Platelet-mediated cytotoxicity. Role of antibody and C3, and localization of the cytotoxic system in membranes.

Platelets are one of several cell types capable of mediating antibody-dependent cellular cytotoxicity. We have developed a plasma-free system in which washed mouse platelets lyse washed antibody and complement-sensitized SRBC targets in the presence of EDTA. The dose-response curve is concave to the abscissa, indicating that lysis is a one-hit reaction. Determination of the actual number of platelets required to lyse a target shows that each platelet could lyse a single target. A limited degree of lysis is observed when platelets are incubated with SRBC sensitized with monoclonal IgG2a alone, but no lysis occurs with SRBC bearing comparable amounts of other isotypes. In the presence of C1 through C3, but not C1 through C2, efficient lysis is triggered by complement-fixing monoclonal IgG2a, IgG2b, and IgG3. In contrast, IgM and non-complement-fixing IgG1 and IgE are inactive. To achieve efficient lysis, it appears that platelets require both target cell-bound antibody and C3 fragments in close proximity. It is unlikely that proteases, pore-forming proteins, or toxic oxygen metabolites are involved in platelet-mediated lysis. Freezing and thawing of platelets, sonication, or sonication followed by hypotonic shock causes severe depletion of cytoplasmic and granular contents, as shown by electron microscopy and marker assays. However, the membrane fraction of these preparations retains cytolytic activity. When platelets are treated with trypsin or heated, lytic activity is eliminated, indicating that at least one component of this system is protein. These findings, as well as the fact that platelets do not lyse unsensitized innocent bystander SRBC, suggest that the complete cytotoxic system of platelets capable of specific recognition and lysis resides in their membranes.

A requirement for membrane-associated phospholipase A2 in platelet cytotoxicity activated by receptors for immunoglobulin G and complement.

Platelets are potent antibody- and complement-dependent cytotoxic effector cells. We showed previously that a single platelet can lyse a target cell sensitized with immunoglobulin G (IgG) and complement components up to C3 (C integral of 3b denotes the target cell-bound fragment of complement up to C3; the precise nature of the bound C3 fragment has not been established), and that the complete cytotoxic system capable of specific recognition and lysis resides in platelet membranes. To define the components of platelet membranes required for cytotoxicity, a set of inhibitors of phospholipase A2 (PLA2) that act by different chemical mechanisms was tested. The lytic reaction is blocked at appropriate concentrations of bromophenacylbromide, mepacrine, and manoalide. When platelets are treated with bromophenacylbromide, inhibition of cytolytic activity and that of PLA2 enzymatic activity occur in parallel. Platelets release arachidonate when incubated with target cells bearing IgG and C integral of 3b, confirming that Fc gamma R and complement receptor trigger both PLA2 action and efficient lysis. Inhibition by thimerosal of a reverse reaction, i.e., reacylation catalyzed by acyltransferase, causes increased target cell lysis, presumably by increasing the products of PLA2 action. Platelet cytotoxicity is increased when platelets are pretreated with some products of PLA2: exogenous lysophospholipids and not free arachidonic acid increase cytotoxicity. Electron microscopy suggests that platelets and target cells may fuse, possibly as a result of the formation of lysophospholipids which are well-known membrane fusogens. Fixation with paraformaldehyde does not affect platelet cytotoxicity, suggesting that the complete cytotoxic system resides as a preformed complex in platelet membranes. The results indicate that platelet membrane-associated PLA2, together with receptors for Fc and complement, are required for platelet cytotoxicity.

Inhibition or activation of human T cell receptor transfectants is controlled by defined, soluble antigen arrays.

We present evidence that direct T cell receptor (TCR) occupancy by antigen can either activate or inhibit T cells, depending upon whether or not a threshold number of local TCRs are crosslinked by multivalent arrays of the antigen. Variants of Jurkat cells were previously transfected with TCR alpha and beta chains that bind fluorescein, yielding FL-TCR+ human T cells. The transfectants are activated upon binding soluble multivalent antigen arrays at concentrations well below those required for monovalent interactions. This activation, measured by calcium fluxes and interleukin 2 (IL-2) production, indicates the superior binding avidity of multivalent ligands. Smaller, less multivalent arrays do not activate the cells, but antagonize larger arrays, demonstrating that antigen can bind TCR as either agonist or antagonist. The balance between activation and inhibition depends upon antigen array size, ligand valence, and concentration, indicating that a threshold extent of receptor crosslinking, and not individual perturbations of single TCR, is required for activation by antigen. Approximately 100 stimulatory arrays specifically bind per FL-TCR+ cell at concentrations where IL-2 production is half-maximal.

Spectrin immunofluorescence distinguishes a population of naturally capped lymphocytes in situ.

Immunofluorescence analysis of mammalian lymphocytes using antiserum directed against chicken erythrocyte alpha-spectrin revealed a lymphocyte population in which spectrin antigen was arranged in the form of a discrete cap (hereafter referred to as capped lymphocytes). This subset could be easily distinguished from other lymphocytes in which the spectrin antigen was diffusely distributed near the plasma membrane (noncapped lymphocytes). The subset of capped lymphocytes could be visualized in situ and in isolated cells in the absence of added ligand. Using frozen sections of lymphoid organs that were fixed in formaldehyde prior to the immunofluorescence procedure, capped lymphocytes were found in characteristic locations depending on the tissue examined. In the thymus, the major population of medullary lymphocytes were capped whereas cortical lymphocytes were mostly noncapped. In Peyer's patches, capped lymphocytes were interspersed with noncapped lymphocytes throughout the tissue. In the spleen, capped lymphocytes were concentrated in the periarterial lymphoid sheath of the white pulp and in lymph nodes they were found predominantly in the paracortical and cortical regions. Capped lymphocytes were not visible in the thymus until just before birth and did not appear in the spleen until 3 d after birth. When lymphocytes were isolated from lymphoid organs, fixed in formaldehyde and prepared for immunofluorescence, capped and noncapped lymphocytes were still identifiable and present in the same relative proportions as seen in situ. Results identical to those described above are obtained using antisera directed against guinea pig fodrin. Natural capping of proteins previously shown to co-migrate with a variety of cell surface macromolecules after cross-linking may be a new means of identifying various stages of lymphocyte activation or differentiation.

Evidence for a causal association between human papillomavirus and a subset of head and neck cancers.

BACKGROUND: High-risk human papillomaviruses (HPVs) are etiologic agents for anogenital tract cancers and have been detected in head and neck squamous cell carcinomas (HNSCCs). We investigated, retrospectively, an etiologic role for HPVs in a large series of patients with HNSCC. METHODS: Tumor tissues from 253 patients with newly diagnosed or recurrent HNSCC were tested for the presence of HPV genome by use of polymerase chain reaction (PCR)-based assays, Southern blot hybridization, and in situ hybridization. The viral E6 coding region was sequenced to confirm the presence of tumor-specific viral isolates. Exons 5-9 of the TP53 gene were sequenced from 166 specimens. The hazard of death from HNSCC in patients with and without HPV-positive tumors was determined by proportional hazards regression analysis. RESULTS: HPV was detected in 62 (25%) of 253 cases (95% confidence interval [CI] = 19%-30%). High-risk, tumorigenic type HPV16 was identified in 90% of the HPV-positive tumors. HPV16 was localized specifically by in situ hybridization within the nuclei of cancer cells in preinvasive, invasive, and lymph node disease. Southern blot hybridization patterns were consistent with viral integration. Poor tumor grade (odds ratio [OR] = 2.4; 95% CI = 1.2- 4.9) and oropharyngeal site (OR = 6.2; 95% CI = 3.1-12.1) independently increased the probability of HPV presence. As compared with HPV-negative oropharyngeal cancers, HPV-positive oropharyngeal cancers were less likely to occur among moderate to heavy drinkers (OR = 0.17; 95% CI = 0.05-0.61) and smokers (OR = 0.16; 95% CI = 0.02-1.4), had a characteristic basaloid morphology (OR = 18.7; 95% CI = 2.1-167), were less likely to have TP53 mutations (OR = 0.06; 95% CI = 0.01-0. 36), and had improved disease-specific survival (hazard ratio [HR] = 0.26; 95% CI = 0.07-0.98). After adjustment for the presence of lymph node disease (HR = 2.3; 95% CI = 1.4- 3.8), heavy alcohol consumption (HR = 2.6; 95% CI = 1.4-4.7), and age greater than 60 years old (HR = 1.4; 95% CI = 0.8-2.3), all patients with HPV-positive tumors had a 59% reduction in risk of death from cancer when compared with HPV-negative HNSCC patients (HR = 0.41; 95% CI = 0.20-0.88). CONCLUSIONS: These data extend recent molecular and epidemiologic studies and strongly suggest that HPV-positive oropharyngeal cancers comprise a distinct molecular, clinical, and pathologic disease entity that is likely causally associated with HPV infection and that has a markedly improved prognosis.

Low molecular weight antigen arrays delete high affinity memory B cells without affecting specific T-cell help.

An ongoing, T-cell dependent, secondary antibody response to an epitope can be suppressed in vivo by low molecular weight, soluble polymers, bearing multiple copies of the same epitope. This study illustrates that such suppressive T-cell independent antigen arrays target the epitope-specific, high affinity, memory B cells for long-term functional elimination. Splenocytes from hyperimmune unsuppressed donors, when adoptively transferred into irradiated recipients will readily reconstitute a secondary anti-hapten response after antigenic challenge. No such response was observed with splenocytes transferred from hyperimmune donors suppressed with antigen arrays. The extent of suppression depended on antigen array dose and duration of exposure in the donor animals. The suppressive antigen array carryover from the donors into the recipients was negligible and insufficient to account for the observed suppression. B cells from hyperimmune mice producing high affinity anti-fluorescein antibodies, generated by multiple fluoresceinated ovalbumin (FL-OVA) injections, were helped efficiently by T cells from hyperimmune donors, which were either unsuppressed or suppressed with antigen arrays. Accordingly, help from T cells, specific for the carrier protein remains intact after such suppression. Neither lipopolysaccharide (LPS), nor additional transferred carrier-primed T cells could reverse the unresponsiveness of adoptively transferred splenocytes from suppressed animals. Flow cytometry showed that the number of hapten-specific B cells was markedly reduced after suppression. Collectively, these data show that the long term elimination of an ongoing T-cell dependent antibody response by suppressive exogenous antigen arrays is due to the functional deletion of high affinity, antigen-specific B cells, even in the presence of adequate T-cell help. The long-term nature of such functional deletion strongly suggests physical deletion of the antigen-specific B cell population.

Durable elimination of high affinity, T cell-dependent antibodies by low molecular weight antigen arrays in vivo.

Ongoing Ab responses to a T cell-dependent Ag can be suppressed in hyperimmune animals by exogenous, multivalent Ag arrays. The pharmacologic basis for this suppression was studied by varying the molecular mass, ligand valence, and dose of Ag arrays, and then determining their efficacy, pharmacokinetics, and tissue distribution. Arrays ranging in molecular mass from 30 to 500 kDa caused initial clearance of specific serum Abs, but only the smaller arrays caused persistent suppression despite their relatively lower binding avidity and shorter retention in vivo. Suppression by the smaller arrays at lower doses was biphasic, implying two distinct modes of Ab elimination. High affinity IgG was eliminated preferentially, as shown by calibrated variable ligand-density ELISA. Suppressive arrays were localized discretely in the splenic germinal centers of hyperimmune animals. These results indicate that Ag array mass, ligand valence, and dose all play critical roles, and histologic compartmentalization may also be a pertinent parameter, in determining suppressive efficacy in vivo.

Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: involvement of a phosphatidylcholine-hydrolyzing phospholipase C.

We have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine [Hoffman, R. D., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3285-3289]. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using [14C]glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis.

A comparison of the immunogenicity of a pair of enantiomeric proteins.

The immunogenicity of a folded, all D-amino acid protein, rubredoxin, has been compared with that for the corresponding L-protein enantiomer. Following multiple administrations with alum adjuvant, the L-protein induced a strong, specific IgG antibody response, whereas the D-protein did not. This relative lack of responsiveness to the D-protein cannot be attributed to rapid excretion, since it is retained at least 4 times longer than the natural L-protein. These observations provide the first direct evidence that a folded D-amino acid protein has low immunogenicity and is long lived in vivo. Proteins with such properties may be useful as molecular platforms in a variety of chemical and pharmacological applications.