SummaryHuman CD3-16 + 56 § natural killer (NK) cells have been shown to display a donaUy distributed ability to recognize major histocompatibility complex (MHC) class I alleles. Opposite to T lymphocytes, in NK cells, specific recognition of MHC class I molecttles appears to induce inhibition of cytolytic activity and, thus, to protect target cells. Since a precise correlation has been established between the expression of the NK-specific GL183 and EB6 surface molecules (belonging to the novel p58 molecular family) and the specificity of NK clones, we analyzed whether p58 molecules could function as receptors for MHC in human NK cells. NK clones displaying the previously defined "specificity 2" and characterized by the GL183 + EB6 + phenotype, specifically recognize the Cw3 allele and thus fail to lyse the Fc"/R + P815 target cells transfected with Cw3. On the other hand, NK clones displaying "specificity 1" and expressing the GL183-EB6 + phenotype failed to lyse Cw4 + target cells. Addition of the F(ab')2 fragments of either GL183 or EB6 mAb as well as the XA141 mAb of IgM isotype (specific for the EB6 molecules) completely restored the lysis of Cw3-transfected P815 cells by the Cw3-specific NK clones EX2 and EX4. Similarly, both the entire EB6 mAb, its F(ab')z fragment and the XA141 mAb reconstituted the lysis of C1R, a Fc'yR-target cell expressing Cw4 as the only serologically detected class I antigen. Thus, it appears that masking of different members of p58 molecules prevents recognition of "protective" MHC class I alleles and thus the delivering of inhibitory signals. Further support to the concept that p58 molecules represent a NK receptor delivering a negative signal was provided by experiments in which the entire anti-p58 mAbs (of IgG isotype) could inhibit the lysis of unprotected Fc'yR + P815 target cells, thus mimicking the inhibitory effect of MHC class I molecules. ecent data have substantially modified the general concept that human CD3-CD16 + CD56 + NK cells (1, 2) represent a homogeneous cytolytic lymphoid population displaying nonspecific, non-MHC-restricted functions. Thus, it has been shown that NK cells are capable of specific functions since they can discriminate among different normal allogeneic target cells (3,4). This ability is clonally distributed as different NK clones derived from single individuals display unique cytolytic patterns against allogeneic target cells (5-7). Thus, five distinct groups of alloreactive clones (each displaying a different specificity) have been identified (7). Genetic analysis (6, 7) and the use of cell transfectants (8) indicated that the target molecules recognized by NK clones are represented, at least in some instances, by HLA class I alleles (8-11). Whereas specific recognition of MHC molecules by alloreactive cytolytic T lymphocytes leads to triggering of their cytolytic function, in the case of NK lymphocytes, a given HLA allele (Cw3) was found to confer specific protection from lysis by a group of NK clones displaying a defined specificity (specific...

SummaryIn previous studies we identified a surface molecule (termed GL183) capable of mediating cell activation and selectively expressed by a subset of human CD3 -CD16+ natural killer (NK) cells. In this study we analyzed whether other subset-specific functional molecules were expressed in GL183 -NK cells . To this end, mice were immunized with the PE29 (CD3-CD16+GL183 -) NK clone. Monoclonal antibodies (mAbs) were selected by screening the hybridoma supernatants for their ability to trigger the cytolytic activity ofclone PE29 against the human myelomonocytic leukemia U937. The EB6 mAb (IgGI) triggered the PE29 clone, but not a GL183+ clone used as a control . EB6+ cells ranged between 1 and 13% of peripheral blood lymphocytes and were largely included in the CD3"CD16+CD56+ cell populations (only <2% of EB6+ cells were CD3+) . Analysis of resting or activated CD3 -CD16+ populations, or clones for the expression ofEB6 or GL183 mAbs, allowed us to identify four distinct, phenotypically stable, NK subsets (EB6+GL183 -; EB6+GL183+ ; EB6 -GL183+ ; EB6 -GL183 -) . Similar to GL183 mAb, the EB6 mAb selectively triggered the NK subset expressing the corresponding surface antigen to lyse human tumor cell lines including U937, IGROVI, M14, and A549. In addition, EB6 mAb sharply inhibited the cytolytic activity of EB6+ clones against P815, M12, and P3U1 murine target cells. In EB6+GL183+ ("double-positive") clones both EB6 and GL183 mAb inhibited the redirected killing of P815 cells induced by anti-CD16, anti-CD2 mAbs and phytohemagglutinin (PHA) . Similar to GL183 molecules, molecules precipitated by EB6 mAb were represented by either single 58-kD chain or double chains of 55 and 58 kD (with no detectable differences in EB6IGL183 -or EB6+GL183+ clones) . In sequential immunoprecipitation experiments using the double-positive clones CEG52 and CA25.50, preclearing of cell lysates with EB6 or GL183 mAb removed only EB6 or GL183 molecules, respectively, thus indicating that the two antigenic determinants are carried by two distinct molecules . Peptide map analysis indicated that EB6 (or GL183) molecules precipitated from double-positive clones were identical to the corresponding molecules isolated from single-positive ones. On the other hand, comparison of the E136 and GL183 maps revealed peptides that were unique to each molecule, although most of the major peptides migrated to identical positions . We further investigated whether correlation existed between the phenotypic assignment of NK clones and their ability to mediate specific lysis of normal allogeneic cells. All mixed lymphocyte culture-derived NK clones displaying the previously defined "1 anti-A" specificity expressed the EB6+GL183 -surface phenotype. In addition, among peripheral blood-derived CD3 -CD16+ clones (with PHA and IIr2), only those expressing the EB6+GL183 -phenotype specifically lysed PHA blasts susceptible to lysis by "1 anti-A" clones. Finally, in individuals susceptible to lysis by "1 antiA" alloreactive clones, EB6+GL183 -NK cells (which would re...

Recognition by natural killer (NK) cells of major histocompatibility complex (MHC) class I molecules on target cells inhibits NK-mediated lysis. Here, inhibition of NK clones by HLA-B*2705 molecules mutated at single amino acids in the peptide binding site varied among HLA-B*2705-specific NK clones. In addition, a subset of such NK clones was inhibited by only one of several self peptides loaded onto HLA-B*2705 molecules expressed in peptide transporter-deficient cells, showing that recognition was peptide-specific. These data demonstrate that specific self peptides, complexed with MHC class I, provide protection from NK-mediated lysis.

The GL183 mAb was obtained by immunizing BALB/c mice with the E57 clone (CD7+CD2+CD3-CD16+CD56+) derived from human peripheral blood NK cells. In human peripheral blood, GL183-reactive cells ranged between 2 and 12% (mean 6.5%) in 10 different donors. Double fluorescence and FACS analysis showed that GL183+ cells were consistently included in the CD56+ or CD16+ cell populations. Moreover, since only a fraction of CD56+ or CD16+ cells (approximately 40%) coexpressed GL183 surface antigen, reactivity with GL183 mAb appears to define two subsets within the CD3- lymphocyte population expressing NK cell markers. Although, the majority of GL183+ cells were CD3-, approximately 1% expressed CD3 surface antigens. As shown by clonal analysis, these infrequent CD3+GL183+ cells coexpressed CD56 and CD16 antigens. Cloning of CD3-GL183+ or CD3-GL183- cell populations under limiting dilution conditions yielded clonal progenies that maintained their original surface phenotype. Therefore, expression or lack of expression of GL183 surface antigens represents a stable phenotypic property of a subset of human CD3- NK cells. Immunoprecipitation experiments and two-dimensional PAGE analysis indicated that GL183-reactive molecules were represented in different clones either by a single 58-kD chain or, more frequently, by two chains of approximately 55 and approximately 58 kD, respectively. Analysis of GL183+ or GL183- NK clones for their ability to lyse human (IGROV I) or murine (P815) tumor target cells indicated that GL183- clones were, on average, fivefold more efficient in inducing target cell lysis. GL183+ and GL183- clones produced comparable levels of TNF-alpha in response to PHA plus PMA or anti-CD16 mAb plus PMA. Importantly, production of TNF-alpha was also induced by stimulation of GL183+ clones with GL183 mAb plus PMA. These data indicated that GL183 antigen could mediate cell triggering. This concept was confirmed by the analysis of Ca2+ mobilization, as GL183 mAb induced (in GL183+ clones) increments of [Ca2+]i comparable with those induced by PHA. Moreover, GL183 mAb, or its F(ab')2 fragments, strongly enhanced the cytolytic activity of GL183+ clones against a panel of human tumor target cells, including U937, Raji, IGROV I, M14, and A549. In contrast, GL183 mAb, but not the F(ab')2 fragments, sharply inhibited the cytolytic activity of the same clones against P815, M12, and P3U1 murine target cells. In this case, the effect of GL183 mAb (inhibition) was opposite that of PHA or of stimulatory anti-CD2 or anti-CD16 mAbs, which consistently enhanced the target cell lysis.(ABSTRACT TRUNCATED AT 400 WORDS)

This study was designed to identify the target molecules of the natural killer (NK) cell-mediated recognition of normal allogeneic target cells. As previously shown, the gene(s) governing the first NK-defined allospecificity (specificity 1) were found to be localized in the major histocompatibility complex region between BF gene and HLA-A. In addition, the analysis of a previously described family revealed that a donor (donor 81) was heterozygous for three distinct NK-defined allospecificities (specificities 1, 2, and 5). HLA variants were derived from the B-Epstein-Barr virus cell line of donor 81 by gamma irradiation followed by negative selection using monoclonal antibodies specific for the appropriate HLA allele. Several variants were derived that lacked one or more class I antigen expressions. These variants were analyzed for the susceptibility to lysis by NK clones recognizing different allospecificities. The loss of HLA-A did not modify the phenotype (i.e., "resistance to lysis"). On the other hand, a variant lacking expression of all class I antigens became susceptible to lysis by all alloreactive clones. Variants characterized by the selective loss of class I antigens coded for by the maternal chromosome became susceptible to lysis by anti-2-specific clones. Conversely, variants selectively lacking class I antigens coded for by paternal chromosome became susceptible to lysis by anti-1 and anti-5 clones (but not by anti-2 clones). Since the Cw3 allele was lost in the variant that acquired susceptibility to lysis by anti-2 clones and, in informative families, it was found to cosegregate with the character "resistance to lysis" by anti-2 clones, we analyzed whether Cw3 could represent the element conferring selective resistance to lysis by anti-2 clones. To this end, murine P815 cells transfected with HLA Cw3 (or with other HLA class I genes) were used as target cells in a cytolytic assay in which effector cells were represented by alloreactive NK clones directed against different specificities. Anti-2-specific clones efficiently lysed untransfected or A2-, A3-, and A24-transfected P815 cells, while they failed to lyse Cw3-transfected cells. NK clones recognizing specificities other than specificity 2 lysed untransfected or Cw3- transfected cells. Thus, the loss of Cw3 resulted in the de novo appearance of susceptibility to lysis, and transfection of the HLA- negative P815 cells with Cw3 resulted in resistance to lysis by anti-2 clones. Therefore, we can infer that Cw3 expression on (both human and murine) target cells confers selective protection from lysis mediated by anti-2 NK clones.

SummaryThe effect of anti-CD69 monodonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16 + natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma ceUs in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16-NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T call receptor (TCR) 3,/~ + clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR cr + cytolytic dones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR 3,/~ + but not TCR ce/~ + thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7 +), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR ot/B + cytolytic cells.

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.

Sillnl'nary GL183 or EB6 (p58) molecules have been shown to function as receptors for different HLA-C alleles and to deliver an inhibitory signal to natural killer (NK) cells, thus preventing lysis of target cells. In this study, we analyzed a subset of NK cells characterized by a p58-negative surface phenotype. We show that p58-negative clones, although specific for class I molecules do not recognize HLA-C alleles. In addition, by the use of appropriate target cells transfected with different HLA-class I alleles we identified HLA-B7 as the protective element recognized by a fraction of p58-negative clones. In an attempt to identify the receptor molecules expressed by HLA-B7-spedfic clones, monodonal antibodies (mAbs) were selected after mice immunization with such clones. Two of these mAbs, termed XA-88 and XA-185, and their F(ab')2 fragments, were found to reconstitute lysis of B7 + target cells by BT-specific NK clones. Both mAbs were shown to be directed against the recently clustered Kp43 molecule (CD94). Thus, mAb-mediated masking of Kp43 molecules interferes with recognition of HLA-B7 and results in target cell lysis. Moreover, in a redirected killing assay, the cross-linking of Kp43 molecules mediated by the XA185 mAb strongly inhibited the cytolytic activity of HLA-B7-specific NK clones, thus mimicking the functional effect of B7 molecules. Taken together, these data strongly suggest that Kp43 molecules function as receptors for HLA-B7 and that this receptor/ligand interaction results in inhibition of the NK-mediated cytolytic activity. Indirect immunofluorescence and FACS | analysis of a large number of random NK clones showed that Kp43 molecules (a) were brightly expressed on a subset of p58-negative clones, corresponding to those specific for HLA-B7; (b) displayed a medium/low fluorescence in the p58-negative clones that are not B7-specific as well as in most p58 + NK clones; and (c) were brightly expressed as in the p58 + clone ET34 (GL183-/EB6 +, Cw4-specific). Functional analysis revealed that Kp43 functioned as an inhibitory receptor only in NK clones displaying bright fluorescence. These studies also indicate that some NK clones (e.g., the ET34) can coexpress two distinct receptors (p58 and Kp43) for different class I alleles (Cw4 and B7). Finally, we show that Kp43 molecules function as receptors only for some HLA-B alleles and that still undefined receptor(s) must exist for other HLA-B alleles including B27.