Differential regulation of surface immunoglobulin and Lyb2 mediated B cell activation: II. cAMP dependent (prostaglandin E2) and independent (IFN-{gamma}) mechanisms of regulation of B lymphocyte activation

We have recently demonstrated that pharmacological agents thatelevated cAMP inhibited sigM but not Lyb2 mediated activationof murine B lymphocytes. In this report we show evidence fordifferential regulation of prostagiandin E₂ (PGE₂), a physiologicalagent that elevated cAMP and IFN- on sigM and Lyb2 mediatedB cell activation. PGE₂ inhibited anti-lgM but not anti-Lyb2induced DNA synthesis in a dose-dependent manner. Interestingly,rlFN- also inhibited anti-lgM but not anti-Lyb2 induced DNAsynthesis. rlFN- exerted its effects directly on B cells sincedepletion of T cells and G-10 Sephadex adherent cells did notalter effects of IFN- on anti-lgM and anti-Lyb2 induced DNAsynthesis. Pretreatment of B cells with IL-4 and/or IL-5 didnotprevent the IFN- mediated inhibition of the anti-lgM response.The inhibitory effect of IFN- was observed during early stagesof B cell activation. Thus IFN- inhibited anti-µ inducedblast transformation and subsequent progression into the G₁phase of the cell cycle. The differential effects exerted byPGE₂ and rIFN- appeared to be mediated by distinct mechanisms.ThusPGE₂ but not rIFN-, at concentrations inhibitory to the slgMresponse, induced elevation of intracellular cAMP levels. Theseresults demonstrate that physiologically relevant immunomodulatorssuch as PGE₂ and IFN- can differentially regulate murine B cellresponses mediated through the antigen receptor and Lyb2 moleculesby cAMP dependent and independent mechanisms. Relevance of thisregulation for the induction of antibody synthesis by T_h1 andT_h2 types of helper T cells is discussed.     

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.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

Emigration of selected subsets of {gamma}{delta}+ T cells from the adult murine thymus

Cells bearing the form of the TCR make up only 1–3% ofT cells in the adult murine thymus and peripheral lymphold organs.Evidence from studies of nude mice suggests that the developmentof at least some T cells is thymus dependent; however, untilnow it has not been directly demonstrated that cells are exportedfrom the thymus. In this paper we have used the technique oflabelling thymocytes in vivo with FITC, followed by flow cytometrlcanalysis to trace cells emigrating from the thymus to the spleen.Using this approach we have been able to demonstrate for thefirst time that T cells are exported from the adult murinethymus to the spleen. We also demonstrate that the cells emigratingto the spleen are a selected subset of thymocytes being heatstable antigen positive, Thy-1⁺, and expressing low levels ofCD44 (Pgp-1). In addition, investigation of TCR V; gene usageamong adult ⁺ thymocytes, recent emigrants, and spleen cells,indicated a selective emigration of cells expressing certainVgenes.     

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.     

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.     

Differential production of IL-12 in BALB/c and DBA/2 mice controls IL-4 versus IFN-{gamma} synthesis in primed CD4 lymphocytes

The profile of cytokines produced by CD4 T cells is profoundlyinfluenced by the presence of IL-12. Here we demonstrate thatduring re-stimulation of antigen-specific immune responses invitro, antigen-primed lymph node cells from DBA/2 mice produced3- to 30-fold more IL-12 than did cells from BALB/c mice, whichare identical at the major histocompatibility locus. The straindifferences in IL-12 production were observed only in antigen-drivenresponses (and not in responses induced by bacterial products),and were dependent upon an interaction between CD4 T cells andlymph node adherent cells. In addition, differences in the quantityof IL-12 produced by DBA/2 and BALB/c antigen-presenting cells(APC) was not dependent on differential production of IFN- byT cells, since APC from DBA/2 mice still produced much greaterquantities of IL-12 than did BALB/c APC when each was culturedwith the same H-2^d-restricted T_h2 clones, in the complete absenceof IFN, or when each was cultured with primed (BALB/c x DBA/2)F₁T cells. The level of IL-12 produced in the cultures criticallyaffected cytokine production in CD4 T cells, since neutralizationof endogenous IL-12 in DBA/2 cultures, which are predisposedtowards developing T_h1 responses, reduced IFN- production andenhanced IL-4 synthesis to levels normally seen in BALB/c cultures,which are predisposed toward developing Th2 responses. We proposetherefore that differential production of antigen-driven IL-12is a mechanism by which the genetic background in DBA/2 andBALB/c mice can affect the pattern of cytokine synthesis byT cells during the development of adaptive immune responses.