Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10

Interleukin-12 (IL-12) induces differentiation of T helper 1 (Th1) cells, primarily through its ability to prime T cells for high interferon-gamma (IFN-gamma) production. We now report that the presence of IL-12 during the first several days of in vitro clonal expansion in limiting dilution cultures of polyclonally stimulated human peripheral blood CD4+ and CD8+ T cells also induces stable priming for high IL-10 production. This effect was demonstrated with T cells from both healthy donors and HIV+ patients. Priming for IL-4 production, which requires IL-4, was maximum in cultures containing both IL-12 and IL-4. IL-4 modestly inhibited the IL-12-induced priming for IFN-gamma, but almost completely suppressed the priming for IL-10 production. A proportion of the clones generated from memory CD45RO+ cells, but not those generated from naive CD45RO- CD4+ T cells, produced some combinations of IFN-gamma, IL-10, and IL-4 even in the absence of IL-12 and IL-4, suggesting in vivo cytokine priming; virtually all CD4+ clones generated from either CD45RO(-) or (+) cells, however, produced high levels of both IFN-gamma and IL-10 when IL-12 was present during expansion. These results indicate that each Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-10) cytokine gene is independently regulated in human T cells and that the dichotomy between T cells with the cytokine production pattern of Th1 and Th2 cells is not due to a direct differentiation-inducing effect of immunoregulatory cytokines, but rather to secondary selective mechanisms. Particular combinations of cytokines induce a predominant generation of T cell clones with anomalous patterns of cytokine production (e.g., IFN-gamma and IL-4 or IFN-gamma and IL-10) that can also be found in a proportion of fresh peripheral blood T cells with "memory" phenotype or clones generated from them and that may identify novel Th subsets with immunoregulatory functions.     

IL-6, IFN-gamma and TNF-alpha production by liver-associated T cells and acute liver injury in rats administered concanavalin A

The relationship between the development of acute hepatitis and the production of TNF-alpha IFN-gamma and IL-6 by liver-associated T lymphocytes following intravenous injection of concanavalin A (Con A) was studied in rats. Following a single injection of Con A, there was a dose and time-dependent correlation in the serum levels of serum alanine aminotransferase (ALT), IL-6, IFN-gamma and TNF-alpha. These increases correlated with an increase in the numbers of CD4+, CD8+ and CD25+ T cells in blood and CD4+ and CD25+ T cells in the liver perfusate, but not with CD8+ T cells in liver perfusate. Increased levels of IL-6, IFN-gamma and TNF-alpha were constitutively produced by liver-associated CD4+ T cells when cultured. In Con A-stimulated cultures, liver-associated CD4+ T cells secreted increasing levels of TNF-alpha in a time-dependent manner following Con A injection, but TNF-alpha production by peripheral blood lymphocytes was transient with peak levels detected at 1 h which then declined over 24 h. Histological examination of the liver revealed fatty change, hepatocyte degeneration and necrosis, with an associated cell infiltrate of neutrophils and CD4+ T cells both in the portal areas and around the central veins. These results support the hypothesis that Con A-induced liver damage is mediated by CD4+ T cells acting within the liver, at least in part through the secretion of TNF-alpha, IFN-gamma and IL-6.     

Characteristic T helper 2 T cell cytokine abnormalities in autoimmune lymphoproliferative syndrome, a syndrome marked by defective apoptosis and humoral autoimmunity

Autoimmune lymphoproliferative syndrome (ALPS) is marked by massive lymphadenopathy, hepatosplenomegaly, autoimmunity and the presence of increased numbers of circulating and tissue TCR-alpha beta, CD4- CD8- T cells. The underlying defect is that of decreased T cell and B cell apoptosis, due in most, but not all, cases to heterozygous mutations of the Fas gene and corresponding defective Fas signaling function. Here we measure in vivo and in vitro cytokine secretion in ALPS to shed light on the relation of apoptosis defects to the development of autoimmunity. In in vivo studies, ALPS patients manifested greatly increased circulating levels of IL-10 (> 100-fold), compared with both healthy individuals and various disease controls; in contrast, their levels of IL-1 beta, IL-4, and IFN-gamma were normal and their levels of IL-2 and TNF-alpha were marginally increased. In parallel in vitro studies, ALPS patients CD4+ DR+ T cells stimulated either with anti-CD3/CD28 or anti-CD2/CD28 produced increased amounts of IL-4 and IL-5 (10 to 20-fold) and decreased amounts of IFN-gamma (4-fold) as compared with those of control CD4+ DR+ T cells. In contrast, ALPS patients' CD4-/CD8- T cells produced very low amounts of cytokines. Finally, ALPS patients' peripheral monocytes/macrophages produced decreased amounts of IL-12 (30-fold) and increased amounts of IL-10 (5-fold). In conclusion, ALPS is marked by the presence of DR+ T cells that exhibit a skewed Th2 cytokine response upon various forms of stimulation. This cytokine response, in the presence of increased circulating IL-10 levels, is likely to define the cytokine milieu that accounts for the humoral autoimmune features of ALPS and, perhaps, of other humoral autoimmune states.     

The CD7- T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA- T cells

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7- T cells express the CD4CD45RO+CD45RA- memory phenotype. We analysed CD4+CD45RA- peripheral blood lymphocytes that were separated into CD7+ and CD7- for their in vitro cytokine secretion in response to different stimuli. The CD4+CD7- subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4+CD7- subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-gamma) secretion was not significantly different in CD7+ and CD7- T cells upon stimulation in vitro. The data indicate that the CD4+CD7- T cell represents the majority of IL-5-secreting cells within the population of CD4+CD45RA- memory T cells. Since CD4+CD7- T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7- T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.     

Active Wegener's granulomatosis is associated with HLA-DR+ CD4+ T cells exhibiting an unbalanced Th1-type T cell cytokine pattern: reversal with IL-10

Wegener's granulomatosis (WG) is a granulomatous vasculitis that affects the upper respiratory tract, lung, and kidney. Since T cells make up a significant proportion of cells infiltrating granulomatous lesions in WG, we investigated the proliferative response and cytokine profile of T cells from these patients. PBMCs were isolated from 12 patients with active WG, 7 patients with inactive disease, and 12 healthy normal donors. PBMCs from clinically active WG patients exhibited increased proliferation following stimulation with either PMA/ionomycin or anti-CD2 and anti-CD28, when compared with normal donors. In addition, these PBMCs exhibited increased secretion of IFN-gamma, but not of IL-4, IL-5, or IL-10. Furthermore, TNF-alpha production from PBMCs and CD4+ T cells isolated from patients with WG was elevated, when compared with healthy donors. In further studies, we investigated the ability of WG patients' monocytes to produce IL-12 and showed that both inactive and active patients produced increased amounts of IL-12. Finally, the in vitro IFN-gamma production by WG PBMC is inhibited in a dose-dependent manner by exogenous IL-10. These data suggest that T cells from WG patients overproduce IFN-gamma and TNF-alpha, probably due to dysregulated IL-12 secretion, and that IL-10 may therefore have therapeutic implications for this disease.     

Signaling through a CD3 gamma-deficient TCR/CD3 complex in immortalized mature CD4+ and CD8+ T lymphocytes

The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD4+ and CD8+ T lymphocytes from a human selective CD3 gamma deficiency using Herpesvirus saimiri. The accessibility of the mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD8+ cells when compared with CD4+ cells, relative to their corresponding CD3 gamma-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-alpha) and late (proliferation and secretion of TNF-alpha), were normal in gamma-deficient cells, despite the fact that their TCR/CD3 complexes were significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 triggering was severely impaired in gamma-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in gamma-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when gamma is absent. They also provide evidence for the specific participation of the CD3 gamma chain in the induction of certain cytokine genes in both CD4+ and CD8+ human mature T cells. These immortalized mutant cells may prove to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex.     

Enhancing effect of interleukin-4 on the secretion of interferon-gamma by alpha s1-casein-specific CD8+ T cells

alpha s1-Casein-specific CD8+ T cell clones expressed the interleukin (IL)-4 receptor, although they did not secrete detectable IL-4. We found that IL-4 significantly enhanced the secretion of interferon (IFN)-gamma by these CD8+ T cell clones. IL-4 also enhanced the secretion of IFN-gamma induced by stimulating the immobilized anti-CD3 antibodies of polyclonal CD8+ T cells which had been isolated from lymph nodes and were stimulated in vitro with the immobilized anti-CD3 antibody and IL-2. In addition, IL-4 added at the time of this first in vitro stimulation induced strong IFN-gamma productivity, as well as IL-4 and IL-10 productivity, which were detectable upon restimulation of these cells. Results are discussed in relation to the inhibitory effects of IFN-gamma production on IL-4-producing cells.     

The mRNA-phenotype of granuloma formation: CD4+ T cell-associated cytokine gene expression during primary murine listeriosis

In murine listeriosis, elimination of bacteria and immunity to reinfection critically depend on Thy1+ CD4- cells, while cell-mediated inflammatory phenomena such as DTH and granuloma formation are mostly mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced, we examined the cytokine gene expression profile associated with the presence or absence of Thy1+, CD4+ and/or CD8+ cells in the livers of mice during a primary infection with L. monocytogenes. The presence of CD4+ cells was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of TNF-alpha and GM-CSF and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, and temporally coincided with the development of granulomatous lesions. In vivo neutralization of TNF-alpha and, to a lesser extent, IFN-gamma resulted in abrogation of granuloma formation. A similar correlation between the presence of CD8+ cells and mRNA expression for any one of the cytokines studied did not exist, pointing to a qualitatively different mechanism of CD8+ T cell mediated cure of listeriosis.     

CD4+ type 1 and CD8+ type 2 T cell subsets in human leishmaniasis have distinct T cell receptor repertoires

The mechanism of protective immunity and immunologic resistance against intracellular pathogens is believed to involve the activation of Ag-specific T cells. The T cells involved in protection/resistance to Leishmania can be studied using localized American cutaneous leishmaniasis (LCL) as a model, because the disease is often self-healing. Our study was undertaken to identify specific T cell populations that had accumulated in LCL lesions on the basis of TCR V beta gene usage. RNA was derived from skin lesions and blood of eight LCL patients, as well as from purified CD4+ and CD8+ subsets from the lesions and blood of three patients. After synthesis of cDNA, V beta gene usage was assessed by polymerase chain reaction. In all eight patients, several V beta gene families were overrepresented in lesions compared to blood. More importantly, the TCR V beta repertoires of both lesional CD4+ and CD8+ subsets were skewed compared to the repertoire of the respective subsets in the blood of the same donor. The overrepresented V beta s in the CD4+ and CD8+ subsets from lesions were in most instances disparate, particularly with the V beta 6 TCR skewed in the lesional CD8+ subset. Not only were the TCR repertoires of the overrepresented V beta in the lesional CD4+ and CD8+ subsets generally distinct, but the cytokine mRNA expressed by these subsets were also discrete. Strikingly, the CD4+ subset was characterized by IFN-gamma mRNA expression and the CD8+ subset by IL-4 and IL-10 mRNA expression. These data indicate that the pathogenesis of human leishmaniasis may be explained by the balance of CD4+ type 1 and CD8+ type 2 T cells, which probably recognize distinct sets of Ag.     

Expression of selectin-binding epitopes and cytokines by CD4+ T cells repopulating scid mice with colitis

Recruitment into the gut of CD4+ T cells and their activation in the colonic lamina propria (LP) are key events in the development of colitis in scid mice reconstituted with CD4+ T cells from immunocompetent, congenic donor mice. This study investigated the expression of cytokines and selectin-binding epitopes by CD4+ T cells repopulating different tissues of the adoptive scid host. Cells from the inflamed colonic LP of transplanted scid mice produced high amounts of IL-12, IFN-gamma and TNF-alpha but only low amounts IL-4 and IL-10. Intracellular cytokine staining confirmed the presence of large numbers of IFN-gamma- and TNF-alpha-producing effector CD4+ T cells in the colonic LP of scid mice with colitis but also in non-inflamed tissues [spleen (S), peritoneal cavity (PC) and mesenteric lymph nodes (mLN)] of the adoptive host. Cells from these tissues furthermore produced large amounts of IL-12. Ligands for endothelial selectins are involved in recruiting T cells into inflamed tissues. We have analyzed the expression of selectin-binding epitopes on CD4+ T cells repopulating different tissues of the adoptive scid host. We found that a large fraction of CD4+ T cells from inflamed colonic LP and from non-inflamed PC, mLN and S expressed high levels of P- and E-selectin-binding epitopes (P-Lhi) in transplanted scid mice, but not in congenic, immunocompetent control mice. Although P-Lhi CD4+ T cells were enriched in IFN-gamma-producing subsets from most (but not all) tissues, we also found large numbers of in vivo generated P-Llo CD4+ T cells producing pro-inflammatory cytokines. This was in contrast to in vitro generated Th1 CD4+ T blasts that were almost exclusively P-Lhi. In this mouse model, production of Th1-type pro-inflammatory cytokines and expression of surface epitopes binding endothelial selectins are hence strikingly up-regulated in CD4+ T cells residing in inflamed and non-inflamed tissues during the development of colitis.     

IL-15 promotes IL-12 production by human monocytes via T cell-dependent contact and may contribute to IL-12-mediated IFN-gamma secretion by CD4+ T cells in the absence of TCR ligation

At inflammatory sites, the number of activated bystander T cells exceeds that of Ag-activated T cells. We investigated whether IL-15, a monocyte-derived cytokine that shares several biologic activities with IL-2, may contribute to bystander T cell activation in the absence of IL-2 and triggering Ag. The addition of IL-15 to cocultures of monocytes and T cells stimulates CD4+ but not CD8+ T cells to produce IFN-gamma. IFN-gamma production requires endogenous IL-12, the production of which in turn is dependent upon CD40/CD154 interactions between CD4+ T cells and monocytes. Indeed, non-TCR-activated CD4+ but not CD8+ T cells express significant levels of CD154. IL-15 may enhance IFN-gamma in this system by up-regulating CD40 expression on monocytes and IL-12Rbeta1 expression on CD4+ T cells. Conversely, using neutralizing anti-IL-15 mAb, we show that the ability of IL-12 to augment IFN-gamma secretion is partly mediated by endogenous IL-15. Finally, in the absence of monocytes, a synergistic effect between exogenous IL-12 and IL-15 is necessary to induce IFN-gamma production by purified CD4+ T cells, while IL-15 alone induces T cell proliferation. It is proposed that this codependence between IL-12 and IL-15 for the activation of inflammatory T cells may be involved in chronic inflammatory disorders that are dominated by a Th1 response. In such a response, a self-perpetuating cycle of inflammation is set forth, because IL-15-stimulated CD4+ T cells may activate monocytes to release IL-12 that synergizes with IL-15 to induce IL-12 response and IFN-gamma production.     

Steroid hormone regulation of cytokine secretion by proteolipid protein-specific CD4+ T cell clones isolated from multiple sclerosis patients and normal control subjects

Steroid hormones have long been known to modulate immune function, and recent studies indicate that one of the means by which they do so involves effects on the secretion of immunoregulatory cytokines. Our laboratory has found recently that estradiol (E2) selectively modifies cytokine secretion in proteolipid protein (PLP)-specific, CD4+ T cell clones isolated from patients with the demyelinating disease, multiple sclerosis, and from normal control subjects. The data suggest that E2 may play a role in regulating the balance between pro- and antiinflammatory conditions, especially at concentrations typical of pregnancy. To determine whether other pregnancy-associated steroid hormones are capable of similar activity, we expanded our testing to include estrone (E1), estriol (E3), progesterone, and dexamethasone. The results indicate that E1 and E3 enhance secretion of Ag- or anti-CD3-stimulated IL-10 and IFN-gamma in dose-dependent fashion, almost identical to that of E2. The effect on IL-10 was more potent than occurred with IFN-gamma. In addition, E1 and E3, like E2, had a biphasic effect on TNF-alphabeta secretion, with low concentrations stimulatory, and high doses inhibitory. None of the estrogens influenced IL-4 or TGF-beta secretion. Progesterone enhanced secretion of IL-4, without affecting any other tested cytokine. Finally, dexamethasone induced TGF-beta secretion, but inhibited IFN-gamma and TNF-alphabeta. This differential effect of steroid hormones on the secretion of cytokines by CD4+ human T cell clones is consistent with the possibility that, collectively, they promote antiinflammatory conditions at high concentrations typical of pregnancy.     

The role of B7-1 and LFA-3 in costimulation of CD8+ T cells

This study compares the ability of LFA-3 (CD58) and B7-1 (CD80) ligands to provide costimulatory signals for superantigen (SAg)-stimulated CD8+ and CD4+ T cells. We show that B7-1 and LFA-3 costimulation activate CD8+ T cells to proliferation, cytokine production (IL-2, TNF, and IFN-gamma), and cytotoxicity. A long-lasting proliferative response was observed after combined DR/B7-1/LFA-3 costimulation. Detailed analysis of SEA-activated CD8+ T cells revealed that maximal production of IFN-gamma was seen in LFA-3-costimulated cells, while production of IL-2 was mainly induced after B7-1 costimulation. A fivefold increase in the IFN-gamma production was observed when activated CD8+ T cells were costimulated with Chinese hamster ovary (CHO)-DR/LFA-3 cells compared with the secretion induced by CHO-DR/B7-1. In contrast, SEA-treated CD4+ T cells costimulated with B7-1 or LFA-3 gave rise to a similar production of IFN-gamma, suggesting a preferential function for the CD2/LFA-3 pathway in the regulation of IFN-gamma in CD8+ T cells. Moreover, the generation of CTL was supported similarly by B7-1 and LFA-3 costimulation, but not by CHO-DR cells. We conclude that ligation of the CD28 and CD2 receptors mediate distinct effect on CD8+ and CD4+ T cell effector functions.     

Differential capacities of CD4+, CD8+, and CD4-CD8- T cell subsets to express IL-18 receptor and produce IFN-gamma in response to IL-18

IL-12 and IL-18 have the capacity to stimulate IFN-gamma production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8+, CD4+, and CD4-CD8- T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8+, CD4+, and CD4-CD8- T cells expressed IL-12R, but not IL-18R, and produced IFN-gamma in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN-gamma production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN-gamma production among T cell subsets. CD4+ cells expressed marginal levels of IL-18R and produced small amounts of IFN-gamma, whereas CD8+ cells expressed higher levels of IL-18R and produced more IFN-gamma than CD4+ cells. Moreover, CD4-CD8- cells expressed levels of IL-18R comparable to those for CD8+ cells but produced IFN-gamma one order higher than did CD8+ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN-gamma production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.     

Role of CD38 and its ligand in the regulation of MHC-nonrestricted cytotoxic T cells

Human CD38 is a type II transmembrane glycoprotein that regulates lymphocyte adhesion, proliferation, and cytokine production. The mAb Moon-1 recognizes a ligand for CD38 (CD38L) and specifically inhibits CD38-mediated cell adhesion. To analyze the role of CD38 and its ligand in MHC-nonrestricted T cell activation, we examined the effects of Moon-1 and the anti-CD38 mAb IB4 on the effector functions of the IL-2-dependent T cell line TALL-104 (CD3/TCR-alphabeta+, CD8+, CD56+) and of LAK cells (90% CD3+). TALL-104 cells were almost 100% reactive with both mAbs, whereas the reactivity of LAK cells for IB4 and Moon-1 ranged from 10 to 60% among different donors. From 78 to 94% of the cytotoxic CD8+/CD56+ LAK subset was CD38L+. Like mAb OKT3 (anti-CD3), and at variance with IB4, Moon-1 drastically enhanced the cytotoxicity of TALL-104 and CD8+ LAK cells against a resistant tumor target. Granule exocytosis did not appear to play a role in Moon-1-induced cytotoxicity. Moreover, neither IB4 nor Moon-1 induced [Ca2+]i mobilization in LAK and TALL-104 cells. Whereas stimulation of CD3 and CD38 resulted in a dramatic induction of cytokine (granulocyte-macrophage-CSF, IFN-gamma, TNF-alpha, and TNF-beta) release by both TALL-104 and LAK cells, ligation of CD38L was not followed by cytokine production in TALL-104 cells. Thus, cytotoxicity and cytokine release are independently regulated, at least in this system. These data demonstrate that CD38 and its ligand can regulate some T cell functions using signaling pathways distinct from those of CD3.     

Study of activation of murine T cells with bacterial superantigens. In vitro induction of enhanced responses in CD4+ T cells and of anergy in CD8+ T cells

Primary and secondary responses of murine CD4+ T cells and CD8+ T cells upon stimulation with staphylococcal enterotoxin E (SEE) bearing superantigenic properties were examined. Both isolated C57BL/6 splenic CD4+ T cells and CD8+ T cells proliferated and produced IL-2 and IFN-gamma upon stimulation with SEE in substantial levels. The amounts of IL-2 were greater in CD4+ T cells and those of IFN-gamma were somewhat greater in CD8+ T cells. SEE-induced CD4+ T lymphoblasts, larger parts of which bore the V beta 11 element in their TCR, proliferated, produced IL-2 and IFN-gamma, and showed toxin-dependent cytotoxicity in substantial levels upon restimulation with SEE. By contrast, SEE-induced CD8+ T lymphoblasts, the larger part of which bore the V beta 11 element, did not show the first two of the three responses at all upon restimulation with SEE, whereas these cells showed greater cytotoxicity. The CD8+ T lymphoblasts did not suppress the reactivity of the CD4+ T lymphoblasts. Both SEE-induced CD4+ T lymphoblasts and CD8+ T lymphoblasts proliferated and produced IL-2 and IFN-gamma in comparable levels upon stimulation with rIL-2 or mAb to CD3 or V beta 11.     

Interleukin-10 dose-dependent regulation of CD4+ and CD8+ T cell-mediated graft-versus-host disease

BACKGROUND: Endogenous interleukin (IL)-10 production has been associated with the lack of graft-versus-host disease (GVHD) in human recipients of MHC-disparate donor grafts. Paradoxically, we have shown that the exogenous administration of high doses (30 microg/dose) of IL-10 to murine recipients of MHC-disparate grafts accelerates GVHD lethality. METHODS: The effects of IL-10 on GVHD mediated by either CD4+ or CD8+ T cells was examined in studies involving exogenous IL-10 administration or the infusion of T cells from IL-10-deficient (-/-) donor mice. The role of interferon (IFN)-gamma on IL-10-induced GVHD acceleration was studied using IFN-gamma-deficient (-/-) donor mice or neutralizing monoclonal antibody. RESULTS: IL-10 was found to have a dose-dependent effect on the GVHD lethality mediated by either CD4+ or CD8+ T cells. High doses of exogenous IL-10 accelerated GVHD lethality. IFN-gamma release was not responsible for the IL-10 facilitation of GVHD lethality. Paradoxically, low doses of IL-10 protected mice against GVHD lethality. The GVHD protective effect of the bioavailability of small amounts of IL-10 was confirmed by demonstrating that the infusion of T cells from IL-10 -/- donors accelerated GVHD lethality. CONCLUSIONS: The results suggest that IL-10 has a dose-dependent effect on the GVHD lethality mediated by CD4+ or CD8+ T cells, such that high doses accelerate lethality, while low amounts of bioavailable IL-10 are protective.     

Peritoneal interleukin-10 increases with decrease in activated CD4+ T lymphocytes in women with endometriosis

This study was performed to determine whether peritoneal T cells are suppressed in the CD4+ or CD8+ T cell subpopulation and whether they are Th1 or Th2 predominant in women with endometriosis. Immune cells in the peritoneal fluid (PF) were obtained from women undergoing laparoscopy for endometriosis or tubal ligation. Three-colour flow cytometry was utilized for immunophenotyping of peritoneal fluid mononuclear cells (PFMC). Concentrations of interleukin (IL)-4, IL-5 and interferon-gamma (IFN-gamma) produced by PFMC with and without mitogen stimulation and concentrations of IL-10 and IL-12 were measured in PF. The peritoneal T lymphocytes were predominantly of the Th1 type that produced much more IFN-gamma but less IL-4 or IL-5 in women with or without endometriosis. The decrease in peritoneal lymphocytes was significant in the HLA-DR+ CD4+ CD3+ subpopulation and the concentrations of peritoneal IL-10 and IL-12 were significantly elevated in women with early stage endometriosis. There was impaired IL-5 production by PFMC after phytohaemagglutinin stimulation in women with advanced stage endometriosis. We concluded that the activated peritoneal CD4+ Th1 cells from the women with endometriosis were decreased in number. The suppression of these T cells may be due to the elevation of IL-10 and IL-12 in the peritoneal fluid.     

Cytolytic T cells with Th1-like cytokine profile predominate in retroorbital lymphocytic infiltrates of Graves' ophthalmopathy

Lymphocytic infiltration of muscular and connective tissues of the retroorbital (RO) space is a histological hallmark of Graves' ophthalmopathy (GO). We have characterized some phenotypical and functional features of T cells derived from RO infiltrates of four GO patients who were submitted to orbital decompression. Fragments of RO tissue were cultured for 7 days in IL-2-conditioned medium in order to generate T cell lines of in vivo activated T cells. Phenotypical analysis of freshly isolated peripheral blood (PB) lymphocytes both from patients and four healthy controls showed a predominance of CD4+ T cells (CD4/CD8 ratios 1.9:2.5), whereas RO-derived T cell lines displayed almost equal proportions of CD4+ and CD8+ cells (CD4/CD8 ratios 0.9:1.2). RO T cell lines and PB T cells from patients and controls were then cloned using a high-efficiency cloning procedure. The phenotypical and functional features of 153 T cell clones (TCC) derived from RO infiltrates were examined and compared with those of 166 and 236 TCC derived from the PB of patients and controls, respectively. CD4/CD8 ratios ranged from 0.8-1.4 in the series of RO-derived TCC and from 1.9-2.2 in the corresponding series of PB-derived TCC. Assessment of lectin-dependent cytolytic activity showed similar proportions of cytotoxic clones in TCC derived from the PB of patients (37%) and controls (38%); most of the cytolytic TCC was CD8+. In contrast, the proportion of cytolytic RO TCC was markedly higher (106/153 = 69%), including 100% of CD8+ and the majority (59/79 = 75%) of CD4+ clones. When compared to TCC derived from the PB of both patients and controls, RO TCC showed remarkably high proportions of both CD8+ and CD4+ clones with a Th1-like cytokine profile, as documented by their ability to secrete IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4 or IL-5. This study provides evidence that cytolytic T cells with Th1 profile of cytokine production predominate in RO infiltrates of GO, a pattern quite similar to those previously described in thyroid infiltrates of Hashimoto's thyroiditis or Graves' disease. The peculiar cytokine secretion profile of RO T cells may be of importance in the pathogenesis of both the tissue alterations and fibrogenic process observed in GO.     

Release of cytokines and soluble cell surface molecules by PBMC after activation with the bispecific antibody CD3 x CD19

Bispecific antibodies (BsAb) consist of two different heavy and light chains and may bind to two different antigens present on different cell types. With their dual specificity BsAb may recognize effector cells (e.g. T cells) on one hand and tumour cells (e.g. malignant B cells) on the other hand. The authors analysed whether T cell activation and subsequent killing of malignant B cells mediated by the bispecific antibody CD3 x CD19 was reflected by the release of cytokines. In addition, the authors investigated whether the in vitro cytokine release was similar to that observed in vivo in the patients treated with BsAb. The in vitro release of cytokines into the supernatant of cell cultures of peripheral blood mononuclear cells (PBMC) and malignant B cells was measured after incubation with either the bispecific antibody CD3 x CD19 or the monospecific anti-CD3 (aCD3) antibody in the presence or absence of interleukin (IL)-2. Release of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-8, IL-10, soluble (s) CD4, sCD8 and sCD25 by PBMC was equal under both conditions and could be used as an indicator for T cell activation. However, the cytokine pattern and level did not correlate with the cytotoxic capacity, which was 4 logs higher with BsAb + IL-2 compared to aCD3 + IL-2. The in vitro pattern of cytokine release was similar to that observed in vivo in the serum of patients treated with BsAb and IL-2, indicating the possibility of predicting cytokine release in future patients with other therapeutic regimens.