336, 76–79.
Killeen, N., Sawada, S. & Littman, D. R. (1993) EMBO J. 12,
Linette, G. P., Grusby, M. J., Hedrick, S. M., Hansen, T. H.,
Glimcher, L. H. & Korsmeyer, S. J. (1994) Immunity 1, 197–205.
Laufer, T. M., DeKoning, J., Markowitz, J. S., Lo, D. & Glim-
cher, L. H. (1996) Nature (London) 383, 81–85.
Agui, T., Oka, M., Yamada, T., Sakai, T., Izumi, K., Ishida, Y.,
Yamada, T., Natori, T., Izumi, K., Sakai, T., Agui, T. & Matsu-
moto, K. (1991) Immunogenetics 33, 216–219.
Molina, T. J., Kishihara, K., Siderovski, D. P., van Ewijk, W.,
Narendran, A., Timms, E., Wakeham, A., Paige, C. J., Hartmann,
K.-U., Veillette, A., Davidson, D. & Mak, T. W. (1992) Nature
(London) 357, 161–164.
Ohno, H., Aoe, T., Taki, S., Kitamura, D., Ishida, Y., Rajewsky,
K. & Saito, T. (1993) EMBO J. 12 4357–4366.
Malissen, M., Gillet, A., Rocha, B., Trucy, J., Vivier, E., Boyer,
C., Koentgen, F., Brun, N., Mazza, G., Spanopoulou, E., Guy-
Grand, D. & Malissen, B. (1993) EMBO J. 12, 4347–4355.
Negishi, I., Motoyama, N., Nakayama, K.-i., Nakayama, K.,
Senju, S., Hatakeyama, S., Zhang, Q., Chan, A. C. & Loh, D. H.
(1995) Nature (London) 376, 435–438.
Wiest, D. L., Ashe, J. M., Howcraft, K., Lee, H.-M., Kemper,
D. M., Negishi, I., Singer, A. & Abe, R. (1997) Immunity 6,
8192Immunology: Dave et al.Proc. Natl. Acad. Sci. USA 95 (1998)