Workshop B: Molecular basis of immune recognition

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  • Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    B. 1 Determination of the affinity for the interaction between CD22 andCD45: Contribution of CD45 protein structure


    Interactions between carbohydrates and peptides are essential to many biological processes. N-and O-glycans and glycolipids often carry sialic acids as their terminal sugars. However only alimited number of mammalian proteins have been identified to use sialic acids in cellular recog-nition events. The best characterized are members of the selectin- and the siglec family. Siglecs(previously known as the sialoadhesin family) are I-type lectins that mediate cell-cell interactionsthrough the recognition ofspecific sialylated glycoconjugates as their counter receptors. Whetherthe protein structure of the counter receptor contributes to the affinity of this interaction isunknown.

    CD22 (siglec-2) is a B cell restricted cell surface molecule and is involved in mediating the Bcell receptor signal and in the homing of B cells back into the bone marrow. It has been shownto bind to the T- and B cell surface molecule CD45 through recognition of N-linked a2,6-linked sialic acid. We studied the affinity of this interaction using surface plasmon resonancespectroscopy. Furthermore, we investigated the importance of the CD45 protein structure forthis affinity. The results of this study provide insight in the characteristics of siglec interactionsand will be useful in the search for ligands.

    IClinical Research Unit for Rheumatology, Albert-Ludwigs University, Freiburg, Germany and2Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan

    B. 2 Cell surface expression of a HSC73-like molecule by CD14+ monocytes

    Heat shock proteins (HSP) are usually localized intracellularly. There was no interest for theirpossible expression on the cell surface until the late 1980s, when it became evident that HSPsoccasionally are also expressed on the plasma membrane. Though the mechanism for transport-ing HSPs to the plasma membrane is still to be revealed, it is now generally accepted that vari-ous members of the HSP70 family are expressed on the cell surface of different tumors wherethey act as immunodominant antigens, but not on normal cells. In addition, it is known thatHSPs act as molecular chaperones and playa role in the uptake and processing of exogenous pro-teins or peptides.

    We reported that the monoclonal antibody NT22 recognizes an extracellularly localized epi-tope of a 70 kDa heat shock cognate (HSC73)-like molecule on EBV-transformed B cell lines,

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    like Daudi and Raji cells [Hirai et. al. (1998) Cell Struc. Func. 23:153-158]. By using severalrecombinant forms of the HSC73 protein, we showed that NT22 detects an epitope whichrequires amino acids 350-372 in the N-terminal stretch of this molecule. This region is highlyconserved among the HSP70 family proteins of prokaryotic and eukaryotic cells.

    We now studied peripheral blood mononuclear cells of healthy donors and patients withrheumatoid arthritis or Morbus Wegener using two-colour flow cytometry (FACS). Surprisingly,the majority of CD14+ monocytes, but only a small percentage of CD19+ B cells, expressed theepitope. Very few CD3+, CD4+ or CD8+ T cells were also positive. At present, we analyse if themolecule detected on monocytes is identical to the one expressed by B cell lines and if it isinvolved in antigen presentation.

    Supported by grants of the DFG (Me 604) and the Hans-Hench Stiftung.

    Departments of 1Medicinal Chemistry, Utrecht Institute of Pharmaceutical Sciences and2Immunology, Institute of Infectious Diseases and Immunology, Utrecht University, Utrecht,The Netherlands

    B. 3 MHC class II binding of peptoid-peptide hybrids


    Altered peptide ligands (APLs) are peptides with a changed interaction with the T cell receptor(TCR), leading to an altered activation pattern of the T cell. They have been successfully appliedin the inhibition of experimental autoimmune diseases. In this study, the major histocompatibil-ity complex (MHC) class II binding characteristics of peptidomimetics (possible APLs) areinvestigated. Recently, protein structures involved in the MHC class II-peptide-TCR interactionhave been elucidated by X-ray crystallography. Using these structures, a computer model of theMHC class II molecule RT1.BL complexed to the peptide MBP72-85 was constructed. Thethree amino acids in the peptide pointing towards the TCR were, one by one, altered into cor-responding peptoid residues. The side chain in a peptoid residue is present on the N atom asopposed to the Ca atom in an amino acid residue. This alteration could possibly lead to an APLfunction, while increasing the metabolic stability by creating a peptoid-peptide hybrid.

    RT1.BL binding of the peptoid-peptide hybrids was determined using a direct MHC-peptidebinding assay. Surprisingly, two of the three hybrids showed a large decrease in MHC bindingaffinity, and the third displayed a slight decrease. The large loss in affinity was unexpected, sincethe three peptoid positions were chosen in order to change the interaction with the TCR, leav-ing the interaction with the MHC undisturbed. To explain the observed effects, derivatives withAla, Gly and NAla (peptoid residue corresponding to Ala) substitutions were synthesized andtested. The individual contributions to MHC binding affinity of the side chain (position), theabsence of a hydrogen bond donor, and the flexibility will be discussed.

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    Department of Immunobiology, Biomedical Primate Research Center, Rijswijk, TheNetherlands

    B. 4 Differential evolutionary MHC class II strategies in humans and Rhesusmacaques


    The rhesus macaque is an important model in clinical and biomedical research in transplanta-tion and in investigations of chronic and infectious diseases. Therefore a well characterised rhe-sus macaque major histocompatibility complex (MhcMamu) is needed. In contrast to humans,the rhesus macaque displays unprecedented polymorphism of the -DRB region configurations,however, with low allelic variation. Sequencing of exon 2 of about 210 pedigreed rhesusmacaques allowed the detection of 16 Mamu-OQA1, 24 -OQB1, and 14 -OPB1 alleles.Segregation analyses revealed 27 OQA1/DQB1 pairs, coupled with one or more -DRB haplo-types. Particular -DRB haplotypes can be linked as well to one or more DQA1/DQB1 pair,whereas nearly no linkage is observed between -DQ/DR and -DPB1. 37 different Mamu-DQB1/DQA1/DRB haplotypes could be discriminated, which is more than described for HLA-DQB1/DQA1/DRB1 in an equivalent human study. In comparison to humans the amount ofallelic variation for Mamu-DQA1 and -DQB1 is high, whereas the degree of freedom of recom-bination is even lower. In contrast to the Mamu-DQA1/DQB1loci, the Mamu-DPB1locus dis-plays only minor allelic variation whereas the HLA-DPB1 locus is highly polymorphic.Furthermore, neither equivalents of the HLA-DQA2 and -DQB2 genes nor variation of the-DPA1 gene can be detected in Old World monkeys. Together with data of the divergent organ-isation of the HLA- and Mamu-DRB regions these observations underline the conclusion thatboth species used different evolutionary strategies to create diversity at the MHC class II loci inorder to cope with pathogens.

    Abteilung Klinische Immunologie, Medizinische Hochschule, Hannover, Germany

    B. 5 The functional role of the glycosylation of FcyR1l1


    FcyRIII (C016) is a low affinity receptor for IgG. There are two different isoforms of this pro-tein: CO16A (transmembraneous, expressed on natural killer cells and on macrophages) andCD16B (glycosylphosphatidylinositol-linked, expressed on neutrophilic granulocytes). Bothforms of the protein have a variable glycosylation pattern. The NA1 allele of CD 16B has fourasparagine(N)-linked glycosylation sites. One of them (N163) is localized in the ligand bindingsite of domain II, which has been confirmed by analysis of the crystal structure. This site isshared by the NA2 allele and CD 16A.

    To examine the functional role of the glycosylation we mutated the four glycosylation sites ofthe NA1 allele (N39, N75, N163, N170) into glutamine(Q). HEK293 cells were stably trans-fected with the single mutants and wildtype CD16 as control. We determined the binding ofhuman IgG to the transfected cells using immunofluorescence studies with an anti-hIgG anti-

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    body. Monomeric IgG bound to N 163Q transfectants with higher affinity than to the other celllines showing that the glycosylation in N163 influences the affinity of CD16 to its ligand.Consistent with this, preincubation of PBL with Tunicamycin (inhibition of N-glycosylation)resulted in an increased binding of monomeric IgG. To test if the modulation of the glycosyla-tion of CD 16 is a physiologically relevant mechanism of regulating receptor affinity we proceedto determine the glycosylation pattern of CD 16 on NK cells obtained from patients with chron-ic inflammatory diseases and from healthy controls.

    Supported by SFB244/A09

    Laboratory of Molecular Biology, Children's Hospital, Charite, Humboldt-University, Berlin,Germany

    B. 6 A novel Bcell superantigen-like interaction of IVIG is suggested by thefavored selection of IgG and IgM VH derived from germline segments3-23 and 3-30/3-30.5


    Intravenous immunoglobulin preparations (IVIG) represent the IgG repertoire of several thou-sand healthy donors. The beneficial use ofIVIG in certain immunodeficiencies and autoimmunediseases has been proven, but it is not clear how IVIG functions at the molecular level. Wesequenced 104 IgG and IgM clones specifically reacting with IVIG molecules derived by phagedisplay and IVIG-panning from patients with autoimmune thrombocytopenia (AITP), SLE,Kawasaki disease (KD), Stevens-Johnson-Syndrome, and a healthy individual. Sequencingrevealed that the most frequently used VH germline gene segments of all IVIG-selected immu-noglobulins (70 of 104=67%) were 3-23 and 3-30/3-30.5. In contrast, only 3 out of 33 (90/0)unselected controls originated from those genes. Comparing two libraries from a patient withKD prepared before and after IVIG treatment, the reactivity with IVIG was higher for clonesgenerated after the therapy. The combined results suggest a favoured interaction of a subset ofIVIG - respectively normal immunoglobulin repertoires - with B cell receptors derived from3-23 and 3-30/3-30.5 gene segments. Importantly, those are the most frequently rearrangedVH germ-line genes among human B cells. As light chains, antigen-specificity and the high vari-ation in CDR3 showed only little influence on the selection by IVIG, this type of interaction ischaracteristic for a B cell superantigen and may significantly shape the B cell repertoire. Furtherexperiments revealed that despite possible homologies, at least some of the contact residues onFab fragments for IVIG must be different from those for the model superantigens Staphylococcalprotein A and HIV gp120. The IVIG-selected Fab fragments may now be used to clone humanantibodies representative of this IVIG-subset to study their regulatory influence on the B cellrepertoire during normal development and disease.

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    Max-Planck-Institute of Immunobiology, Freiburg, Germany

    B. 7 Tcell receptor a-chain determines antigen and restriction specificity ofa nickel-reactive human Tcell clone


    Nickel as well as several other heavy metals may induce severe allergic diseases by activatingmetal-specific a,~ T cells. However, the molecular interactions underlying this activation, andthe nature of metal-induced allergenic determinants are poorly understood. We have establisheda system to functionally express human T cell receptors (TCR) in mouse hybridoma, allowingfor site directed mutagenesis as well as for combinations of a- and ~-chains isolated from differ-ent TCR. In this way, we recently identified a CD4+, Ni-reactive human T cell clone (SE9)whose VA22+ TCR a-chain combined with related or unrelated ~-chains (even from murineTCR), retaining its specificity for nickel as well as a highly promiscuous restriction for variousHLA-DR alleles. Mutations within the original ~-chain or its replacement by ~-chains fromother TCR occasionally altered the relative efficacy of nickel recognition in the context of differ-ent HLA-DR alleles, but never totally extinguished nickel-reactivity. We conclude from thesedata that the major contact sites of the SE9 TCR for nickel as well as for HLA-DR determinantsmust be situated in its a-chain with the ~-chain serving mainly to conserve the structural integ-rity of the TCR. Preliminary experiments with other TCR expressing the same VA22 elementappear to exclude a superantigen like reactivity of Ni-ions with VA22. We are presently perform-ing mutational analyses to identify amino acids essential for nickel- as well as for HLA-DR-con-tacts. Our data favour a nickel coordination complex bridging amino acids of the TCR a-chainwith non-polymorphic residues of the HLA-DR molecule.

    Supported by the Klinische Forschergruppe Pathomechanismen der allergischen Entziindung,BMBF FKZ 01GC9701/7

    Max-Planck-Institute of Immunobiology, Freiburg, Germany

    B. 8 Specificity of thymus-selected T cells expressing two antigen receptors


    Rearrangement ofT cell receptor a-genes is thought to be regulated by positive selection in theabsence of allelic exclusion, resulting in substantial numbers of peripheral T cells expressing twodifferent TCR. It has been proposed that only one of those TCR needs to be positively selected,and hence only one of them might determine antigen specificity in dual TCR T cells. We havecharacterized two CD8+ murine CTL clones, expressing two productively rearranged TCR a-chains. Both clones were hapten-specific and H-2Kb-restricted. They were induced with the Kb_binding octapeptides* M4L-TNP (clone E6) or M4L-DNP (clone Dl), and crossreacted with04-TNP or with 04-DNP and M4L-TN~ respectively, i.e. either with different carriers or witha different hapten on the same carrier. Hybridomas produced from both clones by fusion withCD8a expressing BW5147 cells lost reactivity to the inducing antigens, but each kept one of the

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    crossreactive specificities (hybridoma HyE6 reacted to 04-TNP but not to M4L-TNB HyDl toM4L-TNB but not to M4L-DNP or 04-DNP). This was in contrast to hybridomas from sin-gle TCR T cells which always reflected the fine specificity of the clones of origin. Antibody stain-ings revealed a marked reduction of surface expression of one of the two TCR a-chains inhybridoma HyE6, but comparable levels of mRNA for both. Preliminary experiments express-ing the individual TCRs of clone E6 by gene transfection identified one of them as selectivelyresponding to Kb/04-TNP but not to M4L-TN~ Hence, in contrast to the above assumptions,both TCR must have been positively selected on H-2Kb, either individually or in the form of aheterodimeric complex. Future studies aim at identifying the role of hetero-TCR complexes indefining antigen specificity.* M4L=SMQKFGEL, 04=SIIKFEKL.Supported by the Deutsche Forschungsgemeinschaft, SFB 388

    Abteilung Strukturforschung, Max-Planck-Institut flir Biochemie, Martinsried, Germany

    B. 9 Crystal structure of Fcy receptor III (CD16): A comparison with other Fcreceptor structures


    The described structure of sFcyRIII (CD16) is a paradigm for immune complex recognition ingeneral. FcyRIII folds into two Ig-like domains that are arranged in a steep angle forming a heartshaped structure. The high degree of amino acid conservation between sFcyRIII and other Fc-receptors and similarly between hFcl and related immunoglobulins suggests similar structuresand modes of association and allows an explanation of the vastly differing affinities of otherFcyR-IgG complexes and the FcRIa-IgE complex. We modeled, based on the FcyRIII/Fc-frag-ment structure the respective complexes for FcyRI, FcyRII and FcRIa. The obtained modelsare consistent with the observed biochemical data and may explain the observed specificity andaffinities. Additionally, a surface analysis reveals alternative binding sites located on the N-termi-nal domain of the FcRs.

    Department of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    B. 10 Changing T cell specificity by retroviral TCR display


    The diversity of the T cell receptor (TCR) repertoire is limited due to the processes of positiveand negative T cell selection. In order to obtain T cells with specificities beyond the immunesystem's capacity, we have developed a strategy for retroviral TCR display. In this approach, alibrary ofT cell variants is generated in vitro and introduced into a T cell receptor negative T cell

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    line by retroviral transfer. We document the value ofTCR display by the creation of a library ofan influenza A-specific TCR and the subsequent in vitro selection ofTCRs that either recognizethe parental influenza epitope, or that have acquired a specificity for a different influenza Astrain. The resulting in vitro selected TCRs induce efficient T cell activation upon ligand recog-nition and are of equal or higher potency than the in vivo generated parent receptor. RetroviralTCR display should prove a useful strategy for the circumvention of T cell tolerance by gener-ating novel TCRs, which have tumor or tumor-lineage antigen specificities. We are currentlyfocussing on lineage-antigens that are expressed on B cell lymphomas, e.g. the B cell specificCD20 antigen. To this purpose, we have isolated a murine TCR that recognizes an MHC-boundpeptide that is structurally related to a CD20 T cell epitope. This TCR will be used as a tem-plate for the generation ofTCR display libraries in order to create CD20-specific TCRs that pos-sess efficient anti-tumor activity.

    Molecular Immunology, Department of Cell Biology and Immunobiology, GBF,Braunschweig, Germany

    B. 11 Analysis of the immunoglobulin heavy chain repertoire of B1a lym-phocytes in A2 MOPC315 transgenic mice


    We have recently established a mouse line - called L2 - transgenic for the A2 light chain of theplasmacytoma MOPC315. The high and age independent expression of the transgene in L2mice results in nearly complete exclusion of endogenous light chain isotypes. However, the mostinteresting feature of this line is the predominance of B lymphocytes that exhibit all characteris-tic cellular features of CD5+ B1 cells. Here, we evaluated the influence of the exclusive expres-sion of the A2MOPC315 L chain on the H chain repertoire of B cells present in L2 mice. We per-formed a comparative molecular analysis ofVDJ rearrangements of peritoneal Bla lymphocytesand fetal!neonatalliver derived B cells from transgenic L2 and control mice. The low frequencyof N nucleotide insertions clearly confirmed the B1a character of peritoneal B cells from adultL2 mice. In contrast to control mice, a high frequency of independent VDJ rearrangements withidentical amino acid sequence in both B cell populations from L2 mice was observed. In addi-tion, a dramatic preferential JH4-usage was found for peritoneal B1a cells from adult L2 mice,which was not observed in case of fetal!neonatal liver deriyed B cells. Thus, the exclusive expres-sion of the transgenic L chain led to a dramatic bias of the expressed Ig H chain repertoire. Blalymphocytes from L2 mice seem to have an oligoclonal Ig H chain repertoire, which is stronglyselected by antigen. Together, these findings provide strong evidence on molecular level for amodel of B cell development, in which both autoantigens and exogenous antigens are involvedat different timepoints in positive selection events shaping the antibody repertoire of B1a lym-phocytes.

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    1Department of Immunology, University Medical Center, Utrecht, The Netherlands and2Department of Immunobiology, DNAX Research Institute, Palo Alto, CA, USA

    B. 12 LAIR-1, a human ITIM-bearing inhibitory receptor that can regulateimmune cell function


    Leukocyte Associated Immunoglobulin-like Receptor-I (LAIR-I) is an ITIM-bearing inhibito-ry receptor constitutively expressed on the majority of human peripheral blood mononuclearleukocytes. The LAIR-I gene is located on chromosome I9qI3.4, in the same region as the Ig-like Transcripts (ILTs) and the Killer cell Ig-like Receptors (KIRs). In the proximity of LAIR-I,the LAIR-2 gene is located, which encodes for a secreted protein with ~80% homology toLAIR-I. Cross-linking of the LAIR-I antigen by mAb results in strong inhibition of the cyto-toxic activity of NK cells and of effector T cells in vitro. LAIR-I has a differential expression onB cells. It is highly expressed on naive B cells while germinal center B cells completely lackLAIR-I expression. Simultaneous cross-linking of LAIR-I and Ig-receptors on naive B cellsresults in decreased intracellular calcium concentrations compared to Ig cross-linking alone.LAIR-I is expressed on and capable of inhibiting the function ofmany different cell types, whichsuggests a regulatory role for LAIR-I in a variety of immune responses. We are currently tryingto identify the natural ligand of LAIR-I to be able to better asses the biological role of this inhib-itory receptor in vivo.

    Immunobiology, Biomedical Primate Research Center, Rijswijk, The Netherlands

    B. 13 Extensive MHC-DQB variation in humans, great apes and monkeyspecies


    Non-human primates are often used in biomedical research, as model for studies on chronic andinfectious diseases, and in transplantation immunology. The application of these primates inresearch on immune-related diseases necessitates the characterization of their MHC system. Inparticular the MHC class II region of primates has been the subject of molecular biological stud-ies in recent years. In our study the emphasis was on sequencing of exon 2 in Mhc-DQBl genesof Macaca fascicularis (crab eating macaque or cynomolgus monkey). The analyses on a randomsample of 65 animals yielded 23 Mafa-DQBl exon 2 sequences, that were not published before.With the aim to classify these novel alleles in lineages, and to assign names, phylogenetic analy-ses were performed on these exon 2 sequences together with all primate (human and non-human) DQBl exon 2 sequences that are published thus far. The resulting phylogenetic treedemonstrates that some lineages are shared among species and predate speciation. Other lineag-es, in particular those of new world monkeys, appear to be more species specific.

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    IAbteilung Strukturforschung, Max-Planck-Institut fUr Biochemie, Martinsried, Germany and2Department of Biochemistry and Microbiology, University of Port Elizabeth, Port Elizabeth,South Africa

    B. 14 A structural basis for immune complex recognition: The 3.2A crystalstructure of the human IgG1 Fc-fragment-FcyRIlI complex


    The immune response depends on the binding of opsonized antigens to cellular Fc receptors andthe subsequent initiation of various cellular effector functions of the immune system. Here wedescribe the crystal structures of a soluble Fcy-receptor (sFcyRIII, CD16) in complex with a Fcfragment from human IgG1 (hFc1). In the 1: 1 complex the receptor binds to the two halves ofhFc1, where it contacts residues of both Cy2 domains and the hinge regions. Upon complex for-mation the angle between the two sFcyRIll domains increases significantly and the Fc fragmentopens asymmetrically. The Fc-fragment is recognized through loops of the C-terminal receptordomain. These loops are found in differing conformations in the isolated and in the complexedstructure of FcyRIII and are flexible. The main interaction of both molecules are of hydrophobicnature and hydrogen bonds are only made by the hinge peptide of the Cy2-B domain. The asym-metrical arrangement of the complex and the involvement of the same residues of the homodi-meric Fc fragment prevents a 2:1 stoichiometry between the FcyRIII and the Fc fragment.

    Department of Immunology, University of Kiel, Kiel, Germany

    B. 15 Chimeric dimeric HLAB7 recombinant molecules regulate alloreactiveC08+ T cells


    Soluble monomeric MHC molecules specifically regulate the function of alloreactive T cells invitro. Aggregation of monomeric MHC molecules potentiates binding and recognition of Tcells. To further improve the TCRlMHC interaction, we generated an MHC dimer. The extra-cellular domains of the HLA-B7 molecule, and the constant region of the human IgG1 heavychain, were amplified by PCR and cloned into a human expression vector. The vector contain-ing the construct of the fusion-protein was used to transfect a human B cell lymphoma cell-line.Dimeric HLA-B7 expressing cell-clones were identified by a conformation-dependent ELISA.Immunoprecipitation and Western blot analysis revealed the dimeric molecule at 170 kD and amonomeric form at 66 kD. More than 900/0 of HLA-B7 specific CTL, but not Jurkat controlcells, showed specificity for the dimer, as indicated by flow cytometry. Binding of the solubleMHC molecule was selective for CD8+, but not for CD4+ T cells, which implies specificity forCD8+ T cells and possible involvement ofCD8 in the recognition ofdimeric HLA-B7 by TCRs.The chimeric dimeric HLA-B7 molecule abrogated the cytotoxicity ofHLA-B7 specific CTL ina concentration-dependent manner. The use of dimeric MHC class I molecules open a new ave-nue for characterization and regulation of alloreactive T cells in vitro and can be used to regulateT cells and study MHC-induced signaling in alloreactive CD8+ T cell.

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    1Institute ofTransplantation Diagnostics and Cell Therapeutics, Heinrich Heine UniversityMedical Center, DUsseldorf: Germany, 2Nuffield Department of Surgery, University ofOxford, Oxford, UK, 3IRIS Research Center, Chiron S.p.A., Siena, Italy, 4Department ofMicrobiology and Immunology, UCSF, San Francisco, and 5Department of Structural Biology,Stanford University School of Medicine, Stanford, USA

    B. 16 Sequential expression of KIR extensively diversifies autologous Tcellpopulations having identical a~ Tcell receptors


    The NK cell receptors (NKR), KIR and CD94:NKG2A are expressed by subpopulations ofTcells. We determined the pattern and diversity of T cell expression of NKR from two donorswhose NK-cell expression of NKR was already well defined.

    T cell clones generated from peripheral blood of both donors expressed multiple NKR in dif-ferent combinations and used the range of receptors expressed by NK cells. However, a~ T cells,yfJ T cells and NK cells were all distinguished by the frequency with which certain receptors,notably CD94:NKG2A and HLA-C-specific KIR, were expressed. In contrast to NK cells notall NKR+ T cells expressed an inhibitory receptor for autologous HLA class 1. This lack of spe-cific inhibitory NKR was especially apparent on a~ T cells of one donor. Analysis ofTCR rear-rangement revealed a dominant subset of NKR+ T cells sharing identical TCRa and ~ chains.Most striking, 18 different KIR phenotypes were represented among the cells of one T cell clon-otype. Common to all T cell clones was expression of KIR2DL4, a receptor of yet unknownfunction. Analyses of serologically KIR-negative T cells of the same donors show that KIR2DL4mRNA expression was detectable in the absence of any other KIR.

    Our results are compatible with a sequential model of KIR acquisition on antigen-experiencedeffector T cells starting with upregulation of KIR2DL4. These data demonstrate the consider-able potential for KIR expression to modulate or diversify T cell responses to antigen.

    1Immunotherapy Laboratory, Department of Immunology, University Medical Center and2Medarex Europe, Utrecht, 3Department of Pathology, University Medical Center, Nijmegen,The Netherlands, and 4Department of Pathology, Harvard Medical School, Boston, MA, USA

    B. 17 Mac-l (COll b/C018) is essential for Fc receptor-mediated neutrophilcytotoxicity and intercellular cleft formation


    Receptors for human IgG and IgA initiate potent cytolysis of antibody (Ab)-coated targets bypolymorphonuclear leukocytes (PMN). Mac-I (complement receptor type 3, CDIIb/CDI8)has previously been implicated in receptor cooperation with Fe receptors (FeR). In this study, wecharacterized the role of Mac-I in FeR-mediated lysis of tumor cells by studying normal humanPMN, Mac-I-deficient mouse PMN, and mouse PMN transgenic for human FeR.

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    All PMN efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellularcytotoxicity (ADCC) was abrogated in Mac-I-I- PMN, and in human PMN blocked with anti-Mac-I mAb. Mac-I-I- PMN were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking differencein intercellular cleft formation between Mac-I-I- and wild type PMN. Also, respiratory burstactivity could be measured outside membrane-enclosed compartments using Mac-I-I- PMNbound to Ab-coated tumor cells, in contrast to wild type PMN.

    In summary, these data document an absolute requirement of Mac-I for FcR-mediated PMNcytotoxicity towards tumor targets. Mac-I-I- PMN exhibit defective spreading on Ab-coated tar-gets, impaired formation of immunologic synapses, and absent tumor cytolysis.

    1Department of Immunology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht,The Netherlands and 2Department of Pathology, Johns Hopkins University School ofMedicine, Baltimore, USA

    B. 18 Production of soluble rat MHC class 11-19 dimeric molecules


    MHC class I tetramers have been instrumental to study the expansion and homeostasis ofCD8+T cells. To study the role of CD4+ T cells in autoimmunity MHC class II tetramers would bevery helpful. Classical MHC class I tetramers are produced using bacterial expression systems.Similar production of MHC class II tetramers is however severely hampered due to in vitrorefolding problems.

    To circumvent the in vitro folding difficulties and to improve heterodimerization we chooseto construct chimeric molecules composed of the extracellular parts of rat MHC class II and theconstant parts of murine IgGI.

    Rat RTI.B1MHC class II a and ~ coding regions were cloned together with the murine IgG 1heavy chain and IgK light chain coding regions into a dual promoter baculovirus expression vec-tor (pFastBacDUAL), which was used to generate recombinant baculovirus.

    FACS analysis of virus infected insect cells showed positive staining using anti RTI.B (OX6),anti IgGI (A8S-I) and anti- IgK (X36) mAbs. This showed proper folding of a dimeric MHCclass II-Ig complex. Analysis of supernatants from virus infected cells by sandwich ELISA usingthe above mentioned anti-IgG1 lanti- IgK mAb pair showed that dimeric MHC class II-Ig com-plexes were also released in the medium. To enable further analysis of dimeric RTl.B1-Ig mole-cules, large-scale cultures and purifications are currently performed.

    To further improve the stability and the peptide loading of the MHC class II-Ig complexes,peptide linked MHC-Ig molecules are produced as well. In conclusion, soluble RTl.B1-Ig mole-cules have been successfully produced. Next, we will focus on their usability to study antigen spe-cific CD4+ T cells in experimental autoimmune models such as Adjuvant Arthritis.

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    Institut ftir Virologie und Immunbiologie, Universitat Wtirzburg, Wtirzburg, Germany

    B. 19 Expression of the CD8a~ heterodimer on y~T cells is neither a markerfor MHC class I restricted antigen recognition nor for MHC class I driv-en positive selection


    On a~T cells, CD8a~ serves as a coreceptor for MHC class I restricted antigen recognition andis essential for positive selection by MHC class 1. We addressed the possible function ofCD8a~on yoT cells of mice and rats. Analysis ofCD8a~ expression on yOT cells from C57BL/6 miceand C57BL/6 mice deficient in Tcr~ or ~2m revealed no differences in the proportion of

    CD8a~ positive yOT cells. Both, mutant and wild type strains expressed CD8a~ on approxi-mately 100/0 ofsplenic yoT cells, suggesting that CD8a~ expression is not a consequence ofpos-itive selection by a ~2m binding class Ia or class Ib molecule. In contrast to mice, rats express

    CD8a~ on nearly 800/0 of splenic yOT cells. Although we found the molecule to be functionalwith respect to costimulatory potential and lck binding, analysis of the yoTCR sequences arguesagainst the possibility that CD8a~may be indicative ofa subpopulation ofMHC class I restrict-ed yoT cells. Spectratyping and determination of TCRo sequences revealed usage of longCDR30 by CD8a~ positive and negative yOT cells. CDR3 of such length have been previous-ly found in yOT cells of mice, man and sheep and are predicted to prevent MHC restricted pep-tide antigen recognition. Consequently, CD8a~ on yoT cells may have functions not related toantigen recognition and its expression appears to be differentially controlled in a~ and yOT cells.

    1Laboratory of Neuroimmunology, Department of Neurology and 3Institute of OrganicChemistry, University ofTtibingen, Ttibingen, and 2Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Germany

    B. 20 Promiscuous peptide libraries to decipher antigen recognition require-ments of autoreactive human Tcells restricted by different HLA-DRmolecules


    Synthetic peptide libraries have been shown to represent valuable tools in elucidating molecularinteractions in the ternary HLA-peptide-T cell receptor (TCR) complex. HLA class II-allele-spe-cific Binding Patterns describing the peptide-HLA-interaction were obtained for a series of HLAclass II molecules by applying undecapeptide libraries (O/X10) in competition assays. However,corresponding activation of human T cell clones (TCCs) by these highly heterogeneous librariesis difficult to obtain due to the low concentration of recognized epitopes by a given TCR.

    Therefore we designed and synthesized new promiscuitive peptide libraries with a million-foldreduced complexity capable to bind to several HLA-DR haplotypes (DRl, DR2a, DR17) and,at the same time, able to stimulate TCCs with different HLA class II restrictions.

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    To guarantee a reliable T cellular read-out system in our model and to focus our attention tothe HLA-peptide-TCR-interaction, we generated TCR-transfectants derived from human auto-reactive TCCs. Human a/B-TCRs from 2 myelin-basic-protein (MBP) specific TCCs werestably transfected into a murine hybridoma (58a-B-). Interestingly, one clone recognizes MBP139-151 in the context of two HLA-DR molecules (DR1 and DR2a). Another clone recogniz-es MBP 86-105 with HLA-DR2a. TCR-transfectants paralleled their parental human TCCs infine specificity as shown by substitution analogues of the native epitope. PBMCs or L-cells trans-fected with HLA-DR1 or -DR2a were used as antigen presenting cells and T cell stimulation wasmeasured by the production of IL-2. Peptide libraries were designed based on published DR-binding motifs, synthesized by Fmoc/GtBu-chemistry and analyzed by Edman-degradation andmass spectrometry. HLA-binding was assessed in cell-free competition assays with affinity-puri-fied DR-molecules.

    Usage of those promiscuitive peptide libraries allows to obtain detailed Recognition Patterns andstimulation requirements for any DR-restricted TCR. These information will allow the identifi-cation of yet unknown peptide epitopes and new T cell agonists as a basis for the developmentof novel immunotherapeutic concepts.

    lInstitute of Animal Physiology, Ludwig-Maximilians-University, Munich, Germany and'2Institute of Animal Health, Compton, UK

    B. 21 Characterization of the C-type lectin B-NK, a putative chicken NK cellreceptor


    NK cells have been the focus of intense research and the receptors involved in target cell recog-nition have been characterized as C-type lectins and immunoglobulin-superfamily members inmouse and man, respectively. The chicken C-type lectin B-NK was identified by its close link-age to the chicken MHC gene cluster. The close linkage of a receptor with its putative ligand,the presence of a cytoplasmic ITIM motif and the mRNA expression in IL-2 activated splenicNK cells lead us to conclude that B-NK may serve as an important chicken NK cell receptor.The chicken T cell line LSCC-UG9 was stably transformed with a construct harboring the B-NK cDNA fused to a C-terminal V5 epitope. The B-NK expression was monitored by cell sur-face staining of the V5 epitope, proofing the predicted type II transmembrane orientation. Uponcell lysis and Western blot using the anti-V5 antibody the B-NK molecule was characterized asa 80 kDa homodimer composed of two glycosylated 40 kDa monomers. B-NK specific mabswere generated using the stable transformed cell lines for immunization. Several mabs werefound to specifically stain B-NK transfected cells only. The mAbs also reacted with a stable trans-formed cell line expressing the B-NK protein without epitope tag, demonstrating that the mabsare not reactive with the V5 epitope. These mabs are now used to determine tissue distributionof B-NK positive cells and the function of the B-NK molecule. As expected from the mRNAexpression data of B-NK preliminary flow cytometry data suggests that the B-NK molecule isonly expressed on a minute splenic and blood lymphocyte subset, thus indicating the low fre-quency of B-NK positive NK cells in these tissues. The B-NK mabs will be important tools tofurther investigate the NK cell phylogeny.

    This work was supported by the DFG Grant GO 489/3-1.


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