TNF superfamily member TL1A elicits type 2 innate lymphoid cells at mucosal barriers.

Immune responses at mucosal barriers are regulated by innate type 2 lymphoid cells (ILC2s) that elaborate effector cytokines interleukins 5 and 13 (IL5 and IL13). IL25 and IL33 are key cytokines that support ILC2s; however, mice deficient in these pathways retain some functional ILC2s. Analysis of human and murine cells revealed that ILC2s highly express tumor necrosis factor (TNF)-receptor superfamily member DR3 (TNFRSF25). Engagement of DR3 with cognate ligand TL1A promoted ILC2 expansion, survival, and function. Exogenous protein or genetic overexpression of TL1A activated ILC2s independent of IL25 or IL33. Dr3(-/-) mice failed to control gut helminthic infections, and failed to mount ILC2 responses in the lung after nasal challenge with papain. Our data demonstrate a key role for TL1A in promoting ILC2s at mucosal barriers.

Modulation of blood fluke development in the liver by hepatic CD4+ lymphocytes.

We have identified an alternate developmental pathway in the life cycle of the trematode pathogen Schistosoma mansoni. This pathway is used in immunodeficient hosts in which the parasite fails to receive appropriate signals from the host immune system. Helminth development is altered at an early stage during infection, resulting in the appearance of attenuated forms that prolong survival of host and parasite. Hepatic CD4+ T lymphocyte populations are an integral component of the immune signal recognized by the parasite.

Early transcription and silencing of cytokine genes underlie polarization of T helper cell subsets.

Naive CD4+ T cells activated through TCR/CD28 under Th1 or Th2 conditions expressed canonical cytokine patterns irrespective of cell division. Only cells that had divided fewer than four times were capable of reexpressing alternative cytokines when restimulated under opposing conditions. Although T cells transcribed both IFN-gamma and IL-4 within hours in a Stat4-/Stat6-independent manner, neither T-bet nor GATA-3 was induced optimally without Stat signals, and polarized cytokine expression was not sustained. Cytokine genes were positioned apart from heterochromatin in resting T cell nuclei, consistent with rapid expression. After polarization, the majority of silenced cytokine alleles were repositioned to heterochromatin. Naive T cells transit through sequential stages of cytokine activation, commitment, silencing, and physical stabilization during polarization into differentiated effector subsets.

Effect of localized cytokine dysregulation: accelerated rejection of IL-2-expressing skin grafts.

Transgenic mice were created in which a sheep keratin promoter directed the expression of IL-2 into the dermis. These KIL-2 transgenic mice were used to investigate the effects of localized IL-2 dysregulation on immune responses. Peripheral tolerance to skin antigens was not broken by in situ IL-2 expression because syngeneic KIL-2 skin grafts were not rejected. However, MHC Class I-disparate skin grafts from KIL-2 donors were rejected faster (median survival time (MST) 12 days) than grafts of non-transgenic littermate skin (MST 18 days). In contrast, the kinetics of KIL-2 H-Y-disparate skin graft rejection (MST 14 days) did not differ significantly from controls (MST 16 days), suggesting that upregulation of IL-2 at the effector site could affect CD4+ T cell- independent, but not CD4+ T cell-dependent, responses. No effect on rejection kinetics was observed when wild type allogeneic skin was grafted onto transgenic mice that expressed bcl2 constitutively in their lymphocytes (MST of 14 days, both sets), indicating that this was not simply due to increased longevity of T cells within the IL-2 expressing graft. We therefore suggest that aberrant expression of IL-2 can accelerate helper-independent CD8+ T cell responses by increasing proliferation and/or differentiation of cytolytic T cells at the effector site.

Chronic schistosomiasis: dendritic cells generated from patients can overcome antigen-specific T cell hyporesponsiveness.

Chronic infection with helminths is associated with down-regulated antigen-specific T cell responses. In order to determine whether schistosome-specific T cells are present, yet functionally unresponsive, or absent in the periphery of chronically infected persons, autologous granulocyte-macrophage colony-stimulating factor and dendritic cells (DC) derived from interleukin (IL)-4 were used as highly efficient antigen-presenting cells (APC). Peripheral blood mononuclear cells (PBMC) from persons harboring Schistosoma haematobium infection and hyporesponsive to parasite antigen were cocultured with autologous DC in the presence of adult worm antigen (AWA). In contrast to PBMC alone, DC-supplemented cultures responded to AWA by proliferation and by IL-4 and IL-5 production and, in some patients, by production of interferon-gamma. Thus, schistosome-specific T helper cells are present in the periphery but are functionally hyporesponsive during active infection with schistosomes. Cytokine responses representing the Th1 and (more importantly) Th2 types can be restored in vitro when DC are used as APC.

Cross-reactivity of myelin basic protein-specific T cells with multiple microbial peptides: experimental autoimmune encephalomyelitis induction in TCR transgenic mice.

Activation of autoreactive T cells is a crucial event in the pathogenesis of autoimmune diseases. Cross-reactivity between microbial and self Ags (molecular mimicry) is one hypothesis that could explain the activation of autoreactive T cells. We have systematically examined this hypothesis in experimental autoimmune encephalomyelitis using mice bearing exclusively myelin basic protein (MBP)-specific T cells (designated T+ alpha-). A peptide substitution analysis was performed in which each residue of the MBPAc1-11 peptide was exchanged by all 20 naturally occurring amino acids. This allowed the definition of the motif (supertope) that is recognized by the MBPAc1-11-specific T cells. The supertope was used to screen protein databases (SwissProt and TREMBL). By the search, 832 peptides of microbial origin were identified and synthesized. Of these, 61 peptides induced proliferation of the MBPAc1-11-specific transgenic T cells in vitro. Thus, the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only two native MBP-residues were sufficient to activate MBPAc1-11-specific T cells in vitro, and experimental autoimmune encephalomyelitis could be induced by immunizing mice with a mimic peptide with only four native MBP residues.

T1/ST2 expression is enhanced on CD4+ T cells from schistosome egg-induced granulomas: analysis of Th cell cytokine coexpression ex vivo.

Th cells are categorized into subsets based on the cytokine production of in vitro-differentiated Th populations. For in vivo-differentiated Th subsets, little is known about the heterogeneity of cytokine production in single cells. We recently described a molecule, T1/ST2, that is preferentially expressed on the surface of Th2 cells. Here we combined high-gradient magnetic cell separation with four-color single-cell cytometry to analyze simultaneously three intracellular cytokines and T1/ST2 surface expression on CD4+ cells from lungs containing granulomas induced by Schistosoma mansoni eggs. T1/ST2 was highly up-regulated on CD4+ T cells from hepatic granulomas and granulomatous lungs. T1/ST2+ cells from granulomatous lungs preferentially produced type 2 cytokines ex vivo. In the total CD4+ population, coexpression of type 1 and type 2 cytokines occurred frequently. However, such coproduction was drastically reduced in T1/ST2+ cells compared with T1/ST2- cells. Coexpression of type 1 and type 2 cytokines was also rare in cells simultaneously producing two cytokines of one type. These findings indicate that individual CD4+ T cells in vivo have different levels of commitment to a certain Th phenotype. Coexpression of two type 2 cytokines or production of one type 2 cytokine together with surface expression of T1/ST2 indicate advanced commitment to the Th2 phenotype.

The effect of anti-IL-10 on proliferation and cytokine production in human schistosomiasis: fresh versus cryopreserved cells.

In this study we neutralized endogenous IL-10 in PBMC from individuals chronically infected with Schistosoma haematobium by using anti-IL-10 MoAbs, and examined the effect and adult worm antigen (AWA)-specific responses in both fresh or cryopreserved cells. Anti-IL-10 alone increased background proliferation of PBMC, but did not augment the AWA-specific responses in either fresh or frozen cells. In freshly isolated PBMC, IFN-gamma production in response to AWA was enhanced significantly in the presence of anti-IL-10. In cryopreserved cells, the augmentation of IFN-gamma in the presence of anti-IL-10 was four-fold less than in the freshly-isolated cells. Neutralization of IL-10 had no effect on IL-4 production. These data show that IL-10 plays a role in specifically down-regulating Th1-but not Th2-type responses and, in contrast to previous reports, anti-IL-10 does not augment proliferation to parasite antigen in chronic schistosomiasis.

T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function.

T helper (Th) cells can be categorized according to their cytokine expression. The differential induction of Th cells expressing Th1 and/or Th2 cytokines is key to the regulation of both protective and pathological immune responses. Cytokines are expressed transiently and there is a lack of stably expressed surface molecules, significant for functionally different types of Th cells. Such molecules are of utmost importance for the analysis and selective functional modulation of Th subsets and will provide new therapeutic strategies for the treatment of allergic or autoimmune diseases. To this end, we have identified potential target genes preferentially expressed in Th2 cells, expressing interleukin (IL)-4, IL-5, and/or IL-10, but not interferon-gamma. One such gene, T1/ST2, is expressed stably on both Th2 clones and Th2-polarized cells activated in vivo or in vitro. T1/ST2 expression is independent of induction by IL-4, IL-5, or IL-10. T1/ST2 plays a critical role in Th2 effector function. Administration of either a mAb against T1/ST2 or recombinant T1/ST2 fusion protein attenuates eosinophilic inflammation of the airways and suppresses IL-4 and IL-5 production in vivo following adoptive transfer of Th2 cells.

Antigen-specific proliferation and interferon-gamma and interleukin-5 production are down-regulated during Schistosoma haematobium infection.

Antigen-specific cellular immune responses were examined in persons previously infected with Schistosoma haematobium and who were, 2 years after chemotherapy, either free from infection (n = 17) or reinfected (n = 20). Proliferation to adult worm antigen (AWA) was significantly higher in uninfected than in reinfected subjects (P = .02), whereas responses to soluble egg antigen (SEA) remained low in both groups. Interleukin (IL)-5 production in uninfected persons in response to AWA and SEA was higher than in infected subjects (P = .05 and P < .001, respectively), while IL-4 and IL-13 release was similar in the 2 groups. Levels of interferon-gamma to AWA and to SEA were inversely correlated with egg output (P = .03 and P = .02, respectively). These data indicate that the presence of schistosome infection leads to T cell proliferative hyporesponsiveness and that both typical Th1 and Th2 cytokines can be down-regulated by active infection.

Anti-schistosome IgG4 and IgE at 2 years after chemotherapy: infected versus uninfected individuals.

Specific IgG4 and IgE responses to adult worm antigen (AWA) and soluble egg antigen (SEA) were examined in 37 subjects from an area in which schistosomiasis is endemic, who were previously infected with Schistosoma haematobium and who became reinfected or remained free of infection 2 years after chemotherapy. The reinfected group was significantly younger (median age, 11 years) than the uninfected group (median age, 24 years). Posttreatment levels of IgG4 to egg antigens (IgG4-SEA) were significantly correlated with reinfection intensity (r = .74, P < .0001), and 13-fold lower levels of IgG4-SEA were observed in uninfected subjects compared with reinfected subjects. Although no correlation was observed between posttreatment IgE to AWA or to SEA, pretreatment IgE-AWA was inversely correlated with the level of reinfection (r = -.39, P = .02).

Elevated proliferation and interleukin-4 release from CD4+ cells after chemotherapy in human Schistosoma haematobium infection.

Cellular immune responses to schistosomal antigens were examined in 110 Schistosoma haematobium-infected individuals before and 5 weeks after treatment with praziquantel. Chemotherapy resulted in significant decrease in worm load as measured by egg output and circulating antigens. The proliferative responses to adult worm antigen (AWA) increased significantly after treatment (p < 0.001) whereas purified protein derivative of tuberculin or phytohemagglutinin responsiveness was unaffected. Interleukin (IL)-4 production in response to both AWA and soluble egg antigen (SEA) increased markedly after treatment (p < 0.001), but IL-5 remained unchanged. None of the studied subjects released any measurable IL-2 and only 21% produced interferon (IFN)-gamma in response to parasite antigens. The deficiency in either IFN-gamma or IL-2 release was not restored by chemotherapy. Thus chronic infection with S. haematobium is associated with the reversible down-regulation of T cell proliferative responses and IL-4 release. Results from cell depletion experiments indicated that CD4+ T cells were the target of this down-modulation.

Antischistosome IgG4 and IgE responses are affected differentially by chemotherapy in children versus adults.

Specific IgG4 and IgE responses and polyclonal cytokine profiles were studied in 110 Schistosoma haematobium-infected persons before and 5 weeks after chemotherapy. Pretreatment IgG4 responses to soluble egg antigen (SEA) correlated with intensity of infection. After chemotherapy, a significant decrease in egg output and circulating anodic antigen was associated with a substantial drop in the IgG4 response to SEA (IgG4-SEA) in adults and children, suggesting that egg laying is a major stimulus for IgG4-SEA. After chemotherapy, IgG4 and IgE to adult worm antigen and IgE to SEA increased in children but remained unchanged in adults. This indicates that the immunoregulatory mechanisms operative in S. haematobium-infected adults differ from those in infected children. The effect of treatment on cytokine profiles was determined following stimulation of whole blood with anti-CD3 antibodies. A significant decrease in interleukin-4 production after treatment indicated that reduction in helminth load may lead to a reduced number of Th2-type cells.

CD30 expression does not discriminate between human Th1- and Th2-type T cells.

CD30 is a member of the TNF receptor superfamily that is commonly used as a marker for Hodgkin and Reed-Sternberg cells in Hodgkin's disease. More recently, it has been proposed that CD30 is preferentially up-regulated on Th2-type human T cells. We analyzed regulation of CD30 expression on both peripheral blood T cells and T cell clones. In short-term culture, CD30 expression could be induced on T cells by Ags that elicit Th2-type responses (Schistosoma haematobium, adult worm Ag, and Toxocaria canis, excretory/secretory Ag) and Th0-type responses (tetanus toxoid), as well as Th1-type responses (tuberculin purified protein derivative). Moreover, simultaneous measurement of membrane phenotype and cytokine production showed that CD30-expressing cells can produce IFN-gamma. Finally, within panels of randomly generated as well as Ag-specific T cell clones, CD30 expression was found on Th0-, Th2-, and Th1-type clones. We conclude that induction of CD30 on activated T cells is not related to differentiation in Th0-, Th1-, or Th2-type cells.

Transforming growth factor-beta modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells.

Transforming growth factor-beta (TGF beta) produced by osteoblasts is present in high levels in bone and influences bone formation, replication of bone cells, and expression of osteoblast protein products. Interactions between bone active hormones and locally released and activated TGF beta were studied by examining the influence of TGF beta preincubation on PTH, calcitonin (CT), and vitamin D receptors in an osteoblastic cell line (UMR 106-06). Preincubation of UMR 106-06 cells with 1 ng/ml TGF beta for 3 days increased specific binding of [125I]PTH-related protein (PTHrP)(1-84) to 140% of that in control cells, but [125I]salmon CT binding decreased to 50% of controls. Binding isotherms indicated that the changes in binding were due to altered receptor numbers since affinities for 125I-labeled PTH and CT remained unchanged. The effect on receptor levels was time dependent, requiring 24 h preincubation with TGF beta for measurable changes, and dose dependent, with maximal effects seen with 1 ng/ml TGF beta. Binding of [3H]1,25(OH)2 vitamin D3 was increased to 130% of control in cytosolic extracts of UMR 106-06 cells pretreated for 3 days with 1 ng/ml TGF beta. Scatchard plots suggested an increase in receptor number without change in affinity. The adenylate cyclase response to PTH increased to 150% of control cells after 3 days of treatment with 1 ng/ml TGF beta; however, the adenylate cyclase response to CT was little changed. Forskolin- and cholera toxin-stimulated adenylate cyclase responses were increased by TGF beta treatment to 130-160% of control, indicating an increase in the stimulatory subunit of the G protein. Increased abundance of both Gs and Gi proteins were indicated by increased cholera toxin- or pertussis toxin-dependent [32P] NAD ribosylation of 47-kilodalton (kDa) and 42-kDa or 40-kDa proteins, respectively, in TGF beta-treated cells. Our data support a complex regulatory effect of TGF beta on UMR 106-06 cells with increases in PTH receptors, vitamin D receptors, and G proteins, whereas there is an apparent down-regulation of CT receptors. TGF beta might induce a more differentiated osteoblast phenotype of these cells, which already express differentiated features such as high alkaline phosphatase activity, PTH and vitamin D receptors, and collagenase production. Since low doses of PTH stimulate bone formation in vivo, TGF beta released or activated at sites of new bone formation might locally modulate PTH activity be allowing increased PTH receptor and postreceptor effectiveness.