©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
IV(NeuGc28NeuGc)-GgCer Is Restricted to CD4 T Cells Producing Interleukin-2 and a Small Population of Mature Thymocytes in Mice (*)

(Received for publication, July 11, 1994; and in revised form, November 9, 1994)

Kyoko Nakamura (1) Hidenori Suzuki (2) Yoshio Hirabayashi (3) Akemi Suzuki (1)(§)

From the  (1)Department of Membrane Biochemistry and (2)Department of Cardiovascular Research, Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo, 113, Japan and the (3)Laboratory for Glyco Cell Biology, Frontier Research Program, The Institute of Chemical and Physical Research, Wako-shi, Saitama, 351-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Monoclonal antibody YK-3 was established by immunization with IV^3alpha(NeuGcalpha2-8NeuGc)-Gg(4)Cer (G(NeuGc-NeuGc-)), and its epitope was determined to be NeuGcalpha2-8NeuGcalpha2-3Galbeta1. 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.


INTRODUCTION

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(^1)was demonstrated to occur on mouse T cells(1) , and Gg(4)Cer (^2)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(4)Cer is expressed on cytotoxic T cells (5) and activated macrophages (6) as well. The association of Gb(3)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^3alphaNeuGc-Gg(4)Cer (G(NeuGc)), IV^4betaGalNAc, IV^3alphaNeuGc-Gg(4)Cer (GalNAc-G(NeuGc)), and IV^4beta(Galbeta1-3GalNAc),IV^3alphaNeuGc-Gg(4)Cer (Gal-GalNAc-G(NeuGc)) in mouse spleen (13, 14) and IV^3alpha(NeuGcalpha2-8NeuGc)-Gg(4)Cer (G(NeuGc-NeuGc-)) (Table 1) in mouse thymocytes(15) . These gangliosides are all synthesized by extension of Gg(4)Cer, and activation of the biosynthetic pathways for Gg(4)Cer is a unique characteristic of mouse immune tissues. Schwarting and Gajewski (16) also indicated the presence of gangliosides containing Gg(4)Cer and sialidase-susceptible sialic acids in mouse lymphocytes. Müthing et al.(17) have reported restricted expression of GalNAc-G(NeuAc) and IV^3alphaNeuAc, III^6alphaNeuAc-Gg(4)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 (^3)or Th1-like cells.


EXPERIMENTAL PROCEDURES

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).

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^3alpha(NeuGcalpha2-8NeuGc)-Gb(5)Cer (V^3(NeuGc-NeuGc)-Gb(5)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-).

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.).

Anti-G monoclonal antibody was generated by immunization of the purified G(NeuAc,NeuAc), and the characterization will be published elsewhere. (^4)

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 times 10^9 cells) or thymocytes (1.3 times 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). 10^6 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(NeuGc-NeuGc-) Positive and Negative Cells and Concanavalin A (ConA) Stimulation

A single cell suspension of splenocytes (4 times 10^8 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 Cellectbulletplus 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 times 10^7 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 times 10^6 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 times 10 ^6 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(3) 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.).


RESULTS

Characterization of YK-3

The monoclonal antibody YK-3 is an IgMkappa. The ELISA data (Fig. 1A) indicate that YK-3 reacts most strongly with NeuGcalpha2-8NeuGcalpha2-3Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1-Cer (G(NeuGc-NeuGc-)) and less strongly with NeuGcalpha2-8NeuGcalpha2-3Galbeta1-4Glcbeta1-Cer (G(NeuGcNeuGc-)) and NeuGcalpha2-8NeuGcalpha2-3Galbeta1-3GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-Cer (V^3(NeuGc-NeuGc)-Gb(5)Cer). Titration of antigen (Fig. 1B) demonstrated that the three gangliosides had similar affinity for the antibody. The reactivity of NeuGcalpha2- 8NeuGcalpha2-3Galbeta1-3GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-Cer clearly indicates that the -3Galalpha1- structure is not involved in the epitope. The binding of NeuGcalpha2-8NeuGcalpha2-3Galbeta1-4Glcbeta1-Cer confirms that the fourth sugar residue from the terminal is not required for reactivity.


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 NeuGcalpha2-8NeuGcalpha2- structure binds most strongly, NeuAcalpha2-8NeuGcalpha2- and NeuGcalpha2-8NeuAcalpha2- bind less well, and NeuAcalpha2-8NeuAcalpha2- does not bind at all. This is supported by the result with G, which carries a NeuAcalpha2-8NeuAcalpha2- terminus and does not react at all.

These results together suggest that the epitope of YK-3 is the NeuGcalpha2-8NeuGcalpha2-3Galbeta1- structure. The results of TLC immunostaining were completely consistent with those of ELISA (data not shown).

Detection of G(NeuGc-NeuGc-) in Ganglioside Fractions of Splenocytes and Thymocytes

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(4)Cer 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.


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(2) (55:45:10, v/v), and those in panel B with a solvent system of chloroform, methanol, 5 M NH(4)OH, 0.4% CaCl(2) (55:50:4:6, v/v).



Although YK-3 can react with G(NeuGc-NeuGc-) and V^3(NeuGc-NeuGc)-Gb(5)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^3(NeuGc-NeuGc)-Gb(5)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-).

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.


Figure 3: Immunohistochemical staining of mouse lymphoid tissues with YK-3. A, thymus; B, spleen; C, mesenteric lymph node. The bar represents 200 µm.



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-).


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).

IL-2 and IL-4 Production by G(NeuGc-NeuGc-)-positive and Negative T Cells

Fig. 5presents three independent experiments using different strains of mice. The cells in fraction A, the CD4-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


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.



YK-3-dependent Complement Killing Analysis

Treatment of CD4 splenocytes with YK-3 and complement depleted 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.




DISCUSSION

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^3alpha(NeuAcalpha2-8NeuAc)-Gg(4)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(4)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 alpha2-3 sialylation on the terminal Gal of the Galbeta1-3GalNAc- structure of O-linked carbohydrate chains (41) . Gillespie et al.(42) demonstrated that this change is mediated by the induction of alpha-2,3-sialyltransferase activity through an increase in its mRNA. This sialyltransferase and other alpha-2,3-sialyltransferases cloned thereafter were reported to be responsible for the alpha2-3-sialylation of glycosphingolipids(43, 44) , so it would be interesting to determine which sialyltransferase is responsible for the sialylation of Gg(4)Cer to G, the precursor for G, and to study its mechanism of induction. For the expression of G, another sialyltransferase, alpha-2,8-sialyltransferase, is required, and cloning of its cDNA is required to study the relationship between alpha2-8 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 Siaalpha2-8Siaalpha2-3Galbeta1- structure was also demonstrated in human T lymphocytes. Structural analysis of human spleen and lymphocyte gangliosides indicated the occurrence of G(NeuAc-NeuAc-), IV^3alpha (NeuAcalpha2-8NeuAc)-nLc(4)Cer, and IV^3alpha(NeuAcalpha2-8NeuAc)-nLc(6)Cer(47, 48) . The terminal trisaccharide structure, NeuAcalpha2-8NeuAcalpha2-3Galbeta1-, was recognized by three different monoclonal antibodies grouped into a new T cell cluster, named CD(w)60. CD(w)60 is able to define a T cell subpopulation that includes not only T helper but also cytotoxic T effector cells. Recently, CD(w)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.


FOOTNOTES

*
This work was supported in part by Grant-in-Aid for Scientific Research in Priority Areas 05274107 from the Ministry of Education, Science, and Culture of Japan and a grant from the Human Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Membrane Biochemistry, Tokyo Metropolitan Inst. of Medical Science, 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3823-2101 (ext. 5483); Fax: 81-3-5685-6607; asuzuki{at}rinshoken.or.jp.

(^1)
The nomenclature for glycolipids follows the recommendations (53) of the IUB, and the ganglioside nomenclature of Svennerholm (54) was used. The sialic acid species of gangliosides are indicated in parentheses.

(^2)
The abbreviations used are: Cer, ceramine; Gg(4)Cer, Galbeta13GalNAcbeta1-4Galbeta1-4Glcbeta1-Cer; Gb(3)Cer, Galalpha1-4Galbeta1-4Glcbeta1-Cer; nLc(4)Cer, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-Cer; nLc(6)Cer, Galbeta1-4(GlcNAcbeta1-3Galbeta1-4)(2)Glcbeta1-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.

(^3)
Definition of naive T cells follows Fitch et al.(35) .

(^4)
Furuya, S., Irie, F. Hashikawa, T., Nakazawa, K., Kozakai, A., Hasegawa, A., Sudo, K. and Hirabayashi, Y.(1994) J. Biol. Chem.269, 32418-32425.


ACKNOWLEDGEMENTS

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.


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