B220+ cells from schistosome-infected mice: A ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 91, pp. 18-22, January 1994 Immunology

Oligosaccharide-specific induction of interleukin 10 production by B220+ cells from schistosome-infected mice: A mechanism for regulation of CD4+ T-cell subsets (lacto-N-fucopentaose III/cytokines/Thl-Tb2 shift)

PALANIVEL VELUPILLAI AND DONALD A. HARN* Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115

Communicated by Elkan Blout, September 16, 1993

saccharides from egg antigens (12): (i) lacto-N-fucopentaose III (LNFP-III), which contains the Lewisx trisaccharide, and (ii) the nonfucosylated homologue, lacto-N-neotetraose (LNT). Lewisx is a weak ligand for P-selectin (13, 14) and is also found on the a and 83 chains of the integrin, lymphocyte function-associated molecule 1, which is a ligand for intercellular adhesion molecule 1 (13). Because of these interesting properties we examined whether LNFP-III or structurally related oligosaccharides could be involved in the Th-1 to Th-2 shift that occurs in schistosomiasis, by testing whether these sugars would induce lymphocytes from naive or schistosome-infected mice to proliferate and/or produce factors known to downregulate Th-1 cells. The results suggest that a specific oligosaccharide plays a role in the regulation of CD4+ T-cell subsets in schistosome-infected mice, stimulating non-T cells to proliferate and produce large quantities of IL-10. We suggest that the specific mechanism for the elaboration of IL-10 from LNFP-III-stimulated cells occurs via a separate or secondary signal distinct from the cross-linking of immunoglobulin molecules on the lymphocyte surface and thus demonstrate a ligand-specific mechanism for the elaboration of a cytokine.

ABSTRACT Defining the factors and/or mechanisms that lead to the predominance of a particular CD4W T-cell subset (Th-1 vs. Th-2) is an area of intense investigation. In murine schistosomiasis, Th-2-type T cells become predominant after deposition of eggs. The most immunoreactive egg components are glycoproteins. Previously we identified two interesting oligosaccharides found on schistosome eggs and schistosomula. One, lacto-N-fucopentaose HI (LNFP-l) contains the interesting trisaccharide LewisW, which is a weak ligand for P-selectin and is a sugar also found on the a and .3 chains of the integrin lymphocyte function-associated molecule 1, a ligand for intercellular adhesion molecule 1. Because of the correlation between schistosome egg glycoproteins and Th-2 dominance, the present study examined whether LNFP-HI and structurally related oligosaccharides were lymphostimulatory and/or able to induce factors known to down-regulate Th-1 cells. We. found that LNFP-M and related sugars did induce proliferation of splenic non-T cells, B220+,CD4-,CD8- cells (B cells) of schistosome-infected and naive mice. In contrast to proliferation, LNFP-Ill was the only oligosaccharide that induced spleen cells to produce large amounts of interleukin 10 and prostaglandin E2, two molecules known to down-regulate Th-1 cells. Further, only spleen cells from infected mice produced cytokines after oligosaccharide stimulation. Interestingly, LNFP-Ill stimulation did not induce production of interleukin 4. Thus, a specific carbohydrate ligand has been identified that stimulates B cells to proliferate and produce factors that down-regulate Th-1 T cells. Further, we suggest that identical or structurally related ligands may contribute to the known Th-1 down-regulation in other parasitic diseases and in chronic blood-vascular diseases such as human immunodeficiency virus infection and a number of metastatic carcinomas and that this effect may, therefore, be a general phenomenon.

MATERIALS AND METHODS Parasites. A Puerto Rican strain of Schistosoma mansoni was maintained in our laboratory via passage through Biomphalaria glabrata snails and Swiss-Webster or CBA/J mice. Lymphocyte Proliferation. Spleens were removed from normal, age-matched CBA/J mice or from CBA/J mice 8 weeks after infection with 20 cercariae. Erythrocytes were eliminated by hypotonic lysis, and the lymphoid cells were washed three times in Hanks' balanced salt solution and suspended in Dulbecco's modified Eagle's medium (DMEM)/20% heat-inactivated fetal calf serum (some experiments used 10% fetal calf serum)/50 ,uM 2-mercaptoethanol/ penicillin-streptomycin at 100 international units/ml/4 mM L-glutamine/2 mM sodium pyruvate/L-arginine at 140 mg/ ml/L-asparagine at 40 mg/ml. Unfractionated splenocytes (5 x 105 cells per well) in 150 ,ul of supplemented DMEM were stimulated (10 pg/well) with oligosaccharides conjugated to human serum albumin (HSA): LNFP-III-HSA; LNT-HSA; lacto-N-difucohexaose I (LND)-HSA; Lewisy tetrasaccharide (LeY)-HSA; lacto-N-tetraose (LT)-HSA; or HSA (Sigma) alone. All oligosaccharide-HSA conjugates were obtained from Accurate Chemicals. Cells were also stimulated

During infection with chronic blood-vascular diseases, including human immunodeficiency virus infection and certain metastatic carcinomas, a shift in CD4+ T-cell subsets occurs from a Th-1 to a Th-2 phenotype (1-6). In all cases, this shift in CD4+ T-cell subsets is associated with elevated levels of interleukin 10 (IL-10) (1-8). A striking Th-1 to Th-2 switch is also seen in murine schistosomiasis (6-8) and occurs coincident with the release of eggs into the tissues by female parasites. Injection of isolated eggs into naive mice is sufficient to alter the lymphoid T-cell response from Th-1 to Th-2. Further, schistosome egg antigen(s) are potent enough to elicit Th-2 responses to third-party antigens that alone generate only Th-1 responses (8). Glycoproteins are the most immunoreactive constituents among the various physical moieties in egg homogenates (9-11). Previously, we identified two immunoreactive oligo-

Abbreviations: LNFP-III, lacto-N-fucopentaose III; LNT, lacto-Nneotetraose; HSA, human serum albumin; PGE2, prostaglandin E2; IL-10, interleukin 10; LND, lacto-N-difucohexose I; mAb, monoclonal antibody; LPS, lipopolysaccharide; Ley, Lewisy tetrasaccharide; LT, lacto-N-tetraose. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Immunology: Velupillai and Harn with Con-A (5 Mg/ml) or LPS (50 ,ug/ml) as standard positive control. Cultures were incubated for 72 hr and for an additional 18 hr with 0.5 ,uCi of [3H]thymidine (1 Ci = 37 GBq) per well. Cells were harvested onto fiber filter paper for scintillation counting. For cytokine assays, the supernatants were removed after 24, 48, or 72 hr and frozen at -70°C until needed. Lymphocyte Depletions. Spleen cells were treated with a mixture of antibodies {anti-Thyl.2 [monoclonal antibody (mAb) 30-H12], anti-CD4 (mAb GK-1.5), and anti-CD8 (15)} followed by complement lysis using low-toxicity rabbit complement from Accurate Chemicals. Macrophages were removed by treatment with 5 mM L-leucine methyl ester (Sigma) as described (16). Dead cells were removed by overlaying the suspension onto a Ficoll-Paque gradient (Sigma). Depleted cells were incubated with anti-Thyl.2, -CD4, -CD8, or -Mac-i mAbs followed by goat anti-rat IgGfluorescein isothiocyanate (GIBCO). B220+ cells were stained with fluorescein isothiocyanate-conjugated anti-B220 mAbs (PharMingen). FACScan analysis (Becton Dickinson) was done by the Core Flow Cytometer Facility, Dana-Farber Institute (Boston). Depleted lymphocytes were 90% B220+ and 1-2% positive for Thyl.2, CD4, CD8, or Mac-i. Cytokine and Prostaglandin Assays. Supernatants were harvested 24 hr after the initiation of culture. Two-site ELISA for IL-10 was done as described by Kullberg et al. (8). In brief, polystyrene microtiter plates (Costar) were coated with IL-10-specific mAb (clone SXC-1), donated by DNAX. Culture supernatants were incubated on the SXC-i-coated plates and then probed with biotinylated SXC-1. Serial dilutions of recombinant mouse IL-10 (PharMingen) were assayed simultaneously to construct a standard curve for relative IL-10 concentrations. Avidin-peroxidase conjugate (Sigma) was diluted in phosphate-buffered saline (PBS)/ 10.0% fetal calf serum and added to the wells for 30 min at room temperature. The plates were washed with PBS/0.05% Tween 20 (Fisher), and then tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories) was added; the reaction was stopped with 0.4 M phosphoric acid and read at 450 nm in a UvMax Reader (Molecular Devices). Interleukin 4 was also detected by sandwich ELISA. ELISA plates were coated with anti-interleukin 4 mAbs (mAb llBll, from DNAX) at a concentration of 2 jg/ml (50 ,ul per well) overnight at 4°C. Wells were washed and blocked with PBS/10% fetal calf serum. Wells were then incubated overnight with culture supernatants diluted 1:2 in PBS/10% fetal calf serum with recombinant interleukin 4 (PharMingen). Plates were washed with PBS/0.05% Tween 20, then incubated with avidin-peroxidase, and developed with substrate as described for IL-10. For prostaglandin E2 (PGE2), culture supernatants were collected at 24 hr and assayed in a competitive ELISA using an assay from Advanced Magnetics, Cambridge, MA. In brief, 96-well polystyrene microtiter plates were supplied precoated with goat anti-rabbit IgG (plates were prewashed and blocked). Culture supernatants or known amounts of PGE2 (supplied with kit) were mixed with rabbit anti-PGE2 and then added to the 96-well plates and incubated overnight at 4°C. Plates were washed with PBS/0.05% Tween 20, and then alkaline phosphatase-conjugated PGE2 was added and incubated for 30 min at 4°C. The plates were then washed and developed with p-nitrophenyl phosphate as substrate and read at 410 nm. The amount of PGE2 (pg/ml) in culture supernatants was derived from the standard curve. RESULTS Proliferation of Splenocytes to Oligosaccharides. We tested whether LNFP-III or structurally related sugars (Table 1) could induce proliferation and/or elaborate soluble media-

Proc. Natl. Acad. Sci. USA 91 (1994)

19

Table 1. Structures of oligosaccharides Sequence Oligosaccharide LNFP-III Gal(f31-4)GlcNAc(,31-3)Gal(,31-4)Glc 3 Fuca LNT LT LND

Gal(13l-4)GlcNAc(181-3)Gal(,81-4)Glc Gal(181-3)GlcNAc(,81-3)Gal(p1-4)Glc Fuc(al-2)Gal(13l-3)GlcNAc(181-3)Gal(,81-4)Glc 4

Fuca

Ley

Fuc(al-2)Gal(181-4)GlcNAc(,81-0) 3 Fuca

tors known to down-regulate Th-i cells. Table 2 shows that proliferation to LNFP-III-HSA was significantly increased in spleen cells from infected CBA/J mice, as compared with normals (mean cpm, 58,035 for infected mice and 7,378 for naive mice; P < 0.001). Similar proliferation was obtained in response to the nonfucosylated homologue of LNFP-III, LNT-HSA. In addition to LNFP-III and LNT, other structurally related sugars induced proliferation in spleen cells from normal animals and higher proliferation in spleen cells from infected animals (Table 2). HSA alone did not induce proliferation. The proliferation in response to LNFP-III or LNT could be inhibited by free sugar in a specific manner. However, neither free sugar induced proliferation (Table 3), indicating that cross-linking was required for proliferation. Identification of Responding Spleen Cell Population. To define the nature of the responding spleen cell population, specific depletion studies were done. Depletion with a mixture of mAbs against T-cell surface markers (Thyl.2, CD4, and CD8) resulted in a population of cells that was 90% B220+ by FACScan analysis. This high efficiency of T-cell depletion was verified by the loss of the proliferative response to Con-A, whereas the response to lipopolysaccharide (LPS) Table 2. Proliferative responses to oligosaccharide conjugates Spleen cells, mean cpm ± SEM Infected Normal Antigen Group A 342 420 Medium 2,364 4,922 Con-A 115,713 + 5750 43,572 3540 LNF-HSA 7,378 ± 455 58,035 ± 3801* LNT-HSA 42,130 3001* 5,309 ± 384 HSA 190 2,991 8,189 1260 Group B 865 Medium 384 6,747 3,756 965 LND-HSA 31,422 2788 12,800 LeY-HSA 13,200 2066 26,346 2402 LT-HSA 731 17,571 ± 1521 12,250 256 HSA 17,955 ± 1915 5,819 were from removed normal, age-matched CBA/J mice or Spleens from CBA/J mice 8 weeks after infection with 20 cercariae (17) of a Puerto Rican strain of S. mansoni. Group A, response to LNF and LNT; group B, response to structurally related sugars. LNF, lactoN-fucopentaose III. Con-A was used at a concentration of 5 pg/ml as positive control. Culture medium contained 20% fetal calf serum and was incubated for 72 hr and for an additional 18 hr with 0.5 ,uCi of [3H]thymidine per well. Triplicate wells were used to determine means. *Data differ at a statistical significance of P < 0.001. ±

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Proc. Natl. Acad Sci. USA 91

Immunology: Velupillai and Harn

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(1994)

from infected mice, as compared with cells from noninfected mice (Fig. 3). Stimulation of B-enriched spleen cells with LNFP-III produced PGE2 levels above the generally elevated level. Thus, of all oligosaccharides tested, only LNFP-III induced high IL-10 levels, as well as an increase in PGE2 over the generally elevated level. Cytokine production by oligosaccharide-stimulated B cells was specific for IL-10; there was no IL-4 production from B-enriched splenocytes of infected mice to any oligosaccharide (Table 4). Stimulation of B-enriched splenocytes with LPS, but not with Con-A, gave rise to minimal IL-4 levels.

Table 3. Stimulation/inhibition with free sugars

Stimulating sugar-conjugate LNF-HSA LNT-HSA HSA 57,059 ± 4006 31,760 ± 3201 14,235 ± 1506 9,386 ± 895 29,669 ± 2763 15,214 ± 1655 68,318 ± 7015 35,344 ± 3420 10,359 ± 996 (4 MAM) LNT (11lM) 62,498 ± 5862 5,008 ± 494 12,483 ± 1192 65,015 ± 5017 32,787 ± 3198 12,974 ± 1315 (4 MuM) Proliferation assay was done as described under Table 2. For inhibition experiments, free sugars (LNF, LNT) were added at 0 ,M (medium), 4 ,uM, or 11 MM, to wells treated with sugar-HSA conjugates or HSA alone. Thus, HSA wells indicate the response to free sugars alone. Free sugar added Medium LNF (11 MM)

DISCUSSION The results obtained in this study suggest that specific oligosaccharides, when presented in a cross-linked manner, may contribute to the down-regulation of Th-1 cells that is observed in experimental schistosomiasis. Specifically, activation of B220+, CD4-, CD8- spleen cell populations from infected mice by LNFP-III leads to subsequent production of IL-10 and PGE2. This ligand-dependent hypothesis is one explanation for the observed elevation of Th-2 function with concomitant reduction of Th-1 responses seen in experimental schistosomiasis. Thus, we have demonstrated the ligandspecific elicitation of a cytokine. The negative regulation of Th-1 responses by IL-10 has been attributed to interference of antigen presentation by

remained intact (Fig. 1A). Interestingly, after T-cell depletion, the proliferative response of B220+,CD4- ,CD8- cells (B cells) to LNFP-III-HSA and LNT-HSA was increased as compared with the total population (Fig. 1A). Depletion did not alter proliferation of normal spleen cells (Fig. 1A). These results suggested the presence of a reactive B-cell population in spleens from infected but not in spleens from normal mice. We also found strong proliferative responses in B-cellenriched spleen cells to the structurally related sugars (Fig. 1B). Production of IL-10 and PGE2 by OligosaccharideStimulated Cells. IL-10 and PGE2 responses are both involved in down-regulation of Th-1 responses (17-20). Therefore, we tested whether B cells that were proliferating to LNFP-III and structurally related oligosaccharides produced IL-10 or PGE2. Supematants of B-cell cultures stimulated with oligosaccharides were collected 24, 48, and 72 hr after culture initiation. Stimulation of B cells from infected animals with LNFP-III-HSA induced dramatic IL-10 synthesis at 24 hr as compared with medium control, LPS, or LNT-HSA (Fig. 2A). However, the synthesis of IL-10 was diminished in 48- and 72-hr supematants (data not shown). When structurally related sugars were tested, we found that Ley induced elevated IL-10 in normal spleen cells, but only LNFP-III induced dramatically elevated levels in infected spleen cells (Fig. 2B). Thus, although LPS and all oligosaccharides tested induced proliferation in infected mouse B cells, only LNFPIII induced significant IL-10 levels. PGE2 is another mediator known to negatively influence Th-1 cells (19, 20). When we tested for PGE2 levels, we found a general elevation in supernatants from B-enriched cells

macrophages (17, 18). However, IL-10 does not affect the antigen-presenting function of B cells, the most likely candidates for the B220+ population of responding cells (17, 18). In this regard, Gajewski et al. (21) have shown that B cells stimulated optimal proliferation of Th-2 clones, and RossiBergmann et al. (22) demonstrated that B cells preferentially induce Th-2 responses in experimental leishmaniasis. In addition to B cells, Sher et al. (23) have shown that Th-2 cells from S. mansoni-infected mice produce IL-10 and may, thus, contribute to the observed down-regulation of Th-1 cells. The nature of the LNFP-III interaction with B cells that leads to proliferation and IL-10 production is unknown. Yamashita et al. (24) have recently reported activation of murine B-cell-enriched populations in the absence of adherent cells by carbohydrate moieties on Schistosoma japonicum egg antigens. Thus, proliferation alone may have been triggered by cross-linking of surface immunoglobulin molecules on B cells with the sugar-HSA conjugates. If crosslinking of immunoglobulin molecules leads to proliferation,

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FIG. 1. Proliferation of B-cell-enriched splenocytes to sugar conjugates. (A) B-cell-enriched splenocytes (5 x 105 cells per well) were stimulated with (10 jAg per well) either LNF-HSA, LNT-HSA, or HSA, under culture conditions identical to those described in Materials and Methods and Table 2. (B) B-cell-enriched splenocytes stimulated with LNF-HSA, LNT-HSA, and structurally related oligosaccharides. Con-A (5 ug/ml) and LPS (50 ,ug/ml) were included as positive controls. Vertical bars are the mean cpm (experimental - medium control) of two experiments, each done in triplicate. Error bars represent ± SEM.


Proc. Natl. Acad. Sci. USA 91 (1994)

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then there is a lack of correlation between proliferation and IL-10 and/or PGE2 production, as demonstrated with the structurally related sugars. Thus, LNFP-III may be crosslinking immunoglobulin molecules on B cells; however, there is probably a distinct, or second, signal that leads to IL-10 production. In this regard, O'Garra et al. (25) have reported that LPS or anti-IgM stimulation of B cells did not induce IL-10. Thus, one hypothesis is that LNFP-III, which contains the Lewisx trisaccharide stimulates B cells to secrete these two factors via a previously undescribed nonimmunoglobulin receptor. Alternatively, the B-cell population may need more than one signal for elaboration of IL-10, and surface immunoglobulin cross-linking is one of these. Because LNFP-III contains the Lewisx trisaccharide, we can propose that LNFP-III might activate B-cell production of IL-10 and/or PGE2 by interacting with a fucose-dependent ligand. One such receptor is P-selectin (13, 14), which interacts weakly with Lewisx but to date has not been found on B cells (26). Perhaps LNFP-III is binding through another, as-yet-undescribed selectin. Alternatively, LNFP-III (Lewisx) may induce proliferation of activated (infected) B cells through a phenomenon similar to crosslinking surface intercellular adhesion molecule 1 on B cells. This hypothesis can be supported by the fact that LNFP-III is present on both a and p chains ofthe integrin lymphocyte function-associated molecule 1, a ligand for intercellular adhesion molecule 1 on

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FIG. 3. PGE2 assay with spleen B-cell-enriched culture supernatants. B-cell-enriched splenocyte preparations were prepared from normal and age-matched infected mice as described. Culture conditions were the same as for Fig. 1. The assay for PGE2 is described; vertical bars represent the average (pg/ml) of duplicate wells.

same as

for Fig. 1. The assay for IL-10

B cells (27). Cellular interaction via immune adhesion molecules has been implicated as essential for sustained B-cell activation (28, 29). Further, LNFP-III is a weak ligand for another adhesion molecule, lung endothelial cell adhesion molecule 1 (30). Lastly, the B220+ (B cell) population that produces IL-10 is present in infected, but not in normal, mouse spleens. Thus, infection with schistosomes may lead to an enrichment of a particular B-cell type that have nonimmunoglobulin ligands for LNFP-III (Lewisx) and can produce IL-10. Ly-l+ B cells (B-1) are found in the peritoneal cavities of mice and have been shown to be major IL-10 producers (25). In preliminary studies we have shown an increase in peritoneal B-1 cells beginning at 2 weeks after infection in C3H/HeJ and CBA/J mice (data not shown). Direction of CD4+ T-cell subsets by specific oligosaccharide activation of B cells that subsequently produce IL-10 and PGE2 may be a general phenomenon. Trypanosoma cruzi is a blood-vascular organism that causes an infection characterized by elevated levels of IL-10 production (31, 32). In preliminary studies, we have shown that splenic B cells from T. cruzi-infected C3H/HeJ mice proliferate and secrete high levels of IL-10 after stimulation with LNFP-III (data not shown). Certain metastatic tumors express Lewisx or structurally related sugars, and patients with these cancers have high levels of IL-10 (30, 33). In human immunodeficiency virus infection, the proposed Th-1 to Th-2 shift occurs after seroconversion and is associated with increased numbers of B cells and elevated IL-10 levels (2, 3, 34). Interestingly, Ley Table 4. Production of interleukin 4 and IL-10 by B cells from schistosome-infected mice Cytokine IL-10,* units/ml IL-4,* pg/ml Normal Infected Normal Infected Antigen Medium 1.7 1.5 0 0 Con-A 1.5 1.2 0 0 LPS 38.0 20.0 40 100 LNF 52.0 0 0 1.5 LNT 3.4 3.0 0 0 HSA 3.5 3.0 0 0 Cytokine levels were determined by ELISA as described by using 24-hr culture supematants of a B-cell-enriched spleen cell preparation (culture medium for these experiments contained lo0 fetal calf serum rather than 20% fetal calf serum). *Data are reported per ml of culture supematant.

22


is found on glycoprotein 120 of human immunodeficiency virus (35). In summary, LNFP-III (Lewisx) or structurally related oligosaccharides interacting with B cells probably lead to the production of factors (IL-10 and PGE2) that down-regulate Th-1 cells and support expansion of Th-2 populations in a number of chronic disease situations; this effect may, therefore, be a general phenomenon. We thank Drs. John David, Evan Secor, and Richard Titus for discussions and critical reading of the manuscript. This work was supported by National Institutes of Health Grant AI27448 and by the United Nations Development Program World Bank Program for Research in Tropical Diseases.

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