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(Received for publication, July 11, 1994; and in revised form, November 9, 1994) From the
Monoclonal antibody YK-3 was established by immunization with
IV
Molecular recognition involving molecules on the cell surface
initiates immunological responses. The advantages of carbohydrate
chains as recognition structures have been discussed from the
viewpoints of their enormous structural diversity and abundant
expression on the cell surface. Glycosphingolipids have attracted
attention as molecules that might play a role in cell surface events.
G
In the
course of our studies on gangliosides of mouse lymphocytes, we noted
that G
The gangliosides used for
thin layer chromatography (TLC) standards and for the enzyme-linked
immunosorbent assay (ELISA) as antigens were as follows:
G
Anti-G
Figure 1:
Reactivity of YK-3 with various
gangliosides on ELISA. A, antibody dilution. The wells were
coated with each ganglioside (0.1 nmol/well) and then reacted with a
serial dilution of YK-3 from an initial concentration of 20 µg/ml. B, antigen dilution. The wells were coated with serial
dilutions of gangliosides (from 0.2 nmol/well) and then incubated with
YK-3 (20 µg/ml).
The sialic acid species of the terminal
disialyl structure is an important feature of YK-3 specificity, as
shown in Fig. 1. The NeuGc These results together suggest that the epitope of YK-3 is the
NeuGc
Figure 2:
TLC immunostaining of ganglioside
fractions from splenocytes and thymocytes. A-1 and B-1 were detected with resorcinol-HCl reagent. The other four plates
were subjected to immunostaining: A-2 and B-2, with
YK-3; A-3 and B-3, with
anti-G
Although YK-3 can react with
G
Figure 3:
Immunohistochemical staining of mouse
lymphoid tissues with YK-3. A, thymus; B, spleen; C, mesenteric lymph node. The bar represents 200
µm.
Figure 4:
Expression of
G
As shown in the lower panels of Fig. 4, more than 70% of CD4 To confirm that molecules
recognized by YK-3 are glycolipids and not glycoproteins, trypsin
digestion of splenocytes was performed before FACS analysis. Digestion
with trypsin (0.1%) for 1 h at 37 °C did not change the positive
staining with YK-3 but abolished the anti-CD4 mAb staining.
Furthermore, no band was detected on Western analysis with YK-3 in the
cell homogenates of splenocytes and thymocytes (data not shown). Sialidase treatment of splenocytes before FACS analysis caused a
remarkable decrease in the positive staining with YK-3. In addition,
the surface expression of G
Figure 5:
IL-2 and IL-4 production by
G
Figure 6:
The effect of killing cells with YK-3 and
complement on IL-2 and IL-4 production. A, control fraction
treated only with complement. B, fraction treated with YK-3
and complement. After ConA stimulation, secreted cytokines were
determined. The cell composition of each fraction is shown in the lower panel as in Fig. 5.
Using monoclonal antibody YK-3, we demonstrated that the
expression of G The ganglioside profiles of several mouse
Th1 and Th2 cell lines or clones were compared by Ebel et
al.(19) , and they demonstrated the specific expression of
G Hayakawa and Hardy (36, 37, 38) described mAb SM3G11, which can
distinguish mouse naive T cells or Th1-like cells, both producing IL-2,
from other T cells. Greer et al. suggested that 3G11 antigen
on native mouse lymphocytes is a ganglioside(39) , and quite
recently, Dittrich et al.(40) reported that antigens
recognized by SM3G11 are
IV The change of peanut
agglutinin-positive cells into negative cells during thymocyte
maturation is well documented. The structural basis for this is the
YK-3 stained a small number of rat
thymocytes and many rat CD4 The limited expression of G
Volume 270,
Number 8,
Issue of February 24, 1995 pp. 3876-3881
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(NeuGc
2
8NeuGc)-Gg
Cer
Is Restricted to CD4
T Cells Producing Interleukin-2
and a Small Population of Mature Thymocytes in Mice (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(NeuGc2-8NeuGc)-Gg
Cer
(G
(NeuGc-NeuGc-)), and its epitope was determined to be
NeuGc2-8NeuGc2-3Gal
1. Thin layer
chromatography immunostaining with YK-3 detected only
G(NeuGc-NeuGc-) among the gangliosides of mouse
thymocytes and splenocytes. Immunohistochemical staining with YK-3
visualized the medulla of mouse thymus and T cell-dependent areas of
mouse spleen and mesenteric lymph nodes. Two-color flow cytometry
demonstrated that G(NeuGc-NeuGc-) was expressed on a
quarter of CD3
mature thymocytes and strongly
expressed on three quarters of CD4 T cells in the
spleen, lymph nodes, and peripheral blood but not on CD8 T cells or B cells. G(NeuGc-NeuGc-)-positive cells
and negative cells were separated by panning with YK-3 on Petri dishes
into adherent and nonadherent fractions. Following stimulation with
concanavalin A, adherent cells, predominantly
G(NeuGc-NeuGc-), produced more
interleukin-2 (IL-2) and markedly less interleukin-4 (IL-4) than
nonadherent cells. This conclusion is supported by data obtained by
lysis of cells by YK-3 and complement. These data indicate that the
cell surface expression of G(NeuGc-NeuGc-) is restricted
to a small number of mature thymocytes and a subset of CD4 T cells, which produce abundant IL-2 and very little IL-4,
suggesting that G(NeuGc-NeuGc-) is an excellent marker
for mouse naive T or T helper 1-like cells in vivo.
(
)was demonstrated to occur on mouse T
cells(1) , and GgCer (
)has been accepted
as a surface marker for mouse NK cells (2, 3) and as a
differentiation marker for fetal thymocytes(4) . It was
reported subsequently that GgCer is expressed on cytotoxic
T cells (5) and activated macrophages (6) as well. The
association of GbCer (CD77) with human B cells (7, 8, 9) and sulfoglucuronyl
glycosphingolipid with human NK cells (HNK-1) (10, 11, 12) were also described. Recent
studies have indicated that glycosphingolipids of immunocytes are more
complex than appreciated previously. We reported the presence of unique
gangliosides, IVNeuGc-GgCer
(G
(NeuGc)), IVGalNAc,
IVNeuGc-GgCer
(GalNAc-G(NeuGc)), and
IV(Gal1-3GalNAc),IVNeuGc-GgCer
(Gal-GalNAc-G(NeuGc)) in mouse spleen (13, 14) and
IV(NeuGc2-8NeuGc)-GgCer
(G(NeuGc-NeuGc-)) (Table 1) in mouse
thymocytes(15) . These gangliosides are all synthesized by
extension of GgCer, and activation of the biosynthetic
pathways for GgCer is a unique characteristic of mouse
immune tissues. Schwarting and Gajewski (16) also indicated the
presence of gangliosides containing GgCer and
sialidase-susceptible sialic acids in mouse lymphocytes.
Müthing et al.(17) have reported
restricted expression of GalNAc-G(NeuAc) and
IVNeuAc,
III
NeuAc-GgCer(G
(NeuAc,NeuAc)) (Table 1) in mature or stimulated T cells, which contrasts with
the evidence that gangliosides synthesized from G restricted to B lymphocytes(18) . Furthermore, their
group demonstrated that G
was only detected in mouse T
helper 2 (Th2) cell lines and that G
was preferably
expressed on mouse T helper 1 (Th1) cell lines(19) .
(NeuGc-NeuGc-) is a major disialoganglioside in
thymocytes and occurs in splenocytes as well. We established monoclonal
antibody (mAb) YK-3 by immunizing a mouse with purified
G(NeuGc-NeuGc-) and studied the expression of
G(NeuGc-NeuGc-) in mouse lymphocytes. Here we report that
the expression of G(NeuGc-NeuGc-) in vivo is
limited to a small number of mature thymocytes and CD4 lymphocytes producing interleukin-2 (IL-2), naive T ()or Th1-like cells.
Materials
C3H/He Slc female mice aged 8 weeks,
used for immunization with G(NeuGc-NeuGc-), and BALB/c
CrSlc, C57BL/6 CrSlc, and DBA/2 CrSlc female mice aged 6-10 weeks
were purchased from the Shizuoka Agricultural Cooperative Association
for Laboratory Animals (Shizuoka, Japan).
(NeuAc) purified from dog erythrocytes(20) ;
G
(NeuAc) from the brain of a patient with Tay-Sachs
disease(21) ; G(NeuAc), G(NeuAc),
and G
(NeuAc) from bovine brain; G(NeuGc),
G(NeuGc), G(NeuGc), and
G(NeuGc) from ICR mouse liver(22) ;
G(NeuGc) from mouse spleen(13) ;
G(NeuAc-NeuGc-) and G(NeuGc-NeuGc-) from a
transplanted WHT/Ht mouse thymoma(15) ;
G
(NeuAc, NeuAc) from frog peripheral nerve, donated
by Dr. M. Ohashi, Ochanomizu University(23) ;
G
(NeuAc-NeuAc-), G(NeuAc-NeuGc-),
G(NeuGc-NeuAc-), and G(NeuGc-NeuGc-) from
bear erythrocytes(24) ;
V(NeuGc2-8NeuGc)-Gb
Cer
(V(NeuGc-NeuGc)-GbCer) from mouse kidney (25) ; and a ganglioside mixture from mouse brain. Salmonella minnesota R595, donated by Dr. Kanagasaki
(Institute of Medical Science, University of Tokyo) was used as an
adjuvant for the immunization of mice with
G(NeuGc-NeuGc-).Antibodies
YK-3 mAb was established in our
laboratory according to the method described by Ozawa et
al.(26) , using 40 µg of purified
G(NeuGc-NeuGc-) and 250 µg of S. minnesota R595 powder per mouse. Antibodies in the culture supernatant
produced by the hybridoma were monitored by ELISA with
G(NeuGc-NeuGc-) as an antigen. Large scale production of
YK-3 was performed in a synthetic medium, ASF 104 (Ajinomoto, Tokyo),
and YK-3 was purified by ammonium sulfate precipitation and column
chromatography on Sepharose CL-6B (Pharmacia Biotech Inc.). The
purified YK-3 was biotinylated with biotin hydrazide (Pierce) by the
method of O'Shannessy et al.(27) . The isotype
of the antibody was determined with a mouse monoclonal antibody kit
(Amersham International plc, U. K.).
monoclonal antibody was generated by immunization of the purified
G
(NeuAc,NeuAc), and the characterization will be
published elsewhere. ()ELISA
YK-3 was characterized by ELISA
according to a slight modification of the method of
Holmgren(28) , using biotinylated anti-mouse IgM antibody (5
µg/ml) and a Vectastain ABC solution (Vector Laboratories,
Burlingame, CA)(14) .Preparation of Gangliosides from Splenocytes and
Thymocytes
The spleens and thymuses removed from 80 BALB/c mice
aged 6 weeks were pressed between two frosted glass slides, and then
the resulting cell suspension in phosphate-buffered saline (PBS) was
filtered through a nylon mesh. The cells were washed twice with PBS,
and then the pelleted cells were subjected to lipid extraction. In the
preparation of splenocytes, erythrocytes were lysed by ammonium
chloride treatment before the PBS washing. Gangliosides were extracted
and fractionated into monosialo and disialo fractions by the method
described in the previous paper(15) . Splenocytes (8 
10
cells) or thymocytes (1.3 10
cells)
were used for the extraction.TLC Immunostaining
The binding of YK-3 to various
gangliosides and the occurrence of G(NeuGc-NeuGc-) and
G
(NeuAc, NeuAc) in splenocytes and thymocytes were
analyzed by TLC immunostaining, according to the methods of Hansson et al. (29) and Yoshino et al. (30) with a slight modification(14) . YK-3 or
anti-G
(NeuAc,NeuAc) at a concentration of 20
µg/ml in PBS containing 1% bovine serum albumin (1% BSA-PBS),
biotinylated anti-mouse IgM antibody (5 µg/ml), and the Vectastain
ABC solution were used.Staining of Tissue Sections
Frozen tissue sections
of the spleen, thymus, and lymph node were obtained by a modification
of the method of Barthel and Raymond(31) , using tissue blocks
fixed in 4% paraformaldehyde-PBS at 4 °C overnight and then
embedded in a solution of OCT compound (Miles Laboratories, Elkhart,
IN), 0.6 M sucrose in the phosphate buffer (1:2, v/v). The
sections were mounted on polylysine-coated slides and blocked with
irrelevant mouse IgM at 50 µg/ml in 5% BSA-PBS at room temperature
for 1 h. The sections were then stained with biotinylated YK-3 at 20
µg/ml in 5% BSA-PBS at 4 °C overnight and with fluorescein
isothiocyanate-conjugated (FITC) avidin (Vector Laboratories) at 20
µg/ml at room temperature for 2 h. The stained sections were
mounted with 20% glycerol in PBS containing an anti-quencher and then
photographed under a Zeiss photomicroscope (Carl Zeiss, Germany).Two-color Flow Cytometric Analysis
Under ether
anesthesis, blood was taken into heparinized syringes by means of
cardiac puncture, and then the spleen, thymus, and mesenteric lymph
nodes were removed from BALB/c mice. Single-cell suspensions of
thymocytes, lymph node cells, and splenocytes were prepared. For
splenocytes, ammonium chloride treatment was applied to lyse
erythrocytes. Peripheral blood lymphocytes were prepared by
centrifugation using an M-SMF solution (Japan Immunoresearch
Laboratories Co., Ltd., Takasaki, Japan). 10 cells in 50
µl of PBS containing 1% fetal calf serum (FCS) and 0.05% sodium
azide were incubated with biotinylated YK-3 at 0.4 µg/ml for 30 min
on ice. The cells were then incubated with FITC avidin (Vector
Laboratories) and phycoerythrin-conjugated (PE) anti-mouse CD3 or CD4
mAbs (Pharmingen, San Diego, CA) for 30 min on ice and analyzed by an
Epics-Profile analyzer (Coulter, Hialeah, FL).Separation of G
A single
cell suspension of splenocytes (4 (NeuGc-NeuGc-) Positive and
Negative Cells and Concanavalin A (ConA) Stimulation 10
cells) was
obtained from four mice of each strain, BALB/c, C57BL/6, and DBA/2, as
described above. To enrich CD4 cells, the splenocytes
were applied to an affinity column, the Cellect
plus mouse CD4 kit
(Biotex Laboratories Inc., Edmonton, Canada), according to the
manufacturer's instructions. The CD4 cells were
obtained in the pass-through fraction, and aliquots of the eluent were
subjected to ConA activation and FACS analysis with antibodies against
surface markers for subset characterization. A population of
CD4-enriched cells was prepared by panning with
anti-mouse CD4 mAb (GK1.5). The cells were incubated for 1 h at room
temperature in a plastic dish (Falcon 1001; Becton Dickinson Labware,
Lincoln Park, NJ) that had been coated with anti-CD4 mAb (15
µg/ml). After nonadherent cells had been gently washed out from the
dish, the adherent cells were collected by forceful pipetteting and
subjected to further panning with YK-3. The plastic dish was prepared
by incubation with YK-3 (30 µg/ml in PBS) at 4 °C overnight,
washing three times with 5 ml of PBS, and blocking with 2% FCS-PBS for
30 min at room temperature. The cells suspended in 2% FCS-PBS were
added to the dish at 3 10
cells/dish and then
incubated for 1 h at room temperature. After the incubation,
nonadherent and adherent cells were collected separately from the dish,
an aliquot of each was analyzed by FACS, and the remainder of the cells
were subjected to ConA activation. The cells for activation were
resuspended at 2.5 10 cells/ml in RPMI 1640 medium
supplemented with 5% FCS, L-glutamine, 2-mercaptoethanol, and
antibiotics, applied to a 96-well microtiter plate (200 µl/well),
and stimulated with ConA (Sigma) at 4 µg/ml. After a 2-day
incubation at 37 °C, the culture supernatant was subjected to the
lymphokine assay described below. Cell growth was monitored by WST-1
assay according to the method of Mosmann(32) . For
characterization of subsets, PE anti-mouse CD4 mAb (Pharmingen), PE
anti-mouse CD8 mAb and PE anti-mouse B220 mAb (Caltag Laboratories, San
Francisco, CA) and FITC anti-mouse Mac-1 (Pharmingen) were used.YK-3 Dependent Complement Killing
Assay
CD4 cells from BALB/c and C57BL/6
splenocytes were enriched as described above. 5 10 cells were suspended in 500 µl of CY medium (Cedarlane
Laboratories Ltd., Ontario, Canada) and treated with YK-3 at 10
µg/ml for 1 h on ice. After washing with CY medium, a complement
solution (Low-Tox-M Rabbit Complement, Cedarlane), 1:10 dilution, was
added to the pelleted cells. The cell suspensions were incubated for 1
h at 37 °C and then washed with CY medium. An aliquot of the cells
was analyzed by FACS to obtain population profiles, the rest were
stimulated with ConA, as described above, and the production of
cytokines was analyzed. As a control, the cells treated with only
complement were used.Lymphokine Assay
Secreted IL-2 or IL-4 was
measured by sandwich ELISA technique, using two monoclonal antibodies.
For the IL-2 assay, each well of a 96-well microtiter plate (Easy Wash,
Corning, New York) was incubated with anti-mouse IL-2 mAb (1A12;
Pharmingen) at 6 µl/ml in 0.1 M NaHCO buffer
(pH 8.2) at 4 °C overnight. After washing the plate with PBS
containing 0.05% (v/v) Tween-20 (Tween-PBS), each well was blocked with
1% BSA-PBS at room temperature for 1 h. The supernatants of
ConA-stimulated cells described above or recombinant (r) IL-2 standards
(Pharmingen) at various concentrations were added and incubated for 4 h
at room temperature. After the washing, biotinylated anti-mouse IL-2
mAb (5H4; Pharmingen) at 8 µg/ml in 1% BSA-PBS was added and
incubated for 1 h at room temperature. Finally, the plate was incubated
with the Vectastain ABC solution for 30 min at room temperature and
then subjected to detection of peroxidase activity. The IL-2
concentration of culture supernatants was calculated from a standard
curve obtained with rIL-2. For the IL-4 assay, the same method as
described above was adopted without using anti-mouse IL-4 mAb (1D11;
Pharmingen), biotinylated anti-mouse IL-4 mAb (24D2; Pharmingen), and
rIL-4 as a standard (Life Technologies, Inc.).
Characterization of YK-3
The monoclonal antibody
YK-3 is an IgM
. The ELISA data (Fig. 1A) indicate
that YK-3 reacts most strongly with
NeuGc2-8NeuGc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer
(G(NeuGc-NeuGc-)) and less strongly with
NeuGc2-8NeuGc2-3Gal1-4Glc1-Cer
(G(NeuGcNeuGc-)) and
NeuGc2-8NeuGc2-3Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-Cer
(V(NeuGc-NeuGc)-GbCer). Titration of antigen (Fig. 1B) demonstrated that the three gangliosides had
similar affinity for the antibody. The reactivity of NeuGc2-
8NeuGc2-3Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-Cer
clearly indicates that the -3Gal1- structure is not involved in
the epitope. The binding of
NeuGc2-8NeuGc2-3Gal1-4Glc1-Cer
confirms that the fourth sugar residue from the terminal is not
required for reactivity.
2-8NeuGc2- structure
binds most strongly, NeuAc2-8NeuGc2- and
NeuGc2-8NeuAc2- bind less well, and
NeuAc2-8NeuAc2- does not bind at all. This is supported
by the result with G, which carries a
NeuAc2-8NeuAc2- terminus and does not react at all. 2-8NeuGc2-3Gal1- structure. The results
of TLC immunostaining were completely consistent with those of ELISA
(data not shown).Detection of G
As shown in Fig. 2, A-1 and B-1, more than 10 bands were
detected for both splenocytes and thymocytes (lanes 4 and 5). This complexity is caused by heterogeneity in fatty acid
and sialic acid structures and the occurrence of gangliosides extending
from Gg(NeuGc-NeuGc-) in Ganglioside
Fractions of Splenocytes and ThymocytesCer in addition to the a and b series of
gangliosides. The ganglioside profiles of thymocytes and splenocytes
were quite similar, except that the G concentration is
higher in splenocytes. The bands migrating between G and
G
for splenocytes and thymocytes are probably
G(NeuGc-NeuGc-) and G
(NeuAc,NeuAc) on
the basis of their comigration with the reference compounds (Fig. 2, A-1). The occurrence of
G(NeuGc-NeuGc-) in both splenocyte and thymocyte
gangliosides was confirmed by staining with YK-3, as shown in Fig. 2, A-2 and B-2.
G
(NeuAc,NeuAc) was also detected in both types of
cells, as shown in Fig. 2, A-3 and B-3,
together with a slow migrating faint band, the structure of which has
not been determined.
(NeuAc,NeuAc) mAb. Lanes 1-3 and 6-8 contained reference gangliosides: lane 1,
G(NeuAc), G(NeuAc), and
G(NeuAc); lane 2, G(NeuGc),
G(NeuGc), and G(NeuGc); lane 3,
gangliosides from mouse brain; lane 6, G(NeuGc)
and G(NeuGc-NeuGc-); lane 7,
G
(NeuAc,NeuAc); lane 8,
G(NeuGc), G(NeuAc-NeuGc-), and
G(NeuGc-NeuGc-). Lanes 4 and 5, the
gangliosides from mouse splenocytes and thymocytes, respectively. The
plates in panel A were developed with a solvent system of
chloroform, methanol, 0.2% CaCl (55:45:10, v/v), and those
in panel B with a solvent system of chloroform, methanol, 5 M NHOH, 0.4% CaCl (55:50:4:6,
v/v).
(NeuGc-NeuGc-) and
V(NeuGc-NeuGc)-GbCer, and weakly with
G(NeuGc-NeuAc-) and G(NeuAc-NeuGc-), as shown
in Fig. 1, YK-3 did not visualize these gangliosides in
thymocytes or splenocytes on TLC immunostaining, indicating that the
occurrence of G and
V(NeuGc-NeuGc)-GbCer in both types of cells is
negligible. Thus, it is concluded that the positive staining on FACS
and immunohistochemistry described below is caused by
G(NeuGc-NeuGc-).Staining of Tissue Sections with YK-3
In the
thymus, cells in the medulla were strongly stained, and a network
structure was weakly detected (Fig. 3A). Treatment of a
thymus section with methanol and chloroform, methanol (1:1, v/v)
abolished the positive staining, indicating that the staining is caused
by glycolipids. In the spleen, T cell-dependent areas were brightly
stained, and follicle cells and germinal centers were not stained (Fig. 3B). In the lymph node, T cell-dependent areas
were brightly stained but not follicle cell regions (Fig. 3C). Controls with biotinylated irrelevant mouse
IgM instead of biotinylated YK-3 exhibited weak and diffuse staining.
These results suggest that only mature thymocytes found in the medulla
and mature T cells in the spleen and lymph nodes are
G(NeuGc-NeuGc-)-positive.
FACS Analysis
As shown in the upper panels of Fig. 4, YK-3-positive cells comprise 3% of the total
thymocytes and a quarter of the CD3 thymocytes. The
YK-3-positive cells account for half of CD3 splenocytes, 60% of CD3 lymph node lymphocytes,
and 70% of CD3 peripheral lymphocytes. These results
indicate that immature thymocytes that are CD3-negative or weakly
positive do not express G(NeuGc-NeuGc-) and that a subset
of CD3-positive mature thymocytes express
G(NeuGc-NeuGc-).
(NeuGc-NeuGc-) in CD3- or CD4-positive cells.
Lymphocytes were prepared from various origins: A, thymus; B, spleen; C, mesenteric lymph node; D,
peripheral blood. The proportion (%) of positive cells for each marker
is indicated in each quadrant.
cells in
splenocytes, and lymph node lymphocytes and peripheral lymphocytes are
G(NeuGc-NeuGc-)-positive, and almost none of the
CD4
cells are G(NeuGc-NeuGc-)-positive,
indicating that G(NeuGc-NeuGc-) is consistently expressed
on a part of CD4 cells. on thymocytes was confirmed
by immunoelectron microscopy using ultrathin frozen sections of cells
(data not shown).IL-2 and IL-4 Production by
G
Fig. 5presents three independent experiments using
different strains of mice. The cells in fraction A, the
CD4(NeuGc-NeuGc-)-positive and Negative T Cells-enriched fractions, included
G(NeuGc-NeuGc)-positive and negative cells in the ratio
of almost 1:1. Fraction B, the cells that did not adhere to YK-3-coated
dishes, contained G
(NeuGc-NeuGc-)-positive and negative cells stimulated
with ConA. A, fraction A, cells obtained with an
immunoaffinity column for CD4 enrichment. B,
fraction B, cells nonadherent to the YK-3-coated dish. C,
fraction C, cells adherent to the YK-3-coated dish. Cytokines secreted
into the culture supernatant were determined by ELISA, and the values
are indicated in units, as calculated from a standard curve. The cell
composition of each fraction was analyzed by FACS, and the results are
summarized in the lower row for each
experiment.
YK-3-dependent Complement Killing
Analysis
Treatment of CD4 splenocytes with YK-3
and complement depleted G
(NeuGc-NeuGc-) is limited to a small
number of mature CD3 thymocytes and a subset of
CD4 T cells, which produce abundant IL-2 and little
IL-4. Therefore, G(NeuGc-NeuGc-) should be an excellent
marker for mouse naive T or Th1-like cells in vivo. Although
it is still a matter of controversy how naive T helper cells
differentiate into Th1 or Th2 cells, there appear to be at least two
distinguishable T helper subsets in vitro. One produces mainly
IL-2 and interferon-
to support inflammatory processes (Th1), and
the other produces mainly IL-4 to facilitate B cell activation and
differentiation (Th2)(35) . If specific surface markers
allowing differentiation of these two subsets become available, they
will be very useful for immunological research on Th1 and Th2 cells and
their progenitor cells.(NeuAc,NeuAc) in cultured Th2 cells and the preferential
expression of G
(NeuAc,NeuAc) in cultured Th1 cells.
We examined several cultured lines of Th1 and Th2 cells by FACS with
YK-3 but did not observe any positive staining, indicating that
cultured Th1 cells do not express G(NeuGc-NeuGc-) on
their surface. These results suggest that G(NeuGc-NeuGc-)
is not directly involved in IL-2 production and indicate that the
glycolipid profiles of cultured and cloned cells are different from
those of native cells and that the analysis of structures expressed in
native cells is required.(NeuAc2-8NeuAc)-GgCer
(G(NeuAc-NeuAc-)) and G(NeuGc-NeuGc-). Our
present results indicate that the native antigen of mouse lymphocytes
recognized by YK-3 is G(NeuGc-NeuGc-) but not
G(NeuAc-NeuAc-). This conclusion is supported by the
evidence that G(NeuAc-NeuAc-) is not detected in mouse
splenocytes by TLC with sialidase treatment and anti-GgCer
antibody staining (data not shown).2-3 sialylation on the terminal Gal of the
Gal1-3GalNAc- structure of O-linked carbohydrate
chains (41) . Gillespie et al.(42) demonstrated that this change is mediated by the
induction of -2,3-sialyltransferase activity through an increase
in its mRNA. This sialyltransferase and other
-2,3-sialyltransferases cloned thereafter were reported to be
responsible for the 2-3-sialylation of
glycosphingolipids(43, 44) , so it would be
interesting to determine which sialyltransferase is responsible for the
sialylation of GgCer to G, the precursor for
G, and to study its mechanism of induction. For the
expression of G, another sialyltransferase,
-2,8-sialyltransferase, is required, and cloning of its cDNA is
required to study the relationship between 2-8 sialylation
and thymocyte maturation. splenocytes on FACS
analysis (data not shown). The immunohistochemical staining of rat
thymus with YK-3 demonstrated that mature thymocytes in the medulla
express G(NeuGc-NeuGc-) (data not shown). The occurrence
of G(NeuGc-NeuGc-) in rat thymocytes and splenic T cells
was reported by Nohara et al.(45, 46) .
Interestingly, the occurrence of gangliosides containing the
Sia2-8Sia2-3Gal1- structure was also
demonstrated in human T lymphocytes. Structural analysis of human
spleen and lymphocyte gangliosides indicated the occurrence of
G(NeuAc-NeuAc-), IV
(NeuAc2-8NeuAc)-nLcCer, and
IV(NeuAc2-8NeuAc)-nLcCer(47, 48) .
The terminal trisaccharide structure,
NeuAc2-8NeuAc2-3Gal1-, was recognized by
three different monoclonal antibodies grouped into a new T cell
cluster, named CD
60. CD60 is able to define a T
cell subpopulation that includes not only T helper but also cytotoxic T
effector cells. Recently, CD60 was reported to recognize
acetylated forms of G more strongly(49) .
Anti-G antibodies were reported to induce proliferation of
human T cells and to increase cytotoxicity of human cytotoxic T
cells(50, 51, 52) . These results indicate
that Sia-Sia-Gal structure is also conserved in human mature T cells. (NeuGc-NeuGc-) on a small
subset of mature thymocytes and naive T or Th1-like cells allows us to
speculate that G(NeuGc-NeuGc-) on the cell surface may
act as a functional molecule critical for the differentiation from
thymocytes to a particular subset of Th1-like T helper cells.
))Cer, Gal13GalNAc1-4Gal1-4Glc1-Cer;
GbCer, Gal1-4Gal1-4Glc1-Cer;
nLcCer, Gal1-4GlcNAc1-3Gal1-4Glc1-Cer;
nLcCer,
Gal1-4(GlcNAc1-3Gal1-4)Glc1-Cer; NeuGc, N-glycolylneuraminic acid; Th1, T helper 1; Th2, T helper 2;
IL-2, interleukin-2; IL-4, interleukin-4; mAb, monoclonal antibody;
PBS, phosphate-buffered saline; ConA, concanavalin A; TLC, thin layer
chromatography; ELISA, enzyme-linked immunosorbent assay; BSA, bovine
serum albumin; FITC, fluorescein isothiocyanate-conjugated; FCS, fetal
calf serum; FACS, fluorescence-activated cell sorter; PE,
phycoerythrin-conjugated.))
We thank Dr. S. Kanagasaki (Institute of Medical
Science, the University of Tokyo) for the gift of S. Minnesota R595, Dr. Y. Asano (the University of Tokyo) for the gift of the
mouse Th1 and Th2 cell lines, and Dr. M. Ohashi (Ochanomizu University)
for the gift of the G
(NeuAc, NeuAc) ganglioside. We
also thank Drs. M. Kotani and M. Tanaka (Tokyo Metropolitan Institute
of Medical Science), Dr. T. Tamatani (Pharmaceutical Basic Research
Laboratories, JT Inc.), and Dr. M. Miyasaka (Biomedical Research
Center, Osaka University Medical School) for helpful discussions; Dr.
K. Tanoue (Tokyo Metropolitan Institute of Medical Science) for
immunoelectron microscopic analysis; Dr. T. Yamakawa (Tokyo College of
Pharmacy) for encouragement; and Dr. D. M. Marcus (Baylor College of
Medicine) for assistance with the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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