CD4+ Th2 response induced by Schistosoma mansoni eggs develops rapidly, through an early, transient, Th0-like stage.

Data from recent studies of murine schistosomiasis mansoni have indicated that certain characteristics of this infection, such as eosinophilia and elevated IgE, are due largely to the induction of Th2-like immune responses by parasite ova. The present study was designed to examine more closely the genesis and development of these skewed Th responses to schistosome eggs. Accordingly, eggs isolated from infected mice were injected s.c. into normal mice. After inoculation, draining lymph node (LN) cells were recovered, phenotyped, and tested for their ability to proliferate and secrete IL-2 and IFN-gamma (as markers of Th1 function) and IL-4, IL-5, and IL-10 (Th2 cytokines). The results show a maximal LN enlargement of 40- to 100-fold by day 3 after egg inoculation. The CD4/CD8/B cell ratio at this time is similar to that in LN from normal mice, but increases in numbers of cells expressing very low levels of MEL-14 and high levels of Pgp-1 are evident by days 3 and 10, respectively. Surprisingly, the initial detectable Ag-specific response to schistosome eggs, observed at day 1, is the production of IFN-gamma. By day 3, LN cells are capable of proliferating and making IFN-gamma plus IL-2, IL-4, IL-5, and IL-10 when stimulated with soluble egg Ag and, therefore, appear Th0-like. After 7 to 10 days, IFN-gamma production is severely depressed but the response continues to be characterized by IL-4, IL-5, IL-10, and IL-2. Depletion studies indicate that CD4 cells are the major population responsible for Ag-mediated proliferation and cytokine production. Results show that schistosome eggs are autonomous inducers of vigorous Th2-like effector responses. Further, our data, from a system that utilizes an in vivo priming step, support the contentions that skewed Th responses develop via an intermediate Th0 stage is accompanied by a loss of the MEL-14 surface marker and an increase in Pgp-1 expression.     

The pathogenic Th17 cell response to major schistosome egg antigen is sequentially dependent on IL-23 and IL-1β

CBA/J mice infected with the helminth Schistosoma mansoni develop severe CD4 T cell-mediated hepatic granulomatous inflammation against parasite eggs associated with a robust Th17 cell response. We investigated the requisites for Th17 cell development using novel CD4 T cells expressing a transgenic TCR specific for the major Sm-p40 egg Ag, which produce IL-17 when stimulated with live schistosome eggs. Neutralization of IL-23 or blockade of the IL-1 receptor, but not IL-6 neutralization, abrogated egg-induced IL-17 secretion by transgenic T cells, whereas exogenous IL-23 or IL-1β reconstituted their ability to produce IL-17 when stimulated by syngeneic IL-12p40-deficient dendritic cells. Kinetic analysis demonstrated that IL-17 production was initiated by IL-23 and amplified by IL-1β. Significantly, schistosome-infected IL-12p40-deficient or IL-1R antagonist-treated CBA/J mice developed markedly reduced hepatic immunopathology with a dampened egg Ag-specific IL-17 response. These results demonstrate that the IL-23-IL-1-IL-17 axis has a central role in the development of severe schistosome egg-induced immunopathology.     

Selective delivery of augmented IL-2 receptor signals to responding CD8+ T cells increases the size of the acute antiviral response and of the resulting memory T cell pool

CD8(+) T cells respond to IL-2 produced both endogenously and by CD4(+) Th during an antiviral response. However, IL-2R signals can potentially promote CD8(+) T cell death as well as proliferation, making it unclear whether IL-2R signals provide a predominantly positive or negative effect upon CD8(+) T cell responses to viral infection. To more precisely define the direct role of IL-2R signaling on CD8(+) T cells during the response to a virus, we examined the effect of delivering augmented IL-2R signals selectively to CD8(+) T cells responding to lymphocytic choriomeningitis virus infection. Although naive CD8(+) T cells are competent to produce IL-2, CD8(+) T cells lose this capacity upon differentiation into effector CD8(+) T cells. However, effector CD8(+) T cells do retain the capacity to produce GM-CSF upon Ag stimulation. Thus, to deliver enhanced autocrine IL-2R signals to CD8(+) T cells, we established a transgenic mouse strain expressing a chimeric GM-CSF/IL-2R (GMIL2R). As GM-CSF production is Ag dependent, the GMIL2R delivers an augmented IL-2R signal exclusively to CD8(+) T cells responding to Ag. Following lymphocytic choriomeningitis virus infection, GMIL2R transgenic mice exhibited an increase in both the peak CD8(+) T cell response achieved and the size of the resulting memory pool established. Upon secondary viral challenge, the GMIL2R also enhanced the proliferative response of memory CD8(+) T cells. Thus, our findings indicate that IL-2 delivery to responding CD8(+) T cells is a limiting factor in both the acute and memory antiviral responses.     

CCR8 is expressed by antigen-elicited, IL-10-producing CD4+CD25+ T cells, which regulate Th2-mediated granuloma formation in mice

CCR8 was initially described as a Th2 cell-restricted receptor, but this has not been fully tested in vivo. The present study used ex vivo and in vivo approaches to examine the distribution and functional significance of CCR8 among CD4+ T cells. Populations of cytokine-secreting CD4+ T cells were generated in primed mice with Th1 or Th2 cell-mediated pulmonary granulomas, respectively elicited by i.v. challenge with either Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg Ag (SEA)-coated beads. Cytokine-producing CD4+ T cells were isolated from Ag-stimulated draining lymph node cultures by positive selection. Quantitative analysis of cytokine mRNA indicated enriched populations of IFN-gamma-, IL-4-, and IL-10-producing cells. Analysis of chemokine receptor mRNA indicated that IL-10+ cells selectively expressed CCR8 in the SEA bead-elicited type 2 response. The IL-10+CCR8+ populations were CD25+ and CD44+ but lacked enhanced Foxp3 expression. Adoptive transfer to naive recipients indicated that IL-10+ T cells alone could not transfer type 2 inflammation. Analysis of SEA bead-challenged CCR8-/- mice indicated significantly impaired IL-10 production as well as reductions in granuloma eosinophils. Adoptive transfer of CD4+CCR8+/+ T cells corrected cytokine and inflammation defects, but the granuloma eosinophil recruitment defect persisted when donor cells were depleted of IL-10+ cells. Accordingly, local IL-10 production correlated with CCR8 ligand (CCL1) expression and the appearance of CCR8+ cells in granulomatous lungs. Thus, IL-10-producing, CCR8+CD4+CD25+CD44+ T cells are generated during SEA challenge, which augment the Th2-mediated eosinophil-rich response to the parasite Ags.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

The absence of IL-6 does not affect Th2 cell development in vivo, but does lead to impaired proliferation, IL-2 receptor expression, and B cell responses

The role of IL-6 in Th2 cell differentiation and response development after the injection of eggs from Schistosoma mansoni was investigated using wild-type (WT) and IL-6-/- mice. IL-6 was induced in the lymph nodes (LN) of WT mice within 24 h of egg injection, and IL-4 production by WT LN cells and CD4 T cells isolated 24 h after egg injection and stimulated in vitro was observed. In the absence of IL-6, this early production of IL-4 by LN cells and purified CD4 T cells was not abolished; although the level of IL-4 produced by IL-6-/- LN cells was similar to WT, IL-4 production by purified IL-6-/- CD4 T cells was reduced compared with WT. Despite the difference in CD4 T cell production of IL-4, the development of egg-specific Th2 cells 7 days after egg injection was not affected by the absence of IL-6. Nevertheless, Ab production was impaired and the in vitro proliferative response of whole LN cell populations, CD4 and CD8 T cells, and B cells from IL-6-/- mice was poor compared with WT. The proliferative defect in the IL-6-/- cells correlated with decreased IL-2R expression, and addition of exogenous IL-6 enhanced IL-2R expression as well as proliferation of LN cells from IL-6-/- mice. These studies demonstrate that Th2 differentiation and response development in vivo is not dependent on IL-6, but that Th-dependent and independent B cell responses are. Our results also emphasize the importance of IL-6 for lymphoproliferation, possibly through induction of IL-2R expression.     

CD73 and Ly-6A/E distinguish in vivo primed but uncommitted mouse CD4 T cells from type 1 or type 2 effector cells

Primed CD4 T cells may develop into effector T cells such as Th1 and Th2, or remain uncommitted as Th primed precursor (Thpp) cells that can subsequently differentiate into Th1 and Th2 cells. Although mouse Thpp-like cells have also been identified among spleen and particularly lymph node cells, further characterization of these cells has been difficult without a defining cell surface marker. Using Affymetrix GeneChips followed by FACS analysis, we found that in vitro-derived Thpp cells expressed CD73 but not Ly-6A/E, whereas Th1 and Th2 cells showed the reciprocal pattern. CD73+ Ly6A/E- memory CD4 T cells were identified in normal C57BL/6 mice, and the proportion of these cells was highest in lymph nodes, lower in spleens, and lowest in the lungs. These cells produced IL-2 and MIP-1alpha, but much less IL-4 and IFN-gamma than CD73- Ly6A/E+ cells. Similar results were obtained with additional Ly-6.2 mouse strains, but not Ly-6.1 strains. Restimulation of Thpp-like CD73+ Ly-6A/E- cells in Th1- or Th2-polarizing conditions induced differentiation into populations producing mainly IFN-gamma or mainly IL-4, respectively. In contrast, the effector-like CD73- Ly-6A/E+ population was more committed, and continued to produce both IL-4 and IFN-gamma in both conditions. CD73 and Ly-6A/E expression therefore identify a population of Thpp-like cells in C57BL/6 mice and at least some other Ly-6.2 mice.     

Induction of competence to respond to IL-4 by CD4+ T helper type 1 cells requires costimulation.

Rested murine CD4+ Th1 clones do not produce IL-4, but have previously been shown to be capable of responding to IL-4 if they are first activated with Ag and APC. In this study, we have examined the activation requirements for induction of competence to respond to IL-4 in these clones. TCR occupancy alone (given either as chemically fixed APC and Ag, anti-CD3, Con A, or ionomycin and PMA) was inadequate, but the addition of a source of costimulation to any of these stimuli resulted in complete induction of competence to respond to IL-4. Pretreatment of the Th1 clones with TCR occupancy alone induced an anergic state from which subsequent full stimulation with Ag and APC failed to give IL-4 responsiveness. Pretreatment of the cells with IL-2 alone was an inadequate signal to induce IL-4 responsiveness and only a partial response was obtained when TCR occupancy was combined with IL-2. Addition of anti-IL-2 and anti-IL-2R antibodies during full activation with APC and Ag gave a 50% inhibition of competence induction. These results demonstrate that costimulation, in addition to its role in IL-2 production, is an important second signal for inducing T cells to become competent to respond to IL-4.     

Oxidative stress promotes polarization of human T cell differentiation toward a T helper 2 phenotype

These studies were conducted to determine the effects of oxidative stress on human T cell differentiation and polarization into Th1 or Th2 phenotypes. Highly purified naive CD4+ T cells were isolated from PBMC of healthy, nonatopic donors. CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence or absence of oxidative stress as supplied by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates a low level of superoxide anion. Increases in cellular superoxide were observed by exposure to DMNQ. Exposure of unpolarized CD4+ T cells to IL-12 or IL-4 resulted in a Th1 or Th2 phenotype, respectively. T cells stimulated in the absence of polarizing cytokines secreted modest amounts of IFN-gamma and TNF-alpha. Cells stimulated in the continuous presence of 5 microM DMNQ, displayed a marked up-regulation in Th2 cytokines, including IL-4, IL-5, and IL-13, but not the Th1 cytokine IFN-gamma. Th2 responses were blunted by concomitant exposure to thiol antioxidants. Long-term exposure of T cells to DMNQ resulted in growth of cells expressing CCR4, and a decrease in cells expressing CXCR3, indicating phenotypic conversion to Th2 cells. These results suggest that oxidative stress favors a Th2-polarizing condition.     

The role of B cells in the development of CD4 effector T cells during a polarized Th2 immune response

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.     

Constitutive expression of CIITA directs CD4 T cells to produce Th2 cytokines in the thymus

We generated mice expressing a human type III CIITA transgene (CIITA Tg) under control of the CD4 promoter to study the role of CIITA in CD4 T cell biology. The transgene is expressed in peripheral CD4 and CD8 T cells, as well as in thymocytes. When CD4 T cells were differentiated towards the Th2 lineage, both control and CIITA Tg Th2 cells expressed similar levels of Th2 cytokines. Th1 cells from control and CIITA Tg mice cells produced comparable levels of IFN-gamma. CIITA Tg Th1 cells also expressed IL-4, IL-5, and IL-13 in the absence of Stat6. There was an approximate 10-fold increase in the number of peripheral na?ve CD4 T cells and NK1.1- thymocytes producing IL-4 from CIITA Tg mice compared to control mice. Finally, Th1 cells from irradiated control mice reconstituted with CIITA Tg bone marrow displayed the same cytokine production profiles as Th1 cells from CIITA Tg mice. Together, our data demonstrate that CIITA expression pre-disposes CD4 T cells to produce Th2 type cytokines. Moreover, phenotypic similarities between Th1 cells expressing the CIITA transgene and CIITA deficient Th1 cells suggest that the role of CIITA in cytokine regulation is complex and may reflect both direct and indirect mechanisms of T cell development and differentiation.     

CD4+CD25+ T cells regulate airway eosinophilic inflammation by modulating the Th2 cell phenotype

We used a TCR-transgenic mouse to investigate whether Th2-mediated airway inflammation is influenced by Ag-specific CD4+CD25+ regulatory T cells. CD4+CD25+ T cells from DO11.10 mice expressed the transgenic TCR and mediated regulatory activity. Unexpectedly, depletion of CD4+CD25+ T cells before Th2 differentiation markedly reduced the expression of IL-4, IL-5, and IL-13 mRNA and protein when compared with unfractionated (total) CD4+ Th2 cells. The CD4+CD25--derived Th2 cells also expressed decreased levels of IL-10 but were clearly Th2 polarized since they did not produce any IFN-gamma. Paradoxically, adoptive transfer of CD4+CD25--derived Th2 cells into BALB/c mice induced an elevated airway eosinophilic inflammation in response to OVA inhalation compared with recipients of total CD4+ Th2 cells. The pronounced eosinophilia was associated with reduced levels of IL-10 and increased amounts of eotaxin in the bronchoalveolar lavage fluid. This Th2 phenotype characterized by reduced Th2 cytokine expression appeared to remain stable in vivo, even after repeated exposure of the animals to OVA aerosols. Our results demonstrate that the immunoregulatory properties of CD4+CD25+ T cells do extend to Th2 responses. Specifically, CD4+CD25+ T cells play a key role in modulating Th2-mediated pulmonary inflammation by suppressing the development of a Th2 phenotype that is highly effective in vivo at promoting airway eosinophilia. Conceivably, this is partly a consequence of regulatory T cells facilitating the production of IL-10.     

A pool of central memory-like CD4 T cells contains effector memory precursors

The L51S mutation in the D10.G4.1 TCR alpha-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the beta-chain of I-A(k), and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44(high)CD62L(neg)CD45RB(neg) CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R alpha only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.     

Maturation- and differentiation-dependent responsiveness of human CD4+ T helper subsets.

The human CD45R0+ (memory) CD4+ T cell population can be subdivided into a large (82%) CD27+ and a small (18%) CD27- subset. Within the CD45R0+CD27- subset, cells accumulate that have been persistently stimulated by Ag in vivo. As an apparent consequence, TLC with a differentiated functional phenotype, producing either high levels of IFN-gamma (Th1-like), high levels of IL-4 (Th2-like) or high amounts of both these cytokines (here referred to as Thx) can primarily be generated from the CD27- memory CD4+ T cell subset. In this study we examined the requirements for induction of proliferation of distinct CD4+CD45R0+ Th subsets. Immobilized CD3 mAb induced proliferation with comparable magnitude and kinetics in all types of TLC. However, interference with intracellular signaling pathways in this activation system, resulted in a strong inhibition of proliferation in TLC derived from CD27+ cells whereas, in contrast, TLC from CD27- cells were relatively resistant to elevation of [cAMP]i, inhibition of protein kinase C activation and the immunosuppressive effects of cyclosporin A. Stimulation with CD3 mAb in soluble form resulted in Il-4 secretion by Th2-like and Thx-type TLC but did not induce IFN-gamma or Il-2 secretion in any Th subset. Interestingly, Th2-like cells but not Thx-type cells were able to use endogenously produced Il-4 for proliferation. These data demonstrate a differential sensitivity of CD45R0+CD4+ Th subsets for immune activation and suppression, which correlated with their maturation stage, as reflected by CD27 membrane expression, as well as with their effector phenotype.     

Egg deposition is the major stimulus for the production of Th2 cytokines in murine schistosomiasis mansoni.

To characterize Th cell populations induced by helminth infection, spleen cells from mice infected with Schistosoma mansoni were stimulated with parasite (worm or egg Ag) or mitogen (Con A) and the supernatants assayed for the Th1-specific cytokines IFN-gamma and IL-2 and the Th2-specific cytokines IL-4 and IL-5. Th2 cytokine production was not detected in substantial quantity until the 6 to 8th wk of infection and after reaching peak levels at 8 to 12 wk declined slowly thereafter. The time courses of IL-4 and IL-5 production, whereas differing from each other, closely resembled corresponding published data on IgE and peripheral blood eosinophil levels during murine schistosome infection. In contrast, Th1 cytokine responses occurred only during the first 6 wk of infection and were virtually absent during the peak period of Th2 production. To assess the role of egg deposition in the observed pattern of Th response, cytokine production was assayed in mice carrying unisexual schistosome infections in which parasite eggs are absent. Splenocytes from these animals displayed only marginal Th2 cytokine synthesis but greater Th1 cytokine responses than the corresponding cells from mice with bisexual infections. Moreover, cultures of liver tissue or isolated granulomas from infected mice constitutively produced high levels of IL-4 and IL-5 but failed to synthesize significant amounts of IL-2 and IFN-gamma even when stimulated with egg Ag or mitogen. Taken together the data indicate that egg deposition is the major stimulus of Th2 cytokine response in S. mansoni-infected mice and suggest that T cells belonging to this subset must play a major role in egg granuloma formation.     

IL-4-based helper activity of CD4 T cells is radiation sensitive

The capacity of CD4⁺ T cells to induce IgG synthesis in B cells has been known to be radioresistant for more than 20 years. However, the radiation sensitivity of helper T cells with regard to their ability to induce the synthesis of isotypes other than IgG has not been studied. We therefore irradiated KLH-primed lymph node T cells and examined their capacity to induce IgG, IgM, and IgE synthesis in hapten-primed B cells. We demonstrated that while the capacity of KLH-primed lymph node cells to induce IgG synthesis was not affected by irradiation, the capacity of such T cells to induce IgE synthesis was greatly reduced by γ-irradiation. This was consistent with our observations that IL-4 and IL-5 synthesis in such cells was greatly diminished by irradiation, whereas IL-2 synthesis was only minimally affected. A similar differential sensitivity to irradiation of the helper activity of Th1 and Th2 clones was observed with regard to their ability to induce IgE and IgG synthesis under cognate conditions. Irradiation greatly inhibited the capacity of Th2 clones to induce IgE synthesis, but only minimally affected the capacity of Th1 clones to induce IgG synthesis in primed B cells. The capacity of irradiated Th2 clones to induce IgE synthesis was restored by the addition of IL-4 and IL-5. These results taken together indicated that the sensitivity to irradiation of T helper cells with regard to the induction of IgE but not IgG synthesis was due to the sensitivity to irradiation of the production of IL-4 but not of IL-2. Thus, although some functions of CD4⁺ T cells are resistant to radiation, other functions, particularly those that depend on the production of IL-4 and IL-5, are greatly diminished by ionizing radiation.