Growth inhibition of Mycobacterium bovis by IFN-{gamma} stimulated macrophages: regulation by endogenous tumor necrosis factor-{alpha} and by IL-10

Murine bone marrow-derived macrophages (BMM) are able to inhibitthe intracellular growth of Mycobacterium bovis and Mycobacteriumtuberculosis H37Rv after activation with recomblnant (r) IFNand growth inhibition is mediated by reactive nitrogen intermediates(RNI) derived from L-arglnlne. We now demonstrate that tumornecrosis factor (TNF)- acts as an endogenous cofactor in theinduction of mycobacterial growth inhibition. TNF- was producedby BMM stimulated with rlFN- and infected with mycobacterla,and a specific antlserum to TNF- Inhibited rlFN--lnduced productionof RNI as well as growth inhibition of M. bovis. IL-10, a cytokinewhich suppresses antlmycobacterial macrophage functions, wasalso produced by BMM activated with hFN- and infected with M.bovis. IFN--induced production of TNF- and of reactive nitrogenintermediates as well as mycobacterial growth inhibition wereinhibited by exogenous IL-10, but only when given prior to IFN-stimulation. We conclude that the outcome of mycobacterial infectionis regulated by a coordinate interplay between IFN-, TNF- andIL-10.     

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.     

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.     

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.     

Composition of TCR-CD3 complex in human intestinal intraepithelial lymphocytes: lack of Fc{varepsilon}Rl{gamma} chain

Human intestinal intraepithelial lymphocytes (DEL) are a uniquepopulation of predominantly CD8ß⁺ TCRß⁺lymphocytes and, to a lesser extent, TCR⁺ lymphocytes that proliferatepoorly to anti-CD3 mitogenic signals but display significantcytolytic activity. Studies in mouse model systems have shownthat the chain of the high-CD3 affinity receptor for IgE (FcRl)may substitute for the chain in the TCR-CD3 complex of iIEL.This has suggested that the functional properties of these cellsmay be associated with an altered composition of the TCR-CD3complex. We therefore analyzed the TCR-CD3 complex of normalhuman iIEL. One-and two-dimensional non-reducing/reducing SDS-PAGEanalysis of CD3, CD3, CD3, and FcRr chain immunopreclpitatesof cell surface radiolabeled proteins with subunit-specificantibodies revealed a TCR-CD3 complex without associated FcRrchains. Thus, normal human NEL contain a TCR-CD3 complex thatconsists predominantly of , homodimers in association with theß TCR and CD3, and , similar to the majority of peripherallymphocytes. This indicates that the distinct properties ofhuman DEL are not associated with substitutions of the FcRlchain in the TCR-CD3 complex.     

Bovine {gamma}{delta} T cells express high levels of functional peripheral lymph node homing receptor (L-selectin)

Lymphocyte migration from the blood into specific tissues Isdirected by their expression of adhesion molecules referredto as homing receptors. The homing receptor L-selectln, forexample, directs the migration of lymphocytes into peripherallymph nodes (PLN). Since bovine T cells, a major lymphocytesubset in peripheral blood (25–50%), represent only aminor subset in PLN, we examined whether these cells lack expressionor function of L-selectin. We found that bovine T cells expressedL-selectln at levels higher (2- to 5-fold) than ßT cells and B cells. Furthermore, T cells accumulated alongthe vascular wall of venules that support lymphocyte extravasationinto PLN (MECA-79⁺ venules) in vivo and bound mouse PLN highendothelial cell venules in an In vivo binding assay. In contrastto this primary adhesive event, we directly demonstrate that T cells in vivo do not appreciably extravasate from the bloodinto the parenchyma of lymph nodes. Since the lack of functionalL-selectln expression could not account for the inability of T cells to enter PLN, we tested for other differences between T cells and PLN homing lymphocytes related to the processesfollowing primary adhesion; for instance, the down-regulationof L-selectin expression following short-term activation andthe expression of accessory adhesion molecules necessary fortransendothellal migration. We found that and ß Tcells demonstrate differential down-regulation of L-selectinafter PMA activation. Kinetic analysis revealed that, at alltime points after PMA treatment, L-selectin expression remainedsignificantly higher on T cells and was down-regulated at aslower rate compared with ß T cells. However, theexpression levels of CD44 and CD18 on and ß T cellswere found to be equivalent. This study Is the first to demonstratefor lymphocytes that the expression of L-selectln alone doesnot predict a PLN homing capacity. Our results suggest thatthe T cells' reduced ability to enter PLN may be due to inefficientdown-regulation of L-selectln compared with non- lymphocytes,thus potentially disrupting the dynamics of the extravasationevent.     

TCR isoform containing the Fc receptor {gamma} chain exhibits structural and functional differences from isoform containing CD3{xi}

The structure and function of the TCR-CD3 complex containinga homodimer of the gamma chain of the high affinity receptorfor IgE (FcR) (FcR⁺ TCR) was investigated by transfecting theFcR gene into a CD3^–, CD3^–, FcR^– T cell line.Introduction of FcR, as well as CD3, induced a high expressionof the TCR-CD3 complex on the cell surface. Transfected FCRformed a homodimer and associated firmly with the TCRßdimer but only weakly with the CD3. Stimulation of both FcRand CD3 transfectants by antibodies against TCR or CD3 inducedaccumulation of inositol phosphates, the Ca²⁺ response, IL-2production, and growth inhibition. On the other hand, antigenstimulation of transfectants expressing FcR as well as CD3 inducedIL-2 production, but only the latter exhibited the antigen-inducedgrowth inhibition. In vitro kinase assay suggested that theCD3 dimer but not the FcR dimer associates with the Fyn kinase.These results indicate that the FcR homodlmer Is able to forma functional TCR complex but that the mode of assembly and thesignaling function of FcR⁺ TCR, including its association withtyrosine klnase(s), may differ from the TCR-CD3 complex containingCD3 homodimers (⁺ TCR). This provides an example which illustratesthat different TCR isoforms mediate distinct signals and functions.     

Human {alpha}{beta} and {gamma}{delta} T cells from unexposed individuals respond to protein antigens of the yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, a dimorphic fungus, causes chronicgranuiomatous mycosis in susceptible individuals. Differentreports have shown that cell-mediated immunity is essentialfor protection against systemic mycosis, including paracoccldloidomycosis.We analyzed the reactivity of ß and T cells fromunexposed Caucasian donors to P. brasiliensis yeast form components.Our results indicate: (I) ß and T cells proliferateafter in vitro stimulation with lysates of P. brasiliensis;(II) similar numbers of ß T cells (f = 1/21,000) andof T cells (f = 1/8000) respond to P. braslllensls; (III) P.braslllensls-reactive T cells express the V9V2 TCR; (Iv) thestimulatory activity of P. brasilensis for both ßand T cells primarily resides in a high molecular weight (100kDa) and in a low molecular weight (<<1 kDa) fraction;(v) the ligands responsible for stimulation of both ßand T cells are sensitive to proteinase treatment We concludethat both ß and T cells from healthy individualsrespond to ubiquitous protein antigens of P. brasiliensis.     

Mechanisms of granuioma formation in murine Mycobacterium avium infection: the contribution of CD4+ T cells

Infection with the virulent Mycobacterium avium strain TMC 724caused progressive Infection in C57BL/6 and BALB/c mice, whileinfection with a less virulent strain (M. avium SE 01) resultedIn chronically persistent bacterial loads. Livers of mice Infectedwith TMC 724 were characterized by progressively expanding tumor-likeInfiltrations of epithelloid macrophages, while SE 01 Inducedwell-developed, compact epithelioid granulomas that remainedconstant in size and number for at least 4 months. When C57BL/6mice were depleted of CD4⁺ T cells by i.p. administration ofspecific mAb at the time of infection, their capacity to Initiategranuloma formation was completely abrogated during the first4 weeks of infection. Semi-quantltatlve competitive RT-PCR ofliver homogenates obtained 3 weeks after infection revealedthat depletion of CD4⁺ T cells was accompanied by a 25-foldreduced expression of IFN- mRNA and a 5-fold reduced expressionof tumor necrosis factor (TNF)- mRNA when compared to controlInfected mice. Granuioma morphology in response to either TMC724 or SE 01 was similar in immunodeficlent SCID mice to thatobserved In syngeneic BALB/c mice. However, SCID mice developedgranuiomas In a delayed fashion and were less efficient in surroundingInfected Kupffer cells with an inflammatory infiltration. Thedelayed kinetics of granuioma Initiation In Infected SCID micewas paralleled by a lower mRNA expression for IFN- and TNF-compared to that observed in Infected BALB/c mice. mAb-mediatedneutralization of IFN- In BALB/c mice significantly reducedinflammatory infiltrations and granuioma formation. These datasupport the conclusion that CD4⁺ T cells accelerate granuiomaformation by enhancing the production of TNF- and IFN- at thesite of infection.     

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.     

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.     

Cytokine production by CD16 CD56bright natural killer cells in the human early pregnancy decidua

Using a cell sorter, CD16^–CD56^bright natural killer (NK)cells were sorted from decidual mononuclear cells at an earlystage of pregnancy. These cells were examined by the reversetranscrlptase-polymerase chain reaction (RT-PCR) method fortheir expression of mRNA coding for the following 12 cytokines:IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), macrophage colonystimulating factor (M-CSF), tumornecrosis factor- (TNF-), interferond- (IFN-), and leukemia inhibitoryfactor (LIF). Although mRNA coding for every cytokine was detectedin decidual mononuclear cells, mRNAs coding for only G-CSF,GM-CSF, M-CSF, TNF-, IFN-, and LIF were detected In CD16^–CD56^brightNK cells. Also, the supernatant of CD16^–CD56^bright NKcell cultures was found to contain G-CSF, GM-CSF, M-CSF, TNF-,IFN-, and LIF. These findings indicate that CD16^–CD56^brightNK cells produce many different cytokines and that these cytokinesmay play an important role in a successful pregnancy.     

{alpha}{beta} and {gamma}{delta} T cells in the immune response to the erythrocytic stages of malaria in mice

Mice lacking T cells with ß TCR (TCR ß–/–)or TCR (TCR –/–) were infected with the erythrocyticstages of the malaria parasite, Plasmodium chabaudi chabaudi(AS). Mice without T cells could control and reduce a primaryinfection of P. chabaudi with a slight delay in the time ofclearance of the acute phase of infection and significantlyhigher recrudescent parasitaemias compared with control intactmice. TCR –/– mice had higher levels of both serumIg and malaria-specific antibodies of the isotypes IgG3 andIgG1 compared with control mice. TCRß–/–mice, despite a striking increase in NK1.1⁺ cells and the presenceof T cells, were unable to clear their infection. Althoughthe plasma of TCR ß–/– mice containedall Ig isotypes before and during a primary infection, theywere unable to produce significant levels of malaria-specificIgG antibodies, suggesting that in the absence of ßT cells T cells are not able to provide efficient help forantibody production.     

{gamma}{delta} T cells promote CD4 and CD8 expression by SCID thymocytes

Molecular studies of the TCR, which is expressed by a minorsubpopulatlon of T lymphocytes in all vertebrate species, havedefined a subset which expresses a receptor with extreme junctionaldiversity and a second subset, most commonly found in eplthella,which expresses a receptor of very limited diversity. In thedeveloping murine thymus, T cells appear in an ordered sequenceof specific v rearrangements, V3V, 1 on day 14, V2V1 on day17, and subsequently V4V5, V6, or V7. We demonstrate that thetransfer of expanded populations of cells from newborn thymusand cell lines expressing the invariant V3V1 receptor into SCIDmice, which lack T and B cells, results in the appearance ofCD3^–CD4⁺CD8⁺ thymocytes. Thus, one role of the early appearingV3V1 T cells in thymlc development in vivo is to promote CD4and CO8 surface expression on precursor cells.     

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.