Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells.

Natural killer (NK) cells destroy virus-infected and tumour cells, apparently without the need for previous antigen stimulation. In part, target cells are recognized by their diminished expression of major histocompatibility complex (MHC) class I molecules, which normally interact with inhibitory receptors on the NK cell surface. NK cells also express triggering receptors that are specific for non-MHC ligands; but the nature of the ligands recognized on target cells is undefined. NKp46 is thought to be the main activating receptor for human NK cells. Here we show that a soluble NKp46-immunoglobulin fusion protein binds to both the haemagglutinin of influenza virus and the haemagglutinin-neuraminidase of parainfluenza virus. In a substantial subset of NK cells, recognition by NKp46 is required to lyse cells expressing the corresponding viral glycoproteins. The binding requires the sialylation of NKp46 oligosaccharides, which is consistent with the known sialic binding capacity of the viral glycoproteins. These findings indicate how NKp46-expressing NK cells may recognize target cells infected by influenza or parainfluenza without the decreased expression of target-cell MHC class I protein.

Soluble HLA proteins with bound peptides are released from the cell surface by the membrane metalloproteinase.

The metalloproteinase (MPase)-mediated pathway of MHC class I processing is a distinct cellular mechanism that generates soluble HLA proteins. It has been implicated in modulation of immune responses induced during transplantation events. It is, therefore, important to define the characteristics of soluble HLA species produced by the MPase pathway. We have previously shown that some mutant peptide-conformed beta(2)-microglobulin (beta(2)m) free heavy chains (HC) with lower affinity for beta(2)m can be released into supernatants by the MPase. These soluble conformed beta(2)m-free HC intermediates can re-associate with beta(2)m in solution giving rise to beta(2)m-associated HC. We now demonstrate that also nonmutant soluble conformed beta(2)m-free HC can be detected in supernatants of activated cells. These soluble HC intermediates appear to have bound peptides and readily re-associate with exogenous beta(2)m producing beta(2)m-associated HC that are stable at physiologic temperature. Thus, generation of peptide-conformed beta(2)m-free HC intermediates is an important step, which precedes generation of both soluble beta(2)m-free and beta(2)m-associated HC by the MPase pathway operating in activated cells.

A novel 26 kilodalton antigen expressed on the surface membrane of activated T cells.

We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) 1B10, raised against an activated gammadelta T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb 1B10 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B10+. 1B10 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) alphabeta+ and gammadelta+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were 1B10+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb 1B10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb 1B10 from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb 1B10 recognizes a novel <> appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.

Peptide-conformed beta2m-free class I heavy chains are intermediates in generation of soluble HLA by the membrane-bound metalloproteinase.

Molecular mechanisms of soluble HLA-release by a membrane-bound metalloproteinase (MPase) are not defined. We have investigated the possibility that certain beta2-microglobulin (beta2m)-free heavy chains (HC) retain peptide-induced conformations before and after the cleavage by using mutant HLA-A2.242K HC with reduced affinity for beta2m. We show that dissociation of HC/beta2m complexes on the surface of C1R lymphoblastoid cells generates both conformed and non-conformed beta2m-free HC recognized by conformation-dependent antibodies. Conformed HC, having bound the HLA-A2-specific peptide HTLV-1 tax 11-19, can retain their proper conformations after dissociation of beta2m. Further, conformed and non-conformed surface beta2m-free HC are cleaved by the MPase, and some released HC preserve their conformations. Exogenous beta2m binds only to conformed HC, and protects them from cleavage as effectively as the MPase inhibitor BB-2116. We propose that soluble HLA-release requires generation of peptide-conformed beta2m-free HC intermediates on the cell surface, which are then cleaved by the MPase and in solution may reassociate with beta2m. Given the role of soluble HLA in the indirect allorecognition, the activity of this MPase may be important in transplant rejection.

The metalloproteinase-mediated pathway is essential for generation of soluble HLA class I proteins by activated cells in vitro: proposed mechanism for soluble HLA release in transplant rejection.

We and others have found donor-derived soluble beta2m-associated HLA class I proteins (sHLA/beta2m) in the serum of allograft recipients with acute and chronic rejection. Whether appearance of sHLA/beta2m and upregulated expression of donor cell-bound HLA/beta2m during allograft rejection are related events is unknown. Activation-induced upregulation of in vitro HLA/beta2m expression correlates with the surface expression of another form of HLA class I, namely beta2m-free HLA heavy chains (beta2m-free HC). We have shown that beta2m-free HC, but not beta2m-associated HC, are then cleaved by a specific membrane-bound metalloproteinase and released into supernatants as soluble 36 kDa proteins. We show now that activated peripheral blood lymphocytes produce predominantly the 36 kDa form of sHLA proteins which is present in supernatants as both beta2m-free HC and sHLA/beta2m. Importantly, the metalloprotease inhibitor BB-94 blocked not only the release of soluble beta2m-free HC, but also the appearance of sHLA/beta2m in cell supernatants. Low levels of 36 kDa beta2m-free HC were also present in human plasma of healthy donors. These data suggest an important role for the HLA class I-specific metalloproteinase in vivo in healthy individuals and during allograft rejection in the generation of soluble beta2m-free and beta2m-associated HLA proteins.

Soluble CD4 induces the binding of human immunodeficiency virus type 1 to cells via the V3 loop of glycoprotein 120 and specific sites in glycoprotein 41.

We have previously reported that incubation of human immunodeficiency virus type 1 (HIV-1) at 4 degrees C with soluble CD4 (sCD4) does not block but increases the binding of virions to CD4-positive H9 cells. In this study, we investigated the mechanism of this effect. It appears that sCD4 can induce the binding of HIV-1IIIB to CD4-negative human cells and to H9 cells with downregulated expression of CD4 at both 4 and 37 degrees C. The binding is proportional to the amount of sCD4 associated with virions, and requires the presence of heparan sulfate proteoglycans on the surface of cells. Monoclonal antibody (MAb) 9284 directed at an epitope overlapping with a putative heparin binding motif in the V3 loop of gp120 almost completely blocked the sCD4-induced binding of virions, while MAbs recognizing other sites of V2 or V3 loops had no effect. The binding of sCD4-coated virions to cells was also inhibited by MAbs 50-69 and 98-6 directed at extracellular epitopes of gp41, whose exposure is increased on binding of sCD4 to virions. Therefore, sCD4 potentiates the binding of HIV-1IIIB virions to cells by inducing conformational changes that enable envelope gp120 and gp41 to interact with cell surface components other than the CD4 receptor.

Bathophenanthroline disulfonate and soluble CD4 as probes for early events of HIV type 1 entry.

We report here that a metalloprotease inhibitor, bathophenanthroline disulfonate (Bphe-ds), neutralizes both laboratory-adapted and primary strains of HIV-1. Presaturation of Bphe-ds with zinc does not alter its neutralizing activity, suggesting that the metal-chelating ability of Bphe-ds is not required for neutralization. Bphe-ds blocks infection of CD4+ cells at the stage of viral entry, not through a direct viricidal effect, but by interfering with both binding and postbinding events. This drug interacts with HIV-1 envelope, blocking almost completely the binding of three MAbs that recognize epitopes overlapping the CD4-binding site on gp120, but has no effect on the binding of MAbs directed to the cellular receptor CD4. The exposure of epitopes in the V2 and V3 but not C5 domains of gp120 is partially decreased in the presence of Bphe-ds, suggesting that the drug induces conformational changes in the envelope glycoprotein(s). Binding of both virions and soluble gp120 to CD4+ cells is inhibited by this drug in a dose-dependent manner. This contrasted with the effects of soluble CD4, which actually increased binding of virions to cells at 4 degrees C, while inhibiting the binding of soluble gp120. Bphe-ds also increases shedding of gp120 from cells infected with HIV-1IIIB. Thus, Bphe-ds appears to be an envelope-directed inhibitor of HIV-1 that neutralizes HIV-1 infectivity via multiple mechanisms.

Clustering of class I HLA molecules on the surfaces of activated and transformed human cells.

We surveyed cells for the clusters of class I HLA molecules, HLA-I, which we have previously found on JY lymphoblasts. Two fluorescence techniques, fluorescence resonance energy transfer and electron exchange quenching, detected clustered HLA-I molecules on activated normal B and T cells, on cells of B and T lymphoblast lines, and on transformed fibroblasts. No HLA-I clusters were detectable in the surfaces of resting B or T cells or normal fibroblasts. HLA clustering correlates perfectly with the presence of the HC-10 epitope of beta 2-microglobulin (beta 2m)-free heavy chains at the cell surface although not with the amount of this epitope expressed. Clustering was reversed by exogenous beta 2m, but this did not change the amount of HC-10 bound. This suggests that a form of beta 2m-free heavy chain in equilibrium with both native HLA molecules and fully denatured HC-10-positive heavy chains is involved in HLA-I cluster formation.

Soluble beta 2-microglobulin-free class I heavy chains are released from the surface of activated and leukemia cells by a metalloprotease.

Regulation of the expression of major histocompatibility complex (MHC) class I heavy chains not associated with beta 2-microglobulin (beta 2m) on freshly isolated and in vitro cultured human B and T leukemia cells was analyzed. These beta 2m-free class I heavy chains originate from surface beta 2m-associated MHC class I molecules and are expressed as integral membrane glycoproteins on activated, but not resting, cells. We found that the levels of beta 2m-free class I heavy chains can be regulated by proteolytic cleavage and release into the medium of soluble molecules containing the extracellular domains. The release is mediated by a Zn(2+)-dependent, membrane-bound metalloprotease that does not cleave HLA-DR, CD4, and CD71 surface receptors and can be activated by phorbol myristate acetate. Specific cleavage by the metalloprotease occurs at a site close to the papain cleavage site in the alpha 3 domain of class I heavy chains. This site is not accessible to the metalloprotease in beta 2m-associated MHC class I molecules. The dissociation of beta 2m-associated MHC class I molecules and subsequent cleavage of beta 2m-free class I heavy chains may be partially responsible for controlling the levels of MHC class I molecules on the surface of activated cells.

CD8 and beta 2-microglobulin-free MHC class I molecules in T cell immunoregulation.

Intracellular assembly of MHC class I heavy chains with beta 2-microglobulin occurs prior to the expression of the antigen-presenting complex on the cell surface. The association of beta 2-microglobulin with newly synthesized class I heavy chains is thought to be a strict prerequisite for their transport to the cell surface. However, MHC class I molecules not associated with beta 2-microglobulin (beta 2-microglobulin-free class I heavy chains) have been detected on the surface of activated lymphoid cells. These molecules have different conformations. Therefore, their interactions with other membrane proteins and biological functions may be different from those assigned to beta 2-microglobulin-associated MHC class I molecules. The two forms of MHC class I molecules on the surface of activated cells can self-associate and also form complexes with distinct proteins. Upon interaction with the appropriate ligands these molecular complexes transduce signals regulating cell activation. The ligand for beta 2-microglobulin-free class I heavy chains appears to be soluble CD8. A model is presented describing a novel mechanism of immunoregulation mediated by both soluble and membrane-bound forms of CD8 and beta 2-microglobulin-free class I heavy chains.

The origin and fate of beta 2m-free MHC class I molecules induced on activated T cells.

We report here that the expression of major histocompatibility complex (MHC) class I heavy chains not associated with beta 2-microglobulin is induced on resting human T cells by a variety of stimuli. These beta 2m-free class I heavy chains are not transported as such from the endoplasmic reticulum but originate from surface beta 2m-associated MHC class I molecules. beta 2m-free class I heavy chains are spontaneously released from the surface of activated cells. Cross-linking of beta 2m-free class I heavy chains with specific monoclonal antibodies results in the rapid down-regulation and internalization of these molecules. In contrast, beta 2m-associated MHC class I molecules display a different pattern of modulation. Previously, we reported that beta 2m-free class I heavy chains interact with CD8 molecules expressed on the same activated T cells. We propose that interactions between these molecules are involved in a mechanism regulating the function of activated T cells.

Activation of human CD8-positive T cells via the CD8/HLA class I complex.

Cross-linking of CD8 and HLA class I molecules with appropriate monoclonal antibodies (mAb) and goat anti-mouse Ig (GaMIg) antibody resulted in a marked proliferation of resting human CD8 cells in the presence of interleukin-2 (IL-2). These cells also expressed IL-2 receptor (IL-2R), transferrin receptor, HLA-DR and -DQ antigens. Activation of the cross-linked CD8 cells is apparently independent of accessory monocytes. Various anti-CD8 and anti-HLA class I mAb recognizing nonpolymorphic antigenic determinants were examined for the efficacy of activating CD8 cells. Among mAb specific for HLA class I molecules, PA2.6, MB40.5, BB7.7, A1.4, and W6/32 mAb markedly stimulated the proliferation of cross-linked CD8 cells, whereas BBM.1, Q1/28, and HC10 mAb were found inactive. Footprinting analysis of HLA class I molecules suggested that the activity of these anti-HLA class I mAb appeared to be related to the corresponding peptides they protect from enzymatic digestion. In contrast to the anti-HLA class I mAb, all anti-CD8 mAb examined (C8, OKT8A, and anti-Leu-2a) induced the proliferation of CD8-HLA class I cross-linked cells with similar efficacy. These results suggest that physical interaction between CD8 and at least one specific region of HLA class I molecules can trigger the activation of resting human CD8 cells.

Characterization of the thymus leukemia (TL) product encoded by the BALB/c T3c gene by DNA-mediated gene transfer. Comparison to the T13c product and BALB/c leukemia TL.

Using DNA-mediated gene transfer, we have studied the TL protein products encoded by both the T3c and T13c BALB/c genes. Biochemically, the proteins differed in their m.w. and pI points; serologically, although both molecules were recognized by TL alloantiserum, only the T13c protein was recognized by monoclonal TL antibodies. Interestingly, both proteins were serologically and immunochemically recognized by leukemia-specific TL.4 antiserum. The quantity of cell surface T13 was significantly greater than T3 possibly due to the less efficient splicing of T3 transcripts in the L cell nucleus; both genes directed the synthesis of cytoplasmic RNA containing an unspliced intron 3 as assessed by S1 analysis. In toto, the results suggest that T3c is similar or perhaps identical with the novel TL product previously identified on the surface of certain x-ray-induced BALB/c leukemias.

S152 (CD27). A modulating disulfide-linked T cell activation antigen.

A large subset of normal resting peripheral blood T cells express a protein at low density defined by the murine mAb S152. Reactive T cells are present in both the CD4+ and CD8+ subpopulations. This determinant, however, is not expressed on growth factor-independent T cell lines. After activation of mononuclear cells by either Con A or PHA, greater than 80% of the cells stained at a mean fluorescence intensity that was more than six times that seen on resting cells. When PWM- or mixed lymphocyte reaction-activated cultures were studied, 7 to 22% of S152+ cells stained at high intensity whereas most cells stained at the baseline low intensity. The increased fraction of S152+ cells staining at high intensity after activation paralleled both the increased percentage of anti-Tac+ and 5E9+ cells and cellular proliferation measured by thymidine incorporation. Modulation of the S152 Ag was induced when either Con A- or PHA-activated mononuclear cells were placed into secondary cultures containing S152 mAb. Expression of the S152 Ag began to decrease after 2 h and reached a minimum after 6 h. Resting T cells, however, did not appear to modulate when cultured with S152 mAb. Immunoprecipitation and gel electrophoresis analysis revealed the S152 molecule to have a mobility of 120 kDa before reduction. After reduction the molecule was shown to be composed of two 55-kDa molecules with an isoelectric point of 5 to 6 indicating that the S152 Ag is a disulfide-linked homodimer. These studies confirm that the S152 mAb reacts with the newly defined CD27 molecule. The presence of the S152 Ag on resting and activated cells, its parallel increase with the Tac and 5E9 Ag, and its ability to modulate on activated cells suggest that this molecule may play a functional role during T cell activation.

Physical association between the CD8 and HLA class I molecules on the surface of activated human T lymphocytes.

Immune recognition by cytotoxic effector T cells requires participation of the CD8 and major histocompatibility complex class I antigens. We found that the CD8 molecule is noncovalently associated with the HLA class I heavy chain on the surface of human T cells activated by Con A. Accordingly, anti-CD8 monoclonal antibodies precipitated a heterodimer containing polypeptides of 32 and 43 kDa from the lysates of activated T cells. The 43-kDa chain of this heterodimer can be adsorbed from cell lysates with anti-HLA-A, -B, and -C antibodies. Endoglycosidase F treatment and chymotryptic peptide mapping identified a structural similarity between this 43-kDa molecule and the HLA class I heavy chain precipitated by the anti-HLA-A, -B, and -C antibody W6/32. Analysis of anti-CD8 precipitates under nonreducing and reducing conditions indicated a lack of interchain disulfide bonding between the CD8 and HLA heavy chain molecules. The CD8-HLA heavy chain complex was also detected in mixed lymphocyte cultures and a cloned cytotoxic T-lymphocyte line but not in purified natural killer cells. The present study indicates that CD8 is complexed with HLA heavy chain on the same cells, and the complex may have functional relevance in the T-cell recognition process.

A new HLA-linked T cell membrane molecule, related to the beta chain of the clonotypic receptor, is associated with T3.

The 38 kD molecule is noncovalently associated with beta 2 microglobulin (beta 2m)-free HLA heavy chain-like molecule, and thus forms a second heterodimer distinct from the clonotypic alpha/beta T cell receptor expressed by the same clone of leukemia cells. This second heterodimer (38 kD/HLA) is variably expressed and appears to be associated with the T3 molecule. We suggest, therefore, that it has a functional role in T cell activation.

Unusual association of beta 2-microglobulin with certain class I heavy chains of the murine major histocompatibility complex.

Class I products of the major histocompatibility complex (MHC) comprise a heavy chain of about 45 kDa noncovalently linked to a 12-kDa beta 2-microglobulin (beta 2m) light chain encoded on a different chromosome. We find that class I products of some mouse strains include an additional 62-kDa molecule which on the following evidence consists of a heavy chain linked covalently with beta 2m. Production of the 62-kDa protein invariably accorded with the occurrence of cysteine at position 121 of the heavy chain (Kb,Kbm1,Kbm3,Dd, and Ld). Substitution of arginine at position 121 invariably accorded with absence of the 62-kDa protein (Kbm6,Kbm7,Kbm9,Kd, and Db). On the basis of observed production versus nonproduction of the 62-kDa molecule, predictions are made regarding residue 121 in class I products for which this is not yet known; namely, Kk, Ks, and Dk, which produce the 62-kDa molecule, as compared with Kj, Qa-2, and TL, which do not. Reported differences in immunologic reactivity between Kb mutant strains with Arg-121 in place of Cys-121 imply that the occurrence of 62-kDa class I products in mice of Cys-121 genotype has functional consequences.

Stimulation and expansion of a human T-cell subpopulation by a monoclonal antibody to T-cell receptor molecule.

A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG1,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In long-term cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an alpha-subunit (45-48 kD) and a beta-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pI from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.

Structural characteristics of Tla products.

Biochemical study of thymus leukemia antigen (TL) from thymocytes of various Tla genotypes and from leukemia cells revealed features that, given present evidence, are peculiar to TL among class I products of the H-2:Qa:Tla region of chromosome 17. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of TL from thymocytes of all TL+ mouse strains, precipitated by anti-TL antiserum or monoclonal antibodies, showed two closely migrating bands of equal intensity in the heavy (H) chain position (45-50,000 mol wt). Comparison of these two bands by two-dimensional isoelectric focusing (2D IEF)-SDS-PAGE and 2D chymotryptic peptide mapping showed no differences indicative of protein dissimilarity. Thus, the two components of the H chain doublet may differ only in a feature of glycosylation that does not affect charge. The two leukemias studied gave only a single band in the H chain position. On 2D peptide mapping and 2D IEF-SDS-PAGE, the patterns for TL of Tlaa and Tlae thymocytes, which are closely related serologically, were broadly similar, but clearly different from the pattern typical of Tlac and Tlad thymocytes. 2D peptide maps of TL from Tlaa thymocytes and Tlaa leukemia cells did not differ. Leukemia cells of Tlab origin (thymocytes TL-) gave 2D peptide and 2D IEF-SDS-PAGE patterns of a third type. With the exception of Tlaa, thymocytes of TL+ mice yielded additional TL products of higher molecular weight than the TL H chain.