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D Pende

Publications and source records attributed to D Pende.

At least 55 records · Page 3Linked to original sources
PubMed1992

Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2).

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.

Alleles↗
PubMed1992

Alloantigen recognition by two human natural killer cell clones is associated with HLA-C or a closely linked gene.

Human natural killer (NK) cells with the CD3- CD16+ phenotype recognize allospecificities on normal T-cell blasts. The NK-defined specificity 1 (NK-1) is recessively inherited and has been mapped to the major histocompatibility complex between the complement gene cluster and HLA-A. A gene for NK-1, however, has not been identified. Here we demonstrate that NK-1 and the recently defined NK specificity 2 (NK-2) are reciprocally associated with homozygosity for a diallelic polymorphism at amino acid positions 77 and 80 in the putative peptide-binding site of HLA-C (P less than 10(-5)). NK-cell recognition of allogeneic cells may, therefore, be controlled by HLA-C itself or by a closely linked gene(s), which dominantly prevents (resistance alleles) or recessively permits (susceptibility alleles) recognition of still-unknown target determinants.

Base Sequence↗
PubMed1992

Evidence of a natural killer (NK) cell repertoire for (allo) antigen recognition: definition of five distinct NK-determined allospecificities in humans.

Previous studies indicated that CD3-CD16+ natural killer (NK) cells are capable of specific alloantigen recognition. Thus, alloreactive NK clones lysed normal allogeneic target cells (phytohemagglutinin [PHA] blasts) bearing the stimulating alloantigen but did not lyse autologous cells or the majority of unrelated allogeneic cells. In this study we investigated whether NK cells isolated from single individuals could exhibit different allospecificities. To this end, we derived large numbers of CD3-CD16+ clones (in the presence of PHA) from fresh CD3- peripheral blood lymphocytes. Cloning efficiencies ranged between 5 and 10%. The resulting CD3-CD16+ clones were tested for their reactivity against a panel of allogeneic PHA blasts (derived from six donors). In a given individual (A), four distinct groups of clones could be identified according to their pattern of reactivity (over 400 clones have been analyzed). Clones that could be assigned to one or another group of specificity represented 36% of all clones derived from this donor. The remaining clones did not display cytolytic activity against any of the allogeneic target cells used in the panel. None of the clones lysed autologous (A) PHA blasts, yet, these cells were lysed by the representative clones G10 and H12 specific for donor A. Clones displaying a cytolytic pattern of reactivity identical to that defined for donor A were present in other individuals studied, however not all groups of allospecific clones were necessarily represented in different individuals. Allospecific clones belonging to the various groups were homogeneous in the expression of EB6/GL183-triggering surface molecules, and could thus be assigned to one or another of the previously defined subsets of NK cells. Genetic analysis of the new NK-defined alloantigens was performed in representative families. The corresponding characters were found to segregate independently and, at least for three of them, an autosomic recessive type of inheritance could be demonstrated. Moreover, the comparative analysis of the segregation of the major histocompatibility complex haplotypes and the recessive or dominant alleles of the genes governing the five specificities analyzed indicated that there is no independent sampling between the two genetic traits, thus suggesting that the genes regulating the NK-defined specificities are carried by chromosome 6. Finally, some donors expressed more than one specificity, thus providing evidence for an NK-defined complex haplotype.

Alleles↗
PubMed1992

Novel surface molecules involved in human NK cell activation and triggering of the lytic machinery.

Three new monoclonal antibodies (MAbs) termed 7A6, PP35 and A6/143 were isolated after mouse immunization with CD3- CD16+ NK clones. The screening procedure was based on the ability of MAbs to trigger cytolytic activity of the immunizing clones in a re-directed killing assay against the P815 murine mastocytoma cell line. The 7A6 MAb reacts with 58 kDa surface molecules that appear to belong to the same molecular family defined by the previously described NK-sub-set-specific GL183 and EB6 MAbs. However, unlike from these MAbs, the 7A6 MAb reacted with (and activated) all CD3- NK lymphocytes, independent of their sub-set assignment (based on the expression or lack of expression of EB6, GL183 and CD16). The PP35 MAb reacted with a 70 kDa surface molecule expressed on all CD3- NK cells, as well as on TCR gamma/delta + cells and on a small sub-set of TCR alpha/beta + CD8+ lymphocytes. The PP35 MAb induced activation of essentially all NK cells, although clonal analysis revealed quantitative differences in the magnitude of the cytolytic responses elicited in different clones. Finally, the A6/143 MAb reacted with a molecule of 115 kDa expressed by all human PBL. Similarly to 7A6 and PP35 MAbs, the A6/143 MAb activated all sub-sets of cloned NK cells.

Animals↗
PubMed1991

CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta.

The effect of anti-CD69 monoclonal 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 cells 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 cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. 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 gamma/delta+ but not TCR alpha/beta+ 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 alpha/beta+ cytolytic cells.

Animals↗
PubMed1990

Identification of four subsets of human CD3-CD16+ natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition.

In 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 of clone PE29 against the human myelomonocytic leukemia U937. The EB6 mAb (IgG1) 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 less than 2% of EB6+ cells were CD3+). Analysis of resting or activated CD3-CD16+ populations, or clones for the expression of EB6 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, IGROV-I, 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 EB6+GL183- 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 EB6 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Specific recognition of human CD3-CD16+ natural killer cells requires the expression of an autosomic recessive gene on target cells.

We analyzed the recently defined ability of CD3-CD16+ cells to specifically recognize and lyse normal allogeneic target cells (PHA-induced blasts). The susceptibility to lysis by a given alloreactive natural killer (NK) clone ("1 anti-A") was expressed by PHA blasts derived from 9 of 38 random donors analyzed. In all instances, the specific lysis of "susceptible" target cells was greater than 35% while that of "nonsusceptible" targets was less than 6% at an E/T cell ratio of 5:1. In addition to 1 anti-A, A anti-1 specific CD3-CD16+ clones could also be isolated from the reverse MLC combination. The relationship existing between lysis of normal allogeneic cells or tumor cells by the same CD3-CD16+ effector cell has been investigated: 1 anti-A specific CD3-CD16+ clones lysed PHA blasts of three of six cancer patients, while they lysed fresh tumor cells (ovarian carcinoma) from all six patients. The type of inheritance of the character "susceptibility to lysis" was analyzed in representative families. This analysis revealed that the character is inherited in an autosomic recessive fashion, and it is therefore different from MHC. We further investigated the type of segregation of the opposite character "resistance to lysis" (which is inherited in a dominant mode). The finding that this character segregated in all donors expressing given MHC haplotypes indicated that the gene regulating the expression of the NK-defined alloantigen is present on chromosome 6.

Antibodies, Monoclonal↗
PubMed1990

Susceptibility or resistance to lysis by alloreactive natural killer cells is governed by a gene in the human major histocompatibility complex between BF and HLA-B.

The specificity recognized on normal allogeneic cells by a given alloreactive (1-anti-A) natural killer clone is controlled by a gene locus termed EC1. Because the EC1 locus was previously shown to be located on chromosome 6, families characterized by a recombinant major histocompatibility complex haplotype were analyzed to map this locus more precisely. The breakpoint of recombination was studied by standard HLA typing, complement typing, and restriction fragment length polymorphism analysis of a series of genes located between the complement cluster genes and HLA-B within the major histocompatibility complex region. Three of 10 families analyzed were informative. From the data obtained, the EC1 locus maps between BF and HLA-B and presumably is one of the normal genes recently described in this region.

Antigens, Differentiation↗
PubMed1990

Clonal analysis of joint fluid T lymphocytes in patients with juvenile rheumatoid arthritis.

Synovial fluid (SF) lymphocytes from 4 patients with pauciarticular juvenile rheumatoid arthritis (JRA) and 4 patients with polyarticular JRA were examined for their phenotypic and functional characteristics. In all 8 patients there was a high proportion of activated SF T cells, together with an increased proportion of CD2+CD3- and the presence of CD3+CD4-CD8-WT31- lymphocytes. The functional analysis at the clonal level in 5 patients (427 clones) showed a relevant proportion of cytotoxic T cell clones, which were not confined to typically cytolytic phenotypes, but were also present among CD3+CD4+CD8- cultures. Compared to those with pauciarticular JRA, patients with polyarticular disease had a significantly higher proportion of T cell clones with cytotoxic activity. Although derived from a limited number of patients, our data suggest a direct involvement of T cells in the pathogenetic mechanisms that originate and maintain the articular damage, and the possibility of different or more pronounced T cell reactivities in the clinically more diffuse JRA types.

Adolescent↗
PubMed1989

CD16 surface molecules regulate the cytolytic function of CD3CD16+ human natural killer cells.

Monoclonal (IgG) antibodies (MAbs) directed to CD16 molecules efficiently induced lysis of the IgG-binding P815 target cells. A similar effect was observed with selected anti-CD2 MAbs. While combinations of 2 appropriate anti-CD2 MAbs were required for induction of T lymphocyte activation, single stimulatory anti-CD2 MAbs were sufficient for inducing cytolytic function in CD3- CD16+ lymphocytes. In order to study possible regulatory mechanisms existing in the process of activation and induction of the cytolytic machinery of CD3- CD16+ effector cells, we utilized the anti-CD16 OKNK MAb. Being of IgM isotype, the OKNK MAb does not allow cross-linking between CD3- D16+ lymphocytes and target cells. Pre-treatment of effector cells with OKNK MAb sharply inhibited the target cell lysis induced by either anti-CD16 (IgG) MAbs or stimulatory anti-CD2 MAb. Moreover, a strong inhibitory activity of PHA-induced target cell lysis and even of "spontaneous" lysis (at high effector:target ratio) was observed. In contrast, in CD3+ CD16+ clones, OKNK MAb selectively inhibited the cell triggering induced by anti-CD16 MAbs (but not by anti-CD3, anti-CD2 MAbs or PHA). Our data indicate that CD16 receptor molecules expressed by CD3- CD16+ lymphocytes down-regulate cell responses to anti-CD2 MAbs or PHA, and then exert a regulatory role in the cytolytic function of these cells.

Antibodies, Monoclonal↗
PubMed1989

Specificity of human T lymphocytes expressing a gamma/delta T cell antigen receptor. Recognition of a polymorphic determinant of HLA class I molecules by a gamma/delta clone.

Alloreactive clones expressing T cell receptor (TcR) gamma/delta were derived by limiting dilution from CD3+ CD4- CD8- WT31- populations stimulated in allogeneic mixed lymphocyte culture. These clones specifically lysed phytohemagglutinin-induced blast cells bearing the stimulating alloantigens, whereas they had no effect on autologous or allogeneic unrelated target cells. Analysis of the reactivity with monoclonal antibodies (mAb) specific for two different subsets of TcR gamma/delta (BB3 and delta-TCS-1) showed that five out of nine clones were BB3+, whereas the remaining reacted with delta-TCS-1. Therefore, we can conclude that both subsets of TcR gamma/delta+ cells are able to specifically recognize and lyse allogeneic cells. mAb directed against the CD3-TcR gamma/delta molecular complex strongly inhibited the specific cytolytic activity of TcR gamma/delta+ clones, whereas they had no effect on the lysis of the natural killer-sensitive K-562 target cells mediated by the same clones. An alloreactive delta-TCS-1+ clone (LM12) was further characterized for its specificity. LM12 clone had been derived after stimulation in mixed lymphocyte culture against donor M.M. (HLA typing: Aw68, 24; B35, w55; DR1, 7). The analysis of a large panel of phytohemagglutinin-induced target cells revealed that only the HLA-A24+ target cells were lysed. The direct evidence that the A24 molecule represented the restriction element was provided by experiments using A24-transfected murine P815 target cells. Thus, clone LM12 efficiently lysed A24-transfected P815 cells, but not the same cells untransfected or transfected with the Cw3 gene. Therefore, it appears that polymorphic determinants of class I major histocompatibility complex molecules can be the target of TcR gamma/delta+ alloreactive cell recognition.

Antibodies, Monoclonal↗
PubMed1989

Surface molecules involved in CD3-negative NK cell function. A novel molecule which regulates the activation of a subset of human NK cells.

Natural killer cells are characterized by the lack of CD3/TCR molecules and by the expression of CD16 and CD56 (NKHI or Leu19) surface antigens. In addition to their ability to lyse certain tumor target cells, they release lymphokines including tumor necrosis factor and interferon gamma. Another unexpected functional capability of at least some NK cells is the ability to specifically recognize and lyse certain normal allogeneic cells (PHA-induced blasts). MAbs directed to CD2 or to CD16 surface molecules induced triggering of NK cells leading to target cell (p815) lysis in a redirected killing assay. Importantly, different from induction of T cell activation, single anti-CD2 MAbs were sufficient to trigger NK cell function. Another MAb (GL183) inducing NK cell triggering recognized a novel surface molecules expressed on 20-50% of resting or cultured NK cells. Cloned GL183+ cells displayed a variable degree of cytolytic activity against a number of human target cells of different histotype; moreover, this activity was strongly enhanced by the addition of GL183 MAb. On the other hand, GL183 MAb inhibited lysis of murine lines (including P815). Thus on P815 target cells GL183 MAb has an effect antithetical to that of other stimuli including PHA, anti-CD2 or anti-CD16 MAbs. GL183 MAb, added simultaneously to one or another of the stimuli above, strongly inhibited the target cell lysis induced by these stimuli. Thus, GL183 may represent an important molecule in the process of activation/regulation of phenotypically-defined NK cell subsets.

Antibodies, Monoclonal↗