Cytokine production from murine CD4 and CD8 cells after mannan-MUC1 immunization.

Immunotherapy with oxidized mannan-MUC1 fusion protein (M-FP) leads to a T1 immune response characterized by the generation of cytotoxic T lymphocytes (CTL), few antibodies, secretion of interleukin-2 (IL-2), IL-12, and interferon-gamma and tumor protection. Immunotherapy with reduced M-FP or fusion protein (FP) alone leads to a T2 immune response characterized by the generation of MUC1 antibodies, few CTL, IL-4 secretion, and no tumor protection. In these studies, cytokine production from T cells was measured from cultures containing whole spleens. We now report the cytokine secretion patterns from spleen cells separated into CD4+ and CD8+ T cells obtained from mice immunized with either oxidized M-FP, reduced M-FP or FP, or the simultaneous administration of oxidized M-FP and FP. Immunization with oxidized M-FP led to the secretion of T1 cytokines from CD8+ T cells (IL-2, IFN-gamma, and tumor necrosis factor-alpha [TNF-alpha]) and from CD4+ T cells (IL-2 and IFN-gamma). IL-12 production, presumably from activated macrophages, was observed in CD8+ but not CD4+ cultures. Immunization with either reduced M-FP or FP led to the secretion of predominantly T2 cytokines from CD4+ T cells (IL-4 and IL-10) and IL-2 production in both CD4+ and CD8+ T cell cultures. The simultaneous immunization of both oxidized M-FP and FP led to the production of both T1 and T2 cytokines from CD8+ T cells (IL-2, IFN-gamma, and TNF-alpha) and CD4+ cells (IL-2, IFN-gamma, IL-4, and IL-10) and IL-12 production in CD8+ cultures that is, both types of immune responses could occur together. The results demonstrate that the cellular immune response observed in oxidized M-FP-immunized mice is indeed dependent on the T1 cytokine profile secreted by CD8+ T cells, and the simultaneous production of both T1 and T2 cytokines is not cross-inhibitory.     

Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer

Effector T cells fall into two subpopulations based on cytokine-secretion. Type 1 cells secrete IFN-gamma, whereas type 2 cells secrete IL-4, IL-10, and GM-CSF. NKT cells represent a third subpopulation that secretes similar cytokines and have been associated with immunoregulation. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of tumor-infiltrating lymphocytes (TIL) in mice challenged subcutaneously in the mammary region. Flow cytometric analysis shows that T cells do not infiltrate the primary tumor site until days 7-14 following tumor challenge. Both CD4 and CD8 TILs were predominantly CD44(High) and expressed CD25, CD69, and CD95 cell surface activation markers. Activated CD4/CD44(High) TIL numbers reached peak levels at day 21 that precipitously decreased by day 28 whereas corresponding CD8 cell numbers progressively increased, however, at lower levels and with later kinetics. Intracellular cytokine staining showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFN-gamma-producing Th1 cells. T cells co-expressing DX5 (CD3(+)/DX5(+)) emerged (>21 days), suggesting a recruitment of NK-like T cells at later stages of tumor progression. Moreover, tumors selectively up-regulated TGF-beta, MIF, and IP-10 gene expression at times as early as day 4, with peak levels at day 7 in vivo. Such gene expression remained elevated and correlated with a continued progression in tumor growth suggesting that preferential effector cell recruitment and production of select factors during different stages of tumor maturation may aid in regulating effective endogenous antitumor responses in progressive breast cancer.     

Neem (Azadirachta indica) leaf preparation induces prophylactic growth inhibition of murine Ehrlich carcinoma in Swiss and C57BL/6 mice by activation of NK cells and NK-T cells

We have reported earlier that pretreatment of mice with neem leaf preparation (NLP) causes prophylactic growth inhibition of murine Ehrlich's carcinoma (EC) and B16 melanoma. Using adoptive cell transfer technology, here we have established that NLP-mediated activation of immune cells may be involved in tumor growth restriction. Mononuclear cells from blood and spleen of NLP-activated Swiss and C57BL/6 mice causes enhanced cytotoxicity to murine EC cells in vitro. Fractionation of spleen cells exhibited greater percentage of tumor cell lysis in macrophage and B-cell-depleted NK and T-cell-rich fractions. Flow cytometric analysis revealed in both blood and spleen, NK cells (DX5+ or NK1.1+) and NK-T cells (CD3+/DX5+ or CD3+/NK1.1+) were increased in number in Swiss, C57BL/6 and athymic nude mice after pretreatment with NLP. NLP-stimulated spleen cells showed greater secretion of TNFalpha and IFNgamma. Thus, NLP-activated NK and NK-T cells in mice may regulate tumor cell cytotoxicity by enhancing the secretion of different cytotoxic cytokines.     

Endotoxin-induced gamma interferon production: contributing cell types and key regulatory factors

Gamma interferon (IFN-gamma) is an important mediator of endotoxin (lipopolysaccharide [LPS])-induced immune responses. However, the specific cell types that produce IFN-gamma in response to LPS and the cellular factors that regulate LPS-induced IFN-gamma production have not been fully determined. The present studies were undertaken to characterize the cell populations that produce IFN-gamma after LPS challenge in the spleens of mice and to determine the regulatory factors that modulate LPS-induced production of IFN-gamma. Our studies show that the levels of splenic IFN-gamma mRNA and protein production peak at 6 and 8 h, respectively, after systemic LPS challenge. Approximately 60% of IFN-gamma-producing cells are natural killer (NK) cells (CD3(-)DX5(+)) and 25% are NKT cells (CD3(+)DX5(+)). Most of the remaining IFN-gamma-producing cells are T cells (CD3(+)DX5(-)), macrophages, and dendritic cells. Functionally, interleukin-12 (IL-12) is the major IFN-gamma-stimulating factor after LPS challenge, with costimulation provided by IL-15, IL-18, and B7 proteins. IL-10 is a major inhibitor of LPS-induced IFN-gamma production. Unlike intact heat-killed gram-negative and gram-positive bacteria, the class II major histocompatibility complex did not play a functional role in LPS-induced IFN-gamma production. LPS is a potent stimulus for splenic IL-10, IL-12 p40, and IL-15 mRNA expression, whereas IL-12 p35 and IL-18 mRNAs, as well as B7 proteins, are constitutively expressed in the mouse spleen. Of the factors studied, IL-18 serves as the most potent costimulus with IL-12 for IFN-gamma production, followed by IL-15 and B7 proteins. These data demonstrate that NK cells and NKT cells are the most abundant IFN-gamma-producing cells in the mouse spleen after LPS challenge and that IL-10 and IL-12 are key functional regulators of LPS-induced IFN-gamma production.     

Cellular profile of cytokine production in a patient with visceral leishmaniasis: gammadelta+ T cells express both type 1 cytokines and interleukin-10

The cytokine profile of CD4+, CD8+ T cells, gammadelta+ T cells and natural killer (NK) cells (CD94+CD3-) was studied in a patient with visceral leishmaniasis (VL). The otherwise healthy, human immunodeficiency virus-negative patient acquired the disease in Tuscany, Italy. Diagnosis was made by demonstration of high concentrations of antibodies against Leishmania antigens in serum. Flow cytometry for the detection of intracellular interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-6, IL-10, IL-13 and tumour necrosis factor (TNF)-alpha expression in peripheral blood mononuclear cells stimulated with phorbol 12-myristate 13-acetate and ionomycin was performed, followed by treatment with liposomal amphotericin B. CD4+ cells were identified as major cytokine-expressing cells, capable of producing both type 1 and type 2 cytokines. A high frequency of IL-4- and IL-13-expressing CD8+ cells was noted. NK cells and gammadelta+ T cells, thought to be involved in innate host defences against Leishmania, expressed IFN-gamma and TNF-alpha. Ten per cent of gammadelta+ T cells expressed IL-10, predominantly together with IFN-gamma, suggesting additional immune-regulatory roles for this T-cell subset in VL.     

Primary stimulation of CD4+ cells in the presence of IL-4 or IFN-gamma alters the frequencies of cytokine-producing cells at restimulation.

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8+ cells and of exogenously added cytokines (interleukin (IL)-2, IL-4 and interferon (IFN)-gamma) on the cytokine production in splenic CD4+ T cells. IL-2, IL-4, IL-5 and IFN-gamma production in CD4+ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8+ cells. On day 5 the cells were restimulated with mitogen alone and analysed to evaluate the short-term development of cytokine-producing cells in such cultures. Preactivation in the presence of either exogenous IL-4 or IFN-gamma led to an increased production of IL-4 and IFN-gamma respectively at restimulation, and the effects of both IL-4 and IFN-gamma were augmented by IL-2. After preactivation in the presence of IL-2 and IL-4, every third CD4+ cell could be induced to produce IL-4. Exogenous IL-4 or IFN-gamma further decreased each other's production. Depletion of CD8+ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8+ cells will influence lymphokine production in CD4+ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4+ and CD8+ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines.     

Interleukin-4 receptor alpha chain and STAT6 signaling inhibit gamma interferon but not Th2 cytokine expression within schistosome granulomas

Compared to wild-type (WT) mice, schistosome granulomas in Stat6 knockout (KO) mice lacked eosinophils and had Th1 features. Interleukin-4 (IL-4) acts through Stat6 in assisting Th2 cell development. The importance of Stat6 for Th2-cell development within schistosome granulomas had not been explored. Therefore we studied gamma interferon (IFN-gamma), IL-4, and IL-5 production in granulomas from Stat6 KO and WT mice. Dispersed granuloma cells from Stat6 KO and WT mice made similar amounts of IL-4 and IL-5. Only Stat6 KO granuloma cells released IFN-gamma. Granuloma T cells contained most of the IL-4, IL-5, and IFN-gamma mRNA and secreted these cytokines. In Stat6 KO mice, 16.6% of the granuloma cells were CD4(+). Of these, 10.7% stained for IFN-gamma and/or IL-4 by intracytoplasmic flow analysis. Few CD4(-) T cells stained positively. The IL-4-producing T cells did not stain for DX5 or with labeled alpha-GalCer CD1d tetramer, suggesting an absence of NK T cells. Thus, conventional Th cells in Stat6 KO granulomas produce IFN-gamma and Th2 cytokines. Stat6 limits IFN-gamma production but is unnecessary for Th2-cell development or localization within the granuloma.     

IL-18 stimulates IL-13-mediated IFN-gamma-sensitive host resistance in vivo

IL-4 and IL-13 are up-regulated during in vivo responses to many nematode parasites, but increasing evidence suggests that increases in IL-13 can also occur independently of the IL-4-dominant Th2 response. Blocking B7 after Trichuris muris inoculation inhibits resistance and IL-4 elevations, instead resulting in an IFN-gamma-dominant response associated with susceptibility. However, blocking IFN-gamma under these conditions restores IL-13-dependent resistance. In this study, we examined the mechanism of IL-13 up-regulation and associated protection during this in vivo immune response. CD4+ T cells and DX5+ TCR- cells were identified as the major producers of IL-13, and the DX5+ TCR- cells were phenotyped as NK cells, since they expressed CD11b, IL-2Rbeta and Ly49C but not c-kit or Fc epsilonRI. NK cell-derived IL-13 elevations were T cell-dependent, as CD4+ T cell depletion blocked IL-13 production by mesenteric lymph node cells and induced susceptibility. IL-13 expression was increased independently of IL-12; however, blocking IL-18 function inhibited IL-13 production and increased susceptibility. These results indicate that CD4+ T cells and NK cells are the major sources of IL-13 during the in vivo Th1 response induced by B7 blockade and that under these conditions, IL-18 is specifically required for the in vivo up-regulation of IL-13 production and associated host protection.     

Type 1 cytokine/chemokine production by mouse NK cells following activation of their TLR/MyD88-mediated pathways

It is well established that IL-18R- and toll-like receptor (TLR)-mediated signalings share a common signal pathway mediated by signal adaptor, MyD88, and that IL-18 synergizes with IL-12 for IFN-gamma production by NK cells. Here, we investigated whether TLR agonists can replace IL-18 for production of IFN-gamma by NK cells. Freshly isolated NK cells possessed functional LPS receptor composed of TLR4/MD2 complex and of CD14, and also expressed other various tlrs. Hepatic CD3(-)DX5(+) NK cells produced IFN-gamma in response to TLR2 or TLR7 agonists only when co-stimulated with IL-12, indicating that TLR agonists synergize with IL-12 for IFN-gamma. The tlr2(-/-) or tlr7(-/-) NK cells could not produce IFN-gamma in response to IL-12 plus TLR2 or TLR7 ligands, respectively, indicating requirement of the corresponding TLRs. Furthermore, upon stimulation with these combinations, wild-type NK cells produced type 1 chemokines, such as CCL3, CCL4 and CCL5 as well. NK cells from bacterium (e.g. Propionibacterium acnes)-inoculated rag2(-/-) mice, when compared with those from naive mice, exhibited significantly enhanced capacity to produce these CC chemokines and IFN-gamma, suggesting that microbial infection enhances responsiveness of NK cells to TLR agonists. These results indicate that upon microbial infection, macrophages produce IL-12 that renders NK cells highly responsive to TLR agonists to produce IFN-gamma and chemokines, which might in turn recruit and fully activate macrophages, leading to the development of inflammatory foci presumably necessary for efficient microbial eradication. Thus, NK cells, like T cells, induce orchestrated immune responses in collaboration with macrophages to show potent host defense effects during early infectious phase.     

Determination of cytokine co-expression in individual splenic CD4+ and CD8+ T cells from influenza virus-immune mice.

We have studied the patterns of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) co-expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet-inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4 and IFN-gamma were determined by three-colour flow cytometric analysis of fixed and saponin-permeabilized cells fluorescent-stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL-2/IL-4, IL-2/IFN-gamma and IL-4/IFN-gamma. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus-immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co-expressing combinations of them (i.e. IL-4/IL-2, IL-4/IFN-gamma and IL-2/IFN-gamma), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.     

Decreased IL-4 producing CD4+ T cells in patients with active systemic lupus erythematosus-relation to IL-12R expression

The balance of interferon-gamma (IFN-gamma) and/or interleukin-4 (IL-4) producing T cells and interleukin-12 receptor (IL-12R) expression on T cells were evaluated in patients with active systemic lupus erythematosus (SLE). Assessment of intracellular IFN-gamma and/or IL-4 were conducted with cytoplasmic staining. IL-12R presenting T cells were also assessed by flowcytometry without in vitro stimulation. In SLE, the number of IFN-gamma producing CD4+ T cells was increased, and the absolute number of IL-4 producing CD4+ T cells was significantly decreased. Although the ratio of IL-12R presenting CD4+ T cells was significantly greater, the absolute number did not increase. The ratio of IFN-gamma/IL-4-producing CD4+ T cells correlated with the SLE disease activity index (SLEDAI) and was significantly higher among patients with lupus nephritis. Therefore, the imbalance of IFN-gamma/IL-4 producing CD4+ T cells was due to the decrease in IL-4 producing CD4+ T cells and may play an important pathogenic role in active SLE.     

Peripheral blood CD4+ and CD8+ T cell type 1 and type 2 cytokine production in atopic asthmatic and normal subjects

BACKGROUND: Increased production of IL-4 and IL-5 and decreased production of IFN-gamma by CD4+ T cells has been implicated in asthma pathogenesis. However, CD8+ T cells also produce type 1 and type 2 cytokines and the relative roles of CD4+ and CD8+ T cell cytokine production in asthma have not been previously studied. OBJECTIVE: To determine the production of the type 1 and type 2 cytokines by CD4+ and CD8+ T cell subsets in asthmatic and normal subjects. METHODS: Intracellular cytokine staining for IL-4, -5, -10, -13 and IFN-gamma was analysed in peripheral blood CD4+ and CD8+ T cells from 24 atopic asthmatic and 20 normal subjects. RESULTS: Both subsets of T cells produced all cytokines studied and there were no significant differences between CD4+ and CD8+ T cells in their capacity to produce either type 1 or type 2 cytokines. There were significantly increased frequencies of IFN-gamma-positive CD4+ (13.1 +/- 2.4%, vs. 7.3 +/- 1.4%) and CD8+ (20.0 +/- 2.9%, vs. 9.6 +/- 2.1%) T cells in asthmatic subjects compared with normal subjects (P < 0.05), but not in frequencies of CD4+ or CD8+ T cells staining positively for IL-4, -5, -10 or -13. CONCLUSION: The frequencies of peripheral blood CD8+ T cells producing type 1 and type 2 cytokines are comparable with the frequencies of CD4+ T cells. There was an increased frequency of IFN-gamma producing CD4+ and CD8+ T cells in asthmatic compared with normal subjects. Further studies investigating T cells derived from the airways and investigating various stages within the disease process are required to further elucidate the importance of type 2 and type 1 T cell cytokine production in the pathogenesis of human allergic disease.     

Expression of the DX5 antigen on CD8+ T cells is associated with activation and subsequent cell death or memory during influenza virus infection

The antigen recognized by the DX5 antibody (DX5 antigen) is expressed on all murine NK cells. In the present study we found that a proportion of CD8+ T cells (approximately 5%) also express the DX5 antigen in uninfected mice, and that numbers of CD8+ T cells expressing DX5 are significantly higher in the lungs of influenza virus-infected mice representing up to 50% of all CD8+ T cells on day 10 post infection. The expression of the DX5 antigen on CD8+ T cells was associated with a memory phenotype in uninfected C57BL/6 mice and with an activation phenotype during influenza virus infection. Interestingly, when lymphocytes were isolated from lungs of influenza virus-infected mice on day 10 post infection and adoptively transferred into recombination activating gene-1 (RAG1)-deficient mice, CD8+DX5+ cells could not be recovered from the recipient mice 2 days later. Moreover, CD8+DX5+ cells were not detected when lung cells were removed from day 10 influenza virus-infected mice and cultured in vitro for 2 days. However, CD8+DX5+ cells could be detected when apoptosis inhibitors were added to these cultures, suggesting that the CD8+DX5+ cells underwent apoptosis during cell culture. Furthermore, almost all DX5 expressing CD8+ cells from lungs of mice on day 10 post influenza virus infection stained positively with Annexin-V. Taken together, the data suggest that CD8+ T cells expressing DX5 are associated with an activation/memory phenotype and are biased towards apoptosis.     

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells

IL-18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL-18 can induce IL-13 production in both NK cells and T cells in synergy with IL-2 but not IL-12, suggesting IL-18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL-18 can act as a cofactor to induce IL-4, IL-10 and IL-13 as well as IFN-gamma production in T cells in the presence of anti-CD3 monoclonal antibodies (mAb). IL-18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL-18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL-4 knockout mice. Furthermore, the administration of IL-18 plus IL-2 induced approximately 70-fold and 10-fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL-18 plus IL-2 was eliminated by the pretreatment of mice with anti-CD4 or anti-CD154, but not anti-CD8 or anti-NK1.1 mAb. These results suggest that IL-18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell-dependent, IL-4-independent IgE production.     

IL-16 inhibits IL-5 production by antigen-stimulated T cells in atopic subjects

BACKGROUND: We have previously shown increased expression of the CD4+ cell chemoattractant IL-16 at sites of airway allergic inflammation. Little is known about the significance of IL-16 in allergic inflammation and its role in allergen-driven T-cell cytokine responses. Because IL-16 interacts specifically with CD4+ T cells, we hypothesized that IL-16 released at sites of inflammation may modulate the pattern of cytokines produced by CD4+ T cells. OBJECTIVE: We investigated the effects of exogenous rhIL-16 on cytokine production of PBMCs from atopic and nonatopic subjects in response to antigen and PHA. METHODS: Primary cultures of freshly isolated PBMCs from ragweed-sensitive atopic subjects and nonatopic subjects were stimulated with ragweed or PHA in the presence or absence of rhIL-16. Supernatant levels of IL-4, IL-5, and IFN-gamma were determined by means of ELISA at different time points between 2 and 6 days. Effects of IL-16 on antigen-induced cellular proliferative responses were determined. RESULTS: No IL-4 protein was detected after antigen stimulation of PBMCs from atopic subjects, whereas significant levels of IL-5 were measured on day 6 (median, 534.9 pg/mL). IL-5 secretion was abolished in PBMC cultures depleted of CD4+ cells. The addition of rhIL-16 in antigen-stimulated PBMC cultures significantly reduced the amount of IL-5 released (median, 99.8 pg/mL; P <.001). Detectable levels of IFN-gamma (median, 53.3 pg/mL) were identified after antigen stimulation. The addition of rhIL-16 in antigen-stimulated PBMC cultures significantly increased IFN-gamma levels (median, 255.6 pg/mL; P <.05). Effects of rhIL-16 appear to be specific for antigen-stimulated PBMCs in atopic subjects because rhIL-16 did not alter IL-5 or IFN-gamma production in response to PHA nor did rhIL-16 alter cytokine production in nonatopic normal subjects. CONCLUSION: These studies suggest that IL-16 can play a role in regulating the production of cytokines seen in allergic states in response to antigen.     

Intracellular expression of MICA in activated CD4 T lymphocytes and protection from NK cell-mediated MICA-dependent cytotoxicity

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.     

Analysis of cytokine production in the colon of nude mice with experimental colitis induced by adoptive transfer of immunocompetent cells from mice infected with a murine retrovirus

LP-BM5 murine leukemia virus (MuLV) is known to induce murine AIDS (MAIDS). We have shown that Sj?gren's syndrome (SjS)-like exocrinopathy can be induced in mice with MAIDS and that adoptive transfer of spleen cells from MAIDS mice can induce inflammatory bowel disease-like colitis as well as SjS-like exocrinopathy in nude mice. To assess the role of interferon (IFN)-gamma and interleukin (IL)-10 in the pathogenesis of our experimental model, we tried to identify the cells producing these cytokines and their localization in the colitis lesions in situ. Expression of mRNA for IFN-gamma and IL-10 was assessed by RT-PCR, and protein expression of these cytokines was also analyzed in frozen sections of colon by double-color-staining immunofluorescence (IF). An increase of IFN-gamma and IL-10 mRNA was detected in the colon of mice with colitis, but not in that of control mice. Double-color IF showed that Mac-1(+) cells were positive for IFN-gamma or IL-10 and that most CD4(+) T cells were positive for IL-10, although the population of IFN-gamma-positive CD4(+) T cells was low. In our experimental colitis model, Mac-1(+) macrophages that produce both IFN-gamma and IL-10 might play a crucial role in the pathogenesis of colitis in combination with CD4(+) T cells.     

Immunization with alpha-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis.

We have compared the immune responses of mice immunized either with alpha-galactosylceramide (alpha-GalCer), capable of eliciting a CD1-metiated stimulation of V alpha14+ NK T cells, or with lipoarabinomannan (LAM), a glycophospholipid derived from mycobacteria which is known to be presented by CD1b in humans. Within 24 h, alpha-GalCer induces a burst of IFN-gamma secretion in vivo, and recall with antigen in vitro leads to the synthesis of IL-4 and IL-10 in addition to IFN-gamma. Associated with this in vivo cytokine release is a polyclonal activation of splenic B and T cells. CD1-reactive NK T lymphocytes mediate these events, because none of them are observed in alpha-GalCer-immunized CD1-/- mice. LAM immunization fails to promote similar early responses in vivo. Repeated exposure of mice to alpha-GalCer induces splenic T cells to secrete IL-4 and IL-10 but dramatically reduced levels of IFN-gamma. Such a bias in the cytokine balance triggered by NK T cells stimulated with multiple doses of alpha-GalCer suggests that this compound might be useful in the induction of Th2 immune responses and the prevention of chronic inflammatory conditions mediated by Th1 cytokines.     

Functional and phenotypical characteristics of hepatic NK-like T cells in NK1.1-positive and -negative mouse strains.

We previously reported and partially characterized a unique monoclonal antibody (mAb), U5A2-13, which recognizes a T cell subset similar to NK1.1+ T cells, not only in NK1.1-positive mouse strains but also in NK1.1-negative strains. In NK1.1-positive C57BL/6 mice, U5A2-13+ TCRalphabeta+ cells produced abundant IL-4 as well as extremely high levels of IFN-gamma upon CD3 cross-linking, but this did not occur with U5A2-13- TCRalphabeta+ cells. In NK1.1-negative C3H/He mice, U5A2-13+ TCRalphabeta+ cells produced high levels of IL-4 and IFN-gamma upon CD3 cross-linking, but this was not observed with U5A2-13- TCRalphabeta+ cells. To the best of our knowledge, this is the first direct evidence of the presence of NK-like T cells defined phenotypically by U5A2-13 mAb and functionally by IL-4/IFN-gamma production in NK1.1-negative mouse strains. We also demonstrated that U5A2-13- NK1.1+ T cells and U5A2-13+ NK1.1- T cells in C57BL/6 mice could produce both IL-4 and IFN-gamma. In addition, Vbeta8 or Vbeta7 usage by U5A2-13+ NK1.1- T cells was lower than that by U5A2-13+ NK1.1+ T cells, but remained higher than that by U5A2-13- NK1.1- T cells. Based on the present results, U5A2-13 mAb appears to be a valuable tool in the study of NK-like T cells.