Brief report: serpin Spi2A as a novel modulator of hematopoietic progenitor cell formation.

Prime regulation over hematopoietic progenitor cell (HPC) production is exerted by hematopoietins (HPs) and their Janus kinase-coupled receptors (HP-Rs). For HP/HP-R studies, one central challenge in determining specific effects involves the delineation of nonredundant signal transduction factors and their lineage restricted actions. Via loss-of-function studies, we define roles for an HP-regulated Serpina3g/Spi2A intracellular serpin during granulomyelocytic, B-cell, and hematopoietic stem cell (HSC) formation. In granulomyelocytic progenitors, granulocyte macrophage colony stimulating factor (GMCSF) strongly induced Serpina3g expression with Stat5 dependency. Spi2A-knockout (KO) led to 20-fold decreased CFU-GM formation, limited GMCSF-dependent granulocyte formation, and compromised neutrophil survival upon tumor necrosis factor alpha (TNF-α) exposure. In B-cell progenitors, Serpina3g was an interleukin-7 (IL7) target. Spi2A-KO elevated CFU-preB greater than sixfold and altered B-cell formation in competitive bone marrow transplant (BMT), and CpG challenge experiments. In HSCs, Serpina3g/Spi2A expression was also elevated. Spi2A-KO compromised LT-HSC proliferation (as well as lineage(neg) Sca1(pos) Kit(pos) (LSK) cell lysosomal integrity), and skewed LSK recovery post 5-FU. Spi2A therefore functions to modulate HP-regulated immune cell and HSC formation post-5-FU challenge.

Human mesenchymal stromal cells deliver systemic oncolytic measles virus to treat acute lymphoblastic leukemia in the presence of humoral immunity.

Clinical trials of oncolytic attenuated measles virus (MV) are ongoing, but successful systemic delivery in immune individuals remains a major challenge. We demonstrated high-titer anti-MV antibody in 16 adults with acute lymphoblastic leukemia (ALL) following treatments including numerous immunosuppressive drugs. To resolve this challenge, human bone marrow-derived mesenchymal stromal cells (BM-MSCs) were used to efficiently deliver MV in a systemic xenograft model of precursor B-lineage-ALL. BM-MSCs were successfully loaded with MV ex vivo, and MV was amplified intracellularly, without toxicity. Live cell confocal imaging demonstrated a viral hand-off between BM-MSCs and ALL targets in the presence of antibody. In a murine model of disseminated ALL, successful MV treatment (judged by bioluminescence quantification and survival) was completely abrogated by passive immunization with high-titer human anti-MV antibody. Importantly, no such abrogation was seen in immunized mice receiving MV delivered by BM-MSCs. These data support the use of BM-MSCs as cellular carriers for MV in patients with ALL.

MicroRNAs transfer from human macrophages to hepato-carcinoma cells and inhibit proliferation.

Recent research has indicated a new mode of intercellular communication facilitated by the movement of RNA between cells. There is evidence that RNA can transfer between cells in a multitude of ways, including in complex with proteins or lipids or in vesicles, including apoptotic bodies and exosomes. However, there remains little understanding of the function of nucleic acid transfer between human cells. In this article, we report that human macrophages transfer microRNAs (miRNAs) to hepato-carcinoma cells (HCCs) in a manner that required intercellular contact and involved gap junctions. Two specific miRNAs transferred efficiently between these cells--miR-142 and miR-223--and both were endogenously expressed in macrophages and not in HCCs. Transfer of these miRNAs influenced posttranscriptional regulation of proteins in HCCs, including decreased expression of reporter proteins and endogenously expressed stathmin-1 and insulin-like growth factor-1 receptor. Importantly, transfer of miRNAs from macrophages functionally inhibited proliferation of these cancerous cells. Thus, these data led us to propose that intercellular transfer of miRNA from immune cells could serve as a new defense against unwanted cell proliferation or tumor growth.

Cathepsin B controls the persistence of memory CD8+ T lymphocytes.

The persistence of memory T lymphocytes confers lifelong protection from pathogens. Memory T cells survive and undergo homeostatic proliferation (HSP) in the absence of Ag, although the cell-intrinsic mechanisms by which cytokines drive the HSP of memory T cells are not well understood. In this study we report that lysosome stability limits the long-term maintenance of memory CD8(+) T cell populations. Serine protease inhibitor (Spi) 2A, an anti-apoptotic cytosolic cathepsin inhibitor, is induced by both IL-15 and IL-7. Mice deficient in Spi2A developed fewer memory phenotype CD44(hi)CD8(+) T cells with age, which underwent reduced HSP in the bone marrow. Spi2A was also required for the maintenance of central memory CD8(+) T cell populations after acute infection with lymphocytic choriomeningitis virus. Spi2A-deficient Ag-specific CD8(+) T cell populations declined more than wild-type competitors after viral infection, and they were eroded further after successive infections. Spi2A protected memory cells from lysosomal breakdown by inhibiting cathepsin B. The impaired maintenance of Spi2A-deficient memory CD8(+) T cells was rescued by concomitant cathepsin B deficiency, demonstrating that cathepsin B was a physiological target of Spi2A in memory CD8(+) T cell survival. Our findings support a model in which protection from lysosomal rupture through cytokine-induced expression of Spi2A determines the long-term persistence of memory CD8(+) T cells.

Could CD4 capture by CD8+ T cells play a role in HIV spreading?

CD8(+) T cells have been shown to capture plasma membrane fragments from target cells expressing their cognate antigen, a process termed "trogocytosis". Here, we report that human CD4, the Human Immunodeficiency Virus (HIV) receptor, can be found among the proteins transferred by trogocytosis. CD4 is expressed in a correct orientation after its capture by CD8(+) T cells as shown by its detection using conformational antibodies and its ability to allow HIV binding on recipient CD8(+) T cells. Although we could not find direct evidence for infection of CD8(+) T cells having captured CD4 by HIV, CD4 was virologically functional on these cells as it conferred on them the ability to undergo syncytia formation induced by HIV-infected MOLT-4 cells. Our results show that acquisition of CD4 by CD8(+) T cells via trogocytosis could play a previously unappreciated role for CD8(+) T cells in HIV spreading possibly without leading to their infection.

Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells.

Membrane nanotubes are membranous tethers that physically link cell bodies over long distances. Here, we present evidence that nanotubes allow human natural killer (NK) cells to interact functionally with target cells over long distances. Nanotubes were formed when NK cells contacted target cells and moved apart. The frequency of nanotube formation was dependent on the number of receptor/ligand interactions and increased on NK cell activation. Most importantly, NK cell nanotubes contained a submicron scale junction where proteins accumulated, including DAP10, the signaling adaptor that associates with the activating receptor NKG2D, and MHC class I chain-related protein A (MICA), a cognate ligand for NKG2D, as occurs at close intercellular synapses between NK cells and target cells. Quantitative live-cell fluorescence imaging suggested that MICA accumulated at small nanotube synapses in sufficient numbers to trigger cell activation. In addition, tyrosine-phosphorylated proteins and Vav-1 accumulated at such junctions. Functionally, nanotubes could aid the lysis of distant target cells either directly or by moving target cells along the nanotube path into close contact for lysis via a conventional immune synapse. Target cells moving along the nanotube path were commonly polarized such that their uropods faced the direction of movement. This is the opposite polarization than for normal cell migration, implying that nanotubes can specifically drive target cell movement. Finally, target cells that remained connected to an NK cell by a nanotube were frequently lysed, whereas removing the nanotube using a micromanipulator reduced lysis of these target cells.

Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis.

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

Antigp41 antibodies fail to block early events of virological synapses but inhibit HIV spread between T cells.

OBJECTIVE: Compared with cell-free viral infection, virological synapses increase HIV capture by target cells, viral infectivity and cytopathicity, and are believed to be less sensitive to antibody neutralization. We have evaluated the impact of antibodies against HIV envelope glycoproteins (gp120 and gp41) on cell-to-cell HIV transmission. METHODS: We analyzed the role of trogocytosis in cell-to-cell HIV transmission and the inhibitory mechanisms of antigp120 antibody IgGb12 and antigp41 antibodies 4E10 and 2F5 using cocultures of NL4-3 or BaL-infected MOLT/CCR5 cells with primary CD4 T cells. RESULTS: Analysis of early steps of HIV transmission in these cocultures showed that IgGb12, but not 4E10 and 2F5, inhibited the formation of virological synapses. Consequently, IgGb12 but not antigp41 antibodies blocked the transfer of HIV particles from infected to target cells and the trogocytic transfer of CD4 molecules from target to infected cells. Interestingly, trogocytic transfer of membranes was not detected in the HIV transmission direction. Furthermore, analysis of late events of HIV transmission showed that all neutralizing antibodies blocked productive infection of target cells, suggesting that HIV infection between T cells is transmitted by a neutralization-sensitive mechanism involving HIV budding from infected cells and capture by target cells. CONCLUSION: Despite mechanistic differences, antigp120 and antigp41 antibodies block infectious cell-to-cell HIV transmission. Our data suggest that eliciting high titers of neutralizing antibodies in vivo should be maintained as a main end of HIV vaccine design.

Capture of plasma membrane fragments from target cells by trogocytosis requires signaling in T cells but not in B cells.

Upon recognition of their respective cellular partners, T and B cells acquire their antigens by a process of membrane capture called trogocytosis. Here, we report that various inhibitors of actin polymerization or of kinases involved in intracellular signaling partially or fully inhibited trogocytosis by CD8(+) and CD4(+) T cells, whereas they had no effect on trogocytosis by B cells. Similarly, trogocytosis by T cells was inhibited at 4 degrees C, whereas in B cells it was independent of temperature, indicating that trogocytosis by B cells does not rely on active processes. By contrast, most inhibitors we tested impaired both T-cell and B-cell activation. The differential effect of inhibitors on T-cell and B-cell trogocytosis was not due to the higher affinity of the B-cell receptor for its cognate antigen compared with the affinity of the T-cell receptor for its own antigen, but it correlated tightly with the abilities of T cells and B cells to form conjugates with their target cells in the presence of inhibitors. Trogocytosis thus has different requirements in different cell types. Moreover, the capture of membrane antigen by B cells is identified as a novel signaling-independent event of B-cell biology.

Chemical labels metabolically installed into the glycoconjugates of the target cell surface can be used to track lymphocyte/target cell interplay via trogocytosis: comparisons with lipophilic dyes and biotin.

Trogocytosis, the process whereby lymphocytes capture membrane components from the cells they interact with, is classically evidenced by the transfer of fluorescent lipophilic compounds or biotinylated proteins from target cells to T or B cells. A particular class of molecules, not studied explicitly so far in the context of trogocytosis is glycoconjugates. Here, we used a method to metabolically install chemical labels in target cell glycoconjugates. Working with those target cells, we describe the conditions allowing CTL to be detected based on glycoconjugate trogocytosis triggered by antigen or stimulatory antibodies. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this approach to the detection of CD4(+) T and B cells. Overall, glycoconjugates were transferred between target cells and lymphocytes during trogocytosis with efficiencies comparable or higher than measured for biotinylated proteins or lipophilic dyes incorporated into general membrane lipids. From a technological point of view, our approach can be employed to detect reactive lymphocytes via glycoconjugate trogocytosis. More generally, we believe that the ever-growing ability to employ chemistry in living systems to label particular compounds will be powerful in unraveling the contributions of glycosylation to various aspects of T and B cells biology.

Capture of target cell membrane components via trogocytosis is triggered by a selected set of surface molecules on T or B cells.

Key events of T and B cell biology are regulated through direct interaction with APC or target cells. Trogocytosis is a process whereby CD4(+) T, CD8(+) T, and B cells capture their specific membrane-bound Ag through the acquisition of plasma membrane fragments from their cellular targets. With the aim of investigating whether the ability to trigger trogocytosis was a selective property of Ag receptors, we set up an assay that allowed us to test the ability of many different cell surface molecules to trigger trogocytosis. On the basis of the analysis of a series of surface molecules on CD4(+) T, CD8(+) T, and B cells, we conclude that a set of cell type-specific surface determinants, including but not limited to Ag receptors, do trigger trogocytosis. On T cells, these determinants include components of the TCR/CD3 as well as that of coreceptors and of several costimulatory molecules. On B cells, we identified only the BCR and MHC molecules as potentials triggers of trogocytosis. Remarkably, latrunculin, which prevents actin polymerization, impaired trogocytosis by T cells, but not by B cells. This was true even when the same Abs were used to trigger trogocytosis in T or B cells. Altogether, our results indicate that although trogocytosis is performed by all hemopoietic cells tested thus far, both the receptors and the mechanisms involved can differ depending on the lineage of the cell acquiring membrane materials from other cells. This could therefore account for the different biological consequences of Ag capture via trogocytosis proposed for different types of cells.