Human Th2-like cell clones induce IL-12 production by dendritic cells and may express several cytokine profiles

Previous studies have shown that human T_h2 cells, unlike theirmurine counterparts, retain the ability to produce IFN- uponactivation in the presence of exogenous IL-12. Here we firstextended this notion by showing that T_h2-like cell clones (T_h2C)are also capable of inducing IL-12 production by physiologicalantigen-presenting cells (APC); we next showed that these cellsmay express several distinct cytokine profiles depending uponthe activation signal and the type of APC with which they interact.We have analyzed the production of IL-4, IL-5 and IFN- by T_h2Cstimulated by either anti-CD3 mAb or exogenous IL-2, using peripheralblood monocytes or dendritic cells (DC) as accessory cells.We found that: (i) DC but not monocytes released IL-12 and promotedIL-12-dependent IFN- production upon interaction with anti-CD3-or IL-2-stimulated T_h2C and (ii) ligation of CD3 was requiredfor the production of IL-4 but not of IL-5 or IFN-. Thus, dependingupon the type of APC with which they interacted and the modeof activation, T_h2C, expressed four distinct cytokine profiles:(i) IL-4 + IL-5, in response to anti-CD3 + monocytes; (ii) IL-4,IL-5 + IFN-, in response to anti-CD3 + DC; (iii) IL-5 + IFN-,in response IL-2 + DC; and (iv) IL-5 alone, in response to IL-2+ monocytes. The ability of human T_h2-like cells to induce IL-12production and to release the proinflammatory cytokines IFN-yandIL-5 upon IL-2-driven interactions with APC may contribute toexplain how local infection exacerbates Th2-mediated diseases,like bronchial asthma and atopic dermatitis.     

Cytokine dependence of V{gamma}3 thymocytes: mature but not immature V{gamma}3 cells require endogenous IL-2 and IL-7 to survive evidence for cytokine redundancy

It has previously been described that V3 cells can proliferateextensively in vitro in the presence of different cytokines.Here, the role of cytokines in the maintenance of V3 cells inthe thymus has been determined. Culture of fetal thymocytesin cell suspension for 24 h showed that, whereas immature TCR^lowHSA^highV3cells remained viable, all mature TCR^highHSA_lowV cells died.These cells died by apoptosis since protein synthesis was requiredand flow cytometric analysis as well as DNA gel electrophoresisshowed that the DNA was degraded to oligonucleosomal bands.Addition of IL-2, IL-4 or IL-7 to suspension cultures of fetalthymocytes rescued V3 cells from dying. Addition of IL-1, IL-3,IL-5, IL-6, IL-9, TNE- or IFN- was without effect. Phenotypicanalysis showed that the -chain of the IL-2 receptor (IL-2R)was expressed by part of the immature V3 thymocytes, all matureV3 cells expressed the p-chain of the IL-2 receptor (IL-2RP).Addition of anti-IL-2R mAb to fetal thymic organ culture (FTOC)resulted in a moderate reduction of the cell number of matureV3 thymocytes. Addition of anti-IL-2Ra, anti-IL-4 or anti-IL-7mAb had no effect. The cell number of mature V3 cells was highlyreduced when both anti-IL-2Rp and anti-IL-7 mAb were added toFTOC. These results show that IL-2 and IL-7 are actively involvedin the maintenance of mature V3 cells in the thymus. This cytokinedependence of mature Vthymocytes may explain their selectivelocalization in skin epithelium.     

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.     

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.     

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.     

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.     

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.     

Antigen mediated and polyclonal stimulation of human cytokine production elicit qualitatively different patterns of cytokine gene expression

Polyclonal activators are widely used as surrogate antigensin analysis of human cytokine gene expression. An implicit assumptionis that the T cell activation and cytokine production observedin response to polyclonal activation provides a more intense,but qualitatively identical, reflection of results that wouldbe obtained with antigen. Here we demonstrate that stimulationusing accessory cell independent (immobilized anti-CD3 mAb)or dependent [phytohemagglutinin (PHA) or soluble anti-CD3 mAb]polyclonal activators yields different conclusions from thosethat are obtained in response to antigen-specific T cell activation.Cytokine synthesis in 1–5 day bulk cultures of fresh peripheralblood mononuclear cells (PBMC) from 52 subjects evenly dividedbetween grass pollen sensitive allergic rhinitis subjects andnormal, non-atopic controls were examined. Antigen-specificre-stimulatlon elicited elevated IL-4 and IL-10 production andlower IFN- synthesis among allergic subjects than normal non-atopiccontrol subjects. This commitment of fresh PBMC towards a T_h2-likeresponse in atopies and the dominance of the IFN- response seenin non-allergic subjects was reinforced when the ratio of IFN-:IL-4production in bulk culture was examined. Atoplc individualsexhibited median IFN-:IL-4 values of 0.07, whereas grass pollenstimulated cytokine production by normal subjects yielded aratio of 4.8. In marked contrast, IL-2, IL-4, IL-10 and IFN-production elicited using polyclonal activators, though muchmore intense, did not differ between allergic and non-allergicsubjects (Wilcoxon rank sum test P >> 0.05). Anti-CD3mediated stimulation evoked ratios characteristic of Th1 -dominantresponses in both populations (IFN-:IL-4 ratios of 10 and 14),while PHA elicited T_h2 dominant responses (ratios of 0.40 and0.22) In both normal and allergic subjects. The data Indicatethe Importance of using specific activation signals In characterizingT cell responses     

Brucella abortus induces a novel cytokine gene expression pattern characterized by elevated IL-10 and IFN-{gamma} in CD4+ T cells

Immunization of BALB/c mice with killed Brucella abortus (BA)has previously been shown to Increase serum lgG2a levels andlong-term T cell clones from these mice secrete T_h1-associatedcytokines: IFN- and IL-2 but not IL-4 or IL-5. We analyzed cytokinegene expression following primary immunization with BA to determinewhen CD4⁺ T cells first express cytokine genes and whether specifichypothesized cytokine patterns (e.g. T_h precursor, T_h0) couldbe identified prior to a T_h1-like pattern. Our results demonstrateda highly consistent and novel pattern of T_h 1/T_h2 cytokine geneexpression characterized by elevated IL-10 and IFN- in CD4⁺T cells which rapidly manifests itself and is sustained forat least 10 days after immunization. No elevation in IL-2 cytokinegene expression was observed and treatment of BA-immunlzed micewith blocking anti- IL-2 antibodies had no effect on the cytokinegene expression pattern, although treatment with anti-IFN antibodiesresulted in increased IL-4, IL-5, and IL-9 cytoklne gene expression,In the absence of any change In IFN- or IL-10 as early as 4days after immunization. These results suggest that a wholepathogen may trigger sufficient costimulatory signals to rapidlyinduce effector T cells in the absence of elevated IL-2 andthat IL-10 Is specifically elevated in certain T_h1-like responses.     

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc{gamma}R) encoded by an alternatively spliced transcript of the Fc{gamma}RII gene

Low affinity FcR are a heterogeneous group of glycoproteinswhich exist in transmembrane (TM) as well as in soluble forms.Two membrane isoforms of the murine type II FcR, FcRilb1 andFc;Rilb2, have been described. They result from the translationof alternatively spliced premRNA, FcRilb2 lacking sequencesof the first intracytoplasmic domain (IC1). Soluble forms ofFcR (sFcR) have previously been shown to result from proteolysisof membrane receptors. We report here the identification, inmacrophages, of a mRNA derived from the FCRll gene by splicingexons encoding the TM and IC1 domains, i.e. corresponding toa TM-deleted FcRllb2 mRNA. A soluble protein possibly encodedby this mRNA was identified in macrophage supernatants. In accordancewith FcR nomenclature, we propose to name this new FcRll IsoformFcRllb3. It is the most abundant 8FcR present in serum, as comparedwith 8FcR resulting from cleavage of membrane FcR.     

A unique pattern of lymphokine synthesis is a characteristic of certain antigen-specific suppressor T cell clones

We report that the lymphokines (IFN-) and IL-10 are co-syntheslzedby previously described CD3⁺ TCRß⁺, minor antigen-specificsuppressor T cell clones. IFN- and IL-10 are known to (I) becharacteristically produced by different helper T cell types,T_h1 and T_h2 respectively, and (II) inhibit the function of thereciprocal subset of T cells: IFN- Inhibits the function ofT_h2 and IL-10 that of T_h1 cells. Although Th0 cells are alsoknown to synthesize cytoklnes of both the T_h1- and T_h2-typeT cells, the suppressor T cells described in this report aredifferent from T_h0 cells in that they produce (I) neither IL-2nor IL-4 molecules and (II) stimulation via their CD3-TCR systemseems independent of both IL-2 and IL-4, the typical autocrinemolecules for T cell proliferation. The lymphokine profile ofthese suppressor T (TJ cell clones, as well as those of humanantigen-specific T. cells reported earlier, suggests that co-synthesisof some T_h1-llke and some T_h2-like cytoklnes may be a characteristicof antigen-specific T, cells as opposed to the type of reciprocalinhibition mediated through IFN- or IL-10, which is antigennon-specific.     

A pivotal role of IL-12 in Th1-dependent mouse liver injury

Intravenous injection of Proplonibacterium acnes and llpopolysaccharide(LPS) with a 7 day interval caused CD4⁺ T cell-dependent severeliver injury in the C57BL/6 (H-2^b) mouse strain. In contrast,BALB/c (H-2^d mice were resistant to P. acnes and LPS-inducedliver injury. The different susceptibilities of the two mousestrains to liver injury appeared to be closely correlated withtheir different abilities to produce IFN- after P. acnea priming.Namely, the sensitive C57BL/6 mouse strain produced a significantlevel of IFN- 7–10 days after P. acnes injection, whereasno significant amount of serum IFN- was detected in the resistantBALB/c mouse strain. The important role of IFN- in liver injurywas demonstrated from the finding that In vivo administrationof anti-IFN- mAb abrogated P. acnes and LPS-induced liver injuryin C57BL/6 mice. Moreover, it was demonstrated that In vivoadministration of recombinant IL-12, a key cytokine for theinduction of IFN-, into mice induced P. acnes and LPS-inducedliver injury in the resistant BALB/c mouse strain. Conversely,In vivo administration of anti-IL-12 mAb blocked the developmentof liver injury in the sensitive C57BL/6 mouse strain. Moreover,it was demonstrated that the failure of the induction of liverinjury in BALB/c mice appeared to be derived from the lack ofexpression of IL-12 at the local site of liver in P. acnes-prlmedmice. These results strongly indicated that endogenous IL-12,which stimulates T^h 1-dominant cellular immunity and IFN- production,may be an essential cytokine on the course of T cell-dependentliver injury.     

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.     

Interleukin 12 alters the isotype-restricted antibody response of mice to hen eggwhite lysozyme

Protein antigens elicit humoral responses in mice that consistpredominantly of IgG1 antibodies. We have now investigated theability of IL-12, a cytoklne reported to augment IgG2a antl-haptenresponses through activation of T_h1 cells, to alter antibodyresponses to hen eggwhite lysozyme (HEL). The normal responseof BALB/c mice to HEL is highly restricted to lgG1 expressionand therefore provides an excellent system for determining effectsof cytoklnes on expression of other isotypes. Seven days afterimmunization, IL-12-treated mice demonstrated greatly elevatedHEL-speciflc IgG2a antibody levels and suppressed IgG1 production,while PBS-treated control mice showed a typical lgG1-restrictedresponse. On day 28, IL-12-treated mice showed heightened serumantibody levels of both isotypes. Delaying cytoklne treatmentuntil after the typical IgG1 anti-HEL response had already beenestablished also led to significant elevation of serum IgG2aantibody levels. These effects correlated with increased IFN-production; however, administration of IL-12 plus anti-IFN-had little influence on lgG2a enhancement, although it did relievethe early lgG1 suppression. Furthermore, the differential effectsof IL-12 on Isotype expression did not correlate with time;in fact, IgG2a enhancement correlated with loss of IgG1 suppression.Our findings indicate that (I) IL-12 reproduclbly Induces largeamounts of IgG2a HEL-speclflc antibodies In vivo; (II) it canalter isotype profiles of both primary and secondary responses;and (III) its effects on humoral immunity are not completelyexplained by Induction of T_h1 cell-derived IFN-.