Lewis(x)-containing oligosaccharide attenuates schistosome egg antigen-induced immune depression in human schistosomiasis.

The proliferative and interleukin (IL)-10 responses to Lacto-n-fucopentaose III (LNFPIII) that contains Lewis(x)(Le(x))-trisaccharide was assessed in PBMC from humans infected with Schistosoma mansoni. All patient groups with low, medium, and high egg counts in their feces responded to polyvalent LNFPIII-HSA (where HSA = human serum albumin) conjugate. PBMC of all subjects showed a significant proliferative response to this sugar conjugate. However, the levels of interleukin (IL)-10 induced by LNFPIII-HSA were higher in groups with low and medium egg counts than those with high egg. Soluble egg antigens (SEA) also induced IL-10 production by PBMC from infected patients. Interestingly, the SEA-induced IL-10 production was remarkably inhibited by pretreatment of PBMC with free ligands of LNFPIII (monovalent form). These LNFPIII-pretreated PBMC displayed appreciable increase in the level of proliferation to SEA stimulation. We propose that the observed bystander immune potentiation rendered by free LNFPIII is due to the reduced IL-10 level which, presumably, up-regulate expression of co-stimulatory molecules on APC. The ensemble of results indicates that the Le(x)-containing LNFPIII is a potent immunoreactive epitope in SEA that negatively influences PBMC response against this parasite antigens via IL-10.

Wild-derived inbred mice have a novel basis of susceptibility to polyomavirus-induced tumors.

Polyomavirus induces a broad array of tumors when introduced into newborn mice of certain standard inbred strains, notably those bearing the H-2(k) haplotype. Susceptibility in these mice is conferred by an endogenous mouse mammary tumor virus superantigen (Mtv-7 sag) that acts to delete T cells required for polyomavirus-induced tumor immunosurveillance. In the present study we show that mice of two wild-derived inbred strains, PERA/Ei (PE) and CZECH II/Ei (CZ), are highly susceptible to polyomavirus but carry no detectable Mtv sag-related sequences and show no evidence of Vbeta deletion. C57BR/cdJ (BR) mice, which are H-2(k) but lack the endogenous Mtv-7, are highly resistant based on an effective anti-polyomavirus tumor immune response. When crossed with BR, both PE and CZ mice transmit their susceptibility in a dominant fashion, indicating a mechanism(s) that overrides the immune response of BR. Susceptibility in PE and CZ mice is not based on interference with antigen processing or presentation since cytotoxic T cells from BR can efficiently kill F(1)-derived tumor cells in vitro. The expected precursors of polyomavirus-specific cytotoxic T cells are present in both the wild inbred animals and their F(1) progeny. These findings indicate a novel basis of susceptibility that operates independently of endogenous superantigen and prevents the development of tumor immunity.

Generation of antigen-specific cytotoxic T lymphocytes and regulation of cytokine production takes place in the absence of CD3zeta.

The TCR-associated protein CD3zeta plays a major role in regulating the state of responsiveness to peptide-MHC complexes on the surface of antigen-presenting cells. In this paper the requirement of CD3zeta in the generation of cytotoxic T cells was compared with its requirement in cytokine gene activation in two mutant mice: ZKO mice with a disrupted CD3zeta gene and ZTG mice in which a truncated CD3zeta segment was expressed as a transgene on the ZKO background. Upon infection of ZTG mice with lymphocytic choriomeningitis virus (LCMV), antigen-specific cytotoxic T lymphocyte (CTL) responses were detected, identical to responses in wild-type mice. In addition, antigen-specific CTL responses to allogeneic class I and class II MHC in ZTG animals were indistinguishable from those in wild-type animals. However, CTL responses to the same major antigens were not detectable in ZKO mice. We conclude that the signal transduction pathways leading to CTL development and cytokine production can be triggered through TCR in the absence of functional CD3zeta, provided the remainder of the TCR-CD3 complex is expressed at high levels on the cell surface. Surprisingly, IFN-gamma production in response to LCMV followed the same kinetics in ZKO, ZTG and wild-type mice. However, in vitro studies showed that cytokine production in general was abnormally regulated in T lymphocytes from ZKO mice, in contrast to ZTG T cells. Taken together, these studies support the hypothesis that development of CTL can take place in the absence of functional CD3zeta. However, CTL development requires stronger TCR-initiated signal transduction events than induction of cytokine genes.

B-1 cell (CD5+B220+) outgrowth in murine schistosomiasis is genetically restricted and is largely due to activation by polylactosamine sugars.

Previously, we demonstrated that lacto-N-fucopentaose III, a sugar found on egg Ags of Schistosoma mansoni, stimulated splenic B cells from parasite-infected mice to proliferate and produce IL-10 and PGE2. The major source of B cell IL-10 is the B-1 subset (CD5+B220+). Thus we examined whether levels of peritoneal exudate B-1 cells changed as a consequence of infection. In CBA/J, BALB/c, and C3H/HeJ mice, we observed significant increases in B-1 cells at 2 to 4 wk postinfection, declining to baseline by 6 to 8 wk. In contrast, the percentage of B-1 cells remained unchanged in C57BL/6 and BALB/c X.id mice after infection. B-1 cells were not observed in the spleens of infected mice; however, coincident with peritoneal B-1 cell decline, splenic CD23+B220+ cells increased from 11% to 30%. Peritoneal B-1 cells could also be expanded by injection of soluble egg Ag, but not by its deglycosylated form, suggesting a role for carbohydrates in B-1 recruitment. In addition, these cells secreted in vitro large amounts of IL-10 in response to lacto-N-fucopentaose III. Further, this sugar induced B-1 cell outgrowth in CBA/J and C3H/HeJ mice, but not in C57BL/6 mice. Thus, early activation of polylactosamine-reactive, IL-10-producing peritoneal B-1 and splenic B cells may be related to early dominance of the Th2-type CD4+ T cell subset. The degree to which this occurs may in part explain differences in the degree of granulomatous pathology reported among various strains of mouse.

Interleukin-12 and -10 and gamma interferon regulate polyclonal and ligand-specific expansion of murine B-1 cells.

B-1 cells (CD5+ B220+) are a self-replenishing lineage of B cells which are autoreactive and capable of producing large amounts of interleukin-10 (IL-10). In mice experimentally infected with the human helminth parasite Schistosoma mansoni, expansion of B-1 cells is seen in the peritoneal cavity just prior to egg laying. In naive mice, B-1 cell expansion can be elicited by intraperitoneal injection of saline soluble egg antigens (SEA) or the polylactosamine sugar lacto-N-fucopentaose III (LNFPIII), which contains the Lewis-X trisaccharide. In this study, we demonstrate that LNFPIII is the major stimulus in SEA responsible for expansion of B-1 cells, since SEA-induced B-1 outgrowth is blocked by multiple injections of non-cross-linked free LNFPIII. IL-10 is an autocrine growth factor for B-1 cells, and we show that B-1 outgrowth after SEA and LNFPIII administration is inhibited by injection of anti-IL-10 antibodies. Furthermore, SEA- and LNFPIII-induced expansion of B-1 cells is inhibited by in vivo administration of recombinant murine IL-12 or recombinant gamma interferon. These data suggest that activation and expansion of IL-10-producing B-1 cells are governed via cross-regulatory cytokines.

Regulation of schistosome egg granuloma formation: host-soluble L-selectin enters tissue-trapped eggs and binds to carbohydrate antigens on surface membranes of miracidia.

Immunogenic carbohydrate epitopes are prominent in soluble egg antigens (SEA) of Schistosoma mansoni and in vivo are released through ultramicroscopic pores in the eggshell. Previously, one immunogenic carbohydrate was identified as lacto-N-fucopentaose III (LNFPIII), which contains the biologically important trisaccharide Lewis(x) (Le(x)), a weak ligand for E-, L-, and P-selectins. Selectins are involved in various inflammatory reactions, including recruitment of granulocytes. Further, L-selectin molecules are shed from leukocyte cell surfaces upon activation. These independent observations suggest that selectins may play various roles in granuloma formation and/or regulation. We tested in situ for alterations in expression of host E-, L-, or P-selectin in murine liver tissue at various times postinfection with schistosome cercariae. We found that L-selectin was expressed on cells surrounding egg granulomas, but surprisingly we also found mouse L-selectin on the surface membranes of larval miracidia within the S. mansoni ova. In contrast, neither E- nor P-selectin was found within ova. Antibodies to human or rat L-selectin or 15 other distinct mouse leukocyte surface molecules did not bind to the miracidial surface. The anti-mouse L-selectin staining of miracidia could be inhibited by wash buffer containing sulfated carbohydrates such as sulfated Le(x), heparan sulfate, fucoidan, and carrageenan. The elution studies imply that the miracidial surface and, therefore, schistosomes express sulfated glycans. The binding of soluble L-selectin to miracidia was not restricted by the genetic background of the host, as mouse L-selectin was detected on the surface of S. mansoni miracidia in livers from BALB/C, CBA/J, C57BL/6, and outbred Swiss mice. We also detected soluble mouse L-selectin binding to Schistosoma japonicum miracidia, indicating that these observations can be generalized to all schistosome infections. On the basis of these observations we hypothesize that host-soluble L-selectin traverses through pores in the eggshell and binds to target ligands on the surface membranes of miracidia; this complex formation might ultimately impede the release of soluble antigens from the eggs. The intraovum binding of mouse L-selectin to immunogenic carbohydrate antigens is a novel role for selectins, and this model may, in part, explain the down-regulation in granulomatous pathology observed following the acute phase of the disease.

Oligosaccharide-specific induction of interleukin 10 production by B220+ cells from schistosome-infected mice: a mechanism for regulation of CD4+ T-cell subsets.

Defining the factors and/or mechanisms that lead to the predominance of a particular CD4+ 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 III (LNFP-III) contains the interesting trisaccharide Lewisx, which is a weak ligand for P-selectin and is a sugar also found on the alpha and beta 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-III and structurally related oligosaccharides were lymphostimulatory and/or able to induce factors known to down-regulate Th-1 cells. We found that LNFP-III 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-III 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-III 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.