Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity.

The anamnestic response to infection with Listeria monocytogenes is characterized by the rapid elimination of normally lethal doses of bacteria and accelerated granuloma formation. These phenomena are mediated by listeria-specific memory T cells within the first 24 h after reinfection. In order to elucidate the mechanisms operative during this decisive phase of infection, we conducted a comprehensive kinetic and quantitative analysis of cytokine gene expression in the livers of naive and immune mice. Organs were removed at 30 min, and 1, 2, 6, and 24 h after primary and secondary infections, and PCR3-assisted messenger RNA (mRNA) amplification was performed on matched samples using primers specific for IL-1 beta, IL-6, M-CSF, GM-CSF, TNF-alpha, IFN-gamma, IL-10, IL-4, IL-2, IL-3 and I1-2Rp55. The cytokine pattern characteristic of secondarily infected animals differed qualitatively by the expression of mRNA for IL-2, IL-2Rp55, IL-3, and IL-4, demonstrating the accumulation and activation of specific T cells in the livers as early as 1 to 2 h after reinfection. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection almost completely abrogated the differentiated cytokine profile typical of the anamnestic response. Using competitive PCR for semiquantitative determination of mRNA levels, the amount of IL-1 beta and IL-6 mRNA was found to be very similar during primary and secondary infection, whereas TNF-alpha mRNA was found to be increased by approximately 10-fold 2 h and IFN-gamma mRNA by approximately 50 to 100-fold 6 h after reinfection when compared with a primary challenge. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection resulted in a substantial (approximately 10-fold) decrease in IFN-gamma mRNA expression. To correlate these findings with cytokine secretion, spleen cells from naive and immune as well as normal and CD4+ and CD8+ cell depleted mice infected 6 h previously were cultured for 48 h, and supernatants were analyzed for the amount of the above mentioned cytokines. Semiquantitative PCR-assisted mRNA amplification is demonstrated to be a superior tool in dissociating the mediators of innate resistance from those operative in protective immunity and granuloma formation.     

Bacillus Calmette-Guérin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles

The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.     

In vitro modulation of cytokine production by lymphocytes in human coccidioidomycosis

The modulation of the cytokine response to coccidioidal antigen by lymphocytes from donors with coccidioidomycosis was examined. In initial experiments, samples from 13 healthy immune donors and seven donors with active coccidioidomycosis anergic to the coccidioidal antigen T27K were assessed for CD3 lymphocyte expression of intracellular IFN-gamma using whole blood analysis. Addition of 10 ng/ml of recombinant IL-12 significantly increased response to T27K among immune and anergic subjects (p<0.05), but the percent of cells expressing IFN-gamma was still significantly greater for immune subjects. Among immune donors, the percentage of CD3 lymphocytes expressing IFN-gamma was significantly reduced with the addition of 10 ng/ml of recombinant IL-4, IL-10, TGF-beta, or their combination (for all, p<0.05). Among anergic donors, addition of 10 ng/ml of anti-IL-10 significantly increased IFN-gamma production (p<0.05), but addition of anti-IL-4 or anti-TGF-beta did not. Among immune donors, the percent of both CD3 lymphocytes and NK cells expressing IFN-gamma after 24h of T27K was increased above control (p<0.05), while the percent of NK cells producing TNF-alpha in response to T27K was not greater than control. Depletion of NK cells from peripheral blood mononuclear cells resulted in significant increases in TNF-alpha and IL-10 (for both, p<0.05) but resulted in no significant decrease in IFN-gamma or IL-2. These data demonstrate a differential response to stimulation with the coccidioidal antigen T27K among donors with coccidioidomycosis that can be manipulated by cell type and cytokine environment.     

Suppressive effect of IL-4 on IL-13-induced genes in mouse lung

Although IL-4 signals through two receptors, IL-4R alpha/common gamma-chain (gamma(c)) and IL-4R alpha/IL-13R alpha1, and only the latter is also activated by IL-13, IL-13 contributes more than IL-4 to goblet cell hyperplasia and airway hyperresponsiveness in murine asthma. To determine whether unique gene induction by IL-13 might contribute to its greater proasthmatic effects, mice were inoculated intratracheally with IL-4 or IL-13, and pulmonary gene induction was compared by gene microarray and real-time PCR. Only the collagen alpha2 type VI (Ca2T6) gene and three small proline-rich protein (SPRR) genes were reproducibly induced > 4-fold more by IL-13 than by IL-4. Preferential IL-13 gene induction was not attributable to B cells, T cells, or differences in cytokine potency. IL-4 signaling through IL-4R alpha/gamma(c) suppresses Ca2T6 and SPRR gene expression in normal mice and induces these genes in RAG2/gamma(c)-deficient mice. Although IL-4, but not IL-13, induces IL-12 and IFN-gamma, which suppress many effects of IL-4, IL-12 suppresses only the Ca2T6 gene, and IL-4-induced IFN-gamma production does not suppress the Ca2T6 or SPRR genes. Thus, IL-4 induces genes in addition to IL-12 that suppress STAT6-mediated SPRR gene induction. These results provide a potential explanation for the dominant role of IL-13 in induction of goblet cell hyperplasia and airway hyperresponsiveness in asthma.     

Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4

Studies on cytokine expression in blood cells are commonly performed on cryopreserved cells. Previous studies show that cryopreservation affects cytokine expression, but the findings are not consistent. This may be due to divergent effects of freezing on different cytokines, different stimuli, and different patient groups or to the use of different assays in the studies. This study was designed to investigate the effect of freezing on spontaneous, auto-antigen, allergen, and mitogen induced cytokine secretion from peripheral blood mononuclear cells from several groups of patients expressing different cytokine profiles; multiple sclerosis, atopic children, non-atopic children, and pregnant women. The expression of IFN-gamma, IL-4, IL-5, IL-9, IL-10, and IL-13 was analysed with ELISA, ELISPOT and/or real time RT-PCR. Our data provide evidence that the process of cryopreservation and thawing does affect the expression of cytokines, both at the protein and the mRNA level. Moreover, the effect varied among different cytokines, different stimuli, and different patient groups, which partly may be explained by differences in optimal freezing conditions for non-activated and activated cells. An increase of allergen and PHA stimulated IFN-gamma secretion in atopic children was found following cryopreservation, but no such increase in auto-antigen induced IFN-gamma was seen in MS-patients. The most consistent finding was that expression of IL-4 was generally decreased in spontaneous and auto-antigen/allergen induced expression in cryopreserved cells. In conclusion, this study points out the importance of investigation of the effects of freezing for each cytokine, stimuli and patient group before using frozen cells in studies of in vitro cytokine secretion.     

The kinetics and stimulant dependence of cytokine production by blood and bronchoalveolar lavage T cells evaluated at the single cell level.

We have previously shown that bronchoalveolar lavage (BAL) T cells from human airways predominantly produce interferon gamma (IFN-gamma) and interleukin 2 (IL-2) when stimulated ex vivo. The kinetics of TH1 and TH2 cell cytokine production by T cells from both blood and BAL were studied to establish the optimal time after stimulation either with pharbol myristate (PMA) and ionomycin or with the more physiological stimulus of anti-CD3 for intracellular cytokine detection of IFN-gamma, IL-2, IL-4 and IL-5 in both blood and BAL T cells. The optimal time for positive identification of IL-2 in both blood and BAL was 5 h after PMA/ionomycin stimulation, whereas the first peak for IFN-gamma was found after 5 h in blood but after only 3 h in BAL. T cells from different biological compartments responded differently to each of the stimuli. Whilst anti-CD3 stimulation did not induce TH1 cytokine production in blood T cells, it readily induced both IFN-gamma and IL-2 production in BAL T cells. The kinetics of cytokine production were found to be stimulus dependent. Whilst IL-2 production showed similar kinetics with both stimuli, the kinetics of IFN-gamma production differed between stimuli. We have also examined the effect of five different stimuli on cytokine production by T cells to determine whether different forms of stimulation may selectively stimulate or inhibit different cytokines. Not surprisingly, PMA/ionomycin induced a greater percentage of BAL T cells to produce TH1 cytokines. However, other than modest amounts of the TH2 cytokines IL-4 and IL-5 were not induced by any of the five stimuli.     

A unique mechanism for innate cytokine promotion of T cell responses to viral infections

The kinetics of CD8 T cell IFN-gamma responses as they occur in situ are defined here during lymphocytic choriomeningitis virus (LCMV) infections, and a unique mechanism for the innate cytokines IFN-alphabeta and IL-18 in promoting these responses is defined. Infections of mice with Armstrong or WE strains of LCMV induced an unexpectedly early day 4 IFN-gamma response detectable in serum samples and spleen and liver homogenates. Production of IFN-gamma was MHC class I/CD8 dependent, but did not require IL-12, NK cells, TCR-gammadelta T cells, MHC class II, or CD4 T cells. Peak response required specific Ag recognition, as administration of antagonist peptide partially impaired day 4 IFN-gamma induction, and viral peptide stimulation enhanced CD8 T cell IFN-gamma expression in culture. The IFN-gamma response was associated with IL-18 and IFN-alphabeta expression. Furthermore, both factors augmented peptide-driven IFN-gamma production in culture, and mice lacking IL-18 or IFN-alphabeta functions had reduced day 4 IFN-gamma. Collectively, these results demonstrate that during viral infections, there is a dramatic in vivo CD8 T cell response preceding maximal expansion of these cells, and that the mechanism supporting this response is dependent on endogenous innate cytokines. Because stimulation by microbial products is linked to innate cytokine expression, the studies also suggest a pathway for precisely limiting T cell functions to times of need.     

IL-18 is correlated with type-2 immune response in children with steroid sensitive nephrotic syndrome

Children with steroid sensitive nephrotic syndrome (SSNS) is thought to have dysregulated type-1/type-2 cytokine network. Interleukin (IL)-18 is a cytokine, which may enhance both type-1 and type-2 responses, depending on the cytokines milieu. This prospective study aimed to assess type-1/type-2 cytokine synthesis and production profile in different stages of SSNS and define the potent involvement of IL-18. Twenty-three children with SSNS, aged 2.5-14 years, were studied; 23/23 both in active stage before treatment initiation and in remission still on steroids; 15/23 in remission off steroids as well. Data were compared with those obtained from 25 age-matched controls. The following parameters were assessed: Basic T cell populations, percentages of CD3+/CD69+/IFN-gamma+ and CD3+/CD69+/IL-4+ T cells as well as serum levels of IFN-gamma, IL-2, IL-4, IL-13 and IL-18. No difference in IL-2 levels was found between nephrotic children of all disease stages and controls (p>0.05). Percentage of CD3+/CD69+/IL-4+ T cells and serum levels of IL-4, IL-13 and IL-18 were significantly higher in the active stage of SSNS compared with the remission stages and controls (p<0.05). On the contrary, percentage of CD3+/CD69+/IFN-gamma+ T cells as well as serum IFN-gamma were significantly lower during active disease stage compared with remission stages and controls (p<0.05). In children with SSNS, of all disease stages, serum levels of IL-18 were significantly correlated with both IL-4 and IL-13 (r=0.628 and p<0.0001, r=0.71 and p<0.0001, respectively). It seems that a type-2 cytokine synthesis and production pattern prevails in children with active SSNS and IL-18 expression is significantly correlated with this type-2 immune response.     

Interferon-gamma (INF-gamma), IL4 expression levels and Leishmania DNA load as prognostic markers for monitoring response to treatment of leishmaniotic dogs with miltefosine and allopurinol

In this study, we searched for a connection between Leishmania load and cytokine expression levels in the tissues of Leishmaniainfantum naturally infected dogs and the efficacy of treatment with miltefosine and allopurinol. To this purpose, we exploited a real-time PCR system to detect Leishmania load and the expression levels of IFN-gamma and IL-4 mRNAs at the time of diagnosis and during the follow up post-treatment. In particular, we measured the amount of parasites in blood and lymph node samples, while the expression levels of IFN-gamma and IL-4 cytokines were assessed in the blood of the animals. We employed different targeted real-time PCR assays on 20 naturally infected dogs with clinical signs. Three healthy dogs living in a non-endemic area were selected as negative controls. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels in different kinds of tissue might represent a reliable tool to evaluate the immune response, the efficacy of the therapy and to predict the relapses during the treatment.     

TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells

TL1A, a recently described TNF-like cytokine that interacts with DR3, costimulates T cells and augments anti-CD3 plus anti-CD28 IFN-gamma production. In the current study we show that TL1A or an agonistic anti-DR3 mAb synergize with IL-12/IL-18 to augment IFN-gamma production in human peripheral blood T cells and NK cells. TL1A also enhanced IFN-gamma production by IL-12/IL-18 stimulated CD56(+) T cells. When expressed as fold change, the synergistic effect of TL1A on cytokine-induced IFN-gamma production was more pronounced on CD4(+) and CD8(+) T cells than on CD56(+) T cells or NK cells. Intracellular cytokine staining showed that TL1A significantly enhanced both the percentage and the mean fluorescence intensity of IFN-gamma-producing T cells in response to IL-12/IL-18. The combination of IL-12 and IL-18 markedly up-regulated DR3 expression in NK cells, whereas it had minimal effect in T cells. Our data suggest that TL1A/DR3 pathway plays an important role in the augmentation of cytokine-induced IFN-gamma production in T cells and that DR3 expression is differentially regulated by IL-12/IL-18 in T cells and NK cells.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

Lower expression of mRNA for interferon-gamma in T helper cells of children with newly diagnosed lymphomas

The complex interactions between cancer and host cells are far from being fully elucidated. Assessment of Th1/Th2/Th3/Tr1 balance is an interesting approach to explain immunological disturbances in lymphomas. The aim of our study was to assess mRNA for pro- and anti-inflammatory cytokines in T-cells in 20 children with Hodgkin- and non-Hodgkin lymphomas. CD4+ and CD8+ cells were isolated from whole peripheral blood and four different cytokine mRNA levels (IFN-gamma, IL-10, IL-4, TGF-beta) were determined by real-time PCR technique. Comparing to the control group, we found lower expression of mRNA for IFN-gamma in CD4+ cells at the time of lymphoma diagnosis. It may be one of the pathogenetic mechanisms of impaired immunity in these patients.     

Polarized type 1 cytokine profile in bronchoalveolar lavage T cells of patients with hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is characterized by an inflammatory lymphocytic alveolitis comprised of both CD8+ and CD4+ T cells. Animal models suggest that HP is facilitated by overproduction of IFN-gamma, and that IL-10 ameliorates severity of the disease, indicating a Th1-type response. To determine whether a Th1 phenotype in HP also exists clinically, bronchoalveolar lavage (BAL) and peripheral blood (PB) T cells were obtained from HP individuals and analyzed for Th1 vs Th2 cytokine profiles. It was determined that soluble OKT3-stimulated BAL T cells cocultured with alveolar macrophages produced more IFN-gamma and less IL-10 than PB T cells cocultured with monocytes, but no difference was observed in IL-4 production. The monocytic cells did not account for this difference, as CD80 and CD86 expressions were similar, and coculturing PB T cells with alveolar macrophages resulted in no difference in IFN-gamma production. Similarly, there was no difference in IL-12 production between stimulated BAL or PB T cells; however, addition of rIL-12 significantly increased production of IFN-gamma by BAL T cells, but not by PB T cells. This effect was due to a difference in IL-12R expression. High affinity IL-12R were only present in association with BAL T cells. These studies indicate that clinical HP is characterized by a predominance of IFN-gamma-producing T cells, perhaps resulting from a reduction in IL-10 production and an increase in high affinity IL-12R compared with blood T cells.     

Deficient IL-12(p35) gene expression by dendritic cells derived from neonatal monocytes

To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-gamma to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-gamma production by allogenic adult CD4(+) T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-gamma production by T cells.     

Restricted T cell expression of IL-2/IFN-gamma mRNA in human inflammatory disease

It has been difficult to demonstrate functionally distinct T cell populations in humans on the basis of cytokine secretion. As previous investigators have examined the T cell cytokine profile from immunized animals, we examined whether Th1 or Th2 type T cells could be identified in the peripheral blood or cerebrospinal fluid (CSF) immune compartments from subjects with or without inflammatory diseases. Using limiting dilution analysis and growth with PHA and IL-2/IL-4, we directly cloned a total of 177 T cells from the peripheral blood and CSF of seven subjects, four with inflammatory disease and three control subjects, and examined the cytokine message profile after stimulation with ionomycin and PMA. We found that most clones from both the peripheral blood and CSF express IL-1, IL-2, IL-4, IFN-gamma, or TNF-alpha cytokine mRNA after activation with ionomycin and PMA. All T cell clones tested produced TNF-alpha mRNA, and all but 14 produced IFN-gamma mRNA. As reported previously, Th0 cells, which produced IFN-gamma, IL-2, IL-4, and IL-5 mRNA, were found in most subjects. In striking contrast, Th1 cells, which expressed IL-2 and IFN-gamma but not IL-4 or IL-5 mRNA, were present in both peripheral blood and CSF of subjects with inflammatory disease but not found in peripheral blood or CSF of subjects without systemic inflammation. Th2 cells, expressing IL-4 and IL-5 but not IFN-gamma or IL-2 mRNA, were not found in any subject. These data present the first evidence for Th1 T cell clones in humans that may be associated with systemic inflammation.     

Stat4 regulates multiple components of IFN-gamma-inducing signaling pathways

Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-gamma, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-gamma induction has not been established, experiments were conducted to examine Stat4 activation of IFN-gamma and other genes required for cytokine-induced expression of IFN-gamma. We first examined IL-12 signaling components. Basal expression of IL-12Rss1 and IL-12Rss2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-gamma production are defective in the absence of Stat4. IL-18 induces minimal IFN-gamma production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-kappaB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-gamma production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-gamma.     

CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9(+) lymphocytes in blood from normal donors. A subset of CCR9(+) T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9(-), CCR9(+)CD4(+) peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-gamma and IL-10. IL-10-producing cells were exclusively detected within the CCR9(+) subset of CD4(+) T cells by intracellular staining and were distinct from IL-2- and IFN-gamma-producing cells. Moreover, memory CCR9(+)CD4(+) lymphocytes respond to CD2 stimulation with proliferation and IFN-gamma/IL-10 production, whereas memory CCR9(-)CD4(+) cells were unresponsive. In addition, memory CCR9(+)CD4(+) T cells support Ig production by cocultured CD19(+) B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9(+)CD4(+) T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.     

Sustained expression of CD154 (CD40L) and proinflammatory cytokine production by alloantigen-stimulated umbilical cord blood T cells

Recent data suggests that graft-versus-host disease (GVHD) is initiated by host APCs. Blockade of CD40:CD154 interactions between APCs and T cells in vivo induces T cell tolerance to host alloantigen and dramatically reduces GVHD. Because allogeneic cord blood (CB) transplantation results in a lower incidence and severity of acute GVHD compared with bone marrow transplantation, we have investigated whether CB T cells can express CD154 in response to stimulation by allogeneic monocyte-derived dendritic cells (MDDC) and have used 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling in combination with intracellular cytokine analysis to assess the proliferation and cytokine profiles of alloantigen-responsive cells. CB T cells stimulated with allogeneic MDDC showed stronger proliferation than adult blood T cells. Surface CD154 expression was detected in the actively dividing CFSElow populations of both the CD4+ and CD4- subsets and was brightest in cells that had divided the most. Assessment of supernatants from MDDC-stimulated CB and adult blood T cells showed no significant difference in the levels of either IFN-gamma or TNF-alpha, but CB T cell supernatants did show a significant lack of detectable IL-2. Intracellular cytokine analysis revealed that dividing CB T cells had been primed to produce IFN-gamma, TNF-alpha, and IL-2 on restimulation. Further phenotype analysis showed that 75% of CB T cells producing IFN-gamma were CD8+. These data suggest that MDDC-stimulated CB T cells express functional CD154 and provide enough costimulation for dendritic cells to prime naive CD8+ CB T cells and induce type 1 cytokine production.