Despite the clinical success of anti-CD20 monoclonal antibody (mAb) in the treatment of lymphoma, there remains considerable uncertainty about its mechanism of action. Here we show that the ability of mAbs to translocate CD20 into low-density, detergentinsoluble membrane rafts appears to control how effectively they mediate complement lysis of lymphoma cells. In vitro studies using a panel of anti-B-cell mAbs revealed that the anti-CD20 mAbs, with one exception (B1), are unusually effective at recruiting human complement. Differences in complement recruitment could not be explained by the level of mAb binding or isotype but did correlate with the redistribution of CD20 in the cell membrane following mAb ligation. Membrane fractionation confirmed that B1, unlike 1F5 and rituximab, was unable to translocate CD20 into lipid rafts. In addition, we were able to drive B1 and a range of other anti-B-cell mAbs into a detergent-insoluble fraction of the cell by hyper-cross-linking with an F(ab) 2 anti-Ig Ab, a treatment that also conferred the ability to activate lytic complement. Thus, we have shown that an important mAb effector function appears to be controlled by movement of the target molecule into membrane rafts, either because a raft location favors complement activation by mAbs or because rafts are more sensitive to complement

Tetraspanins interact with a wide variety of transmembrane and intracellular proteins called molecular partners, and modulate their function. In this article, we describe a new partner of tetraspanin web, membrane metalloprotease CD10, which is selectively associated with CD9. By constructing chimeras between tetraspanins CD9 and CD82 (the latter does not interact with CD10) or by using site-directed mutagenesis, we determined that a portion of the large extracellular loop from the CCG motif to transmembrane domain 4, as well as the C-terminal tail of CD9, are involved in the interaction with CD10. The stable expression of wild-type CD9 in K562 CD10-positive cells enhanced the level of CD10 released with exosomes five-fold. In contrast, the expression of chimeric CD9, which contained the cytoplasmic C-terminal domain from CD82, had little effect on CD10 release. Short hairpin RNA knockdown of CD9 expression in Nalm-6 pre-B cells resulted in a two-fold reduction in the amount of endogenous CD10 released with microvesicles. The peptidase activity of CD10 measured either on cells or on exosomes correlated with the level of CD10 expression, and was not significantly modulated by CD9 expression as such. Our data suggest that the interaction of CD10 with tetraspanin CD9 can play an important role in the redistribution of peptidase activity from the cell surface to outer microenvironments. In bone marrow, where CD10 presumably contributes to the maturation of pre-B cells and migration of B cells to the blood circulation, release of CD10 peptidase activity with exosomes may effectively regulate extracellular matrix microenvironments.

Objectives To predict the spread of coronavirus disease (COVID‐19), information regarding the immunological memory for disease‐specific antigens is necessary. The possibility of reinfection, as well as the efficacy of vaccines for COVID‐19 that are currently under development, will largely depend on the quality and longevity of immunological memory in patients. To elucidate the process of humoral immunity development, we analysed the generation of plasmablasts and virus receptor‐binding domain (RBD)‐specific memory B (Bmem) cells in patients during the acute phase of COVID‐19. Methods The frequencies of RBD‐binding plasmablasts and RBD‐specific antibody‐secreting cells (ASCs) in the peripheral blood samples collected from patients with COVID‐19 were measured using flow cytometry and the ELISpot assay. Results The acute phase of COVID‐19 was characterised by the transient appearance of total as well as RBD‐binding plasmablasts. ELISpot analysis indicated that most patients exhibited a spontaneous secretion of RBD‐specific ASCs in the circulation with good correlation between the IgG and IgM subsets. IL‐21/CD40L stimulation of purified B cells induced the activation and proliferation of Bmem cells, which led to the generation of plasmablast phenotypic cells as well as RBD‐specific ASCs. No correlation was observed between the frequency of Bmem cell‐derived and spontaneous ASCs, suggesting that the two types of ASCs were weakly associated with each other. Conclusion Our findings reveal that SARS‐CoV‐2‐specific Bmem cells are generated during the acute phase of COVID‐19. These findings can serve as a basis for further studies on the longevity of SARS‐CoV‐2‐specific B‐cell memory.

Cell-to-cell transmission is an efficient mechanism to disseminate human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic virus type 1 (HTLV-1). However, it has been challenging to quantify the level of cell-to-cell transmission because the virus-producing cells cannot be easily distinguished from infected target cells. We have previously described replication-dependent vectors that can quantify infection events in cocultured cells. These vectors contain an antisense-oriented promoter and reporter gene interrupted by a sense-oriented intron from the human gamma-globin gene. This strategy prevents expression of the reporter gene in the transfected cells but permits its expression in target cells after infection. However, the gamma-globin intron is not efficiently removed by splicing in the aforementioned vectors, thereby reducing the level of reporter gene expression after transduction into target cells. Here, we used two approaches to improve the replication-dependent vectors. First, we improved the splicing events that remove the gamma-globin intron by optimizing the intron insertion site within the reporter gene. Second, we improved the packaging of the spliced RNA without the gamma-globin intron by targeting the introncontaining RNA via microRNA 30 (miR30)-based short hairpin RNAs. Using two optimized fluorescent reporter vectors and flow cytometry, we determined that multiply HIV-1-infected cells were generated at a higher frequency in coculture than in cellfree infection; furthermore, this increase was dependent upon viruses bearing HIV-1 Env. Compared with previously described vectors, these improved vectors can quantify the infection in lymphocytes and in primary cells with a higher sensitivity and allow the detection and quantitation of multiply infected cells, providing better tools to study retroviral cell-mediated infection. IMPORTANCEThe human-pathogenic retroviruses HTLV-1 and HIV-1 can be transmitted more efficiently in vivo via direct contact of infected cells with healthy target cells than through cell-free virion-mediated infection. Despite its importance, cell-to-cell transmission has been difficult to quantify because the previously infected cells and the newly infected cells are mixed together in the same culture. In the current study, we generated vectors that are significantly improved over the previously described replication-dependent vectors. As a result, these improved vectors can efficiently detect and quantify cell-to-cell transmission or new infection events in cells in mixed culture. These luciferase-or fluorescence protein-based reporter vectors can be used to quantify and study HIV-1 or HTLV-1 cell-mediated infection in a simple one-step transfection/infection assay.

Immunoglobulin E (IgE) is the key immunoglobulin in the pathogenesis of IgE associated allergic diseases affecting 30% of the world population. Recent data suggest that allergen-specific IgE levels in serum of allergic patients are sustained by two different mechanisms: inducible IgE production through allergen exposure, and continuous IgE production occurring even in the absence of allergen stimulus that maintains IgE levels. This assumption is supported by two observations. First, allergen exposure induces transient increases of systemic IgE production. Second, reduction in IgE levels upon depletion of IgE from the blood of allergic patients using immunoapheresis is only temporary and IgE levels quickly return to pre-treatment levels even in the absence of allergen exposure. Though IgE production has been observed in the peripheral blood and locally in various human tissues (e.g., nose, lung, spleen, bone marrow), the origin and main sites of IgE production in humans remain unknown. Furthermore, IgE-producing cells in humans have yet to be fully characterized. Capturing IgE-producing cells is challenging not only because current staining technologies are inadequate, but also because the cells are rare, they are difficult to discriminate from cells bearing IgE bound to IgE-receptors, and plasma cells express little IgE on their surface. However, due to the central role in mediating both the early and late phases of allergy, free IgE, IgE-bearing effector cells and IgE-producing cells are important therapeutic targets. Here, we discuss current knowledge and unanswered questions regarding IgE production in allergic patients as well as possible therapeutic approaches targeting IgE.

We describe Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a novel method to select mammalian cells with precise genome modifications that does not rely on cell cloning. SORTS is designed to disrupt the target gene with an expression cassette encoding an epitope tag embedded into human glycophosphatidylinositol (GPI)-anchored protein CD52. The cassette is very short, usually less than 250 nucleotides, which simplifies donor DNA construction and facilitates transgene integration into the target locus. The chimeric protein is then expressed from the target promoter, processed and exposed on the plasma membrane where it serves as a marker for FACS sorting with tag-specific antibodies. Simultaneous use of two different epitope tags enables rapid isolation of cells with biallelic knock-ins. SORTS can be easily and reliably applied to a number of genome-editing problems such as knocking out genes encoding intracellular or secreted proteins, protein tagging and inactivation of HIV-1 provirus.

We used replication-dependent retroviral vectors to identify cell surface antigens involved in the cell-to-cell transmission of human T cell leukemia virus type 1 (HTLV-1). We generated monoclonal antibodies ( H uman T cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that causes two major diseases, adult T cell leukemia (ATL) (29) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (1, 51). Unlike HIV-1, which causes fatal immune deficiency-associated diseases in almost all infected individuals if left untreated, only about 5% of HTLV-1-infected people develop disease 10 to 25 years after the initial exposure. The remarkable feature of HTLV-1 is that transmission is 40% effective with blood lymphocytes but never with a patient's plasma (37). The phenomenon was explained by the extremely low infectivity of free viral particles (10, 13) and by the efficient cell-to-cell transmission of HTLV-1. Therefore, HTLV-1 can serve as an excellent model to study the cell-to-cell transmission of retroviruses in vitro. In 2003, Igakura et al. (20) described the formation of a microtubule-dependent cell-cell contact that was induced by HTLV-1. Based on structural and molecular similarity to the immunological synapse (IS), the cell-cell contact zone was named the virological synapse (VS). Later, the formation of the VS was demonstrated for human immunodeficiency virus (HIV) infection (7,21,22,35). Recently, a number of different and remarkable types of cell-cell interactions have been reported to mediate retroviral transmission, particularly nanotubes (43, 45), mono-or polysynapses (42), biofilm-like structures (39), and conduits (52). Nevertheless, our understanding of the mechanisms of viral transmission at the molecular level remains poor.We have recently complemented microscopy studies of cell-tocell transmission by developing inLuc and inYFP reporter vectors for HIV-1 and HTLV-1, which were designed to quantify cell-tocell infection (32). The signal generated by these vectors depends on the completion of a full cycle of virus replication. They are silent in producer cells but generate a readout in target cells. These vectors helped us to clarify the role of the viral protein Tax and different Env proteins in cell-to-cell infection and demonstrated that HTLV-1 is transmitted more efficiently in lymphoid cells than between nonlymphoid cells. In this study, we aimed to define cell surface antigens (Ags) possibly involved in the cell-to-cell infection of HTLV-1 and HIV-1. For that, we generated mouse monoclonal hybridomas against Jurkat cells and screened the monoclonal antibodies (MAbs) by inLuc infectivity assays. Finally, MAbs that decreased cell-to-cell infection were selected. Most of the HTLV-1-inhibitory MAbs recognized the carbohydrate Tn (T-nouvelle) antigen and precipitated the CD43 and CD45 proteins. We found that both the sialophorin CD43 and the phosphatase CD45 were important for HTLV-1 infection. These antigens are heavily O-glycosylated in normal cells, have a rodlike shape, and ext...

We describe a method for leukocyte sorting by a microarray of anti-cluster-of-differentiation (anti-CD) antibodies and for preparation of the bound cells for morphological or cytochemical examination. The procedure results in a “sorted” smear with cells positive for certain surface antigens localised in predefined areas. The morphology and cytochemistry of the microarray-captured normal and neoplastic peripheral blood mononuclear cells are identical to the same characteristics in a smear. The microarray permits to determine the proportions of cells positive for the CD antigens on the microarray panel with high correlation with flow cytometry. Using the anti-CD microarray we show that normal granular lymphocytes and lymphocytes with radial segmentation of the nuclei are positive for CD3, CD8, CD16 or CD56 but not for CD4 or CD19. We also show that the described technique permits to obtain a pure leukemic cell population or to separate two leukemic cell populations on different antibody spots and to study their morphology or cytochemistry directly on the microarray. In cases of leukemias/lymphomas when circulating neoplastic cells are morphologically distinct, preliminary diagnosis can be suggested from full analysis of cell morphology, cytochemistry and their binding pattern on the microarray.