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L Moretta

Publications and source records attributed to L Moretta.

At least 145 records · Page 8Linked to original sources
PubMed1993

Human CD3-CD16+ natural killer cells express the hGATA-3 T cell transcription factor and an unrearranged 2.3-kb TcR delta transcript.

In this study we analyzed the T cell receptor(TcR) delta transcripts expressed by CD3-CD16+ cells and we investigated whether these cells expressed the hGATA-3 T cell transcription factor and the recombination-activating gene (RAG)-1. Multiple TcR delta transcripts deriving from an unrearranged TcR delta gene were detected in both polyclonal and clonal CD3-CD16+ natural killer(NK) cell lines. Two unrearranged TcR delta transcripts had a size similar to that of the functional TcR delta mRNA (2.3 and 1.3 kb) found in TcR gamma/delta+ T lymphocytes. Sequence analysis of nine different 2.3-kb cDNA clones obtained from NK-derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR delta gene. These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the J delta 1 segment. The sequence located upstream of the J delta 1 segment corresponded to the previously reported germ-line sequence. The J delta 1 segment was correctly spliced to C delta; in addition the four C delta exons were found to be already assembled. Two polyadenylation sites were present in the fourth C delta exon. However, only that located at the 3' end appeared to be utilized in the 2.3-kb cDNA. The expression of hGATA-3, a T cell-specific factor known to be involved in the regulation of the transcription of TcR delta locus, was analyzed by Northern blot, in cultured NK cell population and clones (but not in freshly derived cell populations). All NK clones and cell lines studied were found to express hGATA-3-specific mRNA, suggesting that hGATA-3 may be involved in the regulation of the unrearranged TcR delta gene expression in NK cells. Finally, no transcription of the RAG-1 gene could be detected in all NK cell lines or clones analyzed.

Base Sequence↗
PubMed1993

CD45-mediated regulation of LFA1 function in human natural killer cells. Anti-CD45 monoclonal antibodies inhibit the calcium mobilization induced via LFA1 molecules.

The TA218 and T205 monoclonal antibodies (mAb) were selected on the basis of their ability to inhibit the non-major histocompatibility complex-restricted lysis of the murine mastocytoma P815 cell line mediated by CD3-CD16+ natural killer (NK) cells. Both mAb were found to react with CD45 molecules, as demonstrated by immunoprecipitation after surface iodination and western blot analysis. A panel of tumor target cells susceptible to lysis by polyclonal or clonal CD3-CD16+ NK cells was used to study the mAb-mediated inhibitory effect. The inhibition of cytolysis mediated by TA218 and T205 mAb was found to consistently parallel the inhibition mediated (with the same tumor target cells) by the anti-LFA1 alpha mAb TS.1.22 or by the anti-LFA1 beta mAb TS.1.18. However, different from the anti-LFA1 mAb, T205 or TA218 mAb did not inhibit the binding of activated CD3-CD16+ effector NK cells to the same tumor target cells. This finding supported the concept that the anti-CD45 mAb-mediated inhibition could occur at a post-binding stage. In polyclonal or clonal CD3-CD16+ NK cells T205 or TA218 mAb were found to reduce by 50-70% the intracellular Ca++ ([Ca++]i) mobilization induced by anti-LFA1 alpha or anti-LFA1 beta mAb. On the other hand, TA218 and T205 mAb did not inhibit the Ca++ mobilization induced by anti-CD16 mAb or phytohemagglutinin, thus suggesting that, in NK cells, CD45 molecules may exert a selective inhibitory effect on the signal transduction mediated by LFA1 molecules. In line with this hypothesis, the cytolytic activity of human NK clones was triggered in the presence of the hybridoma cells secreting either anti-CD16 or anti-LFA1 alpha mAb (as "triggering targets"). This effect of anti-LFA1 alpha, but not of anti-CD16 hybridoma was susceptible to inhibition by the anti-CD45 mAb T205 or TA218. Further, experiments on cloned NK cells indicated that T205 or TA218 mAb induced a strong decrease in the constitutive phosphorylation of the LFA1 alpha chain (but not of HLA class I antigens). Taken together, these studies suggest that in human NK lymphocytes, CD45 molecule may regulate both the activation state and the function of the LFA1 molecule.

Animals↗
PubMed1993

Use of anti-CD3 and anti-CD16 bispecific monoclonal antibodies for the targeting of T and NK cells against tumor cells.

To target T lymphocytes against EGF-R+ tumors, we constructed anti-CD3/anti-EGF-R bimAbs either by the generation of a hybrid hybridoma (quadroma) or by a chemical cross-linking method. Analysis of the in vitro functional activity of these two different constructs indicated that the quadroma-secreted bimAb was more efficient in targeting the CD3+8+ clones against EGF-R+ target cells with respect to the bimAb produced by chemical method. In addition, the quadroma-produced bimAb is able to induce cytolysis of EGF-R+ tumor cell lines of PHA-induced lymphoblasts that had been expanded in IL-2-containing medium, whereas tumor cells lacking expression of EGF-R were not lysed. Resting PBL targeted by the bimAb did not display significant cytotoxicity against the relevant tumor. An anti-CD16 hybridoma (IgG1) was fused with an anti-folate-binding protein hybrid (IgG2a) to construct bimAbs to target NK cells against NK-resistant ovarian carcinomas. The hybrid IgG1/IgG2a bimAb triggered the specific lysis of relevant target cells by resting NK cells, but it was ineffective when CD8+TCR alpha/beta+ cultured cell populations were used as effectors. Only marginal increases of cytolytic activity could be induced by the bimAb when IL-2-activated PBL (i.e., LAK cells) were used as effectors due to the high cytolytic activity of these cells against the relevant tumors in the absence of bimAb. The possible use of anti-CD16 or anti-CD3 bimAbs for the development of different cellular immunotherapy strategies against cancer is discussed.

Antibodies, Bispecific↗
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

Functional and morphologic characterization of human T lymphocytes expressing the TCR gamma/delta.

A minor subset of T lymphocytes express a TCR composed of gamma and delta chains. This subset differs from conventional T cells for a number of phenotypic and functional characteristics. TCR gamma/delta+ cells simultaneously lack both CD4 and CD8 antigens. Cloning of CD4-8- peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which efficiently lyse tumor target cells. Formal proofs have been provided that TCR gamma/delta+ cells are able to recognize antigens. For example, they proliferated in response to allogeneic mixed lymphocyte culture (MLC); in addition, MLC-derived TCR gamma/delta+ cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The selection of monoclonal antibodies specific for TCR gamma/delta molecules allowed to identify two distinct subsets of TCR gamma/delta+ cells. Both of these mABs, termed BB3 and delta TCS-1 respectively, induced specific activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca++ and/or lymphokine production or cytolytic activity). Analysis of the distribution of subsets expressing different forms of TCR gamma/delta, showed that the BB3-reactive form is prevalent in the peripheral blood. In contrast, delta-TCS-1-reactive cells are relatively infrequent in peripheral blood but represent the majority of TCR gamma/delta+ cells in tissues.

Humans↗
PubMed1992

Specific functions of human NK cells.

Since natural killer (NK) cells lack both CD3/TCR molecules and surface Ig, it is generally thought that they are unable to recognize antigens. However, CD3-CD16+ cells were found to respond in MLC against irradiated allogeneic mononuclear cells and to lyse normal PHA blasts derived from the stimulating donor, but not autologous cells or cells derived from most allogeneic donors. A similar pattern was obtained with cloned NK cells, thus indicating that the ability to specifically recognize given normal allogeneic cells is a clonally distributed function. Moreover, analysis of NK clones for their ability to lyse either tumor cells or normal PHA blasts (both derived from individual cancer patients) indicated that the two phenomena are distinct. Analysis of a large number of NK clones derived from a given individual for their ability to lyse a panel of allogeneic donors allowed the identification of at least four groups of clones characterized by unique patterns of reactivity ("specificities"). We further studied the mode of inheritance of the various NK-defined specificities: all the characters "susceptibility to lysis" by NK clones (displaying one or another specificity) segregated independently, were inherited in an autosomic recessive manner and were carried by chromosome 6. The finding of clonally distributed specific functions in NK cells suggested the existence of clonally distributed receptor molecules. Along this line mAbs were raised against NK clones, and screened for their ability to trigger the immunizing clones: two mAbs (termed GL183 and EB6) were directed against a novel family of 58-kDa surface molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Clone Cells↗
PubMed1992

Allorecognition by NK cells: nonself or no self?

The issue of antigen recognition by NK cells is complex, fascinating and, as yet, unresolved. This article reviews recent research on the repertoire of human NK cell clones for the recognition of different allogeneic cells, and summarizes the studies, most of which have been performed in mice, that implicate the MHC in NK cell recognition. It goes on to provide a common conceptual framework within which these different systems may be understood.

Animals↗
PubMed1992

Selective increase of a subset of T cell receptor gamma delta T lymphocytes in the peripheral blood of patients with human immunodeficiency virus type 1 infection.

Increased proportions of the small lymphoid subset expressing T cell receptor (TCR) gamma delta occur in different infectious diseases, particularly in mycobacterial infections. In this study the two main subsets of TCR gamma delta+ cells in peripheral blood mononuclear cells (PBMC) of 54 patients with human immunodeficiency virus type 1 infection were analyzed. These subsets were defined by indirect immunofluorescence techniques and FACS analysis using BB3 and A13 monoclonal antibodies, which specifically react with V delta 2- and V delta 1-encoded forms of TCR gamma delta. The proportion of BB3+(V delta 2+) and A13+(V delta 1+) cells was analyzed in purified PBMC populations. Patients were stratified according to Walter Reed (WR) clinical stage. A sharp increase in percentage of A13+(V delta 1+) cells was observed in all stages of the disease. In addition, a strict correlation was found with stage of the disease and percentage of CD8+ PBMC. An inverse correlation was found with the proportion of CD4+ PBMC. An early (WR2) inversion of the V delta 2-to-V delta 1 ratio was consistently detected even before the inversion of the CD4-to-CD8 ratio.

CD4 Antigens↗
PubMed1992

Targeting of T or NK lymphocytes against tumor cells by bispecific monoclonal antibodies: role of different triggering molecules.

MAbs directed against triggering surface molecules expressed by T lymphocytes (CD3, TCR, CD2, CD28) or by NK cells (CD2, CD16) are able to induce the functional program of these cells. These MAbs represent suitable reagents to construct biMAbs directed against TAA, in order to specifically target effector lymphocytes against tumor cells. Anti-CD3/anti-EGF-R biMAbs were constructed to specifically direct T lymphocytes against EGF-R+ tumor cells. Such biMAb are able to induce cytolysis of EGF-R+ tumor cell lines (A431, IGROV, KATO-III and U-87) by cytolytic CD3+ effector lymphocytes while tumor cells having low or absent expression of EGF-R were not lysed. In addition, both cytolytic T (CD8+) cells and non-cytolytic (CD4+) IL-2-expanded lymphocytes were able to secrete lymphokines upon contact with EGF-R+ tumor cells. To target NK cells against NK resistant ovarian carcinomas, we used an anti-CD16 Mab (IgG1) together with an anti-ovarian carcinoma MAb (IgG2a), to construct biMAbs using the hybrid hybridoma technique. The hybrid IgG1/IgG2a biMAb triggered the specific lysis of relevant target cells by resting NK cells and by a subset of NK clones. In addition, some TCR gamma/delta+ clones but not TCR alpha/beta+ clones could be targeted by the biMAb.

Antibodies, Monoclonal↗
PubMed1992

Possible targets on carcinoma for bMAb retargeting of lymphocyte or drug cytotoxicity.

Retargeting of drugs or lymphocyte cytotoxicity through bi-specific monoclonal antibodies (bMAbs) has been proven a therapeutic tool against human carcinoma both in pre-clinical in vitro and in vivo studies. Some of these reagents have already been introduced into clinical trials and preliminary results appear to be promising. However, improvement of the specificity of this approach could be achieved by selecting more suitable target molecules on tumor cells. The research focused on developing MAbs directed against molecules with tumor-restricted distribution and homogeneous expression. Cell-membrane receptors for nutrients or growth factors which operationally represent tumor-specific molecules due to their overexpression, could be considered appropriate targets. Several bMAb anti-nutrients (the folate-binding protein) or growth-factor receptor (c-erbB1, c-erbB2)/anti-triggering molecules have been generated and all were able to efficiently retarget the relevant population of lymphocytes on tumor cells. In order to design a more reliable and selective therapeutic tool, the following parameters were analyzed: correlation between cytotoxicity and antigen level, potency and possible modulation of the target molecule.

Antibodies, Monoclonal↗
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↗
PubMed1991

In vitro proliferation and cloning of CD3- CD16+ cells from human thymocyte precursors.

Purified CD3-4- thymocytes were obtained by depletion of CD3+ and CD4+ cells from fresh thymocyte suspensions. 5-15% of these cells were found to express CD16 antigen, while other natural killer (NK) cell markers were virtually absent. Double fluorescence analysis revealed that 20-40% of thymic CD16+ cells coexpressed CD1, while approximately half were cyCD3+. When cultured in the presence of peripheral blood lymphocytes and H9 leukemia cell line as a source of irradiated feeder cells and interleukin 2 (IL-2), CD3-4- thymocytes underwent extensive proliferation. In addition, after 1-2 wk of culture, 30-50% of these cells were found to express CD16 surface antigen. Cloning under limiting dilution conditions of either CD3-4- or CD3-4-16- thymocytes in the presence of irradiated H9 cells resulted in large proportions (approximately 50%) of CD16+ clones. On the basis of the expression of surface CD16 and/or cyCD3 antigen, clones could be grouped in the following subsets: CD16+ cyCD3+; CD16+ cyCD3-; CD16- cyCD3+; and CD16- cyCD3-. All clones expressed CD56 surface antigen, displayed a strong cytolytic activity against NK sensitive (K562) and NK-resistant (M14) target cells, and produced IFN-gamma and tumor necrosis factor, but not IL-2. Similar to peripheral NK cells, thymic CD16+ cells expressed transcripts for CD16 and for CD3 epsilon (Biassoni, R., S. Ferrini, I. Prigione, A. Moretta, and E.O. Long, 1988. J. Immunol. 140:1685.) and zeta chains (Anderson, P., M. Caligiuri, J. Ritz, and S.F. Schlossman. 1989. Nature [Lond.]. 341:159). Therefore, it appears that cells that are phenotypically and functionally similar to CD3- CD16+ NK cells may arise from immature thymocytes.

Antigens, CD↗