Opposite role for interleukin-4 and interferon-γ on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells

Polarized human type 1 and type 2 T helper cells not only produce different sets of cytokines, but they also preferentially express certain activation markers, such as lymphocyte activation gene-3 (LAG-3) and CD30, respectively. In this study we have examined the LAG-3 and CD30 expression in relation to the lineage commitment of human naive CD4⁺ T cells, as assessed at the single-cell level of committed T cells. Purified CD45RA⁺ umbilical cord blood T lymphocytes were activated with phytohemagglutinin and interleukin (IL)-2 in the absence or presence of interleukin IL-4 or IL-12 and assessed for CD30 and LAG-3 expression, as well as for intracellular cytokine synthesis. Significant numbers of CD30⁺ cells were only found in CD4⁺ and CD8⁺ T lymphocytes of cultures primed with IL-4, which developed into cells able to produce IL-4 and IL-13 in addition to interferon (IFN)-γ. By contrast, LAG-3 expression was strongly up-regulated in CD4⁺ and CD8⁺ T cells from cultures primed with IL-12, which developed into high numbers of IFN-γ producers. The addition of a neutralizing anti-IFN-γ antibody to IL-12-primed CD4⁺ T cell cultures virtually abolished the development of LAG-3-expressing CD4⁺ T cells. Taken together, these data suggest that CD30 expression is dependent on the presence of IL-4, whereas LAG-3 expression is dependent on the production of IFN-γ during the lineage commitment of human naive T cells.     

Interleukin-12 but not interferon-γ production correlates with induction of T helper type-1 phenotype in murine candidiasis

By means of polymerase chain reaction-assisted mRNA amplification, we have monitored message levels of interleukin (IL)-12 in splenic macrophages and of interferon-γ (IFN-γ), IL-4, and IL-10 in CD4⁺ and CD8⁺ T cells using Candida albicans/host combinations that result either in a T helper type-1 (Th1)-associated self-limiting infection (“healer mice”) or in a Th2-associated progressive disease (“nonhealer mice”). The timing and pattern of message detection did not differ qualitatively by the expression of IFN-γ or IL-10 mRNA in CD4⁺ and CD8⁺ cells from healer (i.e. PCA-2 into CD2F1) vs. nonhealer (i.e. CA-6 into CD2F1 or PCA-2 into DBA/2) mice. In contrast, IL-4 mRNA was uniquely expressed by CD4⁺ cells from nonhealer animals. IL-12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen-driven cytokine production by CD4⁺ and CD8⁺ cells was assessed in vitro, while IFN-γ-producing cells were enumerated in CD4^? CD8^? cell fractions. Overall, our results showed that (i) antigen-specific secretion of IFN-γ protein in vitro by CD4⁺ cells occurred only in healing infection; (ii) IL-4- and IL-10-producing CD4⁺ cells would expand in nonhealer mice in the face of high levels of circulating IFN-γ, likely released by CD4^? CD8^? lymphocytes; (iii) a finely regulated IFN-γ production correlated in the healer mice with IL-12 mRNA detection, and IL-12 was required in vitro for yeast-induced development of IFN-γ-producing CD4⁺ cells. Although the mutually exclusive production of IL-4/IL-10 and IFN-γ by early CD4⁺ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL-12 rather than IFN-γ production may be an indicator of Th1 differentiation.     

IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL-4 or IFN-γ. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4⁻ CD8⁻ TCRα β⁺ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-γ in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-γ is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-γ production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-γ secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant up-regulation of the capacity of NK T cells to produce IFN-γ after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.     

Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-γ or IL-12. The resulting CD4⁺ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-γ and tumor necrosis factor (TNF)-β in response to stimulation with phorbol 12-myristate 13-acetate (PMA)+anti-CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4⁺ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-γ, TNF-β or IL-10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN-γ. However, upon addition of exogenous IL-12, the resulting CD4⁺ thymocyte clones produced TNF-β, IFN-γ, and IL-10 in addition to IL-4 and IL-5. These results suggest that CD4⁺ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.     

Schistosoma-specific helper T cell clones from subjects resistant to infection by Schistosoma mansoni are Th0/2

Although T helper cells play a critical role in human immunity against schistosomes, the properties of the T lymphocytes that govern resistance and pathogenesis in human schistosomiasis are still poorly defined. This work addresses the question as to whether human resistance to Schistosoma mansoni is associated with a particular T helper subset. Twenty-eight CD3⁺, CD4⁺, CD8^? parasite-specific T cell clones were isolated from three adults with high degree of resistance to infection by S. mansoni. The lymphokine secretion profiles of these clones were determined and compared to those of 21 CD3⁺, CD4⁺, CD8^? clones with unknown specificity, established from these same subjects in the same cloning experiment. Almost all parasite-specific clones produced interleukin (IL)-4 and interferon (IFN)-γ in large amounts. However, they generally produced more IL-4 than IFN-γ; variations in IL-4/IFN-γ ratios were accounted for by differences in IFN-γ production since IL-4 levels were comparable for the clones from the three subjects. T cell clones of unknown specificity produced significantly less IL-4 and more IFN-γ than parasite-specific T cell clones. Most clones produced IL-2, and IL-2 production did not differ between the two types of clones. Parasite-specific T cell clones from the resistant subjects were compared to specific T cell clones from a sensitized adult from a nonendemic area: T cell clones from this latter subject were the highest IFN-γ and the lowest IL-4 producers, compared to those of resistant subjects. Thus, parasite-specific T cell clones isolated from adults resistant to S. mansoni belong to the Th0 subset and produced more IL-4 than IFN-γ (Th0/2), whereas clones of a sensitized adult from a nonendemic area are also Th0, but produce more IFN-γ than IL-4 (Th0/1). These results support previous conclusions on the role of IgE in protection against schistosomes in humans, and may indicate that IFN-γ is required for full protection.     

Specific expression of surface interferon-γ on interferon-γ producing T cells from mouse and man

Interferon (IFN)-γ is a potent immunoregulatory protein secreted by CD4⁺ and CD8⁺ T cells and by natural killer cells. Here, we show that IFN-γ is specifically displayed at a low concentration on the cell surface of those activated T cells from mouse and man which express IFN-γ. It is transiently expressed on the cell surface with kinetics similar to those of intracellular IFN-γ expression. Detectable surface IFN-γ is not expressed by activated T helper (Th) cells producing other cytokines but which do not express IFN-γ. Thus, surface IFN-γ is the first available marker for live T lymphocytes expressing IFN-γ, e.g. Th1 cells.     

Primary defect in CD8+ lymphocytes in the antibody deficiency disease (common variable immunodeficiency): abnormalities in intracellular production of interferon-gamma (IFN-γ) in CD28+ (‘cytotoxic’) and CD28− (‘suppressor’) CD8+ subsets

We have measured by flow cytometry the ability of subsets of CD8⁺ CD3⁺ lymphocytes within mononuclear cell preparations to make intracellular cytokines (IL-2, tumour necrosis factor-alpha (TNF-α) and IFN-γ) on stimulation in vitro with phorbol myristate acetate (PMA) and ionomycin for 16 h. These CD8⁺ subsets were defined by the presence or absence of CD28 or HLA-DR. Subsets of normal CD8⁺ cells were compared with cells from the antibody deficiency disease common variable immunodeficiency (CVID). In CVID there was a significant increase in the production of IFN-γ in the CD8⁺ CD28⁺ subset (‘cytotoxic’). This reflects a shift in this disease towards an excessive Th1 response away from B cell help. Paradoxically, some CVID patients also showed a reduction in IFN-γ production in the CD8⁺ CD28^? subset (‘suppressor’) which was associated with a failure of these cells to maintain a state of activation after a stimulus in vitro. The B cell problem in this disease is known to be related to a failure of T cell help shown by an inability to produce the antigen-specific CD4⁺ memory T cells needed for successful B cell maturation. The two pathological CD28 subsets of CD8⁺ cells we have found in CVID may both be detrimental to a normal CD4-dependent immune response. The CD28^? suppressor subset expands and is unable to maintain activation and cytokine secretion, and the CD28⁺ cytotoxic subset is over-producing the Th1 cytokine IFN-γ.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

Regulation of Human Cytotoxic T Lymphocytes Development by the Synergistic Effect of IL-7 and sCD23

The present study was undertaken to investigate the interaction of IL-7 and sCD23 on human peripheral blood T cell activation and CTL differentiation. Purified T lymphocytes were stimulated with mitogen plus IL-2 and subcultured for 7 days with IL-7 and/or sCD23. The combination of IL-7 and sCD23 synergistically enhanced the proliferation of both CD4⁺and CD8⁺T cells. CD8⁺T cells, however, were usually more responsive to IL-7 and sCD23. This synergy was observed on both subsets of T cells. Furthermore, these cytokines synergistically augment the CTL activity of CD8⁺T cells in both mitogen- and antigen-activated T cells. MAbs anti-IL-2 or anti-IL-2R (CD25) and anti-IL-12 had no effect on T cell proliferation and CD8⁺cytotoxic activity induced by IL-7 and sCD23. We analyzed the effect on IFN-γ induction by CD8⁺T cells and found that IL-7 alone was incapable of inducing detectable levels of IFN-γ production, but together with sCD23 it enhanced the production of IFN-γ. We also found that IFN-γ was not required for enhanced CTL activity of CD8⁺T cells, because rabbit anti-IFN-γ did not block the synergistic effects of either cytokine. The data demonstrate that the synergistic stimulatory activity of IL-7 and sCD23 may be of significance in the human CTL development and provide an alternative mechanism of stimulating T cells for use in immunotherapy.     

Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions

Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-α, IL-1β and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E₂ to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-α/IL-1/IL-6 or MCM alone induced CD4⁺ T cells that release intermediate levels of interferon (IFN)-γ and no IL-4 or IL-10. Production of IFN-γ was significantly induced by addition of PGE₂, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8⁺ T cells. While MCM conditions only induced IFN-γ^low, IL-4^neg cells, TNF-α/IL-1/IL-6 promoted growth of IFN-γ^intermediate, IL-4^neg CD8⁺ T cells. Addition of PGE₂ again only further polarized this pattern enhancing IFN-γ production by alloreactive CD8⁺ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-α/IL-1/IL-6 can substitute for MCM and that addition of PGE₂ further enhances the yield and quality of DC generated. TNF-α/IL-1, IL-6 + PGE₂-cultured DC seem to be optimal for generation of IFN-γ-producing CD4/CD8⁺ T cells.     

T cell response in murine Leishmanial mexicana amazonensis infection: production of interferon-γby CD8+ cells

The immune response to Leishmania major has been the subject of many investigations. However, Leishmania includes many species with different clinical manifestations. In this report, we studied the Tcell response to L. mexicana amazonensis, a New World species, in a murine model. We found that, similar to L. major, an Old World species, resistant C57BL/6 mice produced a high level of IFN-γ and a low level of IL-4. Conversely, susceptible BALB/c mice produced a much lower level of IFN-γ and higher level of IL-4. Although IFN-γ is one of the important lymphokines that mediate macrophage activation and thus the destruction of the intracellular parasites, which lymphocyte subsets are producing the IFN-γ is still a controversy. Much evidence including the isolation of protective, IFN-γ-producing, CD4⁺ cell lines have confirmed the participation of CD4⁺ Thl cells unequivocally. However, both CD4⁺ and CD8⁺ cells produced IFN-γ. Recently, an increasing body of evidence has appeared suggesting that CD8⁺ cells also play a role in the resolution of murine L. major infection. We found that in the L. m. amazonensis model, when CD8⁺ lymphocytes from resistant C57BL/6 mice were eliminated by anti-CD8 antibody and complement-mediated lysis, the IFN-γ production was reduced by 77%. This indicated that CD8⁺ cells produced a significant amount of the IFN-γ. However, our results also indicate that IFN-γ production by CD8⁺ cells was dependent on CD4⁺ cells.     

Human recombinant interferon-β influences T helper subset differentiation by regulating cytokine secretion pattern and expression of homing receptors

Type I interferons (IFN) are important regulators of both innate and acquired immunity. We have used an in vitro system of human CD4⁺ T cell differentiation to determine how IFN-β influences development of T helper (Th) subsets and homing receptor expression. IFN-β promoted differentiation of CD4⁺ T cells that produce low levels of both IFN-γ and lymphotoxin compared to interleukin (IL)-12-derived Th1 CD4⁺ T cells. IFN-β inhibited production of Th2 cytokines (IL-5 and IL-13) and augmented IL-12-mediated IL-10 secretion. In addition, IFN-β significantly enhanced L-selectin expression on CD4⁺ T cells and synergized with IL-12 to induce expression of cutaneous lymphocyte-associated antigen (CLA). This Th1 L-selectin⁺, CLA⁺ phenotype is characteristic of T cells found in normal human skin and suggests a role for type I IFN in the regulation of Th subset differentiation and tissue-specific homing receptors.     

Regulation of IFN-γ production in granulocyte-macrophage colony-stimulating factor-deficient mice

Granulocyte-macrophage colony-stimulating factor-deficient (GM-CSF−/−) mice produce far lower serum levels of IFN-γ in response to LPS than GM-CSF+/+ mice. CD4⁺ and CD8⁺ T cells from LPS-injected GM-CSF−/− mice showed a deficiency in IFN-γ production and proliferative activity in response to IL-2 and IL-12, whereas IFN-γ production by NK cells was not compromised. These defects of T cells were reversed by administration of GM-CSF in vivo, but not by supplementation with GM-CSF in vitro. GM-CSF−/− mice do not have an intrinsic defect in IFN-γ production, because IL-12 injection induces the same high levels of IFN-γ in GM-CSF−/− and GM-CSF+/+ mice. To investigate the inhibitory effect of LPS on GM-CSF−/− T cells and the indirect restorative activity of GM-CSF, we tested the action of supernatants from cultured dendritic cells (DC). A factor or factors in the DC supernatant normalized serum IFN-γ levels and T cell responses in LPS-injected GM-CSF−/− mice. IL-18 reproduced some but not all of these in vivo and in vitro effects of DC supernatants. Our results indicate that GM-CSF is important in protecting T cells from inhibitory signals generated during immunization or exposure to LPS, and that this effect of GM-CSF is indirect and mediated by factors produced by DC.     

HLA-DQ2-restricted gluten-reactive T cells produce IL-21 but not IL-17 or IL-22

We have analyzed the production of the effector cytokines interleukin (IL)-17, IL-21, and IL-22 in gluten-reactive CD4⁺ T cells of celiac disease patients, either cultured from small intestinal biopsies or isolated from peripheral blood after an oral gluten challenge. Combining intracellular cytokine staining with DQ2-α-II gliadin peptide tetramer staining of intestinal polyclonal T-cell lines, we found that gluten-specific T cells produced interferon-γ (IFN-γ) and IL-21, but not IL-17 or IL-22, even if other T cells of the same lines produced these cytokines. Similarly, in DQ2-α-II-specific T cells in peripheral blood of gluten-challenged patients, very few stained for intracellular IL-17, whereas many cells stained for IFN-γ. We conclude that gluten-reactive T cells produce IL-21 and IFN-γ, but not IL-17. Their production of IL-21 suggests a role for this cytokine in the pathogenesis of celiac disease.     

Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production

Cytokine production in the spleens of mice infected with the protozoan parasite Trypanosoma cruzi was analyzed in three models which differ in the outcome of the infection. Using immunocytochemical techniques to detect cytokine-producing cells, the production of type 1 [interleukin-2 (IL-2) and interferon (IFN)-γ], type 2 (IL-4, IL-5, IL-10), inflammatory [tumor necrosis factor (TNF)-α, IL-1α, IL-6] and regulatory (transforming growth factor-β) cytokines were examined. With the exception of IL-4 and IL-5, cells producing all of the cytokines assayed were detected in both the resistant and susceptible models of T. cruzi infection. Cells producing IL-4 and IL-5 were not detected until later in infection in the resistant mice (>34 days), at about the time animals of the susceptible strain succumb to the infection. Mice of the susceptible model showed a slight delay in the appearance of cells producing the type 1 cytokines IL-2 and IFN-γ and an earlier appearance of TNF-producing cells, in comparison to resistant mice. Cells producing IL-2 or IL-10 were transient in their appearance in the spleen while cells producing IL-1, IL-4, IL-5, IL-6, IFN-γ, TNF, or TGF-β were first detectable in either the acute or post-acute stage of the infection and persisted up to 700 days post infection in two different resistant models of the infection. Cells producing IFN-γ, TNF-α and TGF-β were particularly numerous even very late in the infection. Double-staining techniques were used to show that the vast majority of the IFN-γ-producing cells in the spleen were CD4^?, CD8^? α/β TCR⁺ T cells. This study confirms the transience of IL-2 production in the acute stage of T. cruzi infection and the persistent and simultaneous production of type 1 and type 2 cytokines during the late-acute and chronic stages of the infection. Susceptibility or resistance to T. cruzi infection does not associate with a Th2 pattern of cytokine production in the three models examined in this study. The overlapping pattern of type 1 and type 2 cytokine-producing cells in both the acute and chronic stages of T. cruzi infection demonstrates that longterm infections do not necessarily lead to a dominance of either type 1 or type 2 cytokine production.     

Changes in intracellular cytokine levels in newborn and adult lymphocytes induced by HSV-1

Changes in the expression of intracellular interleukin-2 (IL-2), interleukin-4 (IL-4), interferon (IFN)-γ, and tumor necrosis factor (TNF)-α in newborn and adult lymphocytes induced by herpes simplex virus (HSV)-1 were examined. Cord blood mononuclear cells (CBMC) or adult peripheral blood mononuclear cells (PBMC) were infected with HSV-1 and cultured with phorbol 2-myristate 13-acetate (PMA) plus ionomycin in the presence of monensin for 4 hr. Surface antigen and intracellular cytokines were stained simultaneously and analyzed by flow cytometry. The percentage of cells that expressed IL-2, IFN-γ, and TNF-α was significantly increased in HSV-1-infected CD3⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45R0⁺ lymphocytes compared with uninfected lymphocytes from adult PBMC. The percentage of cells that expressed IL-2 and TNF-α was increased significantly in HSV-1-infected CD3⁺, CD4⁺, CD8⁺, and CD45RA⁺ lymphocytes compared with uninfected lymphocytes from CBMC. IFN-γ was under the detectable level in HSV-1-infected and uninfected lymphocytes from CBMC. Intracellular IL-4 was not detected in HSV-1 or in uninfected lymphocytes from PBMC and CBMC. These results demonstrate that HSV-1 enhances intracellular levels of IL-2, IFN-γ, and TNF-α in adult lymphocytes and defective IFN-γ production in cord blood. J. Med. Virol. 56:145–150, 1998. © 1998 Wiley-Liss, Inc.