- Email: [email protected]
Distinction of mechanism of antigen uptake and stimulation of T cells in cultured bovine dendritic cells
24 June 1997 - Poster presentations
explanation is that the TCR affinity diiers between transgenic and normal T cells.
P.3.05.13
Extended expreselon of MHC claes II genes on mouse macrophapes as8oclated wlth protectlve/suppresslve effects
APCs and the induction of T- and B-cell responses
inhibition of IL2 secretion at limited CD25B7 interaction and by the differential recognition of altered peptkls ligands in the presence of different APC.
P.3.05.15
Btigttte Mtiller, Martin Baumgart, Verena Moos, Diana Schuhbauer. Deufscfres Rheuma ForschunosZentrum. Monbiioustr: 2. D- 10117 Berlin, Germany htroduction: Protective/suppressive MHC dass II alleles have been Identified in man and mouse where thev exert a disease-omtective and immunosuopmssive effect. Various modes of action have been proposed, among them differential expression in different types of antigen-presentfng cells impacting on the Thl-Th2 balance. To test thls possibility, the expression of H-2 molecules from the four haplotypes H-Zb, H-2@,H-2k and H-2q have been determined on bone manow derfved macrophages (BMDM) and splenic B cells. Yaterfale and mode: The mouse strains used in this study are Bl0.A (5R) (H@), BALB/c (H-2’) BDFI of C57BU6 x BALB/c (H-F), BlO.BR (H@), CBA/J (H@), DBAfl (H-2s) and DBA/l x Bl0.Q (H-29). Bone marrow cells were cultured in complete RPM1 1640 medium containing 30% (voiIvol) L-929 cell-conditioned medium for up to 25 days and single cell suspensions of spleens were cultured for up to five days in complete RPM1 1640 medium containing 4.5 &ml LPS. The surface expression of MHC class II on BMDM and splenic B cells was monitored via two-color flow cytornetnc analysis using mAb recognizing the various class II molecules and CD11b or 8220 as cell-type spec5tc markers, respectively. Reeuh The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are exfxessed on almost all CDllb+ cells for five to eight days. In contrast, I-Ad-, I-Ak-, and I-Aq-expression peaks for a shorter period and fewer CD11b+ cells become positive. No differential expression could be detected on LPS-stimulated E cells. Concluskm:Our data suggest that macrophages exert Thl bias via cytoklne secretion, presumabty of 11-12, in response to ligation of their MHC class II molecules andlor facflitated ligation of their CD40 mdecules. The extent of expression of the class II gene would thus gate the back-signal from T cells and in this wav control the level of IL-12 oroduoed bv macmohaoes. This effect mediated by f&morphic non-exon segments of class II MHC genes may well play a role in determining disease susceptibility in man and mouse.
P.3.05.14
Dlfferentlal T cell activation regulated by antlgen presenting cells
P. Gogoldk ’ , 8. Rethy ‘, A. Horvath ‘, G.K. T6th 2, E. Buds 3, L. Cervenak ‘, G. L&zl&’ , ik RajnavMgyi ‘. ‘Dept.of Immunology L. E&v& Univem& G&l, 2Dqt. of Me&alChem&& A. &en&Gy@yi UniverS& Szeged, 3Dept. of Anatomy, Medical School of Deb-n, Hungary
Intrcduction:To obtain quafitatfve and quantitative data on the contribution of antigen presenting cell (APC) mediated signals in T cell triggering, the activation of three MHC class II rest&ted T cell hybridomas of distinct specfficity was studied in the presence of various APC. MaUale end Methods:The three signal transducing receptors of T cells, i.e. TCR, CD28 and CD4, were stimulated by their natural ligands expressed on the surface of a series of APCs wfth characterized phenotypes. The intermediate and late responses of T cells to diierent doses of peptide antigens presented on various APC was monitored by measuring intracellular calcium influx and It2 omductfon. msc&Nelv. The role of CD4 was studied bv using a CD4- T cell subclone, &ntrfbution br CD26 was studied in the presence orabsence of B7-speciffc antibodies or of soluble CTLA4. Reeuftez Analysis of T cell activation revealed that i) calcium mobilization induced by peptlde loaded APC requires rapid conjugate formation highly dependent on the expression pattern of adhesion molecules on APC; Ii) a direct correlation was observed betwwn the magntude of the calcium lnffux and the concentratfon of secreted I12 induced by dlfferentfal TCR ligation modulated by peptfde dose or by the presence or absence of CD4; iii) measured at identical APC/peptide and T/APC ratios, the intermediate and late responses of T cells to different peptide concentrations was similar; iv) a substantial difference in the wnsftfvity of the two methods, used for monitoring T cell actfvatlon, was observed, v) CD4 mediated slgnalling has a cc-stfmulatory effect predominantly at suboptfmal in vi& conditfons: vi) sustained increase of [Ca2+]ras well as the production of high concentrations of IL2 is highly dependent on the appropriate CD2&B7 interaction determined by the APC; vii) the response of a certain T cell to peptfde analogs is highly dependent on APC which can influence the agonistic or antagonistic behavtour of a pepttde. Conclusion: These results demonstrate that distinct peptide doses and the presence or absence of CD4 results in qualitative changes of the T cell response, while the degree of CD28 mediated signalling has a qualitative affect on the outcome of T cell activation. This was revealed by the decreased duration and aftered time kfw of the calcium response, by the complete or partial
267
lmmortallzed dendrltk llke cell lines derived from mice lacking both typesI and type II Interferon receptors, process and present MHC II restrlcted antigen to prlmed and naive T cells, Induce MLR and support virus repllcatlon
R. Nunez, M. Engels, M. Ackermann. M. Suter. institute for Vim/w, Facula)!University of Ziirich, Switzerland
Veterinary
lnterferons (IFN) are potent regulators of the immune response. Mice (AG 129) lacking both type I and II IFN receptors have been generated (Science 2641 1918). However, the role of the IFNs in the function of dendrftic cells (DC) is not fully understood. Therefore, the present study was aimed at generating immortalized (Cncogene 9: 1473) DC lines from AG 129 mice and to study the phenotype and the bldogical functions of the cells obtained. Several immortalized cell lines were obtained from thymus, spleen, brain, eye, lung and skin. These cell lines have cytoplasmatic extensions like dendrites, attach to plastic, have no inhibition of growth by contact and do not require external growth factors for proliferation. Some of the cell lines have been kept in culture for more than one year. The cell surface markers are characteristic for cells of the monocyte macrophage lineage. The cells express Macl+ and have low levels of MHC class II molecules, some are CD45+, F4.80+, CD& and B220-. The skin, brain and thymus derived cell lines express significant levels of Thyl. Functional studies were focused on one skin (named AGlOl) and one brain (named AGl16) derived cell line. The cell lines are able to: i) take up and process soluble antigens, ii) present soluble antfgens to T cell hybrtdomas iii) induce MLR with as few as 100 stimulating cells, iv) and prime naive T cells to soluble antigen in vitro. Preliminary experiments indicate that the cell lines AGlOl and A0116 allow replication of Bovine Herpes Virus. The data indicate that the cell lines obtained from mice with deleted IFN receptors have functional charactenstics of DC and may prove to be useful for the study of virus replication.
P.3.05.16
W;gMlon
of bacterial antigens from human
I.A. Schrijver, M.J. Melief, MA. Hoijer, M.P. Hazenberg. Department of Immunology Erasmus University Rotterdam, The Netherlands Introduction: Rheumatoid arthritis (RA) is considered to be a chronic T cell dependent response against an unknown antigen. We propose that this immune response is directed against bacterial antigens from the intestinal flora. The hypothesis is based (among others) on the presence of antigen presenting cells with peptidoglycan pdysaccharfde complex (PG-PS) in synovia of A;\ patients. PG-PS positive cells were found in spleen from healthy human also. We developed a method to isolate this antigen from human spleen. Recently the method was modified to minimize the amount of protein in the fraction. Materfals and Methods: Human spleen tissue was homogenized, sonificated and extracted with acetic acid. Gel filtration was performed with Sephadex 625. Fractions with muramic acid were pooled and loaded on a Supetdex 200 column connected to a fast pressure liquid chromatography system (FPLC). The presence of PG-PS was determined by ELISA using a PG-PS specific monoclonal antibody and confirmed by the detection of muramic acid by high pressure liquid chromatography (HPLC) with a Sephacil C8 column. Rwult& After Sephadex G25 gel f&ration a high molecular weight fraction with muramic acid (PG-PS) ww pooled. With the Superdex 200 column two fractions could be separated. One with a relatively high and one wfth a relatively low moleculair weight and almost no protein. HPLC analysis confirmed the presence of muramfc acid in both fractions. Conclusion: The described procedure is a useful method to obtain a fraction of bacterial antigens from human spleen containing a minimal amount of protein. The fractions will be used to determine humoral and cellular responses against these endogenous bacterial antigens in healthy subjects and RA patients.
P.3.05.17
Dlstlnctlon of mechanism of antigen uptake and stlmulatlon of T cells In cultured bovine dendrltlc cells
D. Werfing, P. Sopp, G. Taylor, R.A. Collins, C.J. Howard. tnstitute forAnimal Hwltf~, Compton, UK Dendritic cells (DC) exhibit a uniquely potent ability to stimulate antigen-specific responses in vitro and in vfvo. Immature DC are extremely effident in antigen uptake mediated by various endocytfc pathways including non-selective (i.e. fluid phase macropinocytosis) and substance-specific (i.e. C-type lectin receptor mediated) interfortsation. Antigen-specific recognhion and intemalisation
268
24 June 1997 - Poster presentations
APCs and the induction of T- and B-cell responses
capabilities of DC are of interest since they may have profound implications in the elicitation, amplification, and perhaps the diversity of the T cell msponse. Human DC derived from oerfoheral blood monocvtes in the oresence of IL-4 and GM-CSF have been r&o&d totake up FITC-dextran (FITk-DX) and lucifer yellow (LY) using the mannase-receptor or macmpinocytosis. Whereas the uptake of FITC-DX can be blocked either by mannan or antibodies to the mannose receptor, the uptake of LY is constitutive and cannot be influenced by lectins or antibodles. Little is known about the mechanism responsible for efficient antigen caoture in bovine DC. Bovine monocvtes and monocytededved DC cultured in ihe presence of recombinant bovine IL-4 and GM-&F incorporate FITC-DX but did not show a fluid phase uptake of LY by macropinocytosis. In contrast to human DC, blocking of the mannose receptor (MR) with antibodies raised against the human MR did not diminish the uptake of FITC-DX by bovine DC, although these antibodies stained a subpopulatfon of the cultured DC. Frozen bovine DC were ineffective at endocytosing FITC-DX or LY,but were still able to present antigen. Our observations imply that bovine monocyte-derived DC lack the mechanism of a fluid phase uptake by macropinocytosis and use diirent MR to human DC to take up mannosylated antigens. These pathways of antigen uptake are further altered by cryoconservation.
P.3.05.18
Characterisation of a iigand for MyP1, a novel mammaiian giycoprote4ninitiaiiy identified on cattle monocytes and dendritic ceils
G.P. Brooke, C.J. Howard. institute &Animal Health, Compton, UK Co-stimulatory molecules expressed on the surface of antigen presenting cells (APC) are required for the effective activation of T cells. Although several of these molecules have been defined, experimental data implies the existence of others that may be involved in the stimulation of naive T cells. We have identified a novel glycopmtein that is expressed on a subpopulation of cattle dendtftic cells and on other cells of the myeloid lineage. A cDNA encoding this molecule, initially identified by mAbs, has been cloned and sequenced and the predicted molecule is a type I glycopmtein member of the IgSF. We have termed the antioen MvD-1 (Mveloid and Dendritid. A functional role for the molecule has be& indi&d by-the finding that the ‘mAb block stimulation of resting T cells by ovalbumin pulsed APC. The role for MyD-1 in T cell stimulation may be further elucidated by identifying the ligand(s) of MyD-1. We have therefore constructed a fusion protein by combining the DNA encoding the extra-cellular region of MyD-I with the Cyl chain of human immunoglobulin, in the plasmid vector piLN. Purified fusion protein (MyD-llg) was shown to bind to major subpopulations of peripheral blood T cells, B cells and monccytes, but not y/S T cells. The ligand for MyD-1 is curmntlv beino cloned using a COS-7 cell transient expression svstem.
P.3.05.19
Role of interieukin 12 in dendritic ceil-dependent Interferon y production by murine peripheral ivmoh node ceils
R.J. Dearman, I.R. Jowsey, D.M. Kemeny I, I. Kimber. Zeneca Central ToxicologyLaboratory; Alderley Park, Macclesfield, UK, 1King’s College, London, UK Introductton: We have demonstrated previously that the addition of exogenous accessory cells to naive peripheral lymph node cells (LNC) enhanced substantially concanavalin A (con A) induced production of interferon y (IFN-y)‘.We have now investigated the role of in&tie&in 12 (IL-12) in the expression of this cytokine using a neutralizing monoclonal anti-IL-12 antibody. Matertafs and Methoda: A single cell suspension of LNC was prepared from the auricular lymph nodes of naive BALB/c strain mice. Dendritic call enriched (DC+) and deoleted (DC-) fractions were oreoared bv buoyant density centrifug&on imm lymbh n&s is&ted from mice’& ihe same St&n 16 hr aft& topical exposure to the contact allergen oxazolone (1%). LNC (5 x 10s cells/ml) were cultured for 46 hr with 2 &ml con A. various concentrations of DC+ or DC- fractions and in the presence or absence of 20 @ml rat monoclonal anti-IL-12 neutralizing antibody or control antibody. The IFN-)I content of culture supematants was determined by enzyme-linked immunosorbent assay. Resuftez Addition of DC+ to cultures of con A-stimulated naive LNC enhanced IFN-y expression up to 5-fold. Elevation of IFN-y production was observed when exooenous‘DC+ comortsed 0.5% or oreater of the cutture woulation. Antigen-actiiated LNC populaions depleted 2 DC (DC-) did not in~lu&ce IFN-y secretion. Neither DC+ or DC- fractions cultured alone in the presence of con A elaborated detectable levels of this cytokine. The addiion of neutmlizing anti-IL-12 antibody, but not control rat antibody, inhibited almost completely IFN-y production by con A-activated naive LNC and DC+ enhanced expression of this cytokine. Conduefons: These data reveal that within unstimulated peripheral lymph nodes there are insufficient numbers of appropriate accessory cells to support vigorous IFN-y expression. Exogenous allergen-activated DC, via the secretion of 11-12,provide for optimal production of this cytokine.
[I] Hodgson CM., Dearman R.J., Morrs A.G. and Kimber I. (lga6). Imm. Letters, 4@,4455.
P.3.05.20
Human 1 ceil activation by APC: Synergistic effect of accessory signaling by CiMO, IL-10 and IL-12
A. Kasran, X. Peng, J.L. Ceuppens. Laborarory of Expetimenfal /rnrnuno/~, C&ho/k University of Leuven, Belgium Introductfon: It is generally accepted that the CD4O-CD4OLinteraction between B cells and accessory cells on one hand and T helper cells on the other, plays an important role in the regulation of B cell and APC functions. Evidences are accumulating that the CD4&CD4OL interaction is bidirectional and dfmctly costirnulates T cell activation as well. MaterfaIr 8nd Methods: In this study we have analysed functional effects on T cells involving CD40-CD4OL interaction using an activation system with purified resting T cells devoid of accessory cells. Using direct activation of protein kinase C (PKC) by the phorbol ester, phortml 12-my~istate 13acetate (PMA), we measured the effect of a monoclonal antibody to CD4OL (Mg2) in the presence of IL-18 or IL-12 in inducing T cell pmliferatfon and cytokine productfon. Rewitsz(1) T cell proliferation was strongly enhanced when PMA-treated T cells were cultured with anti-CD4OL in the presence of IL-l/l or 11-12.(2) 11-2, 11-5,IL-lo, IL-13 and IFN-y were readily produced when T cells were activated through ccstimulation ofCD4OLwitJ1IL-l/3 or 11-12.Wherethepresence of IL-la favoured the oroductfon of IL-5 and 11-13.the ~msence of IL-12 in the culture favoured the production of 11-2,IL-10 and.lFN-;. (3) Them is synergy between IL-18 and IL-12 in upregulating IL-2R (CD25) expression when PMA-treated T cells were cultured in the presence of CD4OLtriggering. Conclusion:We have added yet another piece of evidence that resting T cells can be costimulated through CD4OL to induce cylokine pmduction and responses to 11-2. Synergy between IL-lg or IL-12 and CD4OL triggering was apparent at the level of proliition and the production of 11-2,IL-S, IL-IO, IL-13 and IFN-y.
P.3.05.21
Regulation of IL-12 production: A regulatory network invoiving CCMOL,IF-, GM-CSF and IL-10
Ahmad Kasran l, MosiK. Bennett ‘, Mark de Boer2, Marfeen Bakkus 3, Krfs Thielemans 3, Jan L. Ceuppens ’ ’ Laboratory of Experimented Immunolog) Catholic University of Leuven, Belgium, 2PanGenetks NY Amsterdam, me Netherlands, 3Laboratory of Physio/ogy-lmmunolcg~ Free University of Brussels, Belgium Introduction: The accessory cell function of monocy&/macrophages is regulated by CD40, a member of the tumor necrosis factor (TNF) receptor family. The ligand for CD40, CD4OL is expressed on ahated T cells. We have studied the regulation of IL-12 production by monocytes through CD4OLand T-cell derived cytokines. MaterlaIr and Methods: Human monocytes in peripheral blood mononuclear cells were isolated in endotoxin-free media. They were cultured with seeded mouse fibroblasts 3T6 cells transfected with human CD4OL (3T6/CD4OL). We studied the effect of CD4OL alone or in the presence of IFN-y, GM-CSF, IL-IO, a blocking rnAb to the IL-10 receptor and a blocking rnAb to CD40 on the cytokine production by monocytes. Rwultrr: (1) CD40 on monocytes was spontaneously upregulated during culture for 24 hours and IFN-y had a stmng enhancing effect. Expression was however completely suppressed in the presence of exogenous IL-IO. (2) When monocytes were cultured in the presence of cD4oL-expressing cells, the presence of IFN-y enhanced TNF and 11-12, bul downregulated the pm duction of IL-10 by rnonocytes. GM-CSF had lii effect on IL-12 production, but enhanced IL-i0 prod&ion. Endogenously produced IL-IO mo&ates the production of IL-12 and TNF, as demonstrated by bloddng of the IL-10 receptor, which resulted in strongly enhanced production of IL-12p40, IL-12~70 and TNF. (3) The role of the CD40-CD4OL interaction in upregulating cytokine production by monocytes was confirmed by the addition of a blocking antibody to CD40 (5012). Concluelon: We have demonstrated that regulation of IL-12 production by monocytes involves CD4O-CD4OLinteraction but also GMCSF and IFN-y. IL-10 oroduction is also strongly enhanced after M stfmulatlon and downr&ulates IL-12 production.-The balance between b&h cytokines (IL-10 and 11-12)might therefore be critical for regulation of Th cell differentiation.
P.3.05.22
Antigen presentation threshold in humans
A.A. Kavokin. Russian State Med&I Uniws~ Russia
Dept. of Immu~
Moscow,
Introduction: Existence of Antigen Presentation Threshdd (APT) was supposed. It means that monocytdmacrophages themselves can manage with an
explanation is that the TCR affinity diiers between transgenic and normal T cells.
P.3.05.13
Extended expreselon of MHC claes II genes on mouse macrophapes as8oclated wlth protectlve/suppresslve effects
APCs and the induction of T- and B-cell responses
inhibition of IL2 secretion at limited CD25B7 interaction and by the differential recognition of altered peptkls ligands in the presence of different APC.
P.3.05.15
Btigttte Mtiller, Martin Baumgart, Verena Moos, Diana Schuhbauer. Deufscfres Rheuma ForschunosZentrum. Monbiioustr: 2. D- 10117 Berlin, Germany htroduction: Protective/suppressive MHC dass II alleles have been Identified in man and mouse where thev exert a disease-omtective and immunosuopmssive effect. Various modes of action have been proposed, among them differential expression in different types of antigen-presentfng cells impacting on the Thl-Th2 balance. To test thls possibility, the expression of H-2 molecules from the four haplotypes H-Zb, H-2@,H-2k and H-2q have been determined on bone manow derfved macrophages (BMDM) and splenic B cells. Yaterfale and mode: The mouse strains used in this study are Bl0.A (5R) (H@), BALB/c (H-2’) BDFI of C57BU6 x BALB/c (H-F), BlO.BR (H@), CBA/J (H@), DBAfl (H-2s) and DBA/l x Bl0.Q (H-29). Bone marrow cells were cultured in complete RPM1 1640 medium containing 30% (voiIvol) L-929 cell-conditioned medium for up to 25 days and single cell suspensions of spleens were cultured for up to five days in complete RPM1 1640 medium containing 4.5 &ml LPS. The surface expression of MHC class II on BMDM and splenic B cells was monitored via two-color flow cytornetnc analysis using mAb recognizing the various class II molecules and CD11b or 8220 as cell-type spec5tc markers, respectively. Reeuh The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are exfxessed on almost all CDllb+ cells for five to eight days. In contrast, I-Ad-, I-Ak-, and I-Aq-expression peaks for a shorter period and fewer CD11b+ cells become positive. No differential expression could be detected on LPS-stimulated E cells. Concluskm:Our data suggest that macrophages exert Thl bias via cytoklne secretion, presumabty of 11-12, in response to ligation of their MHC class II molecules andlor facflitated ligation of their CD40 mdecules. The extent of expression of the class II gene would thus gate the back-signal from T cells and in this wav control the level of IL-12 oroduoed bv macmohaoes. This effect mediated by f&morphic non-exon segments of class II MHC genes may well play a role in determining disease susceptibility in man and mouse.
P.3.05.14
Dlfferentlal T cell activation regulated by antlgen presenting cells
P. Gogoldk ’ , 8. Rethy ‘, A. Horvath ‘, G.K. T6th 2, E. Buds 3, L. Cervenak ‘, G. L&zl&’ , ik RajnavMgyi ‘. ‘Dept.of Immunology L. E&v& Univem& G&l, 2Dqt. of Me&alChem&& A. &en&Gy@yi UniverS& Szeged, 3Dept. of Anatomy, Medical School of Deb-n, Hungary
Intrcduction:To obtain quafitatfve and quantitative data on the contribution of antigen presenting cell (APC) mediated signals in T cell triggering, the activation of three MHC class II rest&ted T cell hybridomas of distinct specfficity was studied in the presence of various APC. MaUale end Methods:The three signal transducing receptors of T cells, i.e. TCR, CD28 and CD4, were stimulated by their natural ligands expressed on the surface of a series of APCs wfth characterized phenotypes. The intermediate and late responses of T cells to diierent doses of peptide antigens presented on various APC was monitored by measuring intracellular calcium influx and It2 omductfon. msc&Nelv. The role of CD4 was studied bv using a CD4- T cell subclone, &ntrfbution br CD26 was studied in the presence orabsence of B7-speciffc antibodies or of soluble CTLA4. Reeuftez Analysis of T cell activation revealed that i) calcium mobilization induced by peptlde loaded APC requires rapid conjugate formation highly dependent on the expression pattern of adhesion molecules on APC; Ii) a direct correlation was observed betwwn the magntude of the calcium lnffux and the concentratfon of secreted I12 induced by dlfferentfal TCR ligation modulated by peptfde dose or by the presence or absence of CD4; iii) measured at identical APC/peptide and T/APC ratios, the intermediate and late responses of T cells to different peptide concentrations was similar; iv) a substantial difference in the wnsftfvity of the two methods, used for monitoring T cell actfvatlon, was observed, v) CD4 mediated slgnalling has a cc-stfmulatory effect predominantly at suboptfmal in vi& conditfons: vi) sustained increase of [Ca2+]ras well as the production of high concentrations of IL2 is highly dependent on the appropriate CD2&B7 interaction determined by the APC; vii) the response of a certain T cell to peptfde analogs is highly dependent on APC which can influence the agonistic or antagonistic behavtour of a pepttde. Conclusion: These results demonstrate that distinct peptide doses and the presence or absence of CD4 results in qualitative changes of the T cell response, while the degree of CD28 mediated signalling has a qualitative affect on the outcome of T cell activation. This was revealed by the decreased duration and aftered time kfw of the calcium response, by the complete or partial
267
lmmortallzed dendrltk llke cell lines derived from mice lacking both typesI and type II Interferon receptors, process and present MHC II restrlcted antigen to prlmed and naive T cells, Induce MLR and support virus repllcatlon
R. Nunez, M. Engels, M. Ackermann. M. Suter. institute for Vim/w, Facula)!University of Ziirich, Switzerland
Veterinary
lnterferons (IFN) are potent regulators of the immune response. Mice (AG 129) lacking both type I and II IFN receptors have been generated (Science 2641 1918). However, the role of the IFNs in the function of dendrftic cells (DC) is not fully understood. Therefore, the present study was aimed at generating immortalized (Cncogene 9: 1473) DC lines from AG 129 mice and to study the phenotype and the bldogical functions of the cells obtained. Several immortalized cell lines were obtained from thymus, spleen, brain, eye, lung and skin. These cell lines have cytoplasmatic extensions like dendrites, attach to plastic, have no inhibition of growth by contact and do not require external growth factors for proliferation. Some of the cell lines have been kept in culture for more than one year. The cell surface markers are characteristic for cells of the monocyte macrophage lineage. The cells express Macl+ and have low levels of MHC class II molecules, some are CD45+, F4.80+, CD& and B220-. The skin, brain and thymus derived cell lines express significant levels of Thyl. Functional studies were focused on one skin (named AGlOl) and one brain (named AGl16) derived cell line. The cell lines are able to: i) take up and process soluble antigens, ii) present soluble antfgens to T cell hybrtdomas iii) induce MLR with as few as 100 stimulating cells, iv) and prime naive T cells to soluble antigen in vitro. Preliminary experiments indicate that the cell lines AGlOl and A0116 allow replication of Bovine Herpes Virus. The data indicate that the cell lines obtained from mice with deleted IFN receptors have functional charactenstics of DC and may prove to be useful for the study of virus replication.
P.3.05.16
W;gMlon
of bacterial antigens from human
I.A. Schrijver, M.J. Melief, MA. Hoijer, M.P. Hazenberg. Department of Immunology Erasmus University Rotterdam, The Netherlands Introduction: Rheumatoid arthritis (RA) is considered to be a chronic T cell dependent response against an unknown antigen. We propose that this immune response is directed against bacterial antigens from the intestinal flora. The hypothesis is based (among others) on the presence of antigen presenting cells with peptidoglycan pdysaccharfde complex (PG-PS) in synovia of A;\ patients. PG-PS positive cells were found in spleen from healthy human also. We developed a method to isolate this antigen from human spleen. Recently the method was modified to minimize the amount of protein in the fraction. Materfals and Methods: Human spleen tissue was homogenized, sonificated and extracted with acetic acid. Gel filtration was performed with Sephadex 625. Fractions with muramic acid were pooled and loaded on a Supetdex 200 column connected to a fast pressure liquid chromatography system (FPLC). The presence of PG-PS was determined by ELISA using a PG-PS specific monoclonal antibody and confirmed by the detection of muramic acid by high pressure liquid chromatography (HPLC) with a Sephacil C8 column. Rwult& After Sephadex G25 gel f&ration a high molecular weight fraction with muramic acid (PG-PS) ww pooled. With the Superdex 200 column two fractions could be separated. One with a relatively high and one wfth a relatively low moleculair weight and almost no protein. HPLC analysis confirmed the presence of muramfc acid in both fractions. Conclusion: The described procedure is a useful method to obtain a fraction of bacterial antigens from human spleen containing a minimal amount of protein. The fractions will be used to determine humoral and cellular responses against these endogenous bacterial antigens in healthy subjects and RA patients.
P.3.05.17
Dlstlnctlon of mechanism of antigen uptake and stlmulatlon of T cells In cultured bovine dendrltlc cells
D. Werfing, P. Sopp, G. Taylor, R.A. Collins, C.J. Howard. tnstitute forAnimal Hwltf~, Compton, UK Dendritic cells (DC) exhibit a uniquely potent ability to stimulate antigen-specific responses in vitro and in vfvo. Immature DC are extremely effident in antigen uptake mediated by various endocytfc pathways including non-selective (i.e. fluid phase macropinocytosis) and substance-specific (i.e. C-type lectin receptor mediated) interfortsation. Antigen-specific recognhion and intemalisation
268
24 June 1997 - Poster presentations
APCs and the induction of T- and B-cell responses
capabilities of DC are of interest since they may have profound implications in the elicitation, amplification, and perhaps the diversity of the T cell msponse. Human DC derived from oerfoheral blood monocvtes in the oresence of IL-4 and GM-CSF have been r&o&d totake up FITC-dextran (FITk-DX) and lucifer yellow (LY) using the mannase-receptor or macmpinocytosis. Whereas the uptake of FITC-DX can be blocked either by mannan or antibodies to the mannose receptor, the uptake of LY is constitutive and cannot be influenced by lectins or antibodles. Little is known about the mechanism responsible for efficient antigen caoture in bovine DC. Bovine monocvtes and monocytededved DC cultured in ihe presence of recombinant bovine IL-4 and GM-&F incorporate FITC-DX but did not show a fluid phase uptake of LY by macropinocytosis. In contrast to human DC, blocking of the mannose receptor (MR) with antibodies raised against the human MR did not diminish the uptake of FITC-DX by bovine DC, although these antibodies stained a subpopulatfon of the cultured DC. Frozen bovine DC were ineffective at endocytosing FITC-DX or LY,but were still able to present antigen. Our observations imply that bovine monocyte-derived DC lack the mechanism of a fluid phase uptake by macropinocytosis and use diirent MR to human DC to take up mannosylated antigens. These pathways of antigen uptake are further altered by cryoconservation.
P.3.05.18
Characterisation of a iigand for MyP1, a novel mammaiian giycoprote4ninitiaiiy identified on cattle monocytes and dendritic ceils
G.P. Brooke, C.J. Howard. institute &Animal Health, Compton, UK Co-stimulatory molecules expressed on the surface of antigen presenting cells (APC) are required for the effective activation of T cells. Although several of these molecules have been defined, experimental data implies the existence of others that may be involved in the stimulation of naive T cells. We have identified a novel glycopmtein that is expressed on a subpopulation of cattle dendtftic cells and on other cells of the myeloid lineage. A cDNA encoding this molecule, initially identified by mAbs, has been cloned and sequenced and the predicted molecule is a type I glycopmtein member of the IgSF. We have termed the antioen MvD-1 (Mveloid and Dendritid. A functional role for the molecule has be& indi&d by-the finding that the ‘mAb block stimulation of resting T cells by ovalbumin pulsed APC. The role for MyD-1 in T cell stimulation may be further elucidated by identifying the ligand(s) of MyD-1. We have therefore constructed a fusion protein by combining the DNA encoding the extra-cellular region of MyD-I with the Cyl chain of human immunoglobulin, in the plasmid vector piLN. Purified fusion protein (MyD-llg) was shown to bind to major subpopulations of peripheral blood T cells, B cells and monccytes, but not y/S T cells. The ligand for MyD-1 is curmntlv beino cloned using a COS-7 cell transient expression svstem.
P.3.05.19
Role of interieukin 12 in dendritic ceil-dependent Interferon y production by murine peripheral ivmoh node ceils
R.J. Dearman, I.R. Jowsey, D.M. Kemeny I, I. Kimber. Zeneca Central ToxicologyLaboratory; Alderley Park, Macclesfield, UK, 1King’s College, London, UK Introductton: We have demonstrated previously that the addition of exogenous accessory cells to naive peripheral lymph node cells (LNC) enhanced substantially concanavalin A (con A) induced production of interferon y (IFN-y)‘.We have now investigated the role of in&tie&in 12 (IL-12) in the expression of this cytokine using a neutralizing monoclonal anti-IL-12 antibody. Matertafs and Methoda: A single cell suspension of LNC was prepared from the auricular lymph nodes of naive BALB/c strain mice. Dendritic call enriched (DC+) and deoleted (DC-) fractions were oreoared bv buoyant density centrifug&on imm lymbh n&s is&ted from mice’& ihe same St&n 16 hr aft& topical exposure to the contact allergen oxazolone (1%). LNC (5 x 10s cells/ml) were cultured for 46 hr with 2 &ml con A. various concentrations of DC+ or DC- fractions and in the presence or absence of 20 @ml rat monoclonal anti-IL-12 neutralizing antibody or control antibody. The IFN-)I content of culture supematants was determined by enzyme-linked immunosorbent assay. Resuftez Addition of DC+ to cultures of con A-stimulated naive LNC enhanced IFN-y expression up to 5-fold. Elevation of IFN-y production was observed when exooenous‘DC+ comortsed 0.5% or oreater of the cutture woulation. Antigen-actiiated LNC populaions depleted 2 DC (DC-) did not in~lu&ce IFN-y secretion. Neither DC+ or DC- fractions cultured alone in the presence of con A elaborated detectable levels of this cytokine. The addiion of neutmlizing anti-IL-12 antibody, but not control rat antibody, inhibited almost completely IFN-y production by con A-activated naive LNC and DC+ enhanced expression of this cytokine. Conduefons: These data reveal that within unstimulated peripheral lymph nodes there are insufficient numbers of appropriate accessory cells to support vigorous IFN-y expression. Exogenous allergen-activated DC, via the secretion of 11-12,provide for optimal production of this cytokine.
[I] Hodgson CM., Dearman R.J., Morrs A.G. and Kimber I. (lga6). Imm. Letters, 4@,4455.
P.3.05.20
Human 1 ceil activation by APC: Synergistic effect of accessory signaling by CiMO, IL-10 and IL-12
A. Kasran, X. Peng, J.L. Ceuppens. Laborarory of Expetimenfal /rnrnuno/~, C&ho/k University of Leuven, Belgium Introductfon: It is generally accepted that the CD4O-CD4OLinteraction between B cells and accessory cells on one hand and T helper cells on the other, plays an important role in the regulation of B cell and APC functions. Evidences are accumulating that the CD4&CD4OL interaction is bidirectional and dfmctly costirnulates T cell activation as well. MaterfaIr 8nd Methods: In this study we have analysed functional effects on T cells involving CD40-CD4OL interaction using an activation system with purified resting T cells devoid of accessory cells. Using direct activation of protein kinase C (PKC) by the phorbol ester, phortml 12-my~istate 13acetate (PMA), we measured the effect of a monoclonal antibody to CD4OL (Mg2) in the presence of IL-18 or IL-12 in inducing T cell pmliferatfon and cytokine productfon. Rewitsz(1) T cell proliferation was strongly enhanced when PMA-treated T cells were cultured with anti-CD4OL in the presence of IL-l/l or 11-12.(2) 11-2, 11-5,IL-lo, IL-13 and IFN-y were readily produced when T cells were activated through ccstimulation ofCD4OLwitJ1IL-l/3 or 11-12.Wherethepresence of IL-la favoured the oroductfon of IL-5 and 11-13.the ~msence of IL-12 in the culture favoured the production of 11-2,IL-10 and.lFN-;. (3) Them is synergy between IL-18 and IL-12 in upregulating IL-2R (CD25) expression when PMA-treated T cells were cultured in the presence of CD4OLtriggering. Conclusion:We have added yet another piece of evidence that resting T cells can be costimulated through CD4OL to induce cylokine pmduction and responses to 11-2. Synergy between IL-lg or IL-12 and CD4OL triggering was apparent at the level of proliition and the production of 11-2,IL-S, IL-IO, IL-13 and IFN-y.
P.3.05.21
Regulation of IL-12 production: A regulatory network invoiving CCMOL,IF-, GM-CSF and IL-10
Ahmad Kasran l, MosiK. Bennett ‘, Mark de Boer2, Marfeen Bakkus 3, Krfs Thielemans 3, Jan L. Ceuppens ’ ’ Laboratory of Experimented Immunolog) Catholic University of Leuven, Belgium, 2PanGenetks NY Amsterdam, me Netherlands, 3Laboratory of Physio/ogy-lmmunolcg~ Free University of Brussels, Belgium Introduction: The accessory cell function of monocy&/macrophages is regulated by CD40, a member of the tumor necrosis factor (TNF) receptor family. The ligand for CD40, CD4OL is expressed on ahated T cells. We have studied the regulation of IL-12 production by monocytes through CD4OLand T-cell derived cytokines. MaterlaIr and Methods: Human monocytes in peripheral blood mononuclear cells were isolated in endotoxin-free media. They were cultured with seeded mouse fibroblasts 3T6 cells transfected with human CD4OL (3T6/CD4OL). We studied the effect of CD4OL alone or in the presence of IFN-y, GM-CSF, IL-IO, a blocking rnAb to the IL-10 receptor and a blocking rnAb to CD40 on the cytokine production by monocytes. Rwultrr: (1) CD40 on monocytes was spontaneously upregulated during culture for 24 hours and IFN-y had a stmng enhancing effect. Expression was however completely suppressed in the presence of exogenous IL-IO. (2) When monocytes were cultured in the presence of cD4oL-expressing cells, the presence of IFN-y enhanced TNF and 11-12, bul downregulated the pm duction of IL-10 by rnonocytes. GM-CSF had lii effect on IL-12 production, but enhanced IL-i0 prod&ion. Endogenously produced IL-IO mo&ates the production of IL-12 and TNF, as demonstrated by bloddng of the IL-10 receptor, which resulted in strongly enhanced production of IL-12p40, IL-12~70 and TNF. (3) The role of the CD40-CD4OL interaction in upregulating cytokine production by monocytes was confirmed by the addition of a blocking antibody to CD40 (5012). Concluelon: We have demonstrated that regulation of IL-12 production by monocytes involves CD4O-CD4OLinteraction but also GMCSF and IFN-y. IL-10 oroduction is also strongly enhanced after M stfmulatlon and downr&ulates IL-12 production.-The balance between b&h cytokines (IL-10 and 11-12)might therefore be critical for regulation of Th cell differentiation.
P.3.05.22
Antigen presentation threshold in humans
A.A. Kavokin. Russian State Med&I Uniws~ Russia
Dept. of Immu~
Moscow,
Introduction: Existence of Antigen Presentation Threshdd (APT) was supposed. It means that monocytdmacrophages themselves can manage with an