By
¶
From the * Department of Medicine, Curriculum in Genetics and Molecular Biology, § Department of
Microbiology and Immunology,
Curriculum in Oral Biology, and ¶ Dental Research Center, University
of North Carolina, Chapel Hill, North Carolina 27599-7455; and ** Progenitor, Inc., Columbus,
Ohio 43212-1566
Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A null mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A-deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A-deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.
Ly-6A (a.k.a., TAP or Sca-1) is a glycosyl phosphatidylinositol (GPI)1-anchored molecule (1) expressed on most
peripheral lymphocytes, thymocytes, and hematopoietic
precursors including stem cells, as well as on nonhematopoietic fibroblasts, kidney epithelial cells, and osteoblasts from
the bone marrow (4). In the peripheral lymphoid organs,
Ly-6A expression is upregulated on activated lymphocytes
(4). Although a ligand of Ly-6A has not yet been determined, cross-linking Ly-6A by mAbs activates T and B lymphocytes in the presence of appropriate secondary signals. For
example, Ly-6A-specific mAbs induce B cells to proliferate
in the presence of IFN- The likelihood that Ly-6A plays a critical role in thymocyte
differentiation is suggested by its regulated expression during thymocyte development. Ly-6A is expressed on bone
marrow-derived prothymocytes which seed the thymic
cortex and are phenotypically differentiated from hematopoietic stem cells by Sca-2 expression (23, 24), but expression
is turned off at an early stage of CD3 To better understand the role of Ly-6A in hematopoietic
development and lymphocyte activation, we have employed the strategy of gene targeting in ES (embryonic
stem) cells to produce mice lacking Ly-6A expression. Ly-6A null mice are apparently normal and contain all hematopoietic lineages. Although the response by thymocytes
to Concanavalin A (Con A) stimulation is not significantly altered between wild-type and mutant littermates, the response by peripheral T cells to antigens and mitogens
which act through the TCR is significantly different. In
contrast to published Ly-6A antisense experiments, including those from our laboratory, splenic T cells derived from
Ly-6A Construction of Targeting Plasmid.
The pl93+ plasmid containing a 4.5-kb EcoRI fragment encoding the Ly-6A.2 chromosomal
gene has been described previously (27). The 1.7-kb fragment
containing exons 1-3 was removed using methylation-sensitive BclI after cycling the plasmid through the dam
Production of Targeted ES cells.
The 129/Ola ES cell line
E14TG2a (29) was cultured on irradiated (3000R) primary embryonic fibroblast feeder layers in DMEM supplemented with
15% (vol/vol) fetal bovine serum (lot tested) and 0.1 mM 2-mercaptoethanol. The fibroblast feeder cells were derived from d14.5
(129/Ola × C57Bl/6J)F2 embryos carrying a copy of a neoR
gene in the PCR Screening and Southern Blot Analysis.
After 48 h, colonies
growing in 24-well plates were trypsinized and half the cells were
removed for PCR analysis. ES cells were pelleted and resuspended in 50 µl lysis buffer (50 mM KCl, 10 mM Tris-HCl, pH
9, 2 mM MgCl2, 0.45% Triton X-100, 0.35% Tween 20). The
samples were boiled for 10 min, cooled, digested with 250 µg/ml
proteinase K for 1 h at 37°C, then boiled for 10 min to inactivate
proteinase K. The forward primer, NeoF (5 and IL-4 (11). Cross-linking Ly-6A molecules on T cells leads to an influx of intracellular
calcium and IL-2 production in the presence of accessory
cells. IL-2 production leads to an upregulation of IL-2R expression and subsequent proliferation via an IL-2-driven autocrine pathway (12, 13). Cross-linking of Ly-6A can also activate T cells to proliferate in the presence of PMA (14).
Several studies suggest that T cell activation by Ly-6A-specific antibodies is directly interrelated with the TCR signaling pathway. When Ly-6A expression is either downregulated by antisense DNA (15, 16) or ablated by mutation
(17), T cell lines cannot be activated via the TCR. Correlatively, loss of TCR expression leads to an inability to activate T cells by anti-Ly-6A crosslinking (18, 19). In addition, downregulation of Ly-6A expression by antisense also
results in downregulation of TCR
chain transcription and
p59fyn activity (16). In contrast, costimulation of T cells
with anti-Ly-6A and anti-CD3 cross-linking can induce downregulation of IL-2 production (20). Thus, the role of Ly-6A
in T lymphocyte activation is complex and unclear.
CD4
CD8
thymocyte
differentiation (5, 25). Ly-6A is reexpressed by mature single-positive medullary thymocytes and peripheral T cells (23,
25). When Bamezai et al. used a human CD2 enhancer- driven transgene to constitutively express Ly-6A at high levels during all stages of thymocyte development (26), thymocyte
development was arrested at the CD3
4
8
44+25+ stage, the
stage at which Ly-6A expression is normally terminated. However, despite the expression analysis and evidence for a
functional role in lymphocyte activation, the biological role
of Ly-6A is largely unknown.
/
mice proliferate more vigorously to antigen and
mitogens than wild-type littermates. Ly-6A mutant splenocytes proliferate at significantly higher levels to stimulation
with Con A, allogenic antigen, and anti-CD3 mAb, but
not when stimulated with PMA plus ionomycin when
compared to wild-type splenocytes. Furthermore, T cells
from mutant mice challenged in vivo with KLH antigen
proliferate at significantly higher levels in response to rechallenge with KLH in vitro compared to T cells from similarly
challenged wild-type littermates. In contrast, antibody levels to KLH in primed Ly-6A mutant mice are significantly
lower than antibody levels to KLH in KLH-primed wild-type littermates.
dcm
GM2163
Escherichia coli strain (E. coli; Genetic Stock Center, Department of
Biology, Yale University, New Haven, CT). Oligonucleotide adapters (XSB-L1, 5
-TACACCCTCCCTTCATAGGAGCT-3
;
XSB-U, 5
-CCTATGAAGGGAGGGTGTACTAG-3
) were used
to anneal XhoI/SalI and BclI ends. XSB-L1 was kinased, annealed to XSB-U, and ligated to the BclI digested pl93+. To construct the pl93neo+ plasmid the 1.1-kb XhoI/SalI fragment of
pMC1neo (Stratagene, La Jolla, CA), containing the neoR gene
driven by the HSV-tk promoter and a polyoma enhancer, was inserted into the adapter-generated XhoI/SalI overhang of pl93+.
In pl93neo+ the neoR transcriptional orientation is the same direction as Ly-6A. The HSV-tk cassette, obtained from Dr. M. Capecchi (Howard Hughes Medical Institute, University of
Utah, Salt Lake City, UT; reference 28), was inserted into the
SalI site in the multiple cloning site of pl93neo+ to generate the
targeting plasmid pLy6ASDI-1 (Fig. 1).
Fig. 1.
Gene-targeting of Ly-6A.2. (A) Strategy of Ly-6A gene targeting. A restriction map of the Ly-6A germ-line locus, the targeting construct, and the targeted locus are depicted. The four exons of Ly-6A
are boxed and labeled. The Ly-6A targeting construct (pLy6ASDI-1) was
prepared by cloning the 4.5-kb EcoRI fragment encoding the Ly-6A.2
gene into the EcoRI site of pBluescript. The BclI fragment encoding exons 1-3 was excised and replaced with the pMC1neo gene. The HSV-tk
gene was cloned into the SalI site of the multiple cloning site of the targeting construct. Probes D, G, and H are PCR fragments using generated
PCR primers corresponding to sequences upstream or downstream from the
targeting construct, and probe B is the pMC1neo insert. The restriction enzyme sites are designated as follows: BamHI (B), BclI (Bc), BglII (Bg),
EcoRI (R), EcoRV (V), HindIII (H), XbaI (X), and XmnI (Xmn). (B and
C) Detection of targeted and endogenous Ly-6A alleles by genomic
Southern blot analysis of EcoRV-digested DNA from a litter from a heterozygous cross using the 5 probe D (B) and the 3
probe H (C). The replacement of the 1.8-kb fragment encoding exons 1-3 with the NeoR reduces the size of the endogenous EcoRV band by 700 bp. The blot was
hybridized with probe H, stripped, and reprobed with probe D. The endogenous (5.2-kb) and targeted (4.5-kb) bands are marked with arrows.
[View Larger Version of this Image (32K GIF file)]
2-microglobulin locus (30) and were able to grow in
media containing G418. Rapidly growing cells were trypsinized and washed in ES media. Approximately 2 × 107 cells were resuspended in 0.4 ml of ES media and incubated for 10 min on ice
with linearized pLy6ASDI-1 at a concentration of 3 nM. Cells were
resuspended again immediately before electroporations. Electroporations were carried out using an electro cell manipulator (600; BTX,
Inc., San Diego, CA) at 160 V, 50 µF, and 360
. Fresh media
were added to electroporated samples which were allowed to recover on ice for 10 min. After electroporation, ES cells were
plated onto feeder layers in 100-mm tissue culture dishes. After
24 h, the media were replaced with media containing 200 µg/ml
G418 (Sigma Chemical Co., St. Louis, MO) in one dish or media
containing 200 µg/ml G418 and 1 µM gancyclovir (GANC) (a gift from Syntex, Palo Alto, CA) in the remaining dishes. After 10-14 d, colonies were picked and transferred to individual wells in 24-well plates seeded with feeder cells.
-ATGGCCTTCTTGACGAGTTCTTCTG-3
), is specific for the 3
end of the
pMC1neo gene of the targeting construct and the reverse primer,
Ly6Aonpa (5
-GGGAGAACAAAGGGTTTATTGGAC-3
), is
specific for the 3
end of the Ly-6A.2 gene and is not contained in the targeting construct (Fig. 1 A). PCR amplifications were carried out using 10 µl of lysates with 50 pmol of each primer and 2 U Taq polymerase (GIBCO BRL, Gaithersburg, MD) in
standard PCR buffer and standard amplification programs in a
Cetus thermocycler. An aliquot of each PCR reaction was fractionated on agarose gels. All PCR screening experiments used
E14TG2a cell lysates as a negative control, and all cell lysates
were tested for
-actin amplification to check for the quality of
DNA template.
phage clone and subsequent cloning
into the pCRII plasmid (Invitrogen, San Diego, CA). The forward primer, 5
-GCACTTGTTGTGGACCATGGTT-3
, and
the reverse primer, 5
-GTATAGGCAGGATCATCACGTG-3
,
were used to amplify the 199-bp probe G. The forward primer,
5
-GCTATCACCATCCACACATAAG-3
, and the reverse
primer, 5
-GGAGAGGTAATGTGGGTGGCT-3
, were used to amplify the 195-bp probe H. Probes were prepared using the random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). Blots were prehybridized and hybridized at 68°C in
Quikhyb (Stratagene, Inc., La Jolla, CA) for 20 min and 1 h, respectively. Blots were washed twice (15 min each) in 2 × SSC
and 0.1% SDS at 65°C, then once for 15 min in 0.2 × SSC and
0.1% SDS at 65°C. Blots were exposed to XAR-2 autoradiographic film (Eastman Kodak Co., Rochester, NY) at
80°C
with intensifying screens.
Generation of Mutant Mice. C57Bl/6J blastocysts were obtained from superovulated females. Uteri were flushed with M2 medium (32) at day 3.5 after conception. Blastocysts were collected and placed in a droplet of M2 medium under paraffin oil. ES cells were trypsinized, washed once with fresh DMEM-H, and diluted to 2 × 106 cells/ml. Cells (5 µl) were added to the droplet containing the blastocysts. Between 8 and 12 cells were injected into the inner cell mass of the blastocysts. After injection, six to nine blastocysts were implanted into each uterine horn of pseudopregnant females B6D2 which had been mated 2.5 d before with vasectomized males. Chimeric progeny were identified by coat color and were bred with C57Bl/6J mice. F1 mice were screened for germ-line transmission of the ES cell line by the presence of agouti coat color. Agouti-positive F1 mice were genotyped by Southern blot analysis as described above from tail biopsies. Mice heterozygous for the Ly-6A mutation were crossed with heterozygous siblings. F2 mice were similarly genotyped.
Simple Sequence Length Polymorphism (SSLP) Analysis and Mapping Linked Mutation. PCR primers for SSLP markers D15Mit3, D15Mit13, D15Mit14, D15Mit17, D15Mit29, D15Mit33, D15Mit34, D15Mit35, D15Mit39, and D15Nds1 were obtained from Research Genetics, Inc. (Huntsville, AL). The distances between loci cited in this manuscript are from the latest maps by National Institutes of Health (Bethesda, MD) and Massachusetts Institute of Technology (reference 33 and www.wi.mit.edu), respectively. PCR was performed using 25 ng tail DNA and 300 pM primer sets in 25 µl reactions with Boehringer Mannheim Taq polymerase and buffers. Using 129 and C57Bl/6 wild-type DNAs, it was determined that all but D15Mit34 and D15Mit39 are polymorphic between 129 and C57Bl/6. PCRs were performed on tail DNAs from all mice which were related to viable Ly-6A null mice and ~25% of the mice which did not generate Ly-6A null mice. The consensus difference between lines of mice was the presence of C57Bl/6 D15Mit33 marker in the mice which could generate Ly-6A null mice.
Antibodies and Flow Cytometry.
The live cells were separated
from dead cells and RBCs by Ficoll isolation (density 1.077g/ml;
Organon Teknika, Durham, NC) using standard procedures. Viable cells were washed twice with RPMI-1640 medium with 5%
FCS, followed by two washes in PBS with 2% FCS, and resuspended in supernatant containing anti-FcR mAb (2.4 G2) (34)
and incubated at 4°C for 30 min to block FcR and thus prevent
nonspecific binding of antibodies. Cells were washed twice in
PBS with 2% FCS and incubated with antibodies at optimally determined concentrations at 4°C for 30 min. Cells were then
washed three times in PBS with 2% FCS and either resuspended
in secondary antibodies or in 1% paraformaldehyde in PBS. Purified anti-Ly-6A.2 (SRT7) was purchased from Serotec Ltd.
(Kidlington, Oxford, UK). FITC- and PE-conjugated streptavidin and biotinylated FITC- and PE-conjugated antibodies against
isotype controls (rat IgG), mouse IgG2a/2b (R2-40), mouse Ig
(polyclonal), B220 (RA3-6B2), CD3 (145-2C11) , CD4 (RM4-5), CD8 (53-6.7), Ly-6A (D7 and E13-161.7), Ly-6C (AL-21),
Ly-6G (RB6-8C5), MAC-1 (M1/70), SCA-2 (MTS 35), TCR-
/
(H57-597), TCR-
/
(GL3), and ThB (49-H4) were purchased from PharMingen (San Diego, CA). Flow cytometry was
performed using a FACScan® (Becton Dickinson, San Jose, CA)
and Lysis II analysis software.
In Vitro Colony Assay. The final concentrations of methylcellulose media are as follows: 1.5% methylcellulose (Sigma Chemical Co.), 15% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 1% BSA (Sigma Chemical Co.), 9 µM monothioglycerol (Sigma Chemical Co.), 50 ng/ml rmIL-3 (R & D Systems, Inc., Minneapolis, MN), 5 U/ml rmEPO (Boehringer Mannheim), and 10 ng/ml rmGM-CSF (Genzyme Corp., Cambridge, MA) in IMDM. Multiple aliquots of single cell suspensions were counted using a hemacytometer to insure identical amounts of input cells. Premixed methylcellulose medium was added to the cells and transferred to 30 × 15 mm dishes (Nunc, Inc., Naperville, IL), and grown at 5-6% CO2, 37°C. Colonies were counted between days 8 and 11 after the initiation of cultures. Cells were stained for hemoglobin expression with 0.1% benzidine (Sigma Chemical Co.) and plates were recounted to confirm erythroid and mixed colonies.
Lymphocyte Activation Assays. In all experiments, sex-matched wild-type littermates were used as controls. Multiple aliquots of single cell suspensions were counted using a hemacytometer to insure identical amounts of input cells. Some experiments used whole splenocyte preparations; however, most experiments used enriched splenic T cell preparations or lymph node cells. Splenic T cells were prepared for proliferation assays by Ficoll isolation followed by B cell depletion using T Cellect columns (BioTex Laboratories, Edmonton, Canada). The enriched T cells were plated in triplicate wells in 96-well plates at 2 × 105 cells/well at various concentrations of ConA (0.25-2.0 µg/ml; see figure legends) or 1.0 µg/ml of PMA and 10 mg/ml of ionomycin in RPMI-1640 media and 10% FCS (Dutchland Laboratories, Denver, PA) at 37°C, 5% CO2 for 48 h. Cell proliferation was assayed by the addition of 1.0 µCi of [3H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA) during the last 18 h of culture. Cells were then harvested and measured for radioactivity in liquid scintillation cocktail. Ficoll separated splenocytes were incubated (in triplicate wells) in 50 µg/ml LPS in RPMI-1640 media and 10% FCS at 37°C, 5% CO2 for 48 h, and cell proliferation was measured as described above. To measure activation of T cells to antibodies, enriched T cells were incubated in supernatant from either C363.29B (anti-CD3; reference 35) or D7 (anti-Ly-6A; reference 12) hybridomas at 106 cells/ml at 4°C for 1 h. The cells were washed and plated in triplicate wells in 96-well plates at 2 × 105 cells/ well in 0.1 ml RPMI-1640 media and 10% FCS. Then, 0.1 ml of goat anti-rat serum (diluted in media) was added to the cells and incubated for 72 h (unless otherwise indicated), and cell proliferation was measured as described above. Enriched T cells (2 × 105/ well) were stimulated (in triplicate wells) with irradiated (2000R) CBA (H-2k) and DBA (H-2d) splenocytes for 5 d at a 2:1 responder/stimulator ratio for MLR assays, and cell proliferation was measured as described above.
C3H (H-2k), Ly-6A+/+ (H-2b), and Ly-6APriming and Response to KLH. 0.1 ml of 1 mg/ml KLH in Freund's adjuvant was injected into each footpad of each mouse. Primary immunizations were performed on day 0 in complete Freund's adjuvant, while secondary and tertiary immunizations were performed on days 10 and 20, respectively, in incomplete Freund's adjuvant. Draining lymph nodes were removed on day 24. Single cell suspensions were prepared, washed, and plated in triplicate wells in 96-well plates at 105 cells/well in RPMI-1640 media, 10% FCS, and 1 µg/ml ConA or 50 µg/ml LPS, or various concentrations of KLH (100, 50, or 10 µg/ml). Mice were bled on day 20 and relative serum concentrations of anti-KLH antibodies were determined by ELISA analysis. ELISA plates were coated with 500 ng (50 µl) of KLH antigen at 37°C for 75 min. 150 µl of 2% BSA in PBS was added and stored overnight at 4°C. The plates were washed three times with 50 mM Tris, pH 7.5, 0.2% Tween 20 (wash buffer). 50 µl of serum (in triplicate wells) was then incubated in plates for 2 h at 37°C. The plates were then washed three times, and 100 µl of biotinylated anti- mouse Ig was added to wells for 1 h at 37°C. The plates were then washed three times, and 100 µl of horseradish peroxidase conjugated-streptavidin (1:8,000 dilution in 2% BSA; Zymed Laboratories, Inc., South San Francisco, CA) was added to wells for 30 min at room temperature. The plates were then washed three times, and 100 µl of tetra-medhyl-benzadione substrate (DAKO Corp., Carpinteria, CA) was added and incubated (in the dark) for 30 min at room temperature. The reactions were stopped with 100 µl of 0.18 M H2SO4. ELISA plates were read using an ELISA plate reader.
The Ly-6A gene is comprised of four exons,
with the sequence encoding the mature protein beginning
in the third exon (27). A targeting plasmid was constructed
by replacing exons 1-3 with the neoR gene in the 5-3
orientation (Fig. 1 A). The targeting construct did not contain a polyadenylation site; which meant that cells containing the targeting construct would not express the neoR
gene unless the targeting construct integrated 5
of a usable polyadenylation site. Using this strategy, fewer random integration events should be G418R, thereby enriching for
homologous recombinants among the G418R colonies. In
addition, the HSV-tk cassette (28) was inserted outside the
homologous sequence of the targeting vector to generate a
positive/negative selection targeting plasmid, pLy6ASDI-1
(Fig. 1). After electroporation with pLy6ASDI-1, E14TG2a
ES cells were selected with G418 and GANC to enrich for
homologous recombinants. In each experiment, between
20 and 100 colonies survived, an ~10-fold decrease in total
colonies compared to electroporated cells selected with
G418 alone. G418R, GANCR colonies were analyzed for
homologous recombinants by a PCR-based assay (36).
From seven electroporation experiments, five colonies were
PCR-positive for homologous recombination. The positive clones were analyzed by Southern blots to verify Ly-6A gene targeting and determine whether there were any
anomalous rearrangements. These are considerable concerns because Ly-6A is a member of a large gene family (18 genes encoded by a single locus on murine chromosome
15; (reference 37) and it is conceivable that other members
of the gene family could be targeted by this construct. Three clones contained the expected RFLP patterns (data
not shown and Fig. 1 B and C). Southern analysis of
EcoRV digested DNA from a litter derived from heterozygous matings is depicted in Fig. 1 B and C. The endogenous Ly-6A gene is 5.2 kb and the mutated allele is 4.5 kb.
Probes derived upstream and downstream as well as a neoR
gene probe were used to determine that the recombination
event occurred as expected. The clones were karyotyped
and found to contain the normal 40 chromosomes. Subsequently, all three clones were injected into C57Bl/6J blastocysts and reimplanted into pseudopregnant females. Chimeras were backcrossed with C57Bl/6J and germ-line transmission was determined in the F1 litters. Only clone
106-60 produced germ-line transmission. Approximately
43% of the agouti F1 mice were heterozygous for the Ly-6A targeting event.
Originally we did not detect any Ly-6A homozygous
mutant mice born from heterozygous matings and we pursued the possibility that Ly-6A null mutation results in embryonic lethality. In fact, Ly-6A protein is expressed during
preimplantation and Ly-6A null embryos died between
embryonic days 3.5 and 6.5 (data not shown). However,
eventually pups sired by multiple founders were born which when bred gave rise to viable Ly-6A/
mice. To
determine the genetic basis differences between the mice
which gave rise to the homozygous mice versus the ones
which did not give rise to viable homozygous embryos,
analysis of chromosome 15 was employed (38). SSLP analysis demonstrated that the difference between the two lines
of mice was that all mice which were able to give rise to viable null pups contained C57Bl/6 markers (D15Mit33) ~0.1 cM distal to the Ly-6 locus (33) instead of 129 markers, suggesting that a crossover event occurred early in the
line which restored function of a gene (or genes) distal to
the Ly-6 locus. All breeders which did not give rise to null
mice contained the 129 D15Mit33 marker distal to the targeted Ly-6A allele, suggesting that these mice have a lethal
mutation distal to the Ly-6 locus. The linked lethal mutation was further mapped proximal to D15Mit14, leaving
~20 cM in which the lethal mutation could have occurred. Additional evidence which suggests that the linked lethal
mutation did not occur in the Ly-6 locus is that the use of
multiple probes which hybridize to all Ly-6 gene family
members did not show any RFLP differences between
wild-type DNA or the 106-60 targeted ES cell line with
the exception of the Ly-6A targeted locus (data not shown).
All the breeders were tested for the C57Bl/6 D15Mit33 marker, and the Ly-6A mouse line was derived from these
mice which are currently at C57Bl/6 backcross eight. Genotypes of these mice do not deviate from the expected
Mendelian frequencies.
Homozygous animals had no apparent health problems, including breeding, in a pathogen-free animal facility. Histological analysis was performed on tissues known to express Ly-6A in wild-type animals. Examination of hematoxylin and eosin stained sections of femur, kidney, liver, lymph node, spleen, and thymus revealed no obvious differences between homozygous mutant and wild-type littermates.
Analysis of Lymphoid and Myeloid Subpopulations. Flow
cytometry was used to verify the absence of Ly-6A expression in homozygous mutant mice (Fig. 2). Approximately
60% of wild-type splenocytes express Ly-6A. Splenocytes
from heterozygous animals show a slight decrease in Ly-6A
expression intensity; however, splenocytes from homozygotes do not stain with any of three antibodies to Ly-6A (Fig. 2). In addition, phenotypic analysis was performed to
determine if the absence of Ly-6A expression in homozygous mutant mice altered the differentiation of various cell
populations. Although there are minor variations between
littermates, as a population Ly-6A null animals (as old as 8 mo) contain normal percentages of B220, TCR-/
,
TCR-
/
, CD3, CD4, CD8, Mac-1, and Ly-6G (Gr-1)
positive cells in the bone marrow, lymph nodes, spleen,
and thymus (data not shown). In addition to Ly-6G, antibodies to other members of the Ly-6 gene family were
used to determine if the lack of phenotypic changes is due
to compensation by other family members. Ly-6C, Sca-2
(TSA-1), and ThB do not appear to be overexpressed in
any hematopoietic tissues (data not shown).
To analyze myeloid precursors, the in vitro colony-forming potential of bone marrow cells from Ly-6A/
was compared to that of wild-type littermate bone marrow.
Table 1 displays the combined total number of colonies
from three experiments. As expected from the FACS®
analysis, the Ly-6A
/
bone marrow contained a normal
number and percentage of all CFU types. Although in each
experiment the total number of colonies was lower, the total percentage of pure granulocyte colonies was lower, and
the total percentage of CFU-granulocyte/macrophage colonies was higher in the Ly-6A
/
cultures, none of these
differences are statistically significant due to the large variation in CFUs within each group of mice.
|
Functional Analysis of Lymphocytes In Vitro. To determine whether the T lymphocytes from Ly-6A null animals
are functionally normal, a Con A response experiment with
two runs was performed in a blinded manner. For each
run, the spleens were removed and coated from four wild-type and four Ly-6A null littermates by one individual and
given to a second individual to perform the experiment. B
cell-depleted splenocytes were stimulated with various concentrations of Con A for a total of 48 h, with an addition of 1 µCi tritiated thymidine deoxyribose ([3H]TdR)
after 24 h, and triplicate cpm were obtained. Fig. 3 is a logarithmic plot of the [3H]TdR incorporation of T cells versus the concentration of Con A from one of the experimental runs. This experiment demonstrates that as a group
the splenic T cells from Ly-6A null animals proliferate at a
much higher rate in response to Con A stimulation than
the T cells from wild-type littermates. For example, the average [3H]TdR incorporation of the T cells from Ly-6A
null animals is 156% of the response of wild-type littermates when stimulated with 2.0 µg/ml Con A (P <0.03).
However, Fig. 3 also demonstrates that there is a significant
variation in response within each group of mice. Therefore, the results of both blind experiments were combined
and independent statistical analysis performed for Con A
levels between 1.0 and 0.5 µg/ml. An ANOVA model was
constructed to examine the separate effects of Con A concentration and genotype adjusted for the run and mouse effects, where mouse was nested within level and genotype.
The effect of each factor was statistically significant. Of particular interest, Ly-6A null mice had higher incorporation
of [3H]TdR (P <0.0001) and a dose-response effect was
found for Con A (P <0.0001).
The response to cross-linked anti-Ly-6A and anti-CD3
by splenic T cells was also evaluated. A representative experiment is depicted in Fig. 4 A and B. As expected, T cells
from Ly-6A null animals do not respond when exposed to
anti-Ly-6A followed by goat anti-rat serum, whereas T cells
from wild-type littermates show normal levels of proliferation (Fig. 4 A). In contrast, when cell surface CD3 was cross-linked by C363.29B mAb followed by goat anti-rat serum,
Ly-6A null T cells showed a 211% increase (P <0.001) in
the level of proliferation when compared to T cells from
wild-type littermates (Fig. 4 B). The average increase of [3H]TdR incorporation by the Ly-6A/
T cells compared
to wild-type littermate T cells from four other experiments
ranged from 61 to 619%.
T cells from Ly-6A deficient and wild-type littermates
were also tested for their response to allogeneic antigen. A
representative experiment is shown in Fig. 4 C, which
demonstrates that splenic T cells from Ly-6A/
(H]2b)
mice generated a significantly higher proliferative response than T cells from wild-type littermate mice. In the experiment shown, the response of Ly-6A
/
T cells was 86%
higher (P <0.01) than the response of Ly-6A+/+ T cells
when stimulated with irradiated spleen cells from CBA (H-2k), and 48% higher (P <0.02) when stimulated with
irradiated cells from DBA (H]2d) mice. The average increase of [3H]TdR incorporation by the Ly-6A
/
T cells
compared to wild-type littermate T cells from other experiments ranged from 12 to 49% (P <0.02) for anti-H-2k response and from 46 to 79% (P <0.01) for anti-H-2d response in three independent experiments.
Unlike antigenic and ConA activation, which stimulate T cells through the TCR complex, the addition of PMA and ionomycin stimulates T cells by activating protein kinase C directly. PMA with ionomycin activates T cells from Ly-6A mutant and wild-type littermates at similar levels. The results from a representative experiment are displayed in Fig. 4 D. In other experiments, whole splenocytes were tested for response to the various stimuli and similar percentage differences between Ly-6A null and wild-type littermate splenocytes results were obtained compared to those results using enriched T cells, although the total [3H]TdR incorporation was lower than the results obtained with the enriched T cells (data not shown). In addition, Con A responses by heterozygous T cells were compared to those of wild-type and null littermates and found to be no different than wild-type responses (data not shown).
The stimulation of thymocytes in response to Con A was
also examined. In contrast to stimulation of splenic T cells,
stimulation of Ly-6A/
thymocytes did not show a statistically significant difference in their ability to respond to
ConA when compared to thymocytes from wild-type littermates. In two experiments involving three mice in each
group, the Ly-6A
/
thymocytes proliferated at a slightly
higher but statistically insignificant rate when compared to
thymocytes from wild-type littermates. The results shown
in Fig. 4 E demonstrate that in the first experiment, the
Con A response by Ly-6A
/
thymocytes was an average
4% higher than wild-type thymocytes (P <0.13), while in
the second experiment Ly-6A
/
thymocytes proliferated by
an average of 16% greater than wild-type controls (P <0.28).
Neither experiment is statistically significant due to the
wide variation of values for each group.
Splenocytes were treated with LPS to determine if there
was a difference between Ly-6A/
and wild-type mitogen-induced B cell proliferation. The results in Fig. 4 F
demonstrate that Ly-6A null splenocytes do not show a significant difference to LPS than splenocytes from wild-type littermates. Fig. 4 F shows the results of two independent
experiments; the first shows a 2% decrease in proliferation
for Ly-6A null splenocytes (P <0.38) when independently
testing three animals in each group, while the second experiment shows the Ly-6A
/
splenocytes proliferated 6%
more (P <0.08%) than wild-type littermate controls when
four animals in each group were tested.
A kinetics experiment was performed to determine the
rate of proliferation at three different time points by splenic
T cells from 4-, 6-, and 8-mo-old Ly-6A mutant and wild-type littermates (three mice from each group). T cells were
activated by cross-linking cell surface CD3 and their proliferation rates were measured at 48, 72, and 98 h by adding
[3H]TdR to the cells 3 h before measuring [3H]TdR incorporation. Fig. 5 demonstrates that Ly-6A/
T cells proliferate at higher rates than the age-matched wild-type T cells
at all time points. In fact, the Ly-6A null T cells appear to
sustain the proliferative response longer than the wild-type T cells; in other words, the percent increase in [3H]TdR
incorporation by mutant T cells over wild-type T cells was 159% at 48 h (P <0.01), 272% at 72 h (P <0.06), and
994% at 96 h (P <0.01).
To determine if the enhanced proliferation activity of
Ly-6A/
T cells was due to an upregulation of autocrine
growth factor production, supernatants and cell lysates were
harvested at 16, 24, 48, and 72 h time points during activation assays and used to determine IL-2,-4, and -6, TNF-
,
and IFN-
production by ELISA analysis. There were no
consistent differences in cytokine production in T cell activation assays between splenocytes from Ly-6A mutant and
wild-type littermates (data not shown). However, it is possible that differences in cytokine production were not detected because the cytokines were used as soon as they
were produced. Therefore, RNA was isolated from Ly-6A
null and wild-type T cells at various time points during activation assays and semiquantitative reverse transcriptionPCR analysis on transcript levels was performed for the
aforementioned cytokines. No differences in cytokine transcription between Ly-6A null and wild-type littermates
were detected (data not shown).
In addition to measuring proliferation responses to antigen and mitogen, splenocytes from wild-type and Ly-6A
mutant littermates were tested for their ability to mediate
an allogenic CTL response. Splenocytes were incubated with
irradiated C3H (H-2k) and syngenic irradiated splenocytes
as stimulators for 5 d. Splenocytes were harvested and counted.
Consistent with the proliferation experiments, in each culture 5-11% more cells were recovered from the Ly-6A null
cultures than the wild-type littermate cultures. Effector cells
were tested for their ability to lyse 51Cr-loaded 6130 (H-2k)
and EL-4 (H-2b) target cells at various effector/target ratios
(Fig. 6). In all three experiments, Ly-6A null splenocytes
efficiently lysed H-2k targets at a slightly higher, but not
statistically significant, rate than did wild-type splenocytes.
Thus, although the total number of lytic units obtained
from the stimulation cultures were higher for the Ly-6A/
splenocytes as compared to wild-type splenocytes, the lytic
activity of these CTLs at a given effector/target cell ratio was
unchanged. Neither wild-type nor Ly-6A mutant splenocytes lysed control H-2b target cells. In addition, Ly-6A
/
and wild-type littermate splenocytes were tested for activation of CTL response against either Ly-6A
/
or wild-type
irradiated splenocytes. Neither group of responders was activated by the syngeneic stimulators, although C3H splenocytes were activated equally well by both Ly-6A
/
and
wild-type irradiated splenocytes to kill H-2b target cells.
Functional Analysis of Lymphocytes In Vivo.
Ly-6A null and
wild-type littermates were challenged with KLH by three
footpad injections of 0.1 mg KLH in Freund's adjuvant, and draining lymph nodes were harvested 4 d after the tertiary immunization. Lymph node cells were then activated
in culture in the presence of LPS, ConA, or KLH, and
their proliferation rate measured by [3H]TdR incorporation. Ly-6A null cells proliferated at significantly higher
rates to KLH and ConA than wild-type lymphocytes (Fig. 7 A). In fact, incorporation of [3H]TdR by Ly-6A/
cells
was 150% higher at 1 µg/ml KLH (P <0.03), 168% higher
at 5 µg/ml KLH (P <0.02), 191% higher at 10 µg/ml KLH
(P <0.01), and 302% higher at 1 µg/ml ConA (P <0.01).
Lymph node cells isolated from animals immunized with
Freund's adjuvant alone did not respond to KLH antigen in
vitro (data not shown). In contrast, LPS activation of cells
did not demonstrate any significant differences (P <0.11) in B cell proliferation between KLH immunized Ly-6A null
and wild-type littermates (Fig. 7 A). However, the anti-KLH antibody response was significantly lower in Ly-6A
null animals in comparison to wild-type animals (Fig. 7 B).
Fig. 7 B summarizes the results from ELISA analysis of serum from 10 d post-secondary immunized littermates (four
in each group) against four dilutions of KLH antigen. At all
dilutions of KLH antigen, the ELISA absorbance was lower
by the serum of the Ly-6A null mice than the wild-type littermates (ranging from 38 to 99% lower; P <0.03).
Our approach to determine whether Ly-6A protein expression was necessary for normal hematopoietic development or T cell activation was to produce Ly-6A null animals by gene targeting. Flow analysis demonstrated that all
normal percentages of hematopoietic lineages were represented in the bone marrow, spleen, lymph node, and thymus, thereby demonstrating that Ly-6A was not necessary
for normal hematopoietic development. In addition, colony forming assays were performed on bone marrow from
Ly-6A null and wild-type littermates. In all three experiments, the total number of colonies was lower, the total
percentage of pure granulocyte colonies was lower, and the
total percentage of CFU-GM colonies was higher in the
Ly-6A/
cultures; however, none of these differences are
statistically significant due to the large variation in CFUs
within each group of mice. Proliferation assays were performed using a variety of mitogens and antigens and Ly-6A
null splenic T cells proliferated at statistically higher levels
compared to T cells of wild-type littermate controls in all
cases except PMA plus ionomycin, which does not activate
through the TCR complex. These results suggest that Ly-6A exerts its effects when T cells are signaled through the
TCR complex. CTL killing activity was found to be similar at various effector/target ratios between Ly-6A null and
wild-type splenocytes, although CTL cultures produced
greater numbers of CTLs from Ly-6A null mice compared
to wild-type littermates, which parallels the results of the
MLR proliferation experiments. The enhanced proliferation is regulated because Ly-6A
/
splenocytes did not respond with increased proliferation or CTL generation
when stimulated with self-antigen. Kinetics experiments determined that the Ly-6A null T cells sustain their proliferation longer than T cells from wild-type littermates. In
contrast, when thymocytes were tested for mitogen-induced
proliferation, Ly-6A mutant thymocytes incorporated between 4 and 16% more [3H]TdR in response to Con A
stimulation than thymocytes from littermate controls in
two experiments, and these differences are not statistically significant due to the wide variation of values for each
group.
Perhaps more importantly, in vivo T cell responses recapitulate the in vitro proliferation data. Lymphocytes were primed in vivo by footpad injections of KLH antigen into Ly-6A null and wild-type littermates. Lymphocytes were harvested and tested in vitro for proliferation to KLH, Con A, or LPS. Ly-6A mutant lymphocytes demonstrated significant increases in proliferation to KLH and Con A compared to lymphocytes from primed wild-type littermates; in contrast, there were no significant differences in LPS response between the two groups, suggesting normal B cell proliferative responses. Interestingly, serum antibody levels against KLH were significantly lower in primed Ly-6A null mice than wild-type littermates, suggesting that the effects of Ly-6A deficiency on immune responses are highly complex.
Bamezai et al. suggest a negative role for Ly-6A in thymic selection (26). Normally, Ly-6A expression during
thymocyte differentiation is strictly controlled. Ly-6A is expressed on the thymocyte progenitor cell which seeds the
thymic cortex (23, 24). During the transition of CD34
8
thymocytes into CD3+4+8+ thymocytes, Ly-6A, CD44, and
CD25 expression are temporally regulated. Ly-6A is expressed on 49% of CD44+25
(stage 1), 34% of CD44+25+
(stage 2), and <4% on CD44
5+ and CD44
25
(stages 3 and 4, respectively) (26). After maturation, Ly-6A is reexpressed on mature, single-positive thymocytes and most CD4+ and ~40% CD8+ peripheral T cells (25, 26, 39). Thymocyte development is arrested in transgenic mice with
constitutive lymphocyte expression of Ly-6A at stage 2, when Ly-6A expression is normally terminated (26). However, thymocyte development appears unaltered in Ly-6A null
mice which have normal percentages of TCR-
/
, TCR-
/
, CD3, CD4, CD8, Sca-2, and ThB positive cells in the
thymus and periphery (data not shown). This suggests that
although overexpression of Ly-6A abrogates thymocyte maturation, Ly-6A expression is not necessary for normal thymocyte development.
Although a ligand of Ly-6A has not been identified, Ly-6A transgenic thymocytes spontaneously adhere to thymocytes, B cells, and T cells, suggesting that these cell types
express a ligand for Ly-6A (40). In addition, there are many
published experiments which suggest that Ly-6A is involved in T cell activation. Ly-6A expression on T cells is
upregulated upon activation or stimulation with cytokines
(41, 42), and cross-linking cell surface Ly-6A leads to IL-2
driven T cell proliferation (12, 43). Several groups have
shown that Ly-6A activation is apparently interrelated with
TCR signaling. For example, TCR mutant cell lines cannot be activated by Ly-6A cross-linking (14, 19), and Ly-6A antisense downregulates TCR-mediated activation of T
cells and T cell lines (15, 16). In fact, cell lines expressing
essentially no Ly-6A due to high expression of antisense
constructs have impaired transcription of TCR- chain
and impaired p59fyn but not p56lck phosphorylation activity
(16). The effect on p59fyn phosphorylation activity is consistent with the data by Stefanova et al. demonstrating that
GPI proteins are weakly associated with protein tyrosine
kinases (44). However, despite the absence of TCR or Ly-6A, PMA plus ionomycin activates these cells via a protein
kinase C pathway. Our results demonstrating that Ly-6A null and wild-type T cells respond similarly to PMA plus
ionomycin but significantly differently to ConA and antigens are consistent with the hypothesis that Ly-6A is involved in signaling via the TCR. However, based on previous antisense experiments, it was surprising that Ly-6A
null T cells proliferated at much higher levels to antigen
than wild-type cells instead of the reverse. The results from
antisense experiments and those with Ly-6A knockout mice appear to give contradictory results; however, they illustrate the difference between a response generated by
cells which never expressed Ly-6A (Ly-6A
/
) and cells
which expressed Ly-6A and then were altered to downregulate Ly-6A expression (antisense). A similar observation
was also demonstrated by the CD2 knockout mice. Although cross-linking cell surface CD2 activates T cells (45),
and mutant cell lines lacking CD2 expression have diminished activation through the TCR (46), the T cells from
CD2-deficient animals do not have any overt altered function compared to wild-type T cells (47). However, finer analysis showed that CD2 regulates positive selection of
CD4
CD8+ T cells (48).
Several approaches were taken to determine the mechanism driving the enhanced proliferation by the mutant T
cells. One possible mechanism of increased proliferation in
mutant cells is increased cytokine production. However,
there were no consistent differences in cytokine production
detected at either the RNA or protein level between splenocytes from Ly-6A mutant and wild-type littermates (data
not shown). Another possible mechanism of enhanced proliferation activity by Ly-6A/
T cells may be that a subpopulation of cells is either present or absent in Ly-6A
knockout animals which normally upregulates or downregulates the T cell response. Although this possibility cannot
be ruled out at this point, the cursory analysis of subpopulations did not show any differences between wild-type and
null thymocytes or T cells, and the T cell activation experiments are consistent with all stimuli which act through the
TCR which were tested. Another possibility is that another
member of the Ly-6 gene family is overcompensating for
the lack of Ly-6A; however, there is no difference in expression of the other cloned members of the Ly-6 gene
family (data not shown). Interestingly, there is growing evidence which suggests that GPI-anchored proteins associate
with Src kinases in caveolae, small invaginations of the
plasma membrane lacking clathrin coats (for review see reference 49). It is possible that GPI-anchored proteins act as
positive or negative regulators of activation by trapping and
concentrating receptors and other signaling molecules. This
model is supported by Romagnoli and Bron, who recently
demonstrated that stimulation of the TCR in GPI-mutant
T cell lines generated reduced activity by the fyn and lck
kinases which resulted in failure to induce tyrosine phosphorylation of the TCR
chain and ZAP-70 (50). The
Ly-6A null mice and antigen-specific T cell lines generated
from these mice (Alexander, R., and P.M. Flood, unpublished results) should be useful to test this model.
The role of Ly-6A on the functional maturation of hematopoietic precursors is also very complicated. In mice which express the Ly-6.2 allele, which includes C57Bl/6 and 129 (the two backgrounds of the Ly-6A null mice and their littermates), Ly-6A is expressed on all hematopoietic stem cells and a significant proportion of committed progenitors (7, 51); however, only 25% of the stem cell activity of the adult bone marrow expresses Ly-6A in Ly-6.1 allele mice (52). Thus, the differences in the expression pattern of the two alleles suggests that Ly-6A is not essential for development of hematopoietic stem cells. This is consistent with normal hematopoietic development in Ly-6A null mice. The CFU assay suggests that there may be some subtle alterations in myeloid precursor activity in Ly-6A mutant bone marrow compared to wild-type bone marrow, similar to CD34-deficient animals (53, 54).
In conclusion, although interactions governing the regulation of the immune responses and peripheral tolerance remain unclear, recent experiments have shed light upon several important regulators of T lymphocyte responses. For example, IL-2-deficient and IL-2R-deficient mice have demonstrated the critical role which cytokines and cytokine receptors may play in regulating T cell responses and self-tolerance (55). Other studies have shown that molecules such as CTLA-4 may provide an important negative signal to downregulate T lymphocyte activity (60, 61). In addition, members of the TNF/TNFR family also contribute to the activation and elimination of lymphocytes during an immune response (62). This report suggests that Ly-6A may play an important role in regulating T lymphocyte responses. Although activation and proliferation of Ly-6A null thymocytes is normal, in vitro and in vivo activation of peripheral T cells from Ly-6A-deficient mice generates higher and more sustained proliferative responses than T cells from wild-type littermates. These data suggest that Ly-6A acts to downmodulate the T lymphocyte response to antigen. Further analysis of the Ly-6A-deficient mice will provide valuable insight into the signaling pathway of Ly-6A and how molecules act in concert to maintain homeostasis and peripheral tolerance.
Address correspondence to Dr. Patrick M. Flood, Department of Microbiology and Immunology, Curriculum in Oral Biology, Dental Research Center, University of North Carolina, Chapel Hill, NC 27599-7455. Phone: 919-966-1538; FAX: 919-966-3683; E-mail: patflood{at}dentistry.unc.edu. W.L. Stanford's present address is Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
Received for publication 8 April 1997 and in revised form 7 July 1997.
1 Abbreviations used in this paper: Con A, Concanavalin A; ES, embryonic stem; GANC, gancyclovir; GPI, glycosyl phosphatidylinositol; SSLP, simple sequence length polymorphism.We thank Dr. F. Fiedorek, Dr. N. Luetteke, and Dr. R. Mannon for their technical advice; Dr. P. Ohashi, Dr. D. Barber and Dr. S. Renfold for critically reading the manuscript; Dr. M. Schell for statistical analysis; and Dr. L. Arnold and J. Vincent for flow cytometry technical support; G. Bowman and B. Garges for general technical assistance; and Dr. C. Ito and Dr. B. Davis for inspiration.
This work was supported in part by grants from National Institutes of Health (NIH) R01DK-4351701 (H.R. Snodgrass), PO1DK-38103 (B.H. Koller), and R01DE-09426 (P.M. Flood) and an NIH-NIAID Training Grant 5 T32 AI-07273.
1. | Reiser, H., H. Oettgen, E.T.H. Yeh, C. Terhorst, M.G. Low, B. Benacerraf, and K.L. Rock. 1986. Structural characterization of the TAP molecule: a phosphatidylinositol-linked glycoprotein distinct from the T cell receptor/T3 complex and Thy-1. Cell. 47: 365-370 [Medline]. |
2. | Hammelburger, J.W., R.G.E. Palfree, S. Sirlin, and U. Hammerling. 1987. Demonstration of phosphatidylinositol anchors on Ly-6 molecules by phospholipase C digestion and gel electrophoresis in octylglucoside. Biochem. Biophys. Res. Commun. 148: 1304-1311 [Medline]. |
3. |
Stiernberg, J.,
M.G. Low,
L. Flaherty, and
P.W. Kincade.
1987.
Removal of lymphocyte surface molecules with phosphatidylinositol-specific phospholipase C: effects on mitogen
responses and evidence that ThB and certain Qa antigens are
membrane-anchored via phosphatidylinositol.
J. Immunol.
138:
3877-3884
|
4. | Shevach, E.M., and P.E. Korty. 1989. Ly-6: a multigene family in search of a function. Immunol. Today. 10: 195-200 [Medline]. |
5. | Spangrude, G.J., S. Heinfeld, and I.L. Weissman. 1988. Purification and characterization of mouse hematopoietic stem cells. Science (Wash. DC). 241: 58-62 [Medline]. |
6. | van de Rijn, M., S. Heimfeld, G.J. Spangrude, and I.L. Weissman. 1989. Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc. Natl. Acad. Sci. USA. 86: 4634-4638 [Abstract]. |
7. |
Uchida, N., and
I.L. Weissman.
1992.
Searching for hematopoietic stem cells: evidence that Thy-1.1loLin![]() |
8. | Ikuta, K., and I.L. Weissman. 1992. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc. Natl. Acad. Sci. USA. 89: 1502-1506 [Abstract]. |
9. | Blake, P.G., J. Maderas, and P.F. Halloran. 1993. Ly-6 in kidney is widely expressed on tubular epithelium and vascular endothelium and is up-regulated by interferon gamma. J. Am. Soc. Nephrol. 4: 1140-1150 [Abstract]. |
10. | Horowitz, M.C., A. Fields, D. DeMeo, H.Y. Qian, A.L. Bothwell, and E. Trepman. 1994. Expression and regulation of Ly-6 differentiation antigens by murine osteoblasts. Endocrinology. 135: 1032-1043 [Abstract]. |
11. |
Codias, E.K., and
T.R. Malek.
1990.
Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation
through Ly-6A/E molecules.
J. Immunol.
144:
2197-2204
|
12. | Malek, T.R., G. Ortega, C. Chan, R.A. Kroczek, and E.M. Shevach. 1986. Role of Ly-6 in lymphocyte activation. II. Induction of T cell activation by monoclonal anti-Ly-6 antibodies. J. Exp. Med. 164: 709-722 [Abstract]. |
13. |
Yeh, E.T.H.,
H. Reiser,
J. Daley, and
K.L. Rock.
1987.
Stimulation of T cells via the TAP molecule, a member in a
family of activating proteins encoded in the Ly-6 locus.
J. Immunol.
138:
91-97
|
14. | Sussman, J.J., M. Mercep, T. Saito, R.N. Germain, E. Bonavini, and J.D. Ashwell. 1988. Dissociation of phosphoinositide hydrolysis and Ca2+ fluxes from the biological responses of a T-cell hybridoma. Nature (Lond.). 334: 625-628 [Medline]. |
15. | Flood, P.M., J.P. Dougherty, and Y. Ron. 1990. Inhibition of Ly-6A antigen expression prevents T cell activation. J. Exp. Med. 172: 115-120 [Abstract]. |
16. | Lee, S.-K., B. Su, S.E. Maher, and A.L.M. Bothwell. 1994. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity. EMBO (Eur. Mol. Biol. Organ.) J. 13: 2167-2176 [Abstract]. |
17. | Yeh, E.T.H., H. Reiser, A. Bamezai, and K.L. Rock. 1988. TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor. Cell. 52: 665-674 [Medline]. |
18. |
Sussman, J.J.,
T. Saito,
E.M. Shevach,
R.N. Germain, and
J.D. Ashwell.
1988.
Thy-1 and Ly-6 mediated lymphokine
production and growth inhibition of a T cell hybridoma require co-expression of the T cell antigen receptor complex.
J.
Immunol.
140:
2520-2526
|
19. | Bamezai, A., H. Reiser, and K.L. Rock. 1988. T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP. J. Immunol. 14: 1423-1428 . |
20. |
Codias, E.K.,
J.E. Rutter,
T.J. Fleming, and
T.R. Malek.
1990.
Down-regulation of IL-2 production by activation of
T cells through Ly-6A/E.
J. Immunol.
145:
1407-1414
|
21. |
Izon, D.J.,
L.A. Jones,
E.E. Eynon, and
A.M. Kruisbeek.
1994.
A molecule expressed on accessory cells, activated T
cells, and thymic epithelium is a marker and promoter of T
cell activation.
J. Immunol.
153:
2939-2950
|
22. | Izon, D.J., K. Oritanik, M. Hamel, C.R. Calvo, R.L. Boyd, P.W. Kincade, and A.M. Kruisbeek. 1996. Identification and functional analysis of Ly-6A/E as a thymic and bone marrow stromal antigen. J. Immunol. 156: 2391-2399 [Abstract]. |
23. |
Spangrude, G.J.,
Y. Aihara,
I.L. Weissman, and
J. Klein.
1988.
The stem cell antigens SCA-1 and SCA-2 subdivide
thymic peripheral T lymphocytes into unique subsets.
J. Immunol.
141:
3697-3707
|
24. | Wu, L., M. Antica, G.R. Johnson, R. Scollay, and K. Shortman. 1991. Developmental potential of the earliest precursor cells from the adult mouse thymus. J. Exp. Med. 174: 1617-1627 [Abstract]. |
25. |
Yeh, E.T.H.,
H. Reiser,
B. Benacerraf, and
K.L. Rock.
1986.
The expression, function and ontogeny of a novel T
cell-activating protein, TAP, in the thymus.
J. Immunol.
137:
1232-1238
|
26. |
Bamezai, A.,
D. Palliser,
A. Berezovskaya,
J. McGrew,
K. Higgins,
E. Lacy, and
K.L. Rock.
1995.
Regulated expression of Ly-6A.2 is important for T cell development.
J. Immunol.
154:
4233-4239
|
27. | Stanford, W.L., E. Bruyns, and H.R. Snodgrass. 1992. The isolation and sequence of the chromosomal gene and regulatory regions of Ly-6A.2. Immunogenetics. 35: 408-411 [Medline]. |
28. | Mansour, S.L., K.R. Thomas, and M.R. Capecchi. 1988. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature (Lond.). 336: 348-352 [Medline]. |
29. | Hooper, M., K. Handy, A. Handyside, S. Hunter, and M. Monk. 1986. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature (Lond.). 326: 292-295 . |
30. |
Koller, B.H.,
P. Marrack,
J.W. Kappler, and
O. Smithies.
1990.
Normal development of mice deficient in ![]() |
31. | Miller, S.A., D.D. Dykes, and H.F. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16: 1215 [Medline]. |
32. | Quinn, P., C. Barros, and D.G. Whittingham. 1982. Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J. Reprod. Fertil. 66: 161-169 [Abstract]. |
33. | Huppi, K., D. Siwarski, J.T. Eppig, and B.A. Mock. 1996. Mouse chromosome 15. Mamm. Genome. 6(Suppl.):S256- 270. |
34. | Unkeless, J.C.. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150: 580-596 [Abstract]. |
35. |
Portoles, P.,
J. Rojo,
A. Golby,
M. Bonneville,
S. Gromkowski,
L. Greenbaum,
C.A. Janeway Jr.,
D.B. Murphy, and
K. Bottomly.
1989.
Monoclonal antibodies to murine CD3
epsilon define distinct epitopes, one of which may interact
with CD4 during T cell activation.
J. Immunol.
142:
4169-4175
|
36. | Kim, H.S., and O. Smithies. 1988. Recombinant fragment assay for gene targeting based on the polymerase chain reaction. Nucleic Acids Res. 16: 8887-8903 [Abstract]. |
37. | LeClair, K.P., M. Rabin, M.N. Nesbitt, D. Pravtcheva, F.H. Ruddle, R.G.E. Palfree, and A. Bothwell. 1987. Murine Ly-6 multigene family is located on chromosome 15. Proc. Natl. Acad. Sci. USA. 84: 1638-1642 [Abstract]. |
38. |
Dietrich, W.,
H. Katz,
S.E. Lincoln,
H.-S. Shin,
J. Friedman,
N.C. Dracopoli, and
E.S. Lander.
1992.
A genetic map of the
mouse suitable for typing intraspecific crosses.
Genetics.
131:
423-447
|
39. | Rock, K.L., H. Reiser, A. Bamezai, J. McGrew, and B. Benacerraf. 1989. The Ly-6 locus: a multigene family encoding phosphatidylinositol-anchored membrane proteins concerned with T-cell activation. Immunol. Rev. 111: 195-224 [Medline]. |
40. | Bamezai, A., and K.L. Rock. 1995. Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding a ligand expressed on lymphoid cells. Proc. Natl. Acad. Sci. USA. 92: 4294-4298 [Abstract]. |
41. | Dumont, F.J., R. Dijkmans, R.G. Palfree, R.D. Boltz, and L. Coker. 1987. Selective up-regulation by interferon-gamma of surface molecules of the Ly-6 complex in resting T cell: the Ly-6A/E and TAP antigens are preferentially enhanced. Eur. J. Immunol. 17: 1183-1191 [Medline]. |
42. |
Dumont, F.J., and
R.D. Boltz.
1987.
The augmentation of
surface Ly-6A/E molecules in activated T cells is mediated by
endogenous interferon-![]() |
43. | Rock, K.L., E.T.H. Yeh, C.F. Gramm, S.I. Haber, H. Reiser, and B. Benacerraf. 1986. TAP, a novel T cell activating protein involved in the stimulation of MHC-restricted T lymphocytes. J. Exp. Med. 164: 709-722 [Abstract]. |
44. | Stefanova, I., V. Horejsi, I.J. Ansotegui, W. Knapp, and H. Stockinger. 1991. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science (Wash. DC). 254: 1016-1019 [Medline]. |
45. | Meuer, S.C., R.E. Hussey, M. Fabbi, D. Fox, O. Acuto, K.A. Fitzgerald, J.C. Hodgdon, J.P. Protentis, S.F. Schlossman, and E.L. Reinherz. 1984. An alternative pathway of T-cell activation: a functional role for the 50kd T11 sheep erythrocyte receptor protein. Cell. 36: 897-906 [Medline]. |
46. |
Makni, H.,
J.S. Malter,
J.C. Reed,
S. Nobuhiko,
G. Lang,
D. Kioussis,
G. Trinchieri, and
M. Kamoun.
1991.
Reconstitution of an active surface CD2 by DNA transfer in CD2![]() |
47. | Killeen, N., S.G. Stuart, and D.R. Littman. 1992. Development and function of T cells in mice with a disrupted CD2 gene. EMBO (Eur. Mol. Biol. Organ.) J. 11: 4329-4336 [Abstract]. |
48. |
Teh, S.-J.,
N. Killeen,
A. Tarakhovsky,
D.R. Littman, and
H.-S. Teh.
1997.
CD2 regulates the positive selection and
function of antigen-specific CD4![]() |
49. | Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes. Nature (Lond.). 387: 569-572 [Medline]. |
50. | Romagnoli, P., and C. Bron. 1997. Phosphatidylinositol-based glysolipid-anchored proteins enhance proximal TCR signaling events. J. Immunol. 158: 5757-5764 [Abstract]. |
51. | Trevisan, M., and N.N. Iscove. 1995. Phenotypic analysis of murine long-term hemopoietic reconstituting cells quantitated competitively in vivo and comparison with more advanced colony-forming progeny. J. Exp. Med. 181: 93-103 [Abstract]. |
52. | Spangrude, G.J., and D.M. Brooks. 1993. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood. 82: 3327-3332 [Abstract]. |
53. |
Cheng, J.,
S. Baumhueter,
G. Cacalano,
K. Carver-Moore,
H. Thibodeaux,
R. Thomas,
H.E. Broxmeyer,
S. Cooper,
N. Hague,
M. Moore, and
L.A. Lasky.
1996.
Hematopoietic
defects in mice lacking the sialomucin CD34.
Blood.
87:
479-490
|
54. |
Suzuki, A.,
D.P. Andrew,
J.A. Gonzalo,
M. Fukumoto,
J. Spellberg,
M. Hashiyama,
H. Takimoto,
N. Gerwin,
I. Webb,
G. Molineux, et al
.
1996.
CD34-deficient mice have
reduced eosinophil accumulation after allergen exposure and
show a novel crossreactive 90-kD protein.
Blood.
87:
3550-3562
|
55. | Schorle, H., T. Holtschke, T. Hunig, A. Schimpl, and I. Horak. 1991. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature (Lond.). 352: 621-624 [Medline]. |
56. | Strober, W., and R.O. Ehrhardt. 1993. Chronic intestinal inflammation: an unexpected outcome in cytokine or T cell receptor mutant mice. Cell. 75: 203-205 [Medline]. |
57. | Willerford, D.M., J. Chen, J.A. Ferry, L. Davidson, A. Ma, and F.W. Alt. 1995. Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity. 3: 521-530 [Medline]. |
58. | Horak, I., J. Lohler, A. Ma, and K.A. Smith. 1995. Interleukin-2 deficient mice: a new model to study autoimmunity and self-tolerance. Immunol. Rev. 148: 35-44 [Medline]. |
59. |
Suzuki, H.,
G.S. Duncan,
H. Takimoto, and
T.W. Mak.
1997.
Abnormal development of intestinal intraepithelial
lymphocytes and peripheral natural killer cells in mice lacking
the IL-2 receptor beta chain.
J. Exp. Med.
185:
499-505
|
60. | Tivol, E.A., F. Borriello, A.N. Schweitzer, W.P. Lynch, J.A. Bluestone, and A.H. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity. 3: 541-547 [Medline]. |
61. | Waterhouse, P., J.M. Penninger, E. Timms, A. Wakeham, A. Shahinian, K.P. Lee, C.B. Thompson, H. Griesser, and T.W. Mak. 1995. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science (Wash. DC). 270: 985-988 [Abstract]. |
62. | Nagata, S., and P. Golstein. 1995. The Fas death factor. Science (Wash. DC). 267: 1449-1456 [Medline]. |
63. | Zheng, L., G. Fisher, R.E. Miller, J. Peschon, D.H. Lynch, and M.J. Lenardo. 1995. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature (Lond.). 377: 348-351 [Medline]. |
64. | Sytwu, H.K., R.S. Liblau, and H.O. McDevitt. 1996. The roles of Fas/APO-1 (CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice. Immunity. 5: 17-30 [Medline]. |
65. | Speiser, D.E., E. Sebzda, T. Ohteki, M.F. Bachmann, K. Pfeffer, T.W. Mak, and P.S. Ohashi. 1996. Tumor necrosis factor receptor p55 mediates deletion of peripheral cytotoxic T lymphocytes in vivo. Eur. J. Immunol. 26: 3055-3060 [Medline]. |