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(1,3)-Fucosyltransferase VII and (2,3)-Sialyltransferase IV Are Up-Regulated in Activated CD4 T Cells and Maintained After Their Differentiation into Th1 and Migration into Inflammatory Sites¹

Section of Immunobiology, Yale University School of Medicine and Howard Hughes Medical Institute, New Haven, CT 06520

	Abstract

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Activated Th1 CD4 T cells bind to P-selectin and migrate into inflamed tissue, whereas Th2 cells do not. We show that (1,3)-fucosyltransferase VII (FucT-VII) and (2,3)-sialyltransferase IV (ST3GalIV), which are crucial for the biosynthesis of functional P-selectin ligands, are absent in naive CD4 T cells, but are rapidly up-regulated upon activation. Th1 or Th2 differentiation in the presence of polarizing cytokines leads to down-regulation of FucT-VII mRNA selectively in Th2 but not in Th1 cells. Influencing the differentiation by varying the priming dose of antigenic peptide results in similar FucT-VII down-regulation only in Ag-specific Th2 cells. ST3GalIV levels remain elevated. FucT-VII and ST3GalIV mRNAs are also up-regulated by Th1 cells primed in vivo and recruited into the lymph nodes draining delayed-type hypersensitivity sites. We identify FucT-VII gene expression as a principal difference between Th1 and Th2 cells, and underscore the importance of FucT-VII and ST3GalIV expression for the biosynthesis of functional selectin ligands.

	Introduction

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Naive CD4 T lymphocytes are most likely to first encounter their stimulating peptide/MHC class II ligands in the lymph nodes draining the sites of antigenic exposure. The consequent differentiation of the T helper lymphocyte into Th1 and Th2 is influenced by several factors, including the cytokines secreted by the activated APCs and the strength of the signal received through the TCR upon engaging its peptide/MHC ligand (1).

Since their first demonstration, the major difference between Th1 and Th2 subsets has been their cytokine secretion profile (2, 3). Whereas Th1 cells produce IL-2, IFN-, and TNF-ß, Th2 cells produce IL-4, IL-5, and IL-13. A second major difference between these two subsets was recently shown to lie in their differential trafficking to peripheral sites. Whereas Th1 cells migrate selectively to sites of delayed-type hypersensivity (DTH),³ Th2 cells do not (4). This suggested that the site of Th1/Th2 differentiation was probably distinct from the site of effector function, and that specific mechanisms existed that enabled these cells to selectively traffic to different peripheral sites (5). These mechanisms include differences in the responses of Th1 and Th2 cells to chemokines as well as differences in their ability to bind to P- and E-selectins. Both chemokines and selectins play a central role in the extravasation of cells from the bloodstream into peripheral tissues (6, 7). Among the chemokine receptors expressed by T cells, Th1 cells express CXC chemokine receptor-3 and CCR5, whereas Th2 cells express CCR3, CCR4, CCR7, and CCR8 (6, 8). The P- and E-selectins, alternatively, are carbohydrate binding adhesion molecules that are up-regulated on inflamed endothelia, bind to their respective ligands on T cells, and mediate their extravasation into the subendothelial matrix (7). Here, we address the mechanisms underlying the ability of inflamed endothelia to mediate the extravasation of Th1 cells with the selective exclusion of Th2 and naive CD4 T cells.

The C-type lectin at the amino terminus of P-selectin allows recognition of a tetrasaccharide moiety sialyl Lewis x (sLe^x ) on the ligand P-selectin glycoprotein ligand-1 (PSGL-1) (9). Whereas both Th1 and Th2 cells express PSGL-1, only Th1 cells bind P-selectin (10). sLe^x is crucial for binding to P-selectin, and it consists of a fucose linked in an (1, 3) linkage to GlcNAc in the NeuNAc-(2, 3)-Gal-ß(1, 4)-GlcNAc trisaccharide moiety (11). The biosynthesis of sLe^x involves the enzymatic actions of two enzymes: (1, 3)-fucosyltransferase VII (FucT-VII) and (2, 3)-sialyltransferase IV (ST3GalIV) (12, 13). FucT-VII mediates the transfer of the (1, 3) fucose, whereas ST3GalIV mediates the transfer of the (2, 3) NeuNAc (12). Strong support for the participation of FucT-VII in selectin ligand synthesis comes from several studies. Leukocytes from mice deficient in FucT-VII cannot bind to E- and P-selectin chimeric proteins and have defects in adhesion, rolling, and extravasation (12). Transfection of FucT-VII cDNA into cells results in their expression of E-selectin ligands (14, 15). More recently, FucT-VII was shown to drive the expression of both E- and P-selectin ligands in cultured human T lymphoblasts (16). The role of ST3GalIV in the synthesis of P- and E-selectin ligands has not been studied as extensively as that of FucT-VII. However, ST3GalIV has clearly been shown to be involved in the biosynthesis of sLe^x (13, 17, 18).

We investigated the effects of both TCR triggering and Th1/Th2 differentiation on the expression of FucT-VII and ST3GalIV. Both FucT-VII and ST3GalIV are absent in naive T cells but are rapidly up-regulated upon activation. Subsequent differentiation in the presence of either polarizing cytokines or various doses of specific peptide/MHC ligand resulted in the down-regulation of FucT-VII expression selectively in Th2 cells. In contrast, ST3GalIV expression remained high in both Th1 and Th2 cells. We further confirmed the in vivo expression of FucT-VII and ST3GalIV in a Th1-dominant cutaneous DTH model. The up-regulation of enzymes that posttranslationally modify selectin ligands for high-affinity binding strongly implicates their direct involvement in dictating the migration patterns of Th1 and Th2 cells. These enzymes may be important in vivo for recruiting and maintaining the appropriate differentiated T cell at the peripheral site where its effector function is necessary.

	Materials and Methods

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Naive, activated, type 1, and type 2 CD4 Th cells

Splenic cells from 6- to 8-wk-old C57BL/6J mice were enriched for CD4 T lymphocytes by magnetic bead depletion after staining with the Abs TIB 146 (anti-B220), TIB 210 (anti-CD8), 2.4.G2 (anti-Fc receptor), and Y3JP (anti-I-A^b) followed by anti-mouse IgG-, anti-rat IgG-, and anti-rat IgM-coated magnetic beads (BioMag, PerSeptive Biosystems, Farmingham, MA). CD4 T cells were cultured in Click’s modified Eagle’s Ham’s amino acids medium with 5% FCS (Gemini Bioproducts, Calabasas, CA), 1 mM penicillin-streptomycin, and 2 mM glutamine (Life Technologies, Grand Island, NY), 2.5 µg/ml Con A (Amersham-Pharmacia, Piscataway, NJ), irradiated syngeneic splenocytes, and either IL-12 (5 ng/ml) and anti-IL-4 11B11 (10 µg/ml) or IL-4 (100 U/ml) and anti-IFN- XMG1.2 (10 µg/ml). A total of 20 U/ml IL-2 were added to IL-12-containing cultures on day 3. The cells were restimulated with Con A on day 7. Naive CD4 T cells were sorted with a FACStar^Plus (Becton Dickinson, San Jose, CA) after staining with CD44-PE and CD45RB (16A)-FITC (PharMingen, San Diego, CA). CD45RB^high and CD44^low CD4 cells were collected and cultured on plate-bound anti-CD3 Ab 2C11. Ag-specific Th1 and Th2 CD4 T cells were established by culturing D10.TCR transgenic CD4 cells (19) with irradiated syngeneic B10.BR splenocytes, 25 U/ml IL-2, and either 0.01 µM of conalbumin-derived peptide CA-WT (HRGAIEWEG) for Th2 or 10 µM of CA-WT peptide for Th1. Cells were rested without cytokines on day 4 and restimulated on day 6 with 10 µM of CA-WT peptide. Cultures were terminated on day 10. The presence of IL-4 or IFN- in the culture supernatants was determined by ELISA using capturing anti-IL-4 Ab (11B11) and anti-IFN- Ab (HB-170), followed by detection with biotinylated anti-murine IL-4 or IFN- (PharMingen), peroxidase-conjugated streptavidin (Zymed Laboratories, San Francisco, CA), and visualization after addition of o-phenylenediamine dihydrochloride from tablets (Sigma, St. Louis, MO). Absorbance at 492 nm was measured on an ELISA reader (Elx800, Biotek Instruments, Luton, U.K.).

RT-PCR

Total cellular RNA was isolated from equal numbers (5 x 10⁶) of Th1 or Th2 CD4 T cells using Trizol (Life Technologies). The amount of RNA isolated was measured by taking spectrophotometric absorbance readings at 260 nm, where an absorbance of 1.0 is equivalent to 40 µg of RNA. Equal microgram amounts of RNA were then taken and used as templates for RT-PCR with superscript II RT (Life Technologies), Taq DNA polymerase (Life Technologies), and the following primers: IL-4, forward: 5'-CATCGGATTTTGAACGAGGTCA-3'; IL-4, reverse: 5'-CTTATCGATGAATCCAGGATCG-3' (PCR product length is 240 bp); IFN-, forward: 5'-CATTGAAAGCCTAGAAAGTCTG-3'; IFN-, reverse: 5'-CTCATGAATGCATCCTTTTTCG-3' (PCR product length is 267 bp). FucT-VII (GenBank accession number U45980), forward: 5'-CACCATCCTTATCTGGCACTGGCCTTTCACC-3'; FucT-VII, reverse: 5'-TCAAGCCTGGAACCAGCTTTCAAGGTCTTC-3' (PCR product length is 888 bp). ST3GalIV (GenBank accession number D28941), forward: 5'-TCTCCAGAGAGGCTATGTCCC-3'; ST3GalIV, reverse: 5'-TGAACATCCTGGGACCAGCCC-3' (PCR product length is 473 bp); and hypoxanthine- guanine phosphoribosyltransferase (HPRT), forward: 5'-GTTGGATACAGGCCAGACTTTGTTG-3'; HPRT, reverse: 5'-GAGGGTAGGCTGGCCTATTGGCT-3' (PCR product length is 353 bp). The resultant cDNA was then measured by spectrophotometric absorbance readings at 260 nm, where an absorbance of 1.0 is equivalent to 50 µg/ml cDNA. PCR amplification of cDNA was conducted with different dilutions of template and different numbers of amplification cycles. Similar band intensities of HPRT-amplified PCR products were taken as a further indication of similar cDNA levels, and the starting template DNA amount was adjusted as such. A total of 30 cycles were found to be below the plateau phase of amplification for all primers giving an accurate reflection of the relative concentration of mRNA. Optimal PCR conditions were 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min on a Hybaid OmniGene Thermocycler (National Labnet, Woodbridge, NJ). Amplified bands were visualized after 1% agarose (Life Technologies) gel electrophoresis of the PCR products.

DNA sequencing of PCR products

FucT-VII and ST3GalIV PCR products amplified with the FucT-VII and ST3GalIV primers described above were subjected to DNA sequencing and confirmed as FucT-VII- and ST3GalIV-specific sequences as follows. The PCR products were cut from agarose gels, purified using QIAquick Gel Extraction Kits (Qiagen, Santa Clarita, CA), and ligated into pCR-Topo TA vector (Invitrogen, Carlsbad, CA). After transforming competent DH5 Escherichia coli (Life Technologies), the plasmid DNA was purified (QIAquick PCR Purification kit, Qiagen) and sequenced with the M13 forward and reverse primers using the DNA sequencing kit with AmpliTaq DNA polymerase (Perkin-Elmer Applied Biosystems, Warrington, U.K.) and an Applied Biosystems International 373A DNA sequencer (Perkin-Elmer). Sequences were analyzed with MacVector software (Oxford Molecular Group, Campbell, CA).

Flow cytometry FACScan analysis

Th1 or Th2 cells were stained with anti-sLe^x (mouse IgM anti-human CD15s, clone KM-93) (Accurate Chemical and Scientific, Westbury, NY) followed by secondary FITC-conjugated polyvalent anti-mouse IgA, IgM, IgG Ab (Sigma). Stained cells were analyzed on a FACScan (Becton Dickinson). Anti-CD4 Quantum Red-conjugated Ab (Sigma) was used to confirm the enrichment and purity of CD4 lymphocytes.

Induction of cutaneous DTH in mice

C57BL/6J, BALB/cJ, or C3H/HeJ mice of 3–4 wk of age were sensitized on days -21 and -20 with 100 µl of 0.5% 2,4-dinitrofluorobenzene (DNFB) (Sigma) in 4:1 acetone:olive oil on the abdominal area. On day -1, sensitized mice were challenged with 0.2% DNFB on the ears.

Migration of Th1 cells to DTH sites

Sensitized mice were challenged with 0.2% DNFB only on the right ears. After 24 h, the mice received an i.v. injection of 10⁷ syngeneic Th1 CD4 cells via the retro-orbital plexus. The syngeneic Th1 CD4 cells had been differentiated in vitro for 6 days on Con A in the presence of IL-12 and anti-IL-4 Ab as described above. Before injection, these cells were >99.9% CD4⁺, expressed surface sLe^x epitopes, and were confirmed as Th1 cells by RT-PCR for IFN- mRNA and ELISA measurement for active IFN- production. These Th1 cells were labeled before injection with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, OR). The mice were sacrificed at 24 h postinjection. Both challenged (right) and unchallenged (left) ears were removed, and epidermal sheets were prepared and observed under a fluorescent microscope.

Isolation of CD4 T cells from lymph nodes draining DTH sites

Eight sensitized mice were challenged with 0.2% DNFB on both ears. Age and sex-matched control mice were treated similarly, but without sensitization and challenge. All mice were sacrificed 48 h at postchallenge, and lymph nodes draining both ears were removed. Single-cell suspensions were prepared, and lymph node cells were enriched for CD4 T cells as described above by magnetic bead depletion of CD8 T cells, B cells, and class II⁺ Fc receptor⁺ cells. Enriched cells were >85% CD4⁺ by FACS analysis for both the challenged mice and the untreated controls. Cellular RNA was isolated, and RT-PCR was performed as described above with primers specific for HPRT, FucT-VII, and ST3GalIV.

	Results

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FucT-VII and ST3GalIV are not expressed in naive T cells and are up-regulated after TCR stimulation

We examined the expression of FucT-VII and ST3GalIV in naive CD4 T cells and after TCR triggering and T cell differentiation. After CD4 enrichment and before FACS sorting of naive cells, 88% of splenic lymphocytes were CD4⁺ (Fig. 1A). Naive CD4 cells were collected by gating on CD44^low and CD45RB^high lymphocytes (Fig. 1B). Postsort lymphocytes were >99% naive CD4 (Fig. 1, C and D). These lymphocytes were stimulated with plate-bound anti-CD3 Ab 2C11 for 24, 48, or 72 h. RNA was isolated at each timepoint for comparison with RNA from uncultured naive CD4 cells. RT-PCR was performed using specific primers to HPRT, FucT-VII, and ST3GalIV. No FucT-VII and ST3GalIV mRNAs were detected in uncultured naive cells (Fig. 2A, lanes 2 and 3). At 24 h, ST3GalIV (Fig. 2B, lane 3) but no FucT-VII mRNA (Fig. 2B, lane 2) was detected. FucT-VII mRNA was detected at 48 h (Fig. 2C, lane 2), with ST3GalIV remaining at similar levels (Fig. 2C, lane 3). No changes from 48 h were observed at 72 h poststimulation. The identity of the FucT-VII and ST3GalIV PCR products was confirmed by DNA sequencing.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. CD4 lymphocytes before and after sorting of naive cells. Splenic lymphocytes were enriched for CD4 T cells and stained either before sorting (A and B) or after sorting (C and D). A and C, staining with CD4-QR. B and D, staining with CD44-PE and CD45RB-FITC. Data are representative of three separate experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. FucT-VII and ST3GalIV mRNAs appear after T cell stimulation. Naive CD4 T cells were stimulated with anti-CD3; RT-PCR was performed for 30 cycles on isolated RNA with primers to HPRT (lane 1), FucT-VII (lane 2), and ST3GalIV (lane 3). A, Naive cells; B, 24 h after anti-CD3; C, 48 h after anti-CD3. Data are representative of three separate experiments.

We subsequently determined the effect of CD4 T cell differentiation on the expression of FucT-VII and ST3GalIV mRNAs. Murine CD4-enriched splenic lymphocytes were cultured in the presence of Con A and either Th1-polarizing conditions (IL-12 and anti-IL-4) or Th2-polarizing conditions (IL-4 and anti-IFN-) for 14 days. Cellular RNA was isolated on different days after Con A stimulation, and RT-PCR was performed using specific primers to HPRT, IL-4, IFN-, FucT-VII, and ST3GalIV. As described above for stimulation with anti-CD3, the up-regulation of FucT-VII and ST3GalIV mRNA levels followed the same pattern: ST3GalIV mRNAs appeared first, followed by FucT-VII. Initially, up to day 3, this up-regulation was accompanied by a similar up-regulation of both IL-4 and IFN- mRNAs. On day 4, the cells began to show polarization in their IL-4 and IFN- expression (Fig. 3, lanes 2 and 3). At that time, both FucT-VII and ST3GalIV mRNAs were up-regulated (Fig. 3, lanes 4 and 5), despite the beginning of divergent IL-4/IFN- expression in the differentiating cells. However, on day 14 after differentiation, FucT-VII levels were different between Th1 and Th2 cells. FucT-VII mRNA levels remained up-regulated in Th1 cells (Fig. 4A, lane 4), whereas they were markedly down-regulated in Th2 cells (Fig. 4B, lane 4). The ST3GalIV mRNA levels remained similar for both Th1 and Th2 cells (Fig. 4, A and B, lane 5). This pattern emerged after day 5 of culture and persisted up to day 14.

View larger version (57K): [in this window] [in a new window]   FIGURE 3. The up-regulation of FucT-VII and ST3GalIV mRNAs after T cell activation is maintained before commitment to Th1/Th2 differentiation. CD4 T cells were cultured with Con A and either IL-12 and anti-IL-4 mAb (A) or IL-4 and anti-IFN- mAb (B). RT-PCR was performed for 30 cycles on RNA isolated on day 4 using primers to HPRT (lane 1), IL-4 (lane 2), IFN- (lane 3), FucT-VII (lane 4), and ST3GalIV (lane 5). Data are representative of at least three separate experiments.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. FucT-VII mRNA is down-regulated in IL-4-induced Th2 but not in IL-12-induced Th1 cells, leading to loss of sLex expression. CD4 T cells were cultured with Con A and either IL-12 or IL-4. RT-PCR was performed for 30 cycles on RNA isolated on day 14 using primers to HPRT (lane 1), IL-4 (lane 2), IFN- (lane 3), FucT-VII (lane 4), and ST3GalIV (lane 5). A, Th1; B, Th2; C, staining on day 7 with anti-sLex Ab KM-93. Data are representative of at least three separate experiments.

Based on the differential expression of FucT-VII mRNA between Th1 and Th2 cells, we predicted that Th1 cells would express sLe^x, whereas Th2 cells would not. This prediction was based on previous studies showing that the up-regulation of FucT-VII activity resulted in the synthesis of sLe^x epitopes on cultured human T cells and FucT-VII-transfected cells (15, 16). Surface sLe^x expression was detected by staining the cells with the murine anti-human anti-CD15s mAb KM-93. KM-93 has been characterized as specific for sLe^x epitopes on human carcinomas (20). We stained murine T cells with KM-93 and detected staining on Th1 but not on Th2 cells (Fig. 4C). Th1 cells polarized in the presence of IL-12 and anti-IL-4 expressed higher levels of surface sLe^x-containing carbohydrates than Th2 cells polarized in the presence of IL-4 and anti-IFN-. This pattern of staining began on day 5 after Con A stimulation and persisted up to day 14. The exact nature of the saccharide moiety on murine cells reacting with KM-93 is not clear at this time; however, considering the specificity of KM-93, it is possible that a variant of the tetrasaccharide sLe^x moiety on murine cells is recognized by KM-93. This finding suggests that up-regulation of FucT-VII mRNA results in increased synthesis of sLe^x-related carbohydrate moieties on murine T cells as well.

Differentiation of CD4 T cells after specific peptide/MHC recognition results in down-regulation of FucT-VII in Th2 cells and maintenance of FucT-VII expression in Th1 cells

We activated D10.TCR transgenic CD4 T cells in the presence of a low (Th2-inducing) or a high (Th1-inducing) dose of their specific ligand CA-WT peptide bound to I-A^k. This allowed us to specifically stimulate CD4 T cells with Ag and follow their differentiation without the need for the addition of polarizing cytokines. Naive D10.TCR transgenic CD4 cells were primed with either 0.01 µM of CA-WT to induce differentiation into Th2 or 10 µM of CA-WT peptide to induce differentiation into Th1. As shown in Table I, priming with 0.01 µM of CA-WT peptide led to 1098 pg/ml IL-4 and no detectable IFN-, whereas priming with 10 µM of CA-WT peptide led to high levels of IFN- and no detectable IL-4. Th1/Th2 differentiation was also evident in the levels of IL-4 and IFN- mRNA as shown by RT-PCR (Fig. 5). Th1 D10 cells (Fig. 5A) expressed mainly IFN- (lane 3) and showed higher levels of FucT-VII (lane 4) than Th2 D10 cells (Fig. 5B), which expressed mainly IL-4 (lane 2) and almost undetectable levels of FucT-VII (lane 4). The levels of ST3GalIV were similar (lane 5). Therefore, the differentiation of Ag-specific CD4 T cells into Th2 in the absence of exogenous polarizing IL-4 still results in the down-regulation of FucT-VII.

View this table: [in this window] [in a new window]   Table I. IL-4 or IFN- cytokine release by D10.TCR transgenic Th1 or Th2 CD4 T lymphocytes

View larger version (66K): [in this window] [in a new window]   FIGURE 5. FucT-VII mRNA is down-regulated in Ag-specific Th2 but not Th1 cells. CD4-enriched lymphocytes from the spleens and lymph nodes of D10.TCR transgenic mice were primed with either 10 µM CA-WT peptide (A) or 0.01 µM CA-WT peptide (B). RT-PCR was performed for 28 cycles on RNA isolated on day 10 of culture using primers to HPRT (lane 1), IL-4 (lane 2), IFN- (lane 3), FucT-VII (lane 4), and ST3GalIV (lane 5). Data are representative of at least three separate experiments.

Th1 cells migrate selectively to sites of cutaneous DTH responses (4). In addition, the percentage of E- and P-selectin binding CD4 T cells increases in the lymph nodes draining the DTH site, especially after rechallenge (21). Most of these cells are IFN--producing Th1 cells. We used a Th1-dominant murine model of cutaneous DTH to investigate whether in vivo-generated endogenous Th1 cells in the lymph nodes draining DTH sites also showed expression of FucT-VII and ST3GalIV. DNFB-sensitized mice were challenged with DNFB on the ears. The ears were chosen as the site of challenge because leukocyte rolling in the ear microcirculation has been shown to depend mainly upon P-selectin interactions (22). The lymph nodes draining both ears were removed 48 h later. Lymph nodes draining the ears were also removed from unsensitized control mice for comparison. The lymph nodes from the challenged mice were considerably larger than the controls. CD4 T cells were isolated from single-cell suspensions, and cellular RNA was prepared from equal cell numbers from both challenged and unchallenged nodes. FucT-VII and ST3GalIV mRNAs were detectable by RT-PCR in lymph node CD4 T cells from both challenged and unchallenged nodes (Fig. 6A). However, there was up-regulation of FucT-VII mRNA specifically in the challenged group (Fig. 6A, lanes 2). This suggests that the expression of these mRNAs could be detected in vivo in the lymph nodes draining the DTH site, and that endogenously primed Th1 cells were the most likely source for the increased FucT-VII levels.

View larger version (61K): [in this window] [in a new window]   FIGURE 6. Th1 cells migrate specifically to the site of DTH challenge and lead to up-regulation of FucT-VII and ST3GalIV mRNA in the draining lymph nodes. DNFB-sensitized C57BL/6 mice were challenged on the ears; control mice were left untreated. A, At 48 h postchallenge, cellular RNA from the lymph nodes draining the challenged ears and control unchallenged ears was subjected to RT-PCR for 30 cycles using primers to HPRT (lane 1), FucT-VII (lane 2), and ST3GalIV (lane 3). A separate group of mice were challenged on the right ears; the left ears were untreated. The mice were injected i.v. with DiI-labeled Th1 cells and sacrificed at 48 h postchallenge. Epidermal sheets were prepared and observed by fluorescence microscopy. DiI-labeled cells were detected only in the challenged ears (B), not in unchallenged ears (C). Light microscopy was superimposed on C to show background cells. Data are representative of at least three separate experiments.

To confirm that Th1 cells specifically accumulate at the DTH site in the Th1-dominant murine model that we have used, we i.v. injected labeled Th1 cells and followed their migration to the DTH site in the challenged ears. Because we could not follow the migration of the in vivo-primed Th1 cells to a DTH site, this method, which had been used previously for the same purpose (4), provided us with a means to track the specific migration of a population of in vitro-differentiated Th1 cells. To do this, Th1 CD4 T cells were differentiated in vitro in the presence of IL-12; their differentiation was confirmed by IFN- production both at the mRNA and protein levels (data not shown). These Th1 cells were >99% CD3⁺, CD4⁺. They were then injected into syngeneic recipient mice that had been sensitized and challenged with DNFB only on the right ears. After 48 h, the transferred Th1 cells localized exclusively to the epidermis of the challenged right ears (Fig. 6B). The unchallenged left ears were devoid of the labeled Th1 cells (Fig. 6C). This selective migration confirmed that Th1 cells in this model were recruited only to the DTH site. Thus, we may conclude that the increased size of the draining lymph nodes was most likely due to the accumulation of endogenous Th1 cells responding to antigenic challenge.

	Discussion

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Selectins are a family of C-type lectins that are involved in cell-cell adhesion and include P-selectin, E-selectin, and L-selectin (7). All selectins recognize common sialylated and/or fucosylated tetrasaccharide structures such as sLe^x (23), yet selectins bind to their ligands with exquisite specificity. This specificity is most likely generated by presentation of the relevant tetrasaccharide moiety on designated carrier protein ligands. The dominant ligand for P-selectin is the homodimeric glycoprotein PSGL-1 (24). PSGL-1 also serves as a ligand for E-selectin, although the region involved in E-selectin binding is distinct from that involved in P-selectin binding. In addition, specific ligands such as E-selectin ligand-1 have also been identified for E-selectin. Lymphocyte-restricted L-selectin has multiple ligands on endothelial cells including glycosylation-dependent cell adhesion molecule-1, CD34, and mucosal addressin cell adhesion molecule-1 (reviewed in Refs. 25 and 26).

Because specific recognition of carbohydrate moieties on selectin ligands is crucial for their binding, the posttranslational modifications of these protein ligands become a potential mechanism for regulating selectin-ligand interactions. These modifications may have important consequences on dictating the type of cell-cell interactions that are mediated through the selectins. One such impact is directly relevant to the differential ability of Th1 and Th2 CD4 T cells to migrate into sites of inflammation. Although both types of differentiated CD4 T cells express PSGL-1 protein, only that expressed by the Th1 cells mediates binding to endothelial cell P-selectin and subsequent extravasation into inflamed tissue (10). In this study, we addressed the possibility that the glycosylation of PSGL-1 is different in Th1 and Th2 cells, and that this is directly due to the influence of differential expression of cellular glycosyltransferases.

Among the glycosyltransferases that have been described, two were of interest here: an (1, 3)-fucosyltransferase and an (2, 3)-sialyltransferase. These enzymes transfer the relevant fucose and sialic acid residues in the correct linkage to the Gal-ß(1, 4)GlcNAc precursor disaccharide to form the sLe^x tetrasaccharide moiety. Among the (1, 3)-fucosyltransferases, only FucT-VII has been shown to synthesize functional P- and E-selectin ligands (12, 14, 15, 16) and was therefore considered a strong candidate for study. Of the four (2, 3)-sialyltransferases, only ST3GalIV fits the above substrate specificity (reviewed in Ref. 27). For example, ST3GalIII transfers a sialic acid in the (2, 3) linkage but does so to a substrate for which Gal is linked to GlcNAc in a ß(1, 3) linkage rather than the ß(1, 4) linkage present in sLe^x (28). ST3GalI and ST3GalII transfer sialic acids exclusively to a Gal-ß(1, 3)GalNAc precursor disaccharide. Furthermore, ST3GalIV has been shown to increase the level of sLe^x expression in transfected cells (13) and was therefore the only candidate among the cloned (2, 3)-sialyltransferases that was reasonable to pursue. In addition, its expression in T lymphocytes had not been investigated.

Based on this study, we conclude that the mRNAs for FucT-VII and ST3GalIV are not detectable in naive CD4 T cells, and that they are sequentially up-regulated after T cell activation. ST3GalIV mRNA is expressed in tissues such as brain, heart, liver, spleen, and thymus, but at very low levels in the colon and testis (27). Our results show for the first time the expression of ST3GalIV by CD4 T lymphocytes. ST3GalIV mRNA appeared within 24 h of TCR stimulation, followed by FucT-VII within 48 h. This sequence of up-regulation correlates with the described activities of these enzymes, where the earlier expressed ST3GalIV may create the substrate necessary for the later expressed FucT-VII (12).

We expected that the differentiation of CD4 T cells would have a marked effect on the expression of FucT-VII and ST3GalIV for two main reasons. First, these two enzymes are involved in the biosynthesis of the sLe^x present on selectin ligands that are essential for leukocyte migration and extravasation (12, 13, 14, 15, 16, 17, 18). Second, Th1 and Th2 cells exhibit distinct migration patterns in vivo (4). We generated Th1 and Th2 cells in two different ways. First, CD4 T cells were stimulated nonspecifically with Con A and forced to differentiate into either Th1 or Th2 with IL-12 or IL-4, respectively. Second, naive D10.TCR transgenic CD4 T cells were stimulated with their specific peptide/MHC ligand, CA-WT peptide bound to I-A^k, at different doses of Ag. Changing the dose of peptide Ag in priming has been shown to influence T cell differentiation and result in a different pattern of functional T cell responses (29, 30, 31). Using these Th1 and Th2 cells, we found that although both up-regulate and maintain ST3GalIV expression, FucT-VII up-regulation is maintained only in Th1 cells. These results are observed in differentiated cells after nonspecific stimulation with Con A as well as after specific stimulation with peptide/MHC.

Because FucT-VII and ST3GalIV are involved in the biosynthesis of the tetrasaccharide moiety sLe^x (12, 13), we predicted that the surface expression of sLe^x would be different between Th1 and Th2 cells. The available mAbs directed to sLe^x have all been generated in mice, forcing us to use a mouse mAb to stain the murine Th1/Th2 cells described in this study. The mAb we used was KM-93, which was derived by immunizing BALB/c mice using a novel immunization procedure (20). Neonatal mice were rendered tolerant to normal human lung tissue and subsequently immunized with squamous cell carcinoma from human lung. Our finding that KM-93 stains murine Th1 cells but not Th2 cells suggests the existence of sLe^x-containing carbohydrate epitopes on murine T cells related to those described on human T cells. Although KM-93 is recognized as an anti-sLe^x mAb, the exact nature of the saccharide residues that it recognizes on murine cells is not presently known.

The differential expression of (1, 3)-fucosyltransferases in in vitro-differentiated Th1 and Th2 cells has been described. For example, Van Wely et al. (32) have shown that FucT-VII mRNA levels were higher in Th1 than in Th2 cells, and that the (1, 3)-fucosyltransferase enzyme activity was elevated in Th1 cells. Recently, Wagers et al. (33) showed that stimulation of CD4 T cells activated in the presence of IL-12 augmented FucT-VII expression and binding to E- and P-selectin, but that activation in the presence of IL-4 had an inhibitory effect. We find that after CD4 T cell stimulation with Con A, the mRNA levels for both IL-4 and IFN- increase, but that the increased IL-4 does not prevent the subsequent up-regulation of FucT-VII mRNA. The initiation of these cultures involves the addition of a moderately high level of active IL-4 protein sufficient to drive the cells toward Th2 differentiation. IL-4 is present in the early cultures driven toward Th2 differentiation, but its presence does not inhibit the appearance of FucT-VII mRNA. Indeed, on days 3 and 4 before commitment to differentiation, the FucT-VII levels are similar in T cells from either IL-12-driven or IL-4-driven cultures. It is thus unlikely that activation in the presence of IL-4 in and of itself results in the inability to up-regulate FucT-VII gene expression. Rather, the resultant differentiated Th2 cell specifically down-regulates this mRNA by an as yet undefined mechanism.

Were our observations in vitro relevant to Th1 cells that were primed and recruited to inflammatory sites in vivo? To investigate this question, we used a Th1-dominant DTH response in a murine model. The exclusive migration of Th1 cells to cutaneous inflammatory DTH sites has been firmly established (4, 9). We found that CD4 T cells isolated from the lymph nodes draining active DTH sites had prominent expression of FucT-VII and ST3GalIV. Compared with CD4 T cells isolated from lymph nodes draining normal skin, only the FucT-VII mRNA level was significantly increased. This was consistent with the expected predominant Th1 cell infiltration into the area of inflamed skin and draining lymph nodes. The presence of ST3GalIV mRNA in the control lymph nodes was not surprising. Our results show that ST3GalIV expression is present in both Th1 and Th2 cells, and it has been reported previously to be ubiquitously present in lymphoid organs such as the spleen and thymus (27). Furthermore, because CD4 T cells were isolated from draining lymph nodes rather than inflamed skin, the presence of other activated T cells in the lymph node is expected. Isolating CD4 T cells from inflamed skin is not technically feasible.

We conclude that naive CD4 T cells up-regulate the expression of two glycosyltransferases, FucT-VII and ST3GalIV, after TCR stimulation. Although the precursor (2, 3)-sialylated substrate may be present in both Th1 and Th2 cells as suggested by the continued expression of ST3GalIV mRNA, differentiation along the Th2 path results in down-regulation of FucT-VII. This provides a molecular explanation for the inability of PSGL-1 on Th2 cells to interact with P-selectin. Differentiation along the Th1 path maintains FucT-VII mRNA expression that, along with ST3GalIV expression, leads to the successful synthesis of sLe^x. The presence of this moiety directly influences the ability of Th1 cells to bind to P-selectin on endothelial cells and successfully extravasate into the subendothelium. The presence of FucT-VII and ST3GalIV mRNA in vivo, in the lymph nodes draining the sites of DTH challenge, further supports this conclusion. Finally, the expression of sLe^x determinants provides a new phenotypic tool for distinguishing Th1 from Th2 cells.

	Acknowledgments

 
We thank Tom Taylor for expert technical assistance with FACS cell sorting, Elizabeth E. Eynon for assistance in the preparation of epidermal sheets from murine ears, and Eve Robinson for help with sequencing PCR products.

	Footnotes

 
¹ This work was supported by a Howard Hughes Medical Institute fellowship (to J.M.B.) and by National Institutes of Health Grant 5-R37-AI14579-20 (to C.A.J.).

² Address correspondence and reprint requests to Dr. Ruslan Medzhitov, Section of Immunobiology, Yale University School of Medicine, P.O. Box 208011, New Haven, CT 06520-8011. E-mail address:

³ Abbreviations used in this paper: DTH, delayed-type hypersensivity; sLe^x, sialyl Lewis x; FucT-VII, (1,3)-fucosyltransferase VII; ST3GalIV, (2,3)-sialyltransferase IV; HPRT, hypoxanthine-guanine phosphoribosyltransferase; DNFB, 2,4-dinitrofluorobenzene; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate; PSGL-1, P-selectin glycoprotein ligand-1.

Received for publication May 4, 1999. Accepted for publication July 23, 1999.

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