Workshop B: Molecular basis of immune recognition

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<ul><li><p>Sir William Dunn School of Pathology, University of Oxford, Oxford, UK</p><p>B. 1 Determination of the affinity for the interaction between CD22 andCD45: Contribution of CD45 protein structure</p><p>T.R. BAKKER, C. PIPERI, E.A. DAVIES, and EA. VAN DER MERWE</p><p>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.</p><p>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.</p><p>IClinical Research Unit for Rheumatology, Albert-Ludwigs University, Freiburg, Germany and2Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan</p><p>B. 2 Cell surface expression of a HSC73-like molecule by CD14+ monocytes</p><p>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.</p><p>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,</p></li><li><p>30 . Joint Annual Meeting 2000</p><p>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.</p><p>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.</p><p>Supported by grants of the DFG (Me 604) and the Hans-Hench Stiftung.</p><p>Departments of 1Medicinal Chemistry, Utrecht Institute of Pharmaceutical Sciences and2Immunology, Institute of Infectious Diseases and Immunology, Utrecht University, Utrecht,The Netherlands</p><p>B. 3 MHC class II binding of peptoid-peptide hybrids</p><p>E.C. DE HAAN 1, M.H.M. WAUBEN2 , M.C.J.T. GROSFELD-STULEMEYER2 , J.A.WKRUIJTZER1, R.M.]. LISKAMpl, and E.E. MORET1</p><p>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.</p><p>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.</p></li><li><p>Joint Annual Meeting 2000 . 31</p><p>Department of Immunobiology, Biomedical Primate Research Center, Rijswijk, TheNetherlands</p><p>B. 4 Differential evolutionary MHC class II strategies in humans and Rhesusmacaques</p><p>G.G.M. DOXIADIS, N. OTTING, N.G. DE GROOT, R. NOORT, and R.E. BONTROP</p><p>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.</p><p>Abteilung Klinische Immunologie, Medizinische Hochschule, Hannover, Germany</p><p>B. 5 The functional role of the glycosylation of FcyR1l1</p><p>B.E. DRESCHER, T. WITTE, and R.E. SCHMIDT</p><p>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.</p><p>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-</p></li><li><p>32 . Joint Annual Meeting 2000</p><p>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.</p><p>Supported by SFB244/A09</p><p>Laboratory of Molecular Biology, Children's Hospital, Charite, Humboldt-University, Berlin,Germany</p><p>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</p><p>~ FISCHER, s. LEUCHT, A. OSEI, M. HOFFMANN, N. JENDREYKO, H. LERCH, M.M.UTTENREUTHER-FISCHER, and G. GAEDICKE</p><p>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.</p></li><li><p>Joint Annual Meeting 2000 . 33</p><p>Max-Planck-Institute of Immunobiology, Freiburg, Germany</p><p>B. 7 Tcell receptor a-chain determines antigen and restriction specificity ofa nickel-reactive human Tcell clone</p><p>K. GAMERDINGER, J. VOLLMER, S. FIETZECK, and H.U. WELTZIEN</p><p>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.</p><p>Supported by the Klinische Forschergruppe Pathomechanismen der allergischen Entziindung,BMBF FKZ 01GC9701/7</p><p>Max-Planck-Institute of Immunobiology, Freiburg, Germany</p><p>B. 8 Specificity of thymus-selected T cells expressing two antigen receptors</p><p>S. HELLWIG, K. FRICCIUS, U. PFLUGFELDER, B. GERLICH, and H.U. WELTZIEN</p><p>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</p></li><li><p>34 . Joint Annual Meeting 2000</p><p>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 refle...</p></li></ul>


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