25 June 1997 - Oral presentations NK cells 345

10:00-12:oo Room C/D

0.1.09 NK cells

0.1.09.1 A novel surface molecule (p45) specificaiiy identifies and triggers human NK ceils

M. Vitaie I, S. Sivoti *, C. Bottino ‘, FL Augugliaro ‘, L. Sanseverino ‘, A. Moretta 2~3.1 CBA, lstituto Scientificv Turnon’ Genova, Italy * lstituto di lstdogia ed Embdologfa Genera/e Univemti di Ganova, It& 3 Dipattimento di Science Biomediche Biotacnolcgiche Universik4 di Brsscia Italy

introduction: The cytoiytic activity mediated by NK cells is regulated by a number of inhibitory and activatory receptors which specifically recognize HLA class I molecules. However NK ceils are aiso equipped with receptors that allow recognition and killing of MHC-negative target ceils. in this study we have identified a new candidate receptor involved in the mechanism of non MHC-restricted cytotoxicity.

Materids and Methods: Generation of monoclonal Abs against doned NK ceils. Selection of mAbs able to trigger NK ceil function. Analysis of the effect of mAbs on NK cytototicity, limphokines production and <Ca++si mobilization. Biochemical characterization on surface iodinated cells.

Reeuit% We have isolated a mAb (BAB261) which recognizes on the NK ceils surface a 46 Kd molecule (p46). The p46 molecule is selectively expressed on CD3-negative NK ceils and its distribution appear to identify NK ceils more precisely than ail available markers. Anti-p46 tn;Ab triggers the-cytolytic activity of all Nk clones in redirected killing assay, induces IFNy and TNFar release by NK ceils and <Ca++zi mobiiizationaffter &es-linking with second reagents. The triggering function of p46 is down-regulated by inhibitory HLA-specific receptors. However efficient inhibition can be achieved only when the two receptor types are co-polarized at the NK ceil surface.

Conolusbn: The p46 molecule represents a new marker for human NK ceils. More importantly this moiecuie is involved in the activation of ail NK cells. So far no evidence could be obtained for the ability of p46 to recognize MHC molecules; thus it might act prtmareiy as a triggering receptor for non-MHC iigands expressed by NK-susceptible target cells.

1 . . .] 0 1 09 2 NKdK~Rtpxicity mediated by CD40 and CD50

E. Catbone ‘, G. Ruggiem ‘, G. Terrazzano ‘, C. Palomba ‘, S. Fontana4, C. Manzo3, H. Spitss, K. K&re2, S. Zappawsta’. ‘Calf&a di Immunologia, Dipartimento di Biologia e Patologia C&/are e Mokolare, lJnive&& di NaDoli Federico II. Na&s. Itak 4 Cents di Endocrinolwia ed Oncologia Spkimantab del kNi?, N&l&,. Ita& 3 Oncologia Sperimentale C, - Imnwnoloaie. lstituto dei Tumori di Napoli. Naples, Italy 5 The Netherlands Cancer In&ute, Amsterdam, Holland; 2 Microbiology &d T.snotbiology Center Kamiinska Institutet, Stockholm, Sweden

Introduotion: NK recognition is regulated by a delicate balance between pos- itive signals, initiating their effector functions, and inhibitory signals preventing cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. Our study is focused on the analysis of the role of CD4O/CD60 molecules in the activation of NK mediated cytotoxicity.

Material and Methods: Human NK lines, clones and rll-2 activated CD56+CD3- lymphocytes were tested for their ability to kill P615 NK resistant tamet or its CD40-CD60 transfectant in a chromium release assays. Classical im&nofluorescence techniques were used for phenotype analysis.

Reeulb: Human NK lines and clones kill CD4O/CD60 P615 transfected ceils, but not the P615 parental line. Freshly CD56+ CD3- lymphocytes activated with rll-2 were found to be able to recognize CD40 or CD60 expressing target cells. The CD40 recoonition aooears to be mediated bv CD40L engagement since NK clones, lines and rll-2 activated CD56+ CD3- lymphocytes e&ess CD4OL. The NK susceptible T2 ceil line expresses on the ceil surface CD4O/CD60 moiecuies in the pre&ce of low level of MHC ciasa I antigens, due to a TAPl-2 defect. T2/TAPl-2 transfectant maintains similar amount of CD40 and CD60 antigens but shows high surface levels of MHC class I. mAbs directed against CD40 and CD60 inhibit NK killing of T2 ceil line, however the reinduction of MHC class I in the TUTAPl-2 transfectants prevents the NK-target recognition.

Conoiusions: We demonstrate that CD4UCD4OL interaction can represent a novel activation pathway for NK human lymphocytes in vitro. The function is likely to bs mediated by the expression of CD40 iigand (CMOL). This molecule is expressed on human NK lines and clones and can be induced on the surface of rll-2 activated human CD%+ CD3- lymphocytes. Furthermore MHC class I antigens have been shown to down regulate the NK susceptibility of CD40 expressing target cells.

0.1.09.3 Cloning and characterization of BY55, an NK and cytoiytic T lymphocyte specific ceil surface protein

A. Bensussan ‘, A. Anurnanthan 2, L. Boumaeii I, S. Vosa2, M.J. Robertson ‘, L.M. Nadier2, G.J. Freemans. ’ INSERM U. 448, Facufi~ ds Mdecine de Cr&ei/, Cr&ei/, Fence, 2 Dana-Farber Cancer Institute, Boston, MA, USA

NK cells, most TCR y8+ lymphocytes and some TCR &I+ lymphocytes medi- ate MHC-unrestricted cylotoxicity. To date, such cytdytic ceils are functionally defined since few if any cell cell surface molecules uniquely expressed on cy- tolytic populations have been defined. Expression of the protein identified by the BY55 rnonocionai antibody has been shown to be tightiy associated with MHC- unrestricted cytoiytic activity. Such BY55+ populations comprise lymphocytes largely of NK (CD3-) and a significant population of CD3+CD6+ phenotype, most of which are TCR yS+. The BY55 mAb, an IgM, did not inhibit or enhance NK mediated cytolysis. BY55 expression was reduced immediately fdiowing activation. Moreover, BY55 was expressed on the CD56dim, CDI6+ subset of NK ceils which have high cytoiytic activity. in contrast, BY55 was not expressed and could not be induced on the CD56b”DM, CDl6-t subset of NK cells, a subset with both high proliferative and low cytoiytic capacity. In an attempt to determine the function of this molecule, we cloned the gene encoding BY55 by COS ceil expression cloning. We identified cDNAs of 1.3 and 1.4 kb with an identical protein coding region and which differed only in the presence of an extra 106 bp intron-like sequence in the 5’ untranslated region of the 1.4 kb cDNA. A GENEBANK search revealed no homologies to previously described sequences. RNA blot analysis revealed BY55 mRNAs of 1.4 and 1.5 kb whose expression was expressed in the NKL NK-iike tumor cell line but not in a variety of T, B, or myeloid tumor ceil lines. In human tissues, BY55 mRNA was expressed only in spleen, thymus, peripheral blood lymphocytes, and small intestine (pre- sumably gut lymphocytes) and not in prostate, testis, ovary, colon, heart, brain, placenta, lung, liver, skeletal muscle, kidney, or pancreas. The sequence pre- dicts a cysteine-rich protein of 161 amino acids with two N-linked glycosylation sites and anchorage to the membrane by GPI linkage. lmmunoprecipitation of BY55 protein from the NKL cell line and from transfectants revealed a 70 kD major band and a 30 kD minor band. The 30 kD band intensified when the immunoprecipitate was first reduced and alkylated with iodoacetamide before SDS-PAGE, consistent with a tightly linked homodimer structure. Although this unique sequence does not predictfunction, GPI anchorage may explain how BY55 can be rapidly shed following activation. Consistent with this notion is the high expression of BY55 in iymphoid tissue of gut origin where this molecule might be readily shed. Therefore, our working hypothesis is that BY55 may be involved in the mobilization of cytolytic cells within distinct microenvironment%

10.1.09.4 1 Peptides isolated from HLA-Cw4304 confer different degrees of protection from natural killer ceil-mediated iysis

F. Zappacosta, F. Borrego, A.G. Brooks, K.C. Parker, J.E. Coiigan. National Institutes of Heatth, National institute of A//ergy 81 Infectious Diseases, Labotatov of Molecular Structure, Rockvif~e, MD, USA

Introduction: Human Natural killer (NK) ceils also bear receptors (KIR) for MHC class I molecules. These receptors have been shown to recognize particular groups of class I molecules and in general transmit a negative signal to the NK cells, resulting in the protection of the MHC class i-beating ceil from NK-medi- ated iysis. The role of peptide in this interaction is still a subject of debate. In the case of HLA-B’2705, some but not all endogenous peptides have been shown to protect TAP-deficient target ceils expressing HiA-B27 from NK-mediated ly- sis, suggesting that the receptor that interacts with HLA-B27 is capable of some level of discrimination between endogenous peptkies bound to HlA-B27. In contrast to these data, NK ceils have been reported to recognize empty HLA-C molecules.

MatsdaIs and Methods: in order to help clarify these issues, we have isolated and characterized by mass spectrometry a number of endogenous peptides that were bound to HlA-Cw’0304, and assessed their ability to confer protection from NK-mediated iysis by NK ceil clones.

Fteeub Functional studies indicated that peptides am an integral part of the HiA complex recognized by HLA-Cw3- specific NK ceil clones as HLA-Cw’O304 expression alone cells does not confer protection on TAP deficient target ceils. Not all HlA-Cw’0304 endogenous peptides confened the same degree of resistance to lysis, and moreover, the ability of peptides to confer resistance to lysis did not correlate with the stability for particular HlA-peptide complexes.

Conoiusion: These studies indicate that the ability to distinguish between subsets of peptidea may be a general feature of HlA class I recognition by NK cells.