Irreversible inhibition of human natural killer cell natural cytotoxicity by modification of the extracellular membrane by the adenine nucleotide analog 5'-p-(fluorosulfonyl)benzoyl adenosine.

Extracellular adenine nucleotides are inhibitors of the human natural killer cell line NK3.3 natural cytotoxicity activity. Natural cytotoxicity was inhibited approximately 26% by 1 mM ATP and 21% by 1 mM ADP. 5'-Adenylyl imidodiphosphate, a nonhydrolyzable ATP analog, inhibited natural cytotoxicity by 41% at a concentration of 1 mM and > 97% at a concentration of 10 mM. In contrast, AMP was not inhibitory. Adenosine was a weak inhibitor of natural cytotoxicity and may represent an alternate regulatory pathway. Removal of the nucleotides resulted in the restoration of control levels of natural cytotoxicity activity. The affinity label 5'-p-(fluorosulfonyl)benzoyladenosine (5'-FSBA) is a synthetic analog of ATP or ADP containing an electrophilic fluorosulfonyl group capable of covalently modifying proteins at adenine di- and triphosphate nucleotide-binding sites. Natural cytotoxicity was irreversibly inhibited by modification of the extracellular membrane of NK3.3 cells by 5'-FSBA. This inhibition was concentration dependent with an I50 approximately 100 microM and complete inhibition at 1 mM. Modification of NK3.3 by 5'-FSBA did not affect the formation of effector-target cell conjugates; however, granule release was inhibited. This targets the site of inhibition by 5'-FSBA modification to a pathway preceding granule release. Irreversible, covalent modification of surface adenine nucleotide-binding proteins by 5'-FSBA provides a probe to study the role of specific adenine nucleotide-binding proteins in the extracellular regulation of natural killer cytolytic activity by adenine nucleotides.

Recognition of different Schistosoma mansoni antigens by specific human T cell clones.

Human T lymphocyte clones (TLC) sensitized to Schistosoma mansoni soluble egg antigens (SEA) were developed from chronic schistosomiasis mansoni patients in order to investigate the regulatory mechanisms involved with in vitro granulomatous hypersensitivity to this parasite. All clones studied displayed CD4+ phenotype and required antigen-presenting cells in antigen-driven proliferation and granuloma formation. Each T lymphocyte clone has been shown to proliferate and generate in vitro granulomas in response to SEA, adult worm antigens (SWAP), or cercaria antigens (CAP). In contrast, no proliferation was observed in any of these T cell clones when unrelated S. mansoni antigens were used. Some SEA-generated TLC were not able to proliferate in the presence of SEA; however, S. mansoni SEA-reactive ones were able to recognize epitopes in Schistosoma japonicum SEA, indicating cross-reactivity between these two species. Using IFN-gamma ELISA, it was shown that TLC cells stimulated with SEA can secrete appreciable amount of this lymphokine. Further in vitro studies with these SEA-TLC will help us to understand in more depth the role of regulatory T cells in the human granulomatous hypersensitivity to S. mansoni eggs.

Association of the p56lck protein tyrosine kinase with the Fc gamma RIIIA/CD16 complex in human natural killer cells.

The multimeric Fc gamma RIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50-70-kDa transmembrane glycoprotein Fc gamma receptor (CD16), the T cell receptor (TCR)-zeta chain, and the Fc epsilon RI gamma chain. Cross-linking Fc gamma RIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the zeta subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the Fc gamma RIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human Fc gamma RIIIA is complexed with p56lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several Fc gamma RIIIA-associated proteins are identified in immune complex kinase assays including the TCR-zeta subunit, a p70-90 zeta-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56lck is physically associated with Fc gamma RIIIA (CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.

Identification and partial characterization of an ectoATPase expressed by human natural killer cells.

An extracellular membrane-associated ectoATPase has been identified on the human natural killer cell line NK3.3. The enzyme is distinct from other classes of ATPases, kinases, and phosphatases. NK3.3 ectoATPase demonstrated a Km for ATP of 41 microM and a Vmax of 0.2 mumol/min and required both Ca2+ and Mg2+ for maximal activity. Purine and pyrimidine nucleotides were competitive inhibitors of the catalytic reaction. Inhibition increased with the addition of increasing negative charge of the phosphate side chain and was also dependent on contributions from the nucleoside. NK3.3 ectoATPase activity was inhibited by reaction with the affinity label [p-(fluorosulfonyl)benzoyl]-5'-adenosine (5'-FSBA), which is shown to modify the enzyme at or near the ATP-binding domain. Photoaffinity labeling of intact NK3.3 cells with [alpha-32P]-8-azidoATP demonstrated an ATP-binding protein of 68-80 kDa unique to NK3.3 cells. A positive correlation was observed between the ability of the various nucleotides to block photoincorporation into the 68-80-kDa protein and their ability to inhibit ectoATPase activity. NK3.3 cells which were made ectoATPase-deficient by reaction with 5'-FSBA demonstrated that this enzyme does not have a major role in the protection of this cytolytic effector cell from the possible lytic effects of extracellular ATP.

Anti-idiotypic T cells in human schistosomiasis.

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of granulomatous hypersensitivity in patients infected with S. mansoni. Our previous studies have shown that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding antigen-conjugated polyacrylamide beads; and 2) this reactivity is a CD4 T cell dependent, macrophage dependent, B cell independent response which is antigenically specific for parasite egg antigens. We report here on idiotype - anti-idiotype receptor interactions involved in the regulation of granulomatous hypersensitivity in patients and former patients infected with S. mansoni. Five human monoclonal antibodies specific for parasite egg antigens were used to activate CD4 and CD8 T cell subsets. These activated anti-idiotypic T cells were then assayed for regulatory effector functions in an autologous in vitro granuloma model.

Polypeptides immunologically related to band 3 are present in nucleated somatic cells.

Band 3, the major transmembrane polypeptide of erythrocytes, mediates the exchange of anions (chloride and bicarbonate) across the membrane. We suspected that band 3 was present on nucleated somatic cells as well as erythrocytes because the senescent cell antigen that is immunologically related to band 3 is present on lymphocytes, platelets, adult liver cells, and embryonic kidney cells; and antibodies prepared against the senescent cell antigen isolated from leukocytes react with erythrocyte band 3. For this reason, we examined human fibroblasts, lung cells, neutrophils, mononuclear leukocytes, squamous epithelial (mouth) cells, lung squamous epithelial carcinoma, mouse neuroblastoma cells, and rat hepatocytes for immunoreactive forms of band 3 by using monospecific antibodies to erythrocyte band 3. The results demonstrated that polypeptides sharing common antigenic determinants with erythrocyte band 3 are present in nucleated somatic cells as determined by immunofluorescence, immunoelectron microscopy, and immunoautoradiography. Peptide mapping revealed substantial sequence homology between erythrocyte band 3 and the band 3-like protein of leukocytes. Immunofluorescence studies indicate that the band 3-like proteins in nucleated cells participate in antibody-induced cell surface capping.