Defective TCRG gene rearrangement in interleukin-7 receptor knock out mice

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Antigen receptors

10 .1 .09 .I5

Role of NK cells relevant to cord blood transplantatlon

Sanaa ELMarsafy, Eliane Gluckman, Armand Bensussan, Edgardo Carosella. Service de Recherche en HBmatoimmunologie (LIRB-DRM-DSV) CEA. Centre Hayem H6pitaI Si Louis. 1, Av. Claude Vellefaux 75475 Paris cedax 10, France, INSERM U448 Faculte de MBdecine de Cretei/ 94000 Cr&reil, France Graft-versus-host disease (GVHD) is one of the significant obstacles that still limit the benefits of bone marrow transplantation. However, its incidence is much lower following cord blood transplantation. The outcome of transplantation and the occurance of cmplications such as infections and GVHD may be influenced by the non specific cytotoxicity and regulatory mechanism of NK cells. The present study clarifies the role of these cells. We have developed a mAb termed BY55 that only labeles NK cells of cord blood. Using this mAb, BY55+ NK cells were sorted using FACS. Other lymphoid cells including T cells were sorted as BY55 cells. Various models of mixed lymphocyte culture (MLC) using irradiated mononuclear cells of peripheral blood (PBL) as stimulator cells were performed. As responding cells, BY55- cells of either CB or PBL were used. Addition of CB BY55+ cells to the MLC resulted in a significant inhibition of 3H- thymidine uptake in a dose dependent fashion, whether the responder BY55-cells were derived from the same CB or from PBL. Locally produced IL-2 by the responder cells results in activation of NK cells with subsequent release of various immunosuppressor factors such as TNFa that would contribute, in part, to the recorded non-MHC restricted suppressor activity. Our results might be of relevance in understanding the mechanism behind the decreased incidence of GVHD after CB transplantation where the activated NK cells can suppress the generation of GVHD in vivo by acting as veto cells.

( 0.1.09.6

1 Association of human NK cell surface receptors NKR-Pl and CD94 wlth Src-family protein klnases

J. Cemg1,3, A. FiSerovh2, 0. HorvBth, K. BezouSkaz,3, M. Posplsi12, V. Hofejsl 1,3.’ /n&it&e of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech RepoMic, zInstitute of Microbiioog~ Academy of Sciences of the Czech Republic, Prague, Czech Republic, 3Faculty of Sciences, Charles University,Prague, Czech Republic Introduction: Human NK cells express on their surface several members of the C-type lectin family such as NKR-Pl, CD94, NKG2 and CD69 that are probably involved in recognition of target cells and delivety of signals modulating NK-cell cytotoxicity. To elucidate the mechanism involved in signalling via these receptors we examined whether they are associated with protein tyrosine kinases. Materials and Methods: In vitro cultured human NK cells were sdubilized by a mild detergent Brij-58. Receptor complexes containing the NRK-PI or CD94 molecules, respectively, were immunoprecipitated by specific mAbs and in vitro kinase assays were performed on these immunoprecipitates. Identity of the receptor-associated proteins was detenined by re-precipitation and by Western blotting. Involvement of the associated kinases in signalling in vivowas examined by rnAb-mediated cross-linking of the NKR-PI and CD94 receptors on the cell surface and following of tyrosine phosphotylation. Resuf& NKR-PI and CD94 were found to be present in separated very large complexes (as judged by gel chromatography on Sepharose 48) containing the Lck, Lyn and Fyn kinases. These kinases apparently phosphorylated in vitro tyrosines in intracellular domains of the respective C-type lectins. In the case of CD94, the protein phosphorylated in vitro by the associated Src-family kinases is probably an NKGP subunit of the CD94-NKG2 heterodimer. Cross-linking of NKR-PI on the NK cell surface induced transient phosphorylation of at least one cellular protein. Conclusion: NK-cell surface receptors NKR-Pi and CD94 are associated within very large receptor complexes resistant to a mild detergent (membrane microdomains) with Src family kinases Lck, Fyn and Lyn and may use them for signal transduction.

( 0.1.09.71

Leukocyte inhibitory receptor (LIR) Is a novel Ig-superfamlly member expressed on human leukocytes that delivers an Inhibitory slgnal to natural killer cells

Linde Meyaard, Gosse J. Adema, Chiwen Chang, Lewis L. Lanier, Joseph H. Phillips. Departments of Human lmmuno/ogy and Molecular Biology, DNAX Research lnstitote of Molecular and Cellular Biology; Palo Alto, CA, USA Human NK cells express inhibitory receptors recognizing HLA that prevent lysis of targets expressing specific HIA alleles. These killer cell inhibitory receptors (KIRs) are members of the lg superfamily and contain ITIM motifs. Here we reoort the identification and characterization of leukocvte inhibitolv receptor (LIR): LIR is a widely expressed molecule. In healthy donor PBMd, CD4t T cells (70-80%), CD8+ T cells @O-90%), NK cells (95-100%). B cells (SO-SO%)and monocytes (99-100%) all expressed the LIR molecule. LIR, how-

25 June 1997 - Oral presentations

ever was not expressed on granulocytes, platelets or red blood cells. Anti-LIR mAb inhibited NK clone killing of EBV-transfomed B cell lines transfected with human FcR, but did not inhibit lysis of targets lacking FcR expression. This suggests that signaling through LIR delivers a negative signal to NK cell clones. In agreement with this, lysis of P815 cells, a FcR-sxpressing mouse mastocytoma cell line, which is killed in vitro bv human NK cell clones upon simultaneous cross linking of CD2, CD16, CD66 or DNAM-I antigens, w& also inhibited by anti-LIR mAb. Similarly, CDlSinduced killing of P815 by NK cells freshly is lated from peripheral blood was inhibited by &ti-LIR mAb. Thus, LIR delivers a potent inhibitory signal to NK cells. In order to identify the LIR protein, a cDNA encoding for a protein recognized by LIR mAb was expression cloned from an NK cell cDNA library. The plasmid container the LlRcDNA was found to contain an insert of 1728 bp. It contains an 865 bp open reading frame encoding a type I membrane protein with a 21 aa leader sequence, 149 aa extracellular domain, 16 aa transmembrane domain and a 101 aa cytoplasmic region. The protein contains one lg-like domain, classifying LIR as a member of the lg superfamily. LIR has up to 53% homology to known KIRs. The cytoplasmlc domain contains two aa sequences fitting the consensus for ITIMs. These motifs are also present in the cytoplasmic domain of inhibitory KlRs and recruitment of SHP-I to phosphorylated ITIM sequences inhibits cytotoxic function of NK cells. Most likely, the negative signal mediated through LIR is due to binding of phosphatases to one of these phosphorylated ITIM sequences. In conclusion, LIR is a novel inhibitory receptor, with homdogy to KIRs, that is broadly expressed on lymphocytes and monocytes. Unlike KIRs, however, LIR does not appear to bind HIA class I molecules.

10.1.09.8 1 Partlal loss of Fas-mediated lytlc actlvlty and ability to fragment DNA In NK cells exposed to Fas-posltlve and Fas-negatlve target cells I. Csipe 1.2,A.H. Monte1‘, J.A. Hobbs ‘, P.A. Morse ‘, G.y. Szegedi *, 2. Brahmi ‘. ’ Indiana Univ Sch, of Med., lndianepdis IN, Hungar)! ‘3rd Dept. of Med., Univ. Med. Sch. of Deb-n, Debnxen, Hungary Recently a granule independent cytolytic mechanism involving the interaction of the Fas ligand with Fas antigen on the surface of the target cells (TC) has been revealed. We have previously shown that natural killer (NK) cells exposed to sensitive TC temporarily loose their lytic ability via the granule mediated pathway and that this loss is recovered after an incubation in medium supplemented with 112. In this study we investigated the fate of Fas lytic pathway in NK cells exposed to sensitive TC. We exposed NK cells to K562 (Fas-) or Jutkat (Fas+) cells for 6 hours, then TC were separated from NK cells. The loss of lytic activity was asses& against K562, Jurkat (Fas+) and Jurkat (Fas-) cells. Fas lytic activity was determined in the absence of calcium ions, in the presence or absence of two Fas-blocking antibodies or a Fas.Fc fusion protein. In addition, the extent of DNA fragmentation in the various TC was also assayed by JAM test. Our results indicate, that (a) NK cells exposed to either Fas+ or Fas- targets lose most of their lytic potential due to granule mediated pathway, (b) but only partially lose their Fas lytic pathway and their ability to fragment DNA, (c) the reactivation of Nki cells lead to the recovew of both the granule as well as the Fas lytic pathways, (d) the Fas lytic activiiy found after-inactivation is almost completely inhibited by the M3 and 284, two Fas-blocking antibodies, as well as by the Fas.Fc fusion protein.

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Antigen receptors

0.1 .l0.1 Defective TCRG gene rearrangement In Interleukln-7 receptor knock out mice S. Candbias 1.2.J.J. Peschon 3. K. Mueaoe 2. S.K. Durum ‘. ’ CEA-G. DBMS-ICMNiERM U238,38&4 GreGUeCedex, France, 2SAIC, NCI-FCRDC, Frederick, MD, USA, 31mmunexCorp., Seattle, WA, USA, 4Laburatory of Molecular Immunoregulation, NC/, N/H, Frederick. MD, USA Introductfon: Interteukin-7 receptor knockout (IL-7R-‘-) mice are severely deficient in mature B lymphocytes and cr@T cells, and completely lack the yS T cell lineage. In these mice, thymccyte development is arrested at a very early stage (DN CD44+CD25-). Because this arrest is eartier than in mice with a block in VDJ recombination (RAG-‘-), we examined the rearrangement status of TCR genes in thymocytes from IL-7R-‘- mice.

Antigen receptors

25 June 1997 - Oral presentations Meterlaband Methods:Thymocyte DNA prepared from IL-7R-‘- mice, wild type age-matched controls, TCRD-‘- mice or splenocyte DNA prepared from a RAG-2-/- animal was analyzed by PCR with different combinations of primers for the presence of gene rearrangement at the TCRB, TCRG and TCRD loci. Reeuh We found identical patterns of TCRB gene rearrangement in IL-7R-‘- thymocytes and wild type controls, with only a slight reduction in the former. Similarly, TCRD rearrangements were found to be normal in IL-7R-‘mice for all the gene combinations analyzed. In sharp contrast, there is a severe impairment ofTCRG gene rearrangement at all TCRG sub-loci: rearrangements were below the level of detection in most cases. This impairment does not result from the absence of yS T lymphocytes, because TCRG genes are normally rearranged in TCRD-‘- mice, which contains only c@T cells. Conciudons:The results presented here clearly show a specific impairment of TCRG gene rearrangement in IL=IR-I- thymocytes. These findings show that sionalina throuah IL-7R is essential to induce TCRG mne rearranwment, and &&&-that &e (or both) of the two known ligan& of this r&ptor, IL-7 and TSLP, serves as an extrinsic signal to specifically mediate TCRG gene accessibility. Dominance of the AVl and BV17 elements in nickel-specific human T cell receptors relates to severitv of contact sensitivitv C. Moulon I, J. Vollmer’ , M. Fritz’, A. Dormoy2, H.U. Webien ‘. ’ Max-Plan&-lnstitut fiir Immunobiologie, Stkeweg 51, D-79108 Freibug, Germany 2Establishment de Tmnsfusion Sanguine, 10 rue Spielmann, F-67065 Strasbourg, France

Introduction:Hypersensitivity to nickel represents the most common manifestation of contact allergy in humans. The role of metal-specific T cells in this disease is well established, but the molecular interactions involved in their activation are oooriv understood. A wav to estimate the degree of their structural variability is’to aialyze the TCR repe&zire of Ni-reactive &Is. Materials and Methods:We examined the peripheral blood TCR repertoire in T cell lines activated with NiSO., for six allergic patients by FACS staining with a panel of antibodies. T cell clones were isolated from these cell lines for two allergic donors and TCR usage of the Ni-reactive clones was detenined by PCR analysis and sequencing. Reoub For the three most hyperreactive donors, we found a strong overrepresention of the TCR BVI 7 element. TCR sequencing for two of these donors revealed an additional skewing for AVl as well as a selection for an N-region encoded argine at position 93 or 95 of the BV17. Since Arg is not known to participate in Ni complexing, we suppose that this selection is driven by peptide- rather than Nicontacts. In contrast, the CDRI of BV17 contains a unique combination of amino acids (HDA) that bears similarities to known motifs in Ni-binding proteins or peptides. Conclusion:We propose that the severe hypersensitivity reactions found in BV17 over-expressors may be the result of Ni++ ions bridging the germline encoded BV17 CDRI loop to cunesponding sites in the MHC-peptfde complex and thereby creating a sbperantigen-like enhancement of weak TCR-peptide contacts. Supported by the Bundesministerium firr Bildung, Wissenschaft, Forschung und Techndogie, grant 07ALL103.

IO.1 .10.3 1 Differential usage of Va2 family members by CD4+ and CD8+ T cells Margarfda Correia Neves, Christophe Benoist, Diane Mathis. lnstitut de G&&ique et de Biologic Mokkulaire et CeMaire, Strasboug, France

ObJectlw:Certain T cell receptor Va genes are preferentially expressed by CD4+ or CD8+ T lymphocytes. This skewing suggests that individual amino acids within the Vu chain react preferentially either with MHC class I versus class II molecules, or the CD4 versus CD8 co-receptor. Anti-Vcz2monoclonal antibodies have failed to demonstrate a dear bias for the CD4+ or CD8+ compartment However, Vcz2 gene segments form a large family whose members are very similar at the nucleic and amino acid sequences, but have conserved and non-conserved amino acid replacements grouped in discrete areas of the CDRI, CDRP and frame-work regions. There is no information on the distribution of individual family members in the two compartments of mature ceils. We have established a transgenic mouse line expressing a single TCR B-chain (V,!?5),and examined the expression of individual Vu2 family members in CD4+ and CD8+ mature T lymphocytes. Materials and Methods:CD4+and CD8+ peripheral T cells expressing Vu2 TCR were sorted from lymph nodes of TCR p-chain transgenic mice (99% of the peripheral lymphocytes express the VP5 TCR B-chain transgene). RT-PCR has been performed to amplify the Va2 family members expressed in the two populations. The PCR products were cloned and sequenced. Reeults: We have observed a strong bias of certain Vu2 members for CD4+ cells and other Va2 members for the CD8+ population. Preliminary results

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indicate that amino acids located in the CDRI and/or CDRP can be responsible for this bias, regardless of the CDR3 sequence. Conclusions:These results are in agreement with the recently publish crystallographic structure of the TCR/MHC complex. The crystal structure shows a central role for CDR3 in peptide recognition while CDRI and CDR2 contact both the peptide and the a-helical segments of the MHC molecule. These observations mighi be the basis for the preferential usage of certain CDRI and/or CDR2 by CD4+ or CD8+ T calls. Studies are in progress to clearly determine which non-conserved amino acids within the different gene segments of the Va2 family are responsible for this dichotomy.

IO.1 .10.4 1 Structural requlrements for 1 cell receptor Va mediated positive repertoire selection by and alloreactlvity to MHC class I N. Tones-Nagel ‘, B. Mehling I, A. Deutschlander ‘, E. Joly2, T. Hernann ‘, T. Hiinig ’ ’ lnstifutefor Vhubgyand Immunobiology, University of Wkbug, Vsrsbacherstr7. D-97078Wiirzburo. Gennanv. 2Institute for Animal Physiology aid kenetics Reseamh&vaha~‘Hall, Cambridge, UK

Introduction:In rats expressing the RTI’ haplolype, CD8 cells utilizing Var8.2 are tenfold overselected. In addition, CD8 cells alloreactive to RTI’ are enriched in Va8.2. Both phenomena are independent of specific VP pairing. MaterIsIsand Methods:50 V&2+ TCRCZchains from overselected, alloreactive and control populations of CD8 cells were PCR cloned and sequenced. A polymorphic RTI’ class I molecule was PCR cloned and expessed in stimulator cells. Results: RTI’ alloreactive V~r8.2+ CD8 cells use a restricted set of Ja segments which contribute short homologous CDR3 loops, whereas thymic RTl’-overselection had liile effect on Ja usage or CDR3 composition. The novel RTIA’ molecule preferentially expands Va8.2+ CD8 cells. Conclusion: Va8.2-RT1Af interaction suffices for positive selection but requires additional contacts by CDR& for peripheral T-cell activation. The differences between A’ and the very closely related AB map Var8.2-RTI A’ interaction to sites which are in close agreement with the recently published crystal structure of an HLA class I resbfcted human TCR complexed to MHC plus peptide.

1

T cell receptor beta genes of Xenopus

I. ChrBtien, L. Du Pasquier. Base/Institute for Immunoiog~ Grenzachetstrasse 487, Base/, Switzerland cDNAs of the T cell receptor beta (TCRB) have been isolated from the anuran amphibian Xenopus and they show strong structural homology to TCRB sequences of other vertebrates. The total number of V beta genes is in the order of 12 per haplotype. The Xenopus V beta are otganised in numemus families containing few members like mammals but unlike the chicken. The number of J segments is at least five and we found at least two different D segments highly conserved in evolution throughout all classes of vertebrates. This high conservation suggest that the D segments might have a more relevant function al the nucleotide level than at the protein level. Surprisingly only one C beta has been found and it lacks N-linked carbohydrate glycosylation sites. The recombination signal sequences suggest that the mechanism of rearrangements are identical to those of mammals. The locus is under diploid inheritance despite the pseudo tetraploidy of the Xenopus laevis and gi//i used in this study. The present work gives us now the possibility to study T-cell development in Xenopus with a special attention to the larval period where the immune system develops in the absence of MHC class I molecules.

0.1 .10.6

Characterization of the chicken CD3 gene locus and of a CD36 splice variant

T.W.F.Gbbel, W. Wiest. Base1Institute for Immunology, Basel, Switzerland

Introduction:The chicken TCWCDI complex contains two different CD3 chains, CD3& and a CD3 chain with equal amino acid homology to both CD3y and CD36. This analysis clarifies the identity of this second CD3 gene, identifies a splice valiant and analyzes the complete structure of the chicken CD3 locus. Materials and Methods: Long range PCR was used to amplify the CD3 locus and the 5’ ends of the CD3 genes were extended with the promoter finder kit (Clontech). DNA or RNA preparations and RT-PCRwere employed according to standard procedures. Results: The chicken CDly/G-like gene consists of 5 exons which are identicallv omanbed like mammalian CD36 aenes. Thus the aenomic omanization un~uiv&ally identifies the CD3y/6 gene as CD36 homiogue, in contrast to mammalian CD3y, which contains 7 exons. In addition, RT-PCR analysis of thymocyte or peripheral T cell CD36 transcripts revealed a second shorter version in addition to the full length form. Most of the extracellular domain is truncated in the short form, due to the presence of a cryptic splice site in exon 3. This alternatively spliced CD36 version is predicted to yield a 12 kDa, non-glycosy