In vitro and in vivo recovery of IFN-{gamma}, but not IL-2, production by IL-12 in mice with plasma ceIL tumors

We have previously found that T ceILs from mice bearing plasmaceIL tumors (PCT mice) demonstrate decreased proliferation asweIL as decreased production of the T^h 1-associated cytokinesIL-2 and IFN- in response to polyclonal stimulation. In thepresent study, we have examined soluble factors as possibleelements required to rescue this decreased proliferation andcytokine production by splenocytes from PCT mice. We find thatthe addition of supernatants from stimulated normal splenocyteshas no effect on proliferation or IL-2 production by splenocytesfrom PCT mice. In contrast, these supernatants completely restoreIFN- production by splenocytes from PCT mice. We have foundthat IL-12 is responsible for the observed increase in IFN-production because: (i) addition of anti-IL-12 antibody blocksthis recovery of IFN- production by these supernatants, (ii)the addition of recombinant IL-12 to cultures of splenocytesfrom PCT mice results in increased IFN- production and (iii)In vivo treatment of PCT mice in IL-12 also results in increasedIFN- production by the subsequently activated splenocytes, buthas little effect on proliferation or IL-2 production. Theseresults demonstrate that both in vitro and in vivo, IL-12 selectivelyrestores the decreased production of IFN- by splenocytes fromPCT mice.     

Allergen-driven limiting dilution analysis of human IL-4 and IFN-{gamma} production in allergic rhinitis and clinically tolerant individualsz

Difficulties in detecting human IL-4 synthesis in antigen-drivenprimary culture have led to widespread reliance on less physlologicappraoches to T cell activation. Although there is general agreementof a T 2-like blas,l the precise defects in ctokine responsivenessremain controverslal. Analysis of cytokine protein productionby fresh, unselected cell populations in response to gognate,antigen-driven stimulation remains an important goal. Here,limitting dilution analysis (LDA) was sued to evaluate antigen-stimulatedcytokine gene expression by fresh peripheral blood mononuclearcells(PBMC)(.PBMC from 19 grass pollen sensitive, allergicrhinitis subjects and normal, non-atopic contrls were evaluated1 mont after natural relmmunization (the peak of te local grasspollen season). Surprisingly, highly atopic subjects and cllnicallynon-allergic individuals exhiboited virtually equlvalent antigen-specific,CD4-dependent cytokine production in response to short-termculture with these common environmental antigens. As anticipated,pronounced increases in T_h2-like activity were evidnet in tecirculating immune repertolre of grass of 117:1 among normalsubjects versus 4:1 among those with allergic rhinitis (Mann-WhitneyU-test, P=0.0067). This T_h2-like blas reflected both a lowerfrequencey of IFN-prducing cells and a markedly increased frequencyof IL-4-producing cells in the circulatin grass-pollen specificrepertoire of atopic donors. The observation that every atopicand normal subject produced IFN- (±IL-4) following antigenre-stimulation arguses that the decision as to whether allergyor (clinical) tolerance results, hinges not on a geneticallydetermined capacity of whether allergen-reactive T cells canbe stimulated in any given individual by cronic exposure toubiqultous enveronmental antigens, but on the nature of thecokine response that comes to dominate that individual's response     

Dendritic cells and macrophages are required for Th1 development of CD4+ T cells from {alpha}{beta} TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-{gamma} production is IFN-{gamma}-dependent

We have examined the antigen presenting cell (APC) requirementsfor primary T cell activation and T helper (Th) cell phenotypedifferentiation using naive CD4⁺ T cells from ß TCRtransgenic mice. Purified dendritic cells were the principalcell required for induction of primary ovalbumln peptide specificT cell activation and clonal expansion. However, dendritic cellsdid not induce differentiation of T cells toward Th1 or Th2phenotype. Addition of IL-4 during primary dendritic cell stimulationsof T cells resulted in the development of a Th2 phenotype whichproduced high levels of IL-4 during secondary and tertiary stimulation.In contrast, development of Th1 cells producing high levelsof IFN- could not be induced with dendritic cells alone butrequired the addition of appropriately activated macrophages.Addition of splenic or peritoneal B cells did not induce Th1development. Activated splenic macrophages induced Th1 developmentvia a non-MHC restricted mechanism. Thus, requirements for inductionof proliferation of naive CD4⁺ T cells are distinct from thosedirecting Th1 phenotype development. IL-12 could replace therequirement for macrophages to induce Th1 development when Tcells were activated with dendritic cells. Furthermore, thisIL-12 mediated development of Th1 cells producing high levelsof IFN- was dependent on IFN-.     

Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-{gamma} production by anti-tumor T cells

Unfractionated spleen cells taken from tumor-bearing mice 2weeks after tumor implantation contained tumor-primed T cellswhich produced cytokines including IL-2 and IFN- when culturedin vitro. With progressive tumor growth this initial lymphokine-producingcapacity decreased. Here, we investigated the ability of IL-12to (I) restore suppressed IFN- production, (II) cause tumorregression and (II) induce anti-tumor protective immunity. Additionof rIL-12 to spleen cell cultures from 4- to 10-week-old tumor-bearingmice resulted in a striking enhancement in the production ofIFN- compared with cultures of these cells in the absence ofrIL-12 or of normal spleen cells in the presence of rIL-12.Five I.p. injections of rIL-12 into mice bearing s.c. tumorsinduced complete tumor regression. This was found when rIL-12was given at early (1–2 weeks), intermediate (4–5weeks) or even late (7 weeks) stages of tumor growth. Furthermore,IL-12-treated mice which rejected the primary tumor exhibitedcomplete resistance to a rechallenge with the same tumor butdid not reject a second syngenetic tumor. Immunohistochemicalanalyses following IL-12 treatment revealed that CD4⁺ and CD8⁺T cells infiltrate the tumor. More importantly, IFN- mRNA expressionwas observed in fresh tumor masses from tumor-bearing mice receivingIL-12 treatment The importance of IFN- was further demonstratedby the observation that the systemic administration of anti-IFN-mAb prior to IL-12 treatment completely abrogated the anti-tumoreffect of IL-12. Thus, these results indicate that administrationof modest levels of rIL-12 to tumor-bearing mice results intumor regression through mechanisms involving reversal of suppressedIFN- production by anti-tumor T cells and the establishmentof a tumor-specific protective immune response.     

Sharing of the IL-2 receptor {gamma} chain with the functional IL-9 receptor complex

The third subunit, the so-called common (_c) chain, of the IL-2receptor is shared among the receptors for IL-2, IL-4, IL-7and IL-15, and dysfunction of the _c chain is thought to causeX-linked severe combined immunodeficiency (XSCID) ascribed toimpairment of early T cell development. However, cytokines linkedto XSCID are as yet unidentified. A mAb specific for the _c chain,TUGm2, profoundly inhibited cell proliferation in response toIL-9. Another mAb, TUGm3, immunoprecipltated [¹²⁵I]IL-9 cross-linkedwith either the IL-9 receptor or the _c chain. These resultsdemonstrate that the _c chain is included in the functional receptorcomplex for IL-9, which was initially characterized as a T cellgrowth factor and is essential for IL-9-dependent growth signaltransductlon.     

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.     

IFN-{gamma} expression in CD8+ T cells regulated by IL-6 signal is involved in superantigen-mediated CD4+ T cell death

Infection with pathogens containing superantigens (Sags) canresult in massive excessive CD4⁺ T cell activation and deathin such conditions as toxic shock, food poisoning and autoimmunediseases. We here showed how enhancement of IL-6 signaling suppressesSag-mediated activated CD4⁺ T cell death. Sag-induced CD4⁺ Tcell death increased in IL-6 knockout (KO) mice, whereas itdecreased in mice characterized by enhanced IL-6–gp130–STAT3signaling. The serum concentration of IFN- was inversely correlatedwith the magnitude of IL-6 signaling, and IFN- deficiency inhibitedSag-induced activated CD4⁺ T cell death, suggesting that IL-6suppresses CD4⁺ T cell death via IFN- expression. Interestingly,depletion of activated CD8⁺ T cells inhibited Sag-mediated increasesin IFN- expression in IL-6 KO mice as well as the augmentedCD4⁺ T cell death. The results demonstrate that IL-6–gp130–STAT3signaling in activated CD8⁺ T cells contributes to Sag-inducedCD4⁺ T cell death via IFN- expression, highlighting this signalingaxis in CD8⁺ T cells as a potential therapeutic target for Sag-relatedsyndromes.     

TNF-{alpha} regulates IL-4-induced Fc{varepsilon}RII/CD23 gene expression and soluble Fc{varepsilon}RII release by human monocytes

We examined the regulatory effects of TNF- on IL-4-induced geneexpression of the low-affinity receptor for IgE (FcRII/CD23)in human monocytes and IL-4-induced soluble FcRII (sFcRII) releasefrom monocytes. IL-4-induced FcRII expression on the surfaceof monocytes was reduced by TNF- as early as 1 day after cultureand the effect of TNF- increased with prolonged culture. Thepresent analysis was designed to examine whether or not TNF-could suppress IL-4-induced FcRII mRNA expression and enhancedIL-4-induced sFcRII release. The addition of TNF- to monocytecultures with IL-4 significantly reduced FcRII expression onthe surface of monocytes and significantly increased sFcRIIrelease from monocytes. Over time, there was an inverse relationshipbetween the disappearance of cell surface FcRII and the appearanceof sFcRII in culture supernatants. FcRII mRNA expression inmonocytes cultured with IL-4 was not affected by TNF- when examinedat 6 h after cultivation. When the cells were cultured withTNF- for more than 24 h, however, TNF- down-regulated IL-4-inducedFcRII mRNA levels. This correlated with the kinetics of down-regulationof IL-4-induced FCRII expression on the surface of monocytesby TNF-. These results suggest that TNF-dependent reductionof IL-4-;induced FcRII expression on the surface of monocytesresulted, at least in part, from the suppression of FcRII mRNAexpression and the enhancement of sFcRII release.     

Molecular mechanisms underlying IFN-{gamma}-mediated tumor growth inhibition induced during tumor growth inhibition induced during tumor immunotherapy with rIL-12

The present studnt Investigates the molecular by which IFN-produced as a result of in vitroIL-12 addministration exertsits anty-tumor,rIL-12 was administered three or five times intomice bearing CDA1M fibrosarcoma, OV-HM ovarian carcinoma orMCH-1-A1 fibosarcoma. This regimen induced complete regressionof CSA1M and OV-HM tumors but only transient growth inhibitionof MCH-1-A1 tumors. The anty-tumor effects of Il-12 were associatatedwith enhanced induction of IFN-becouse these effects were abrogatedby pretreatment of hosts with anti-IFN- antibody.Exposure inin vitro of the three types of tumor cells to rIFN- resultedin moderate to potent inhibition of tumor cell growth.IFNstimulatedthe expression of mRNAs for an inducible type of NO synthasa(INOS)in CSA1M cells and indoleamine 2,3-dioxygenasa (IDO),an enzyme capable of degrading tryptophan, in OV-HM cells ,but induced only marginal levels of these mRNAs in MCH-I-ALcells. In association withiNOS gene expression, INF--stimulatedCSA1M cells produced a large amount of NO which functioned toinhibit their own growth in vitro. Although OV-HM and MCH-1-A1cells did not produce NO, they also exhibited NO susceptibility.Whereasthe tumor masses from IL-12-treated CSA1M-bearing mice inducedhigher levels of INOS (for CSA1M) or IDO and iNOS (for OV-HM)mRNAs,the MCH-1-A1 tumor mass expressed lower levels of iNOS mRNAalone.Moreover, massive infiltration of CD4⁺and CD8⁺ T cellsand Mac-1⁺ cells was seen only in the CSA1M and OV-HM tumors.Thus, these results indicate that IFN- produced after IL-12treatment induces the expression of various genes with potentialto modulate tumor cells and growth by acting directly on tumorecells or stimulating tumor-infiltrating lymphold cells and thatthe effectiveness of IL12 therapy is assoiated with the operation if these mechanisms.     

Requirement for IFN-gamma in IL-12 production induced by collaboration between v(alpha)14(+) NKT cells and antigen-presenting cells

Two cytokines IL-4 and IL-12 are known to determine the balance between T(h)1 and T(h)2 development. In addition to IL-4 production of V(alpha)14(+) NKT cells, they have recently been demonstrated to have the capacity to stimulate IL-12 production by antigen-presenting cells (APC). This study demonstrates that IFN-gamma is absolutely required for the NKT cell-stimulated IL-12 production. Culture of B cell-depleted spleen cells from C57BL/6 mice with alpha-galactosylceramide (alpha-GalCer) capable of selectively stimulating V(alpha)14/J(alpha)281(+) NKT cells resulted in the production of IL-12 together with IL-4. Whereas IL-4 production occurred in culture of IFN-gamma(-/-) C57BL/6 splenocytes, the same culture failed to generate IL-12 production. While IL-12 production induced during culture of V(alpha)14(+) NKT cells and APC depended on the interaction between CD40 ligand on NKT cells and CD40 on APC, the expression levels of these key molecules were comparable in cells from wild-type and IFN-gamma(-/-) mice. Addition of rIFN-gamma to alpha-GalCer stimulated IFN-gamma(-/-) splenocyte culture, and administration of rIFN-gamma to alpha-GalCer-injected IFN-gamma(-/-) mice resulted in the restoration of IL-12 production in vitro and in vivo. These results illustrate a mandatory role for IFN-gamma in V(alpha)14(+) NKT cell-stimulated IL-12 production by APC.     

The development of dermatitis infiltrated by {gamma}{delta} T cells in IL-7 transgenic mice

Transgenic mice constitutively expressing IL-7 developed severedermatitis with erythroderma and alopecia. The skin lesionswere characterized by massive infiltration of mononuclear cells.Immunofluorescence staining showed that most of the infiltratingcells were T cells with the majority bearing the TCR otherthan the V5 moiety. Furthermore, the number of T cells hadincreased in the lymphold organs of the dermatitis animals.These findings idicate the strong relationship between the expressionof IL-7 and the development of T cells in vivo and the pathologicalinvolvement of proliferated and/or activated T cells in skindisease.     

Ligand-induced autoregulation of IL-2 receptor {alpha} chain expression in murine T cell lines

The IL-2 receptor (IL-2R) is composed of three chains a, ßand . In mice, contrary to the human system, we have previouslydemonstrated that the IL-2Rß complex does not bindIL-2. Therefore, mouse IL-2 response is completely dependenton the expression of the IL-2R gene product. T cell clones expressingmouse IL-2Rß and the human IL-2R transgene have beenstudied. When cells are grown in IL-4, mouse IL-2R is not expressed.However, exposure to IL-2 leads to the expression of the endogenousmurine IL-2R subunit. The T cell line expressing mouse IL-2Rand human IL-2Rß can grow in IL-2 but does not expressendogenous murine IL-2 R. Transfection of these cells with thehuman IL-2R gene restores the capacity to induce murine IL-2R.This result demonstrates that IL-2-IL-2R interactions are requiredfor induction of IL-2R. The kinetics of induction and deinductionof murine IL-2R have been studied using clone 18.III. From negativecells, expression of murine IL-2R is a very slow phenomenon.From cells fully expressing IL-2R, deinduction is a two-stepprocess: after a rapid decrease of IL-2R the cells continueto express, for a long period of time, basal levels of murineIL-2R. When cells expressing basal levels of IL-2R are exposedto IL-2, induction of IL-2R is a very rapid phenomenon. Theautoregulatory loop formed by IL-2-IL-2R therefore displaysdifferent levels of functioning.     

Expression of the IL-2 receptor {gamma} chain on various populations in human peripheral blood

We have established two rat mAbs, TUGH4 and TUGh5, specificfor the human chain of the IL-2 receptor (IL-2R), which isknown to be shared among receptors for IL-2, IL-4 and IL-7.The antibodies bound to cell lines transfected with the human chain gene but not to their parental cell lines, and precipitated65–70 and 80–90 kDa cell surface molecules fromlysates of human T cells surface-labeled with Na¹²⁵I and chemicallycross-linked with ^[125]IL-2 respectively. Flow cytometry withTUGh4 and TUGh5 detected the chain in a wide variety of peripheralblood cell populations including CD4⁺ T cells, CD20⁺ T cells,CD20⁺ B cells, CD56⁺ natural killer cells, CD4⁺ monocytes andgranulocytes, contrasting with expression of the and ßchains of IL-2R.     

IL-15 enhances the function and inhibits CD95/Fas-induced apoptosis of human CD4+ and CD8+ effector-memory T cells

Although the effect of IL-15 has been described on murine cells in vitro and in vivo, its effect on human memory CD8(+) T cells is not well characterized. We show here that IL-15 preferentially enhances the activation and effector function of human effector-memory CD45RA(-)CD62L(-) and CD45RA(+)CD62L(-) CD4(+) and CD8(+) T cells in both healthy and HIV-infected individuals. We find that IL-15 increases 2- to 5-fold both the activation and secretion of the effector cytokines IFN-gamma and tumor necrosis factor (TNF)-alpha by anti-CD3-stimulated purified CD4(+) and CD8(+) T cells and peripheral blood mononuclear cells from healthy and HIV-infected individuals. Furthermore, IL-15 potently inhibits CD95/Fas-induced apoptosis of the effector-memory CD4(+) and CD8(+) T cells from HIV-infected individuals. These findings suggest that in addition to being a growth and survival factor for memory CD8(+) T cells, IL-15 is also a potent activator of human effector-memory CD8(+) T cells both in healthy and in HIV-infected individuals.     

Phenotypic and functional properties of murine {gamma}{delta} T cell clones derived from malaria immunized, {alpha}{beta} T cell-deficient mice

Six murine T cell clones expressing TCR were generated frommalaria immunized, ß T celldeficient mice. Phenotypiccharacterization of these clones has revealed that, in contrastto conventional ß T cells, there is a considerabledegree of heterogeneity among these clones with regard to theirsurface markers and their lymphokine profile. One clone wasfound to display significant anti-parasite activity in vivoupon adoptive transfer. We attempted to determine whether theprotective clone differs in one or more key characteristicsfrom the non-protective clones. Although no obvious patternpeculiar to the protective clone was observed, it appears thatmore than one parameter may, in combination, define a distinctprotective phenotype, and thus explain the functional differencebetween the protective and non-protective clones.     

Up-regulation of Borrelia-specific IL-4- and IFN-gamma-secreting cells in cerebrospinal fluid from children with Lyme neuroborreliosis

The clinical course and outcome of several infectious diseases are dependent on the type of immune response elicited against the pathogen. In adults with neuroborreliosis (NB), a type 1 response with high production of Borrelia-specific IFN-gamma, but no IL-4, has been reported. Since children have a more benign course of NB than adults, we wanted to investigate type 1 and type 2 responses in children with NB. Cerebrospinal fluid (CSF) and blood were collected from children during the acute stage of 'confirmed NB' (n = 34), 'possible NB' (n = 30) and 'non-NB' (n = 10). The number of Borrelia-specific IL-4- and IFN-gamma-secreting cells was measured by enzyme-linked immunospot assay. Borrelia-specific secretion of both IL-4 and IFN-gamma was increased in CSF in confirmed (P < 0.05) and possible (P < 0.01) NB, when compared with non-NB controls. Furthermore, children with NB had significantly higher Borrelia-specific IL-4 secretion in CSF than an adult reference material with NB (P < 0.05). There were no differences in cytokine secretion in relation to onset or recovery of neurological symptoms. Since IL-4 is known to down-regulate the pro-inflammatory and possibly harmful effects of prolonged IFN-gamma responses, the prominent IL-4 response observed in the central nervous system compartment might contribute to the more benign disease course seen in children with Lyme NB.