The evolution of guidelines for the validation of flow cytometric methods.

The recent advances in the field of flow cytometry have resulted in instrumentation with increased capacity which is actually more user-friendly. Thus, the technology has become more valuable to research scientists, the pharmaceutical industry and clinical laboratories. The use of flow cytometry in regulated labs has been hampered by the challenges associated method validation and the lack of official guidance documents on the topic. Thus key stakeholders have published recommendation papers with the hope that these will be incorporated as official guidance documents. This review will focus on the achievements of the stakeholders and a high-level overview their recommendations.

Specificity of two anti-class I HLA monoclonal antibodies that block class I recognition by the NKB1 killer cell inhibitory receptor.

Cytolysis by NK cells that possess the NKB1 killer cell inhibitory receptor is inhibited by target cell expression of Bw4+ HLA-B molecules. The inhibitory effect can be prevented by addition of mAbs which block recognition of class I molecules by NKB1. The epitopes recognized by two anti-class I mAbs, DX15 and DX16, which inhibit the interaction of NKB1 with class I have been characterized. Binding of DX15 and DX16 to class I allotypes was investigated by flow cytometric analysis of transfected cell lines which express just one HLA-A, B, or C allele, and by immunoprecipitation of class I molecules from HLA typed B-lymphoblastoid cell lines, followed by isoelectric focusing. The DX16 mAb recognizes class I allotypes which possess alanine at position 71 of the alpha 1 helix, and therefore has a specificity resembling that of the ME1 mAb but with broader specificity. Class I recognition by DX15 is affected by polymorphisms of the C-terminal part of the alpha 1 helix, and the N-terminal part of the alpha 2 helix. DX15 thus appears to recognize a complex epitope near the end of the peptide binding groove which may be conformationally determined. Both antibodies are as effective as the anti-NKB1 mAb (DX9) in preventing class I recognition by the NKB1 receptor. DX16 also blocked recognition by a B*0702 allospecific CTL clone, whereas DX15 did not.

Human immunodeficiency virus type 1 membrane fusion mediated by a laboratory-adapted strain and a primary isolate analyzed by resonance energy transfer.

Previous studies of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein-mediated membrane fusion have focused on laboratory-adapted T-lymphotropic strains of the virus. The goal of this study was to characterize membrane fusion mediated by a primary HIV-1 isolate in comparison with a laboratory-adapted strain. To this end, a new fusion assay was developed on the basis of the principle of resonance energy transfer, using HeLa cells stably transfected with gp120/gp41 from the T-lymphotropic isolate HIV-1LA1 or the macrophage-tropic primary isolate HIV-1JR-FL. These cells fused with CD4+ target cell lines with a tropism mirroring that of infection by the two viruses. Of particular note, HeLa cells expressing HIV-1JR-FL gp120/gp41 fused only with PM1 cells, a clonal derivative of HUT 78, and not with other T-cell or macrophage cell lines. These results demonstrate that the envelope glycoproteins of these strains play a major role in mediating viral tropism. Despite significant differences exhibited by HIV-1JR-FL and HIV-1LAI in terms of tropism and sensitivity to neutralization by CD4-based proteins, the present study found that membrane fusion mediated by the envelope glycoproteins of these viruses had remarkably similar properties. In particular, the degree and kinetics of membrane fusion were similar, fusion occurred at neutral pH and was dependent on the presence of divalent cations. Inhibition of HIV-1JR-FL envelope glycoprotein-mediated membrane fusion by soluble CD4 and CD4-IgG2 occurred at concentrations similar to those required to neutralize this virus. Interestingly, higher concentrations of these agents were required to inhibit HIV-1LAI envelope glycoprotein-mediated membrane fusion, in contrast to the greater sensitivity of HIV-1LAI virions to neutralization by soluble CD4 and CD4-IgG2. This finding suggests that the mechanisms of fusion inhibition and neutralization of HIV-1 are distinct.

HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.

The beta-chemokines MIP-1alpha, MIP-1beta and RANTES inhibit infection of CD4+ T cells by primary, non-syncytium-inducing (NSI) HIV-1 strains at the virus entry stage, and also block env-mediated cell-cell membrane fusion. CD4+ T cells from some HIV-1-exposed uninfected individuals cannot fuse with NSI HIV-1 strains and secrete high levels of beta-chemokines. Expression of the beta-chemokine receptor CC-CKR-5 in CD4+, non-permissive human and non-human cells renders them susceptible to infection by NSI strains, and allows env-mediated membrane fusion. CC-CKR-5 is a second receptor for NSI primary viruses.

The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor.

Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-B*1502 and B*1513 which differ only at residues 77-83 which define the Bw4 and Bw6 serological epitopes, we tested the hypothesis that the presence of the Bw4 epitope on class I molecules determines recognition by NKB1+ NK cells. HLA-B*1513 possesses the Bw4 epitope, whereas B*1502 has the Bw6 epitope. Lysis by NKB1+ NK cell clones of transfected target cells expressing B*1513 as the only HLA-A, -B, or -C molecule was inhibited, whereas killing of transfectants expressing B*1502 was not. Addition of an an anti-NKB1 monoclonal antibody reconstituted lysis of the targets expressing B*1513, but did not affect killing of targets bearing B*1502. The inhibitory effect of B*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the Bw4 epitope influences recognition of HLA-B molecules by NK cells that express NKB1, and suggest that the NKB1 molecule may act as a receptor for Bw4+ HLA-B alleles. Sequences outside of the Bw4 region must also affect recognition by NKB1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the Bw4 epitope but are in other ways substantially different from HLA-B molecules, was not increased by addition of the anti-NKB1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the Bw4/Bw6 region. The glycosylation site of the Bw4-positive molecule B*5801 was mutated, and the mutant molecules tested for inhibition of NKB1+ NK cells. Inhibition that could be reversed by addition of the anti-NKB1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NKB1+ NK cell clones.

NKB1: a natural killer cell receptor involved in the recognition of polymorphic HLA-B molecules.

Natural killer (NK) cells kill normal and transformed hematopoietic cells that lack expression of major histocompatibility complex (MHC) class I antigens. Lysis of HLA-negative Epstein Barr virus-transformed B lymphoblastoid cell lines (B-LCL) by human NK cell clones can be inhibited by transfection of the target cells with certain HLA-A, -B, or -C alleles. NK cell clones established from an individual demonstrate clonal heterogeneity in HLA recognition and a single NK clone can recognize multiple alleles. We describe a potential human NK cell receptor (NKB1) for certain HLA-B alleles (e.g., HLA-B*5101 and-B*5801) identified by the mAb DX9. NKB1 is a 70-kD glycoprotein that is expressed on a subset of NK cells and NK cell clones. DX9 monoclonal antibody (mAb) specifically inhibits the interaction between NK cell clones and B-LCL targets transfected with certain HLA-B alleles, but does not affect recognition of HLA-A or HLA-C antigens. An individual NK cell clone can independently recognize B-LCL targets transfected with HLA-B or HLA-C antigens; however, DX9 mAb only affects interaction with transfectants expressing certain HLA-B alleles. These findings demonstrate the existence of NK cell receptors involved in the recognition of HLA-B and imply the presence of multiple receptors for MHC on an individual NK clone.

Specificity of HLA class I antigen recognition by human NK clones: evidence for clonal heterogeneity, protection by self and non-self alleles, and influence of the target cell type.

Prior studies using polyclonal populations of natural killer (NK) cells have revealed that expression of certain major histocompatibility complex (MHC) class I molecules on the membrane of normal and transformed hematopoietic target cells can prevent NK cell-mediated cytotoxicity. However, the extent of clonal heterogeneity within the NK cell population and the effect of self versus non-self MHC alleles has not been clearly established. In the present study, we have generated more than 200 independently derived human NK cell clones from four individuals of known human histocompatibility leukocyte antigens (HLA) type. NK clones were analyzed for cytolytic activity against MHC class I-deficient Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) stably transfected with several HLA-A, -B, or -C genes representing either self or non-self alleles. All NK clones killed the prototypic HLA-negative erythroleukemia K562 and most lysed the MHC class I-deficient C1R and 721.221 B-LCL. Analysis of the panel of HLA-A, -B, and -C transfectants supported the following general conclusions. (a) Whereas recent studies have suggested that HLA-C antigens may be preferentially recognized by NK cells, our findings indicate that 70% or more of all NK clones are able to recognize certain HLA-B alleles and many also recognize HLA-A alleles. Moreover, a single NK clone has the potential to recognize multiple alleles of HLA-A, HLA-B, and HLA-C antigens. Thus, HLA-C is not unique in conferring protection against NK lysis. (b) No simple patterns of HLA specificity emerged. Examination of a large number of NK clones from a single donor revealed overlapping, yet distinct, patterns of reactivity when a sufficiently broad panel of HLA transfectants was examined. (c) Both autologous and allogeneic HLA antigens were recognized by NK clones. There was neither evidence for deletion of NK clones reactive with self alleles nor any indication for an increased frequency of NK clones recognizing self alleles. (d) With only a few exceptions, protection conferred by transfection of HLA alleles into B-LCL was usually not absolute. Rather a continuum from essentially no protection for certain alleles (HLA-A*0201) to very striking protection for other alleles (HLA-B*5801), with a wide range of intermediate effects, was observed. (e) Whereas most NK clones retained a relatively stable HLA specificity, some NK clones demonstrated variable and heterogeneous activity over time. (f) NK cell recognition and specificity cannot be explained entirely by the presence or absence of HLA class I antigens on the target cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Receptor properties of two varicella-zoster virus glycoproteins, gpI and gpIV, homologous to herpes simplex virus gE and gI.

The varicella-zoster virus (VZV) genome contains 70 reading frames (ORF), 5 of which encode the glycoproteins gpI, gpII, gpIII, gpIV, and gpV. ORF 67 and 68 lie adjacent to each other in the unique short region of the VZV genome and code for gpIV and gpI, respectively. These two genes, which are contained within the HindIII C fragment of the VZV genome, were subcloned in the correct orientation downstream from the promoter regions of the eukaryotic expression vectors pCMV5 and pBJ. After transfection, 5 to 20% of the Cos cells bound antibody specific for the given glycoprotein. In this study, it was shown that only the cells transfected with the gpI construct bound to the Fc fragment of human immunoglobulin G. Neither the transfected gpIV gene product nor the vector only bound to the Fc fragment. Thus, VZV gpI is confirmed to be the VZV-encoded Fc-binding glycoprotein. Like the wild-type form of gpI expressed in VZV-infected cells, gpI precipitated from transfected cells contained both N-linked and O-linked glycans and was heavily sialated. In addition, the transfected gpI gene product was phosphorylated both in cell culture and in protein kinase assays by mammalian casein kinases I and II. Extensive computer-assisted analyses of the VZV gpI sequence, as well as those of alphaherpesviral homolog glycoproteins, disclosed properties similar to those of other cell surface receptors; these included (i) exocytoplasmic regions rich in cysteine residues, (ii) membrane-proximal regions with potential O-linked glycosylation sites, and (iii) cytoplasmic domains with consensus phosphorylation sites.

Herpesviral Fc receptors and their relationship to the human Fc receptors.

Nearly two decades ago, it was observed that cells infected with herpes simplex virus (HSV) acquired an IgG Fc binding activity. The properties of the viral Fc receptor (FcR) have now been characterized by several laboratories. The Fc binding activity appears on the surface of the infected cell prior to formation of progeny virions. The FcR induced by HSV has been identified as the HSV glycoprotein, gE. When HSV gE forms a complex with a second HSV glycoprotein, gI, the receptor binds IgG with higher affinity. Varicella-zoster virus (VZV), which is closely related to HSV, has also been shown to induce an FcR. Like the HSV FcR, the FcR specified by VZV possesses characteristics common to viral glycoproteins. VZV encodes two glycoproteins, gpI and gpIV, which are the homologs of HSV gE and gI. The VZV glycoproteins have many properties common to cell surface receptors, including O-linked glycans and phosphorylation sites. However, extensive computer-assisted analyses of the amino acid sequences of VZV gpI and gpIV did not uncover regions of homology to the human cellular Fc receptors for IgG.

Cell surface expression of the varicella-zoster virus glycoproteins and Fc receptor.

Varicella-zoster virus (VZV) specifies the synthesis of viral glycoproteins which are important antigens for induction of the host immune response. In this report the technology of laser-activated flow cytometry has been employed to measure the membrane expression of VZV glycoproteins gpI, gpII, gpIII, and gpIV. By use of biotinylated monoclonal antibodies as probes, all four glycoproteins were demonstrated on the infected cell surface. The temporal appearance of the viral glycoproteins was defined in a time course experiment and shown to be maximal about 24 hr postinfection. The issue whether VZV induces the cell surface expression of an Fc receptor (FcR) was investigated with biotinylated nonimmune human IgG, followed by streptavidin-phycoerythrin. By this technique a 10-fold increase in fluorescence intensity was seen in the VZV-infected cells as compared to the mock-infected controls. When the experiment was repeated with purified human Fc fragment rather than whole IgG, a similar degree of binding was seen. Both the VZV glycoproteins and the VZV FcR were exquisitely sensitive to trypsin treatment (1 mg/ml); likewise, the cell surface expression of these VZV products was diminished by treatment of the infected cultures with monensin, an inhibitor of glycoprotein transport. In order to prove that VZV infection was not causing the induction of a cellular Fc gamma R, the VZV-infected and mock-infected cells were stained with monoclonal antibodies directed against each of the three human cellular IgG FcR, but no differences were observed. Therefore, the FcR activity seen in the infected culture was not due to one of the known cellular Fc gamma R.