(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(NeuGc
2-8NeuGc)-Gg
Cer
(G
(NeuGc-NeuGc-)), and its epitope was determined to be
NeuGc
2-8NeuGc
2-3Gal
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.
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(
)was demonstrated to occur on mouse T
cells(1) , and Gg
Cer (
)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 Gg
Cer is expressed on cytotoxic
T cells (5) and activated macrophages (6) as well. The
association of Gb
Cer (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, IV
NeuGc-Gg
Cer
(G
(NeuGc)), IV
GalNAc,
IV
NeuGc-Gg
Cer
(GalNAc-G
(NeuGc)), and
IV
(Gal
1-3GalNAc),IV
NeuGc-Gg
Cer
(Gal-GalNAc-G
(NeuGc)) in mouse spleen (13, 14) and
IV
(NeuGc
2-8NeuGc)-Gg
Cer
(G
(NeuGc-NeuGc-)) (Table 1) in mouse
thymocytes(15) . These gangliosides are all synthesized by
extension of Gg
Cer, and activation of the biosynthetic
pathways for Gg
Cer is a unique characteristic of mouse
immune tissues. Schwarting and Gajewski (16) also indicated the
presence of gangliosides containing Gg
Cer and
sialidase-susceptible sialic acids in mouse lymphocytes.
Müthing et al.(17) have reported
restricted expression of GalNAc-G
(NeuAc) and
IV
NeuAc,
III
NeuAc-Gg
Cer(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) .
In the
course of our studies on gangliosides of mouse lymphocytes, we noted
that G(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.
The gangliosides used for
thin layer chromatography (TLC) standards and for the enzyme-linked
immunosorbent assay (ELISA) as antigens were as follows:
G(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
(NeuGc
2-8NeuGc)-Gb
Cer
(V
(NeuGc-NeuGc)-Gb
Cer) 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-).
Anti-G monoclonal antibody was generated by immunization of the purified
G
(NeuAc,NeuAc), and the characterization will be
published elsewhere. (
)
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 NeuGc2-8NeuGc
2- structure
binds most strongly, NeuAc
2-8NeuGc
2- and
NeuGc
2-8NeuAc
2- bind less well, and
NeuAc
2-8NeuAc
2- does not bind at all. This is supported
by the result with G
, which carries a
NeuAc
2-8NeuAc
2- terminus and does not react at all.
These results together suggest that the epitope of YK-3 is the
NeuGc2-8NeuGc
2-3Gal
1- structure. The results
of TLC immunostaining were completely consistent with those of ELISA
(data not shown).
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(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 NH
OH, 0.4% CaCl
(55:50:4:6,
v/v).
Although YK-3 can react with
G(NeuGc-NeuGc-) and
V
(NeuGc-NeuGc)-Gb
Cer, 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)-Gb
Cer 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-).
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(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.
As shown in the lower panels of Fig. 4, more than 70% of CD4 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.
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 on thymocytes was confirmed
by immunoelectron microscopy using ultrathin frozen sections of cells
(data not shown).
Figure 5:
IL-2 and IL-4 production by
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.
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(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.
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(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.
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(NeuAc
2-8NeuAc)-Gg
Cer
(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-Gg
Cer
antibody staining (data not shown).
The change of peanut
agglutinin-positive cells into negative cells during thymocyte
maturation is well documented. The structural basis for this is the
2-3 sialylation on the terminal Gal of the
Gal
1-3GalNAc- 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 Gg
Cer 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.
YK-3 stained a small number of rat
thymocytes and many rat CD4 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
Sia
2-8Sia
2-3Gal
1- structure was also
demonstrated in human T lymphocytes. Structural analysis of human
spleen and lymphocyte gangliosides indicated the occurrence of
G
(NeuAc-NeuAc-), IV
(NeuAc
2-8NeuAc)-nLc
Cer, and
IV
(NeuAc
2-8NeuAc)-nLc
Cer(47, 48) .
The terminal trisaccharide structure,
NeuAc
2-8NeuAc
2-3Gal
1-, was recognized by
three different monoclonal antibodies grouped into a new T cell
cluster, named CD
60. CD
60 is able to define a T
cell subpopulation that includes not only T helper but also cytotoxic T
effector cells. Recently, CD
60 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.
The limited expression of G(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.