ActA, a membrane protein from Listeria monocytogenes, does not induce a protective immune response

264

24 June 1991- Poster presentations

APCs and the induction of T- and B-cell responses

09:00-18:30/12:00-14:00

P.3.05

Forum lounges

APCs and the induction of T- and B-cell responses

of direct physical interaction between CD43 on T-cells and MHC-I coated beads it was necessary, however, to ligate CD2 on T-cells with a stimulatory pair of CD2 mAb (VIT13 plus TS2118). Condualon: Our results indicate, that CD43/MHC-I and CD2/CD58 interactions act in concert to induce and mediate T-cell conjugate formation with certain cell types.

1P.3.05.03 P.3.05.01

Intvoduatlon: In vitro priming of naive donor T-cells has become feasible with the availability of culture techniques for professional antigen-presenting cells, i.e. dendrftic cells (DC). Thus, naive T-cells can be specifically primed with contact allergens and tested for specificity and potential cross-reactivfties with other, more or less related, compounds. Four different methacrvlates (MA) have recently been identified as frequent contact allergens in different occupations, notably in dentistry: methyl MA (MMA), P-hydroxyethyl MA (HEMA), 2-hydroxypropyl MA (HPMA) and ethyleneglycol diMA (EGDMA). Patch tests in MA hypersensitive patients usually show multiple positive reactions. The primary sensitizer(s) are generally unknown and therefore true cross-reactivity patterns are unclear. Here, therefore, we developed an in vitro T-cell priming protocol for methacrylates, including nickel as an unrelated specificity control sensitizer. Materials & Mathoda: DC were generated by culturing plastic adherent PBMC from non-sensitized volunteers in the presence of GM-CSF (550 U/ml) and IL-4 (1000 U/ml) for 7 days. DC culture efficiencies were controlled by double-staining CDla and CD14 expression on days 0, 4 and 7. Cultured DC were ore-incubated for 4-6 h with either Ni (2 uM) or MMA (1 LLM) before adding autologous PBMC. Cultures were supplemented after 3 days with IL-7 (0.1 nglml), and after 10 days with rll-2 (25 U/ml) and nil-2 (IO U/ml). Wells were split at day 17, and challenged with autologo&, irradiated PBMC, either or not pulsed with various MA’s (30 mM) or Ni (50 mM). Six days later proliferation was assessed by 3H-thymidin incorporation, and stimulation indices (SI = ratio of jsH] thymidine uptake in the presence of contact allergen and in the medium control) were used to assess reactivities. Reaulta: DC quality was confirmed by CDla upregulation, accompanied by the decrease of CD14 expression dudng culturing (CDla+CD14-/ CDla+CD14a+/CDla-CD14+: day 0: l/2/85, day 4: 7154f40, day 7: 82/17/l). Initial specificity experiments using Ni and MMA primed T-cells revealed specific proliferation with the respectiie contact allergens: SI > 2 for Ni 20/26 cultures, median SI: 2.4, (MMAcontrol: 0126,median: 0.9). resp. for MMA: 21127,median: 2.4 (nickel control: O/27, median: 1.I). When evaluating MMA prfmed T-cells for ootential activation bv the other methacrvlates, marked cross-reactivles for all tested MA could be shown: for HEMA: 16/27 (mean relative Sl’s as compared to the MMA-specific SI 80%), HPMA: 16/27 (90%) and EGDMA: 13/27 (65%). Condual&a: Followingthis newly develbped’in vitro T-cell priming protocol for contact allergens highly specific T-cells can be generated. These preliminary results indicate that four clinically relevant methacryfates can generate the same or highly similar epitopes, recognized by MMA-induced T-cells.

P 3 05 02 rr7-J

Th2 responses is associated with enhanced co-stimulatory molecule expression and regulatory cytokine production

Contact allergen cross-reactivity studies using in vitro primed naive T-cells

T. Rustemeyer ‘, B.M.E. von Blomberg ‘, P.J. Frosch *, R.J. Scheper ‘. ’ Departmentof Patho/og)! Free University Hospitel of Amsterdam, Amsterdam, The Netherlands, 2Department of Dermetolog~ Municipal Hospital Dortmund, Dortmund, Germany

CD43 - MHC class I molecule interaction involved in spontaneous Tcell conjugate formation

J. St6ckl, 0. Majdic, P. Kohl, W.F. Pickl, J.E. Menzel, W. Knapp. institute of Immunolcg~ University of Vienna, Vienna, Austria Introduction: Resting T-cells spontaneously adhere in a selective manner to potent accessory ceils, such as dendritic cells (DC) and lymphoblastoid B-blasts (LCL). Matarlal and Methods: T-cell conjugate formation with monocyte derived DC (md-DC) and B-LCL was tested in a rosette formation and analysed microscopically. Reaulte: Here we demonstrate, that leukosialin (CD43) and MHC class I molecules (MHC-I) might play a critical role in this process. T-cell conjugate formation with md-DC and LCL could be strongly inhibited by either preincubating T-cells with Fab fragments of CD43 mAb 6F5 or by preincubating md-DC or LCL with MHC-I mAb W6/32. Intact CD43 mAb 6F5, in contrast to monovalent Fab fragments, enhanced T-cell adhesiveness by transactivating CD2 binding to CD58 molecules. Interestingly, induction of this pro-adhesive signal via CD43 with intact 6F5 mAb was found to revert mAb W6/32 mediated inhibition of T-cell conjugate formation. These observations indicated, that CD43 cross-linkage mimics and monovalent mAb 6F5 inhibits interaction of T-cell CD43 with a stimulatory ligand on opposing cells, presumably MHC-I. For the demonstration

1 The adjuvant action of pertussis toxin for Thl and

Mark Ryan, Leone McCarthy, Rino Rappuoli ‘, Bernard P. Mahon, Kingston H.G. Mills. Infection and Immunity Group, Maynooth Universi& Co. Kifdare, Ireland, 1lstituto Ricerche lmmunobiologiche Siena, 531W Siena, My Introduatlon: Pertussis toxin (PT) is a major virulence factor of Bordetella pertussis which exerts a range of effects on the immune system, including the enhancement of IgE production, DTH reactions and the induction of expedmental autoimmune disease in mice. In this study we examined the mechanisms by which PT can potentiate immune responses mediated by reciprocally regulated Thl and Th2 subpopulations of T cells. Matarlal and Methods: Mice were immunized with KLH or filamentous haemagglutinin (FHA) from 8. p&u&s either alone or with active PT or a range of genetic mutants and detoxified forms of the toxin. Antigen-specific T cell proliferative responses and Thl/ThP cytokine production was assessed using spleen cells removed two weeks after immunization. The levels of IgG sub&sses were determined in the serum by ELISA. The effect of different PT preparations on the expression of the co-stimulatory molecules 87-1, 87-2 and CD28 on purified T cells, B cells and macrophages was assessed by dual-colour flow cytometry. Raaulb: We demonstrated an adjuvant affect of PT for Thl and Th2 responses. Co-injection of FHA or KLH with PT enhanced antigen-specific CD4+ T cell proliferation and the secretion of IFN-y, 11-2, IL-4 and 11-5. Furthemtore a non toxic S-l mutant, devoid of enzymatic activity but still capable of receptor binding, retained its adjuvanticfty, augmenting both Thl and Th2 subpopulations of T cells. In contrast a chemically detoxified PT (PTd) and a genetic mutant with substitutions/deletions in the S-1 and B-oligomer components that abrogate enzymatic and binding activity, displayed no adjuvant properties. The adjuvant properties of PT were found to be associated with increased expression of the co-stimulatory molecules B7-1, B7-2 on B cells and macrophages and CD26 on T cells and enhanced secretion of IFN-y and IL-2 by T cells and IL-I by macrophages. Conclusions: Our findings suggest that the mechanism of adjuvant action of PT involve upregulation of B7-l/67-2 on APC and their counter-receptor CD28 on T cells and the stimulation of T cell and APC/accessory cell cytokines that enhance antigen presentation and T cell activation. Although the immunopotentiating properties of PT for Thl and Th2 cells appear to be largely associated with the receptor binding domain, our findings also suggest that the S-1 and B-oligomer components of PT may have selectiie effects on Thl and Th2 responses through effects on distinct signalling pathways.

P.3.05.04

ActA, a membrane protein from Llsreria

monocvtoaenes, does not Induce a protective immuni reGpon;e D. Bruder l, A. Daji ‘, T. Chakraborty *, J. Wehland ‘, S. Weiss’. ‘Divisionof Cell Biology and Immunology GBF; 38124 Braunschweig, Germany. *Institute for Medical Microbio~logy.University Giessen, 35392 Giessen, Germany Introduction: The poreforming activii of listeriolysin, the hemolysin of Listeria monocyiogenes, allows the introduction of soluble proteins into the cytosol of antigen presenting cells. Thus it is possible to generate CD8+ cytotoxic T cells against purifted proteins and to propagate them in vitro. To avoid concomitant induction of listerfolysin-specific CD8+ T cells, we have established variants of listerlolysin which no longer induce a MHC dass I restricted T cell response against this toxin in BALB/c mice, but retain their hemolytfc activity. Using this system we were able to induce and characterize cytotoxlc T cells against ActA, a protein found on the surface of Listeria monccytogenes. Ma~Iala and Methods:Listenolysin mutants were generated by sitedirected mutagenesis using the overlap extension PCR technique. The mutated hemolysins were overexpressed in the nonpathogenic strain Listeda hnccua and purified by a two step ionexchange chromatography according to a standard protocol. Cytotoxic T cells were generated by i.p. immunization of mice with purified proteins in iFA and restimulation in vitro. Cytotoxic activity was measured by using the JAM assay. Results: Two listenoiysin mutants with amino acid substitutions within the immunodominant MHC class I epitope 91-99 were established. These mutants did not induce a cytotoxic T cell response in BALB/c mice. No activation of cytotoxic T cells against other potential epitopes was observed. The ability

24 June 1997 - Poster presentations

of the toxin to introduce soluble proteins into the MHC class I presentation pathway wasunaltered. Thus we were able to generate CD8+ T ceils against the membrane protein ActA and propagate them k, vi& Wti these ActA specific cytotoxic T cells we could show that in L&&r& infected calls this antigen is not available for MHC class I presentation as long as the pathogen is viable. Furthermore, no protection was observed when ActA-specific CD8+ T cells were adoptively transfered into syngeneic mice. Conciusionr:We have established a system that allows the generation and propagation of cytotoxic T cells against any soluble protein. in addition, our results raise the question whether bacterial membrane proteins represent protective T cell antigens. These finding5 will have a strong influence on the design of subunit vaccines against pathogenic microorganisms.

I..P 3

05 .05

1 Functional characterlxatbn

of T-cells involved in skin silograft rejactlon in case of inherlted &lntegrln deflclency of cattle

Kerstln E. MQiler’, Victor P.M.G. Rutten*, A. Hock*, Cad G. Figdor3. ‘Department of Large Animal Medicine and Nut&ion, InsfiMe of Infectious Diseases and lmmum Utmcht fJniverai& Nijmegen, The Netherlands, * Dqartment of Immunol~, institute of lnfactkws Diseases and Immunw Uttwht lJniwsi& N@sgen, The Netherlends, 3Depatiment Tumor Imnwollogy. Univetafty Hospitel St. Radboud, Nijmagen, The Netherlands

introduction:Rejection of transplanted tissues occurs when naive T-cells activated by encounter wlth donor antigen proliierate and initiate a cascade of cellular interactkms involving effector T-cells which finally damage the graft. The ~&&grin LFA-1 (CDlla/CDl8) has been shown to parttdpate in adhesion-dependent cellular interaction5 of lymphocyte5 such as adhesion to endothelfum and interacticn5 with antigen-presenting cells for antigen-recognition and effector functions. The aim of the present study was to investigate the involvement of LFA-1 in T-lymphocyte interactions with target cells obtained from peripheral blood and graft biopsies after skin transplantations in cattle wlth inherited ~.&tegrtn deficiency (Bovine Leukocyte Adhesion Deficiency/ BLAD). Materlaioand Methods: Skin transplantations were performed in cattle with BfAD and heatthy controts. PBMC and biopsy specimens from skin allografts were obtained at regularintervals. Allograft-infiltrating lymphocytes (GIL) were cultured from skin biopsy specimens. Phenotypic characteristics and donorspeciffc cytotoxicity of PBMC and GIL were investigated by flow-cytometry, immunoprecipitation and in vitro cytotoxicity assays, respectively. Rwut& Graft-infktrating cells were identifted as CD2+4+and CD2+8+T-lyrnphocytes and - in cases of cattte wtth BIAD - showed no detectable amount5 of ,9p-integrin expression on short-term T-ceil lines. Long-term T-cell lines obtained from BlAD recipients showed upregulation of CD18, but not CDlla. PBMC from cattle with BLAD - in contrast to controls - consistently failed to exert cytototic activity, when stimulated in bulk with donor-derived targets. On the other hand, &&tegrtn deffdent GIL from cattle with BLAD showed selective cytototi activky against donor-speciftc antigen5 in the same amount as healthy animals. Donor-specific lysis by allograft derived T-cells being 92% at an ET ratio of 25:15ub5tanQaliy decreased after addition of mAb against CD8 (50%) or against MHC-class I-molecules (73%) to culture. inhibition assays performed on long-term cultured GIL gave similar results. However in the latter experiments, a substantial inhibition of cytototi responses was observed after addition of mAbe against CD18, but not CDlla. Condueions: During rejection of skin aiiografts in cattle with &-integdn deficiency, the frequency of donor-speckic CTL in peripheral blood seems to be low, whereas T-lymphocytes are present in the ailograft in a high frequency which are able to recognize and specifically kill donor cells. P.3.05.06

Commltment of cytoklne expresslon In lndlvldual THl-llke lymphocytes

M. Assenmacher ‘, M. Ldhning *, A. Scheffold *, A. Richter*, S. Miltenyi ‘, J. Schmitz ‘, A. Radbruch *. ’ Miltenyi Biofec GmbH, Be@ch Gladbach, Gemreny, * De&ches Rheuna&chun@entNm, Se& Genany institute for Genetics, University of Cologne, Germany introduobon: Conversion of a THl or TH2 phenotype has been analysed for potarized ceil population5 only. Then it was never dear whether successful conversion was due to real conversion of individual THl or TH2 like cells or due to selective outgrowth of uncommitted precursors. Therefore we have ar&sed indivtduai murine THI-like fymphocytes for their commtknent to expression of interferon gamma (IFN-y) versus interteukfn 4 (IL4) and IL-IO. Mate&t end Methods: Naive (CD82L+) murfne TH (CD4+) cefls were isolated by multiparameter magnetic cell sorting (MACS MuMSott) and were stimulated in vttro with antigen and IL-12 for one week. Using surface IFN-y as a specific marker [Assenmacher et at. (lQQ8) Eur. J. Immunoi. 28: 2831,live IFN-y expressing ceils were Notated from such THl cultures by MACS. lsoiated THI cells were then metfmufated in the presence of exogeneous 114, i.e. under

APO and the induction of T- and B-cell responses

265

converting condii and analysed for expression of 114, IL-10 and IFN-y on the single ceil level. Rwub: Some but not all of the sorted IFN-y expressing cells continue to express IFN-y in the presence of IL4. Many cells respond to IL4 by downregulation of expression of IFN-y. A substantial fraction of the sorted TH cells, expressing IFN-y but not IL4 at the time of sorting, could be induced to express IL4 and/or IL-10 by addftion of exogsneous 114. Concluefon: After 7 days of induction by IL-12 some THl cells are already committed to a pro-inflammatory phenotype. However many cells are funckonally suppressed or converted upon restimulation in the presence of 114. Thus IL4 Is still effective on earty THl cells in suppression of IFN-y expression as well as induction of IL4 and/or IL-10 expression -much more effective than on late THl ceils, but already less than on naive TH cells.

P.3.05.07

A search for peptlde immunodominance In the direct ant1 HLA42 alloresponse

R.J. Baker, H.J. Stauss. Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, Lot&n, UK Introduction: There are two main theories to explain the high T cell precursor frequency for alloantlgens. The “high ligand density” model dictate5 that T cells are stimulated by the extraordinary density of the allo-MHC target such that many T cell clones, even with low TCR affinities, can be activated by cross linking, increasing the overall avidity of the interaction. Alternatively the “multiple detemtinant” model suggests that many different T cell dories can be stimulated since there are many “foreign” targets constkuted by allo_MHC combined with their respective peptides. The peptkies meet frequently associated with human HLA-A2 molecule on the B cell lymphobtastoid cell line Cl R-A.2 have been previously identified by Hunt using tandem mass spectrometry. We have investigated whether these peptides which, in combination with A2, represent some of the most frequently encountered ligands, are frequently mcognised by anti-A2 allomactive CTCs. MWai and Method%PBMC were obtained from HLA-A2 negative individuals and these were stimulated with Cl R-A2 ceils. Stimulation was repeated on a weekly basis until bulk specificity for A2 was obtained. These lines were then cloned by limitlng dilution. Fife of the most prevalent peptkMs identified by mass spectrometry were synthesised and then tested for recognition by A2atloreactive bulk lines and clones. Reeuftr: For those A2-allomacttve bulk lines that showed little killing of the peptide loading deficient cell line T2, and were therefore deemed peptidedependent, them was no evidence for increased killing of T2 when loaded with any of the five peptides. Similarly with the peptide dependent clones, them was no evidence for sensitisation by the five peptides. Conciusion:In this system them was no evidence that five ofthemost pmvalent peptides, identified by mass spechometry, dominate the alloresponse. In other word5 peptii abundance dces not equate wkh peptide immunodominance. However further work is required to charactertse these responses in mom detail and to indeed prove that these peptides are present in high concentrations in our cells.

[ P.3.05.08

( Antigen preaentlng cells and cytoldne expresslon In human papllloma virus associated cervical lesions

S.L. Giannini, W. Al-Saleh, H. Pin-m,J. Boniver, P. Delvenne. Uniwrsity of Liege, Depat?rnent of Pathology B-QOWLiege, Belgium The chronic infection of mucosal epfthelium kerattnocytes of the cervfx by the human papilloma virus (HPV) is associated wtth the development of cervical cancer. Within the epithelium, Langerhans cells am important for the immunosurveillance against mucosal infectfons and cancers, due to their capacky to present antigen and induce a cellular mediated immune response. In contrast, the presentation of antigens by keratinocytes, can result in the tolertzation or the generation of an antibody mediated response. The aim of our study is to determine the role of these potential antigen pmsenttng cells (APC) and how cytokines may influence their function during the development cervical cancer. For our investigation we used a combination of immunohisto&emisby, rtPCR and a mixed lymphocyte epfthelial response assay (MELR). We have demonstrated them is a diminished density of LangMane cells in (pre) neoplastic cervical lesions, compared to normal exocervix biopsies. Moreover, coincident with the pmgmaeion of the lesion the percentage of biopsy specimens whemkeratinocytes express HLA-DR incmaeed from 0% (nomtaf biopsies) to 88% (high grade lesions). We atso found that the epithefial ceils derived from lesionai biopsies were less efficient in indudng an aliogeneic response using

MELFt. Since the depletton and the function of Langethans cells could be influenced by local cytokines we evaluated the expression of several cytokines within the cervical lesions. Usina rt-PCR we have found that the exomssion of IL12 was diminished in high grade lesions compared to the low g&de and normal biopsies. In addiion, the relative expression level of IL10 end TGFB