Reciprocal regulatory effects of IFN-gamma and IL-4 on the in vitro development of human Th1 and Th2 clones.

The effects exerted on the in vitro development of purified protein derivative (PPD)-specific or Dermatophagoides pteronyssinus group I (Der p I)-specific T cell lines (TCL) and T cell clones (TCC) by IL-4 or IFN-gamma addition or neutralization in human PBMC cultures were examined. PBMC from two normal individuals, which were stimulated with PPD and then cultured in IL-2 alone, developed into PPD-specific TCL and TCC able to produce IFN-gamma and IL-2 but not IL-4 and IL-5 (Th1-like). IFN-gamma or anti-IL-4 antibody addition in bulk cultures before cloning did not influence the PPD-specific TCL profile of cytokine production. In contrast, the addition of IL-4 resulted in the development of PPD-specific TCL and TCC able to produce not only IFN-gamma and IL-2 but also IL-4 and IL-5. PBMC from one atopic Der p I-sensitive patient, which were stimulated with Der p I and then cultured in IL-2 alone, developed into Der p I-specific TCL and TCC able to produce IL-5 and large amounts of IL-4 but no IFN-gamma (Th2-like). The addition in bulk cultures, before cloning, of either IFN-gamma or anti-IL-4 antibody markedly inhibited the development of Der p I-specific T cells into IL-4- and IL-5-producing TCL. Accordingly, the development into Der p I-specific Th2-like TCC was significantly reduced by the addition of IFN-gamma in bulk culture and was virtually suppressed by the presence of both IFN-gamma and anti-IL-4 antibody. These data suggest that the presence or the absence of IL-4 and IFN-gamma in bulk cultures of PBMC before cloning may have strong regulatory effects on the in vitro development of human CD4+ T cells into Th1 or Th2 clones.     

Th2 cytokines in susceptibility to tuberculosis

We need to understand what is different about susceptibility to tuberculosis (TB) in developing countries where most TB occurs, and where the current vaccine, Bacillus Calmette et Guérin (BCG) usually fails to protect. The presence of a background mixed IFN-gamma and Th2 response to mycobacterial antigens before infection with M. tuberculosis (Mtb), and the development of a large IL-4 response during progressive TB, are characteristics of individuals in the locations where BCG fails, which are also seen in animal models in the same countries. Recent data suggest that the background Th1 component in developing countries protects from low dose challenge with Mtb in mouse and man, but that following high dose challenge the pre-existing IL-4 component increases and blocks immunity unless the individual's immune system releases IL-4delta2, an antagonist of IL-4, which is raised in the blood of donors with stable latent TB. We outline how IL-4 (and IL-13) can undermine Th1-mediated immunity and drive inappropriate alternative activation of macrophages. The mechanisms of the effects of IL-4 include impaired antimicrobial activity due to reduced TNF-alpha-mediated apoptosis of infected cells, reduced activity of iNOS, increased availability of iron to intracellular Mtb, and increased proliferation of antigen-specific FOXP-3+ regulatory T cells. IL-4 also increases the toxicity of TNF-alpha and drives pulmonary fibrosis, thus enhancing immunopathology. The conclusion is that a vaccine that will work in developing countries might need to do more than enhance the existing Th1 response. In these environments it might be more important to block the Th2 component.     

IL-18 regulates intestinal mastocytosis and Th2 cytokine production independently of IFN-gamma during Trichinella spiralis infection

Expulsion of the gastrointestinal nematode Trichinella spiralis is associated with pronounced mastocytosis mediated by a Th2-type response involving IL-4, IL-10, and IL-13. Here we demonstrate that IL-18 is a key negative regulator of protective immune responses against T. spiralis in vivo. IL-18 knockout mice are highly resistant to T. spiralis infection, expel the worms rapidly and subsequently develop low levels of encysted muscle larvae. The increased speed of expulsion is correlated with high numbers of mucosal mast cells and an increase in IL-13 and IL-10 secretion. When normal mice were treated with rIL-18 in vivo, worm expulsion was notably delayed, and the development of mastocytosis and Th2 cytokine production was significantly reduced. The treatment had no effect on intestinal eosinophilia or goblet cell hyperplasia but specifically inhibited the development of mastocytosis. Addition of rIL-18 to in vitro cultures of bone marrow-derived mast cells resulted in a significant reduction in cell yields as well as in the number of IL-4-secreting mast cells. In vivo treatment of T. spiralis-infected IFN-gamma knockout mice with rIL-18 demonstrated that the inhibitory effect of IL-18 on mastocytosis and Th2 cytokine secretion is independent of IFN-gamma. Hence, IL-18 plays a significant biological role as a negative regulator of intestinal mast cell responses and may promote the survival of intestinal parasites in vivo.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

Human atopen-specific types 1 and 2 T helper cell clones.

Eight representative T lymphocyte clones (TLC) randomly selected from previously described panels of CD4+ housedust mite Dermatophagoides pteronyssinus (Dp)-specific TLC from atopic and nonatopic donors were studied in more detail in a comparative investigation. The TLC from the atopic donors closely resembled murine type 2 Th (Th2) cells by secreting substantial IL-4, IL-5, IL-6, TNF-alpha, and granulocyte-macrophage (GM)-CSF, minimal IFN-gamma, and relatively little IL-2. In contrast, the nonatopic's TLC resembled murine type 1 Th (TH1) cells by secreting substantial IFN-gamma, IL-2, TNF-alpha, and GM-CSF, no IL-4, and little IL-5. A difference with murine Th1 cells was their additional secretion of IL-6. These cytokine profiles were consistent upon stimulation via different activation pathways including stimulation with specific Dp Ag, mitogenic lectins, and antibodies to CD2, CD3, or CD28. The observed differences in IL-2 secretion, however, were most evident upon stimulation with anti-CD28. If TLC cells were cultured with highly purified B cells and stimulated with anti-CD3 in the absence of exogenous IL-4, IgE synthesis was induced only in cultures with the atopics' Th2 clones, which could be completely abrogated by anti-IL-4. The mere presence of exogenous rIL-4, however, did not result in IgE synthesis, nor did unstimulated TLC cells alone. But if unstimulated TLC cells (that proved not to secrete detectable amounts of cytokines) were added together with rIL-4, again IgE synthesis was induced only in cultures with the atopics' Th2 clones, suggesting the involvement of an additional, as yet unidentified accessory helper function of the atopics' Th2 clones for IgE induction. Unstimulated Th2 clones showed a significantly higher expression of CD28 than the Th1 clones, but three days after stimulation, CD28 expression was elevated to comparable levels on both subsets. When added to B cells at this time point, together with rIL-4 and anti-IFN-gamma, still only the atopics' Th2 clones supported IgE synthesis, arguing against a role for CD28 in this accessory helper function. Whereas the atopics' Th2 clones were excellent helper cells for IgE induction, a unique property of the nonatopic's Th1 clones was their cytolytic activity toward autologous APC which could be induced by specific Dp Ag and by anti-CD3. The present data provide clear evidence for the existence of Th1 and Th2 cells in man.     

Heterogeneity of mouse helper T cells. Evidence from bulk cultures and limiting dilution cloning for precursors of Th1 and Th2 cells

Many long term mouse Th clones express either the type 1 or type 2 Th cell (Th1 or Th2) cytokine secretion phenotype. In this report we present two lines of evidence for the existence of additional Th differentiation states. Lectin-stimulated spleen cells secreted moderate levels of IL-2 compared with long term Th1 clones, whereas the levels of other cytokines were more than 100-fold lower than those produced by either Th1 or Th2 clones. This suggests that many spleen cells produce substantial amounts of IL-2 but little or no IL-4, IL-5, IFN-gamma, IL-3, and granulocyte/macrophage-CSF. In contrast to long term Th clones, many short term alloreactive clones displayed cytokine secretion phenotypes intermediate between the Th1 and Th2 patterns. The proportion of recognizable Th1 and Th2 clones at early times in culture was greatly increased by immunization of the mice from which the responder and stimulator cells were derived; Brucella abortus immunization resulted in the isolation of exclusively Th1 clones, whereas infection with Nippostrongylus brasiliensis resulted in a strong trend toward the isolation of Th2 clones. The immunization of mice from which responder cells were derived strongly affected the type of Th clone obtained, whereas the source of stimulator cells had much less effect, suggesting that the commitment of Th cells to the Th1 or Th2 phenotypes occurred mainly in vivo. A model for the possible relationships of the various Th cells is presented.     

IL-18 stimulates the proliferation and IFN-gamma release of CD4+ T cells in the chicken: conservation of a Th1-like system in a nonmammalian species

The phylogeny of Th1 and Th2 subsets has not been characterized mainly due to the limited information regarding cytokines in nonmammalian vertebrates. In this study, we characterize a Th1-like regulatory system focusing on the IL-18-regulated IFN-gamma secretion. Stimulation of splenocytes with chicken IL-18 induced high levels of IFN-gamma secretion. Depletion of either macrophages or CD4(+) T cells from the splenocyte cultures caused unresponsiveness to IL-18. In contrast, PBL were unresponsive to IL-18 in the presence or absence of macrophages, but IFN-gamma secretion was stimulated by suboptimal anti-TCR cross-linking combined with IL-18. Splenocytes from five different chicken lines responded equally well to the IL-18 treatment. LSL chicken splenocytes, however, responded only to IL-18 when stimulated either with optimal TCR cross-linking alone or suboptimal TCR cross-linking combined with IL-18. IL-18 not only induced IFN-gamma secretion, but also stimulated splenocyte proliferation. This IL-18-induced proliferation was compared with the effects observed with IL-2. Both cytokines activated the splenocytes as demonstrated by increased size and MHC class II Ag up-regulation in the case of IL-18. Phenotypic analyses following 6 days of culture revealed that IL-2 mainly affected the proliferation of CD8(+) cells, whereas IL-18 had an opposite effect and stimulated the proliferation of CD4(+) cells. Taken together, these results demonstrate the conservation of Th1-like proinflammatory responses in the chicken; they characterize IL-18 as a major growth factor of CD4(+) T cells and identify two distinct mechanisms of IL-18-induced IFN-gamma secretion.     

Functional maturation of adult mouse resting microglia into an APC is promoted by granulocyte-macrophage colony-stimulating factor and interaction with Th1 cells

A precise knowledge of the early events inducing maturation of resting microglia into a competent APC may help to understand the involvement of this cell type in the development of CNS immunopathology. To elucidate whether signals from preactivated T cells are sufficient to induce APC features in resting microglia, microglia from the adult BALB/c mouse CNS were cocultured with Th1 and Th2 lines from DO11.10 TCR transgenic mice to examine modulation of APC-related molecules and Ag-presenting capacity. Upon Ag-specific interaction with Th1, but not Th2, cells, microglia strongly up-regulated the surface expression of MHC class II, CD40, and CD54 molecules. Induction of CD86 on mouse microglia did not require T cell-derived signals. Acutely isolated adult microglia stimulated Th1 cells to secrete IFN-gamma and, to a lesser extent, IL-2, but were inefficient stimulators of IL-4 secretion by Th2 cells. Microglia exposed in vitro to IFN-gamma showed enhanced expression of MHC class II, CD40, and CD54 molecules and became able to restimulate Th2 cells. In addition to IFN-gamma, GM-CSF increased the ability of microglia to activate Th1, but not Th2, cells without up-regulating MHC class II, CD40, or CD54 molecules. These results suggest that interaction with Th1 cells and/or Th1-secreted soluble factors induces the functional maturation of adult mouse microglia into an APC able to sustain CD4+ T cell activation. Moreover, GM-CSF, a cytokine secreted by T cells as well as reactive astrocytes, could prime microglia for Th1-stimulating capacity, possibly by enhancing their responsiveness to Th1-derived signals.     

IL-4 directs the development of Th2-like helper effectors

Our studies show that the presence of IL-4 during the response of naive Th cells causes precursors to develop into a population comprised largely of "Th2-like" effectors that secrete IL-4 and IL-5, but little IL-2 or IFN-gamma. We find that the levels of IL-4 and IL-2 determine both the level of effectors developed in response to mitogen or Ag and the patterns of lymphokines they secrete when restimulated. IL-2 is required for optimum generation of effectors, and increasing levels of IL-2, augments the expansion of effectors secreting both IL-4/IL-5 and IFN-gamma. In contrast, IL-4 is required for the development of IL-4/IL-5 secreting effectors but suppresses the development of IL-2 and at higher doses IFN-gamma-secreting effectors detected after 4 days. Also dramatic are the effects of the presence or absence of IL-4 evaluated after an additional 1 to 2 wk. When cultures with or without initial IL-4 are cultured in IL-2 alone from days 4 to 11, they retain their distinct patterns of lymphokine production. Those cells that developed in cultures without IL-4 progressively secrete more IL-2 and can be maintained and expanded in IL-2. They continue to produce IFN-gamma, though the levels decrease somewhat with time, but they do not acquire the ability to produce IL-4 or IL-5. These cells thus increasingly resemble Th1 cell lines. In contrast, those cells in cultures initially exposed to IL-4, generate effectors which secrete high levels of IL-4/IL-5 (plus variable levels of IFN-gamma) at days 4 to 5, but the populations of cells developed, are not maintained well on IL-2 alone. Those cells that do survive continue to secrete IL-4 and IL-5 but not IL-2. In addition, IFN-gamma production, if present, falls off with time. Thus the cells in these cultures take on an increasingly Th2-like phenotype. It appears that the effects of low levels of IL-4 in suppressing IL-2 production by day 4 effectors appear to be transient whereas the higher levels appear to drive the development along a distinct pathway which is irreversible. These studies support the concept that different subsets of helper cells, which correspond roughly to Th1 and Th2 subsets, can develop rapidly in short term culture with respectively low vs high levels of IL-4. They support the concept that such distinct phenotypes arise from alternate pathways of differentiation that can be expected to reflect pathways available for helper T cell differentiation in the animal.     

Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

Protein vaccines induce uncommitted IL-2-secreting human and mouse CD4 T cells, whereas infections induce more IFN-gamma-secreting cells

Mouse and human CD4 T cells primed during an immune response may differentiate into effector phenotypes such as Th1 (secreting IFN-gamma) or Th2 (secreting IL-4) that mediate effective immunity against different classes of pathogen. However, primed CD4 T cells can also remain uncommitted, secreting IL-2 and chemokines, but not IFN-gamma or IL-4. We now show that human CD4 T cells primed by protein vaccines mostly secreted IL-2, but not IFN-gamma, whereas in the same individuals most CD4 T cells initially primed by infection with live pathogens secreted IFN-gamma. We further demonstrate that many tetanus-specific IL-2+IFN-gamma- cells are uncommitted and that a single IL-2+IFN-gamma- cell can differentiate into Th1 or Th2 phenotypes following in vitro stimulation under appropriate polarizing conditions. In contrast, influenza-specific IL-2+IFN-gamma- CD4 cells maintained a Th1-like phenotype even under Th2-polarizing conditions. Similarly, adoptively transferred OTII transgenic mouse T cells secreted mainly IL-2 after priming with OVA in alum, but were biased toward IFN-gamma secretion when primed with the same OVA peptide presented as a pathogen Ag during live infection. Thus, protein subunit vaccines may prime a unique subset of differentiated, but uncommitted CD4 T cells that lack some of the functional properties of committed effectors induced by infection. This has implications for the design of more effective vaccines against pathogens requiring strong CD4 effector T cell responses.     

IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis.

Resistance to Leishmania major in mice is associated with the generation of distinct CD4+ Th subsets, termed TH1 and TH2. To define the factors contributing to the genesis of these Th cells, we first investigated when these subsets developed following L. major infection. Lymph node (LN) cells collected 3 days after infection of BALB/c mice secreted IL-4 and IL-5 in vitro, but little IFN-gamma, whereas LN cells from a resistant strain, C3H/HeN, secreted IFN-gamma and no IL-4 or IL-5. Cytokine production was eliminated in both cases by in vivo or in vitro depletion of CD4+ cells, but not after depletion of CD8+ cells. Similar responses were observed after inoculation of killed promastigotes or a soluble leishmanial Ag preparation. These data indicate that the development of Th1- and Th2-like responses can precede lesion formation and does not require a live infection. We next investigated whether IFN-gamma was important in the differentiation of Th1 and Th2 cells. C3H/HeN mice have previously been shown to be susceptible to leishmanial infection after treatment with anti-IFN-gamma. We confirmed this observation and found that the abrogation of resistance was associated with enhanced production of IL-4 and IL-5, and decreased production of IFN-gamma by cells taken from these mice. Conversely, LN cells from BALB/c mice inoculated with parasites plus IFN-gamma produced significantly higher levels of IFN-gamma, and decreased levels of IL-4 and IL-5, than mice infected with parasites alone. Finally, we determined if IFN-gamma might augment vaccine induced immunity. We found that s.c. immunization with soluble leishmanial Ag, the bacterial adjuvant, Corynebacterium parvum and IFN-gamma could protect mice against L. major infection, and that this protection was associated with induction of Th1 responses. From these data we conclude that levels of IFN-gamma at the time of infection or immunization dramatically alters the type of response elicited: high levels of IFN-gamma favor Th1 type responses, whereas low levels promote a Th2 response.     

B cell response to T helper cell subsets. II. Both the stage of T cell differentiation and the cytokines secreted determine the extent and nature of helper activity.

Helper activity of several murine CD4+ T cell subsets was examined. Effector Th, derived from naive cells after 4 days of in vitro stimulation with alloantigen, when generated in the presence of IL-4, secreted high levels of IL-4, IL-5, and IL-6, and low levels of IL-2 and IFN-gamma, and induced the secretion of all Ig isotypes particularly IgM, IgG1, IgA, and IgE from resting allogeneic B cells. Effectors generated with IL-6 secreted IL-2, IL-4, IL-5, IL-6, and IFN-gamma, and induced similar levels of total Ig, 25 to 35 micrograms/ml, but with IgM, IgG3, IgG1, and IgG2a isotypes predominating. Helper activity of these Th was significantly greater than that of effectors generated with IL-2 (10-15 micrograms/ml Ig) and of 24-h-activated naive and memory cells (2-4 micrograms/ml), both of which induced mainly IgM. Unlike other isotypes, IgE was induced only by effector Th generated with IL-4. Blocking studies showed that secretion of all isotypes in response to IL-6-primed effectors was dependent on IL-2, IL-5, and IL-6. IL-4 was required for optimal IgM, IgG1, and IgA secretion, but limited secretion of IgG2a, whereas IFN-gamma was required for optimal IgG2a secretion, and limited IgM, IgG1, and IgA. In contrast, secretion of all isotypes in response to IL-4-primed effectors was dependent on IL-5, although IL-4 and IFN-gamma were also essential for IgE and IgG2a, respectively. Addition of exogenous IL-5 to B cell cultures driven by IL-6-primed effectors did not obviate the requirement for IL-2, IL-4, and IL-6, suggesting that interaction of IL-4-primed effectors with B cells was qualitatively different from that of IL-6-primed effectors, driving B cells to a stage requiring only IL-5 for differentiation. Addition of exogenous factors to IL-2-primed effector Th, particularly IL-4 in the presence of anti-IFN-gamma, resulted in levels of Ig, including IgE, comparable to those induced with other effectors. These results show that functionally distinct Th cell subsets can be generated rapidly in vitro, under the influence of distinct cytokines, which vary dramatically in their levels of help for resting B cells. The cytokines involved in responses to distinct Th cells differ depending on the quality of interaction with the B cell, and the extent of help is strongly determined by the quantity and nature of cytokines secreted by the T cells.     

IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones.

The cytolytic potential of a total number of 118 CD4+ human T cell clones specific for purified protein derivative (PPD) from Mycobacterium tuberculosis, tetanus toxoid, Lolium perenne group I allergen (Lol p I), Poa pratensis group IX allergen (Poa p IX), or Toxocara canis excretory/secretory antigen(s) (TES) was assessed by both a lectin (PHA)-dependent and a MHC-restricted lytic assay and compared with their profile of cytokine secretion. The majority of clones with Th1 or Th0 cytokine profile exhibited cytolytic activity in both assays, whereas Th2 clones usually did not. There was an association between the cytolytic potential of T cell clones and their ability to produce IFN-gamma, even though IFN-gamma produced by T cell clones was not responsible for their cytolytic activity. IL-4 added in bulk culture before cloning inhibited not only the differentiation of PPD-specific T cells into Th1-like cell lines and clones, but also the development of their cytolytic potential. The depressive effect of IL-4 on the development of PPD-specific T cell lines with both Th1 cytokine profile and cytolytic potential was dependent on early addition of IL-4 in bulk cultures. In contrast, the addition in bulk culture of IFN-gamma enhanced both the cytolytic activity of PPD-specific T cell lines, as well as the proportion of PPD-specific T cell clones with cytolytic activity. The addition in bulk cultures before cloning of IFN-gamma or IFN-alpha favored the development of TES-specific and Poa p IX-specific T cells into T cell clones showing a Th0 or even a Th1, rather than a Th2, cytokine profile. Accordingly, most of TES- and Poa p IX-specific T cell clones derived from cultures containing IFN-gamma or IFN-alpha displayed strong cytolytic activity. These data indicate that the majority of human T cell clones that produce IFN-gamma, but not IL-4 (Th1-like), as well as of T cell clones that produce IFN-gamma in combination with IL-4 (Th0-like) are cytolytic. More importantly, they demonstrate that the addition of IFN (alpha and gamma) or IL-4 in bulk cultures before cloning may influence not only the cytokine profile of human CD4+ T cell clones but also their cytolytic potential.     

Lymphokine control of type 2 antigen response. IL-10 inhibits IL-5- but not IL-2-induced Ig secretion by T cell-independent antigens.

A supernatant derived from the Th2 clone D10.G4.1 (D10 supernatant) stimulated high numbers of Ig-secreting cells when added to dextran-conjugated anti-delta-antibody (anti-delta-dextran)-activated B cells but stimulated only marginal Ag-specific responses when added to B cells cultured with TNP-Ficoll. When anti-IL-10 antibody was added to cultures containing D10 supernatant, IL-5, and TNP-Ficoll, there was a significant increase in the numbers of anti-TNP-antibody producing cells, suggesting that at least a part of the inhibitory activity of D10 supernatant is mediated by IL-10. Addition of rIL-10 inhibited both TNP-Ficoll- and anti-delta-dextran-mediated Ig secretion that was stimulated in the presence of IL-5 but had no suppressive effect on IL-2-stimulated responses, indicating that its inhibitory effect was selective for a specific mode of B cell activation. Addition of IL-10 did not, however, inhibit anti-delta-dextran-stimulated B cell proliferation. The IL-10-induced-inhibition of Ig secretion was not due to suppression of IFN-gamma production, because the addition of IFN-gamma did not reverse the inhibition, nor did the addition of anti-IFN-gamma mimic the IL-10-mediated inhibition. These data suggest that a composite of lymphokines secreted by Th cells may contain both inhibitory and stimulatory activities. Sorting out the conditions under which stimulation or inhibition is seen may reveal additional diversity in Ag-stimulated pathways of B cell activation.     

3D computation modelling of the influence of cytokine secretion on Th-cell development suggests that negative selection (inhibition of Th1 cells) is more effective than positive selection by IL-4 for Th2 cell dominance

Th-cell development has been suggested to include selective mechanisms in which certain cytokines select either Th1 or Th2 cells to proliferate and grow. The selective theory is based on the observation that Th2 cells secrete IL-4, a cytokine that promotes Th2 development, whereas Th1 cells secrete interferon-gamma (IFN-gamma) that favours Th1 development, and both positive and negative selective influences have been suggested to operate. In this study, we investigate the role of autocrine secretion and utilization of IL-4 by Th2 cells and address the question of whether an activated Th2 cell can be positively selected by IL-4 secreted from other Th2 cells. We present a spatial three dimensional (3D) modelling approach to simulate the interaction between the IL-4 ligand and its IL-4 receptors expressed on discrete IL-4 secreting cells. The simulations, based on existing experimental data on the IL-4 receptor-ligand system, illustrate how Th-cell development is highly dependent on the distance between cells that are communicating. The model suggests that a single Th2 cell is likely to communicate with possible target cells within a range of approximately 100 microm and that an activated Th2 cell manages to fill most of its own IL-4 receptors, even at a low secretion rate. The predictions made by the model suggest that negative selection against Th1 cells is more effective than positive selection by IL-4 for promoting Th2 dominance.     

Collaboration of Th1 and Th2 T cell clones in specific antibody responses: regulation of the IgM response to phosphorylcholine

Carrier (KLH)-specific type 1 T cell clones (Th1), which are defined by secretion of IL-2 and IFN-gamma but not IL-4, and type 2 (Th2) clones, which secrete IL-4, but not IL-2 or IFN-gamma, have been isolated and analyzed for their ability to collaborate in providing help for B cells to secrete phosphorylcholine-specific IgM antibodies. The resulting antibody responses exhibited a characteristic pattern suggesting two distinct regulatory interactions among the Th1, Th2, and B cells. At low doses of antigen, Th1 cells enhanced the helper function of the Th2 cells, an effect due primarily to IL-2. At high doses of antigen, Th1 cells or IFN-gamma inhibited Th2-dependent antibody responses. The inhibitory effect of Th1 or IFN-gamma affected primarily the hapten-carrier-linked portion of the response. The overall effect was a modulation of the antigen dose-response curve for antibody production, eliminating the sharp increases in dose response mediated by isolated T cell clones. The data suggest that collaborative interactions of Th1 and Th2 cells in antibody production may have important physiological consequences.     

Generation of B cell memory and affinity maturation. Induction with Th1 and Th2 T cell clones.

We used an adoptive transfer system and CD4+ T cell clones with defined lymphokine profiles to examine the role of CD4+ T cells and the types of lymphokines involved in the development of B cell memory and affinity maturation. Keyhole limpet hemocyanin (KLH)-specific CD4+ Th2 clones (which produce IL-4 and IL-5 but not IL-2 or IFN-gamma) were capable of inducing B cell memory and affinity maturation, after transfer into nude mice or after transfer with unprimed B cells into irradiated recipients and immunization with TNP-KLH. In addition, KLH-specific Th1 clones, which produce IL-2 and IFN-gamma but not IL-4 or IL-5, were also effective in inducing B cell memory and high affinity anti-TNP-specific antibody. The induction of affinity maturation by Th1 clones occurred in the absence of IL-4, as anti-IL-4 mAb had no effect on the affinity of the response whereas anti-IFN-gamma mAb completely blocked the response. Th1 clones induced predominantly IgG2a and IgG3 antibody, although Th2 clones induced predominantly IgG1 and IgE antibody. We thus demonstrated that some Th1 as well as some Th2 clones can function in vivo to induce Ig synthesis. These results also suggest that a single type of T cell with a restricted lymphokine profile can induce both the terminal differentiation of B cells into antibody secreting cells as well as induce B cell memory and affinity maturation. Moreover, these results suggest that B cell memory and affinity maturation can occur either in the presence of Th2 clones secreting IL-4 but not IFN-gamma, or alternatively in the presence of Th1 clones secreting IFN-gamma but not IL-4.     

Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells

The regulatory roles of Th1 and Th2 cells in immune protection against Helicobacter infection are not clearly understood. In this study, we report that a primary H. pylori infection can be established in the absence of IL-12 or IFN-gamma. However, IFN-gamma, but not IL-12, was involved in the development of gastritis because IFN-gamma(-/-) (GKO) mice exhibited significantly less inflammation as compared with IL-12(-/-) or wild-type (WT) mice. Both IL-12(-/-) and GKO mice failed to develop protection following oral immunization with H. pylori lysate and cholera toxin adjuvant. By contrast, Th2-deficient, IL-4(-/-), and WT mice were equally well protected. Mucosal immunization in the presence of coadministered rIL-12 in WT mice increased Ag-specific IFN-gamma-producing T cells by 5-fold and gave an additional 4-fold reduction in colonizing bacteria, confirming a key role of Th1 cells in protection. Importantly, only protected IL-4(-/-) and WT mice demonstrated substantial influx of CD4(+) T cells in the gastric mucosa. The extent of inflammation in challenged IL-12(-/-) and GKO mice was much reduced compared with that in WT mice, indicating that IFN-gamma/Th1 cells also play a major role in postimmunization gastritis. Of note, postimmunization gastritis in IL-4(-/-) mice was significantly milder than WT mice, despite a similar level of protection, indicating that immune protection is not directly linked to the degree of gastric inflammation. Only protected mice had T cells that produced high levels of IFN-gamma to recall Ag, whereas both protected and unprotected mice produced high levels of IL-13. We conclude that IL-12 and Th1 responses are crucial for H. pylori-specific protective immunity.