CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells.

The prototypical genetic autoimmune disease is immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a severe pediatric disease with limited treatment options. IPEX syndrome is caused by mutations in the forkhead box protein 3 (FOXP3) gene, which plays a critical role in immune regulation. As a monogenic disease, IPEX is an ideal candidate for a therapeutic approach in which autologous hematopoietic stem and progenitor (HSPC) cells or T cells are gene edited ex vivo and reinfused. Here, we describe a CRISPR-based gene correction permitting regulated expression of FOXP3 protein. We demonstrate that gene editing preserves HSPC differentiation potential, and that edited regulatory and effector T cells maintain their in vitro phenotype and function. Additionally, we show that this strategy is suitable for IPEX patient cells with diverse mutations. These results demonstrate the feasibility of gene correction, which will be instrumental for the development of therapeutic approaches for other genetic autoimmune diseases.

TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma.

Activation of the TCL1 oncogene has been implicated in T cell leukemias/lymphomas and recently was associated with AIDS diffuse large B cell lymphomas (AIDS-DLBCL). Also, in nonmalignant lymphoid tissues, antibody staining has shown that mantle zone B cells expressed abundant Tcl1 protein, whereas germinal center (GC; centrocytes and centroblasts) B cells showed markedly reduced expression. Here, we analyze isolated B cell subsets from hyperplastic tonsil to determine a more precise pattern of Tcl1 expression with development. We also examine multiple B cell lines and B lymphoma patient samples to determine whether different tumor classes retain or alter the developmental pattern of expression. We show that TCL1 expression is not affected by Epstein-Barr virus (EBV) infection and is high in naïve B cells, reduced in GC B cells, and absent in memory B cells and plasma cells. Human herpesvirus-8 infected primary effusion lymphomas (PEL) and multiple myelomas are uniformly TCL1 negative, whereas all other transformed B cell lines tested express moderate to abundant TCL1. This observation supports the hypothesis that PEL, like myeloma, usually arise from post-GC stages of B cell development. Tcl1 protein is also detected in most naïve/GC-derived B lymphoma patient samples (23 of 27 [85%] positive), whereas most post-GC-derived B lymphomas lack expression (10 of 41 [24%] positive). These data indicate that the pattern of Tcl1 expression is distinct between naïve/GC and post-GC-derived B lymphomas (P < 0.001) and that the developmental pattern of expression is largely retained. However, post-GC-derived AIDS-DLBCL express TCL1 at a frequency equivalent to naïve/GC-derived B lymphomas in immune-competent individuals (7 of 9 [78%] positive), suggesting that TCL1 down-regulation is adversely affected by severe immune system dysfunction. These findings demonstrate that TCL1 expression in B cell lymphoma usually reflects the stage of B cell development from which they derive, except in AIDS-related lymphomas.

Monocytes adhering by LFA-1 to placental syncytiotrophoblasts induce local apoptosis via release of TNF-alpha. A model for hematogenous initiation of placental inflammations.

Placental inflammations (villitis) are accompanied by loss of the syncytiotrophoblast, which is the cellular barrier separating maternal blood from fetal tissue in the villous placenta. We propose that syncytiotrophoblast loss is mediated by adhesion of activated maternal monocytes. This hypothesis was tested with a co-culture model of peripheral blood monocytes and placental syncytiotrophoblasts. We find that LPS-activated monocytes adhere to interferon-gamma (IFN-gamma)-treated syncytiotrophoblasts via monocyte LFA-1 for >48 h, during which time the monocytes induce trophoblast apoptosis and subsequent damage of the trophoblast layer. Optimal monocyte-mediated syncytiotrophoblast death requires both lipopolysaccharide (LPS) and IFN-gamma and is inhibited by either anti-tumor necrosis factor (TNF) antibody or epidermal growth factor. Syncytiotrophoblast damage is largely limited to culture surfaces in the vicinity of bound monocytes. These results show that activated maternal monocytes bound to the placental barrier can induce focal damage mediated by the inflammatory cytokine TNF-alpha and suggest a route for maternal leukocyte infiltration into the fetal stroma.

Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex.

Pre-B cell receptor (pre-BCR) expression is critical for B lineage development. The signaling events initiated by the pre-BCR, however, remain poorly defined. We demonstrate that lipid rafts are the major functional compartment for human pre-B cell activation. A fraction of pre-BCR was constitutively raft associated, and receptor engagement enhanced this association. These events promoted Lyn activation and Igbeta phosphorylation and led to the generation of a raft-associated signaling module composed of tyrosine phosphorylated Lyn, Syk, BLNK, PI3K, Btk, VAV, and PLCgamma2. Formation of this module was essential for pre-BCR calcium signaling. Together, these observations directly link the previously identified genetic requirement for the components of this module in B lineage development with theirfunctional role(s) in human preBCR signaling.

In vitro reconstitution of human B-cell ontogeny: from CD34(+) multipotent progenitors to Ig-secreting cells.

We describe a long-term, in vitro culture system initiated with CD34(+) or CD34(+)CD38(-) umbilical cord blood hematopoietic progenitors that supports normal human B-lineage development, including the production of mature Ig-secreting B cells. In the first stage (human B-progenitor long-term culture [HB-LTC]), CD34(+) hematopoietic progenitors are cultured on the murine stromal cell line, S17, leading to the sustained production of large numbers of CD10(+), CD19(+) early B progenitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) and three-parameter flow cytometry for VpreB (surrogate light chain), cytoplasmic mu chain, and surface IgM expression were used to characterize the CD19(+) B progenitors present within these cultures. This analysis showed distinct B-lineage subpopulations, including pro-B cells, cycling pre-B cells, and IgM+, IgD-/+ immature B cells. The limited expansion of IgM+ B cells and the immature surface phenotype of this population (IgM+, IgD+, CD10(+), CD38(+)) suggested that HB-LTC conditions were unable to provide appropriate signals for further differentiation. A second culture stage was used to determine if these immature B cells were functionally competent. Purified CD19(+) cells were transferred onto fibroblasts expressing human CD40-ligand in the presence of IL-10 and IL-4. This lead to cell proliferation, modulation of the IgM+ cell surface phenotype to one consistent with an activated mature B cell, secretion of Ig, and isotype switching. Notably, IgM and IgG producing B cells were also generated using two-stage cultures established with highly purified multipotent CD34(+)CD38(-) hematopoietic stem cell progenitors. This culture model should permit detailed in vitro analysis and genetic manipulation of the major transition points in human B ontogeny, beginning with commitment to the B lineage and leading to development and activation of mature B cells.

Placental trophoblasts resist infection by multiple human immunodeficiency virus (HIV) type 1 variants even with cytomegalovirus coinfection but support HIV replication after provirus transfection.

Whether cell-free human immunodeficiency virus type 1 (HIV-1) can productively infect placental trophoblasts (which in turn could transmit the virus into the fetal circulation) is controversial but essential to know for the evaluation of alternative routes (such as cell-mediated infection or trophoblast damage). We have addressed infection factors such as cell purity, source, culture methods, and activation states as well as virus variant and detection methods to conclusively determine the outcome of trophoblast challenge by free virus. Pure (> 99.98%) populations of trophoblasts from 11 different placentas were challenged at a multiplicity of infection (MOI) as high as 6 with five different HIV-1 variants, three of which are non-syncytium-forming, macrophage-tropic isolates from infected infants, with and without coinfection with cytomegalovirus; these preparations were monitored for productive infection for up to 3 weeks after challenge by five different criteria, the most sensitive of which were cocultivation with target cells that can detect virus at an MOI of 10(-7) and HIV DNA PCR that detects 30 virus copies per 10(5) cells. Infection was never detected. However, molecularly cloned T-cell (pNL4-3)- and macrophage (pNLAD8)-tropic provirus plasmids, when transfected into primary trophoblasts, yielded productive infections, indicating that trophoblasts do not suppress late-stage virus replication and assembly. Because of the purity of the trophoblast preparations, the extended length of the infection culture period, the number of trophoblast preparations and virus types examined, the sensitivity of the bioassays and molecular detection assays, and the observations that trophoblasts can support virus replication from provirus, the results of this study strongly argue that free virus cannot infect primary villous trophoblasts.

ICAM-1-mediated adhesion of peripheral blood monocytes to the maternal surface of placental syncytiotrophoblasts: implications for placental villitis.

Accumulation of maternal monocytes in the villous/intervillous space (villitis) is associated with increased risk of perinatal morbidity and mortality and may initiate in utero transmission of cell-associated infectious agents such cytomegalovirus and HIV-1. We have developed an in vitro model of trophoblast syncytialization and have investigated the adhesive interactions between this tissue and peripheral blood monocytes. We show that monocytes strongly adhere to cultured syncytiotrophoblasts (STs) and that treatment with the inflammatory cytokines interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 alpha greatly increase the number bound. Pretreatment of STs with these cytokines upregulated apical expression of intercellular cell adhesion molecule (ICAM)-1 but not E-or L-selection, ICAM-2 or -3, or various integrins. ICAM-1 expression was cytokine concentration dependent, significantly increased within 6 hours of treatment, peaked after 24 hours, and remained undiminished for 48 hours after cytokine removal from the cultures. Adhesion of monocytes to STs was inhibited > 80% by antibody to ICAM-1 or its cognate ligand LFA-1. ICAM-1 was detected immunohistochemically only in rare foci on intact term placental villi. These results suggest that villous trophoblast expression of ICAM-1 occurs only during an immune inflammatory reaction and that aberrant expression of this molecule may be an important pathological feature in those immunoinflammatory disorders of the placenta characterized by an excessive accumulation of leukocytes in the intervillous/villous space such as spontaneous abortion, perinatal hematogenous infections, and villitis of unknown etiology.

Expression of the human p55 and p75 tumor necrosis factor receptors in primary villous trophoblasts and their role in cytotoxic signal transduction.

Tumor necrosis factor alpha (TNF alpha) induces the apoptotic death of primary villous cytotrophoblasts in culture (Yui et al., Placenta 1994; 15:819). Since both p55 and p75 TNF receptors (TNFRs) localize to the villous trophoblast, we examined their roles in mediating trophoblast apoptosis. Comparison of 125I-TNF alpha binding competition by receptor-specific antibodies revealed 2.7-fold more TNFRp75 than TNFRp55. Immunohistochemical analysis of receptor distribution showed TNFRp75 to be expressed strongly in < 20% of cells and TNFRp55 moderately in approximately 50%. Culture with TNF alpha increased the percentage of cells expressing TNFRp75 to > 40% but had little effect on TNFRp55 expression. Agonistic anti-TNFRp55 antibody and TNFRp55-specific TNF mutant protein stimulated both apoptosis and loss of trophoblast viability. In contrast, TNFRp75-specific mutant TNF alpha protein failed to induce either of these responses. Furthermore, neither cell death nor apoptosis stimulated by wild-type TNF alpha was inhibited by an antagonistic anti-TNFRp75 antibody. Thus, the apoptotic death of primary cytotrophoblasts is mediated almost entirely by TNFRp55, and the p75 receptor appears to have little effect on the process.

Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts.

In the placenta, as in other organs, the development and maintenance of the differentiated phenotype depend on a balance between cell proliferation, maturation, and death. We are interested in the mechanisms that regulate the survival and differentiation of placental trophoblasts and have recently demonstrated that the inflammatory cytokines tumor necrosis factor alpha (TNF alpha) and gamma interferon (IFN gamma) act in concert to induce apoptotic cell death in normal cytotrophoblasts in culture. In this report we show that exposure to epidermal growth factor (EGF), a 6,700 dalton polypeptide that is abundantly expressed in maternal and fetal tissues, blocks the in vitro TNF/IFN-induced cytotoxicity of human cytotrophoblasts and syncytiotrophoblasts from normal term placentas. This antagonistic effect is dose-related (10-10 M EGF, half-maximal) and proceeds via the interruption of an early step in the cytokine-induced apoptotic response. These observations suggest a novel role for EGF in normal placental development and indicate that the interplay between EGF, TNF alpha, and IFN gamma may determine the rate of trophoblast growth and renewal during gestation.

Cytotoxicity of tumour necrosis factor-alpha and gamma-interferon against primary human placental trophoblasts.

Tumour necrosis factor-alpha (TNF-alpha) and gamma interferon (IFN-gamma) are expressed within human placental villi during normal pregnancy, yet their functions remain unknown. Since villous cytotrophoblasts are within the paracrine reach of this expression, the effects of TNF-alpha and IFN-gamma on a purified population of term placental cytotrophoblasts were examined. After 4 days of culture TNF-alpha alone induced a loss of trophoblast viability as measured by both metabolic capacity (MTT reduction) and DNA content. The combination of TNF-alpha and IFN-gamma enhanced the damaging effect. Neutralizing antibodies against TNF receptor p55, but not p75, partially reversed the TNF-alpha-induced cytotoxicity. After 24 h of culture, TNF-alpha and IFN-gamma increased the fraction trophoblasts containing nicked DNA, and after 60 h, increased the detachment of cells characterized by a distorted morphology, lower DNA content, and fragmented DNA. These results suggest that a physiological role of TNF-alpha and IFN-gamma expression in the placental villi may be to regulate the apoptotic death of villous cytotrophoblasts. The studies also predict potential harmful effects on placental development and function following aberrant inflammatory cytokine expression triggered by intravillous infections.

Demonstration of functional cytokine-placental interactions: CSF-1 and GM-CSF stimulate human cytotrophoblast differentiation and peptide hormone secretion.

The placental syncytiotrophoblast (ST) is a terminally differentiated epithelial cell monolayer that constitutes the outermost boundary between fetal and maternal tissues and performs a variety of synthetic, secretory, and transport functions essential for the maintenance of pregnancy. Although it is known that the ST arises from the underlying germinal layer of mononuclear cytotrophoblasts (Langhans' cells) by a process of cell fusion, the molecular mechanisms involved in this process are unclear. In order to address this question, we have investigated the effects of macrophage colony-stimulating factor (CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphohemopoietic cytokines implicated in mammalian placental development, on the in vitro morphological and functional differentiation of human trophoblast. Both CSF-1 and GM-CSF stimulated cytotrophoblast aggregation into large multinucleated structures composed of extensive patches of syncytium interspersed with mononuclear cells. Concomitant with this morphological differentiation was upregulation of the production of the placental hormones placental lactogen and chorionic gonadotrophin. Placental fibroblasts derived from the villous stroma that underlies the trophoblastic epithelium were found to produce both GM-CSF and CSF-1 under the control of the trophoblast-derived cytokines IL-1 and TNF alpha. These observations suggest that a network of interrelated cytokines operates within the basal (fetal) aspects of the villous stroma where they are situated to play a significant role in the morphological and functional development of the human placenta.

Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells.

We have extended previous observations of expression of the trypsin-resistant cell surface antigen CD9 on placental fibroblasts to virtually all cells in the villous stroma and developed a method for eliminating CD9 expressing cells from trypsinized placental preparations. Preparations incubated with the mouse anti-human CD9 monoclonal antibody 50H.19 were passed through a goat anti-mouse immunoglobulin column that captures CD9 expressing cells. Approximately 95 per cent of the eluted cells stained positive with the villous trophoblast specific antibody GB25 and could be cryopreserved and thawed with > 80 per cent recovery in culture. One week cultures contained fewer than 0.3 per cent vimentin positive (mesenchymal) cells and maintained secretion of hCG. Two week cultures remained free of fibroblasts and macrophages. Clusters of trophoblasts that formed spontaneously during the first week of culture were shown by microinjection of carboxyfluorescein and by staining with anti-desmoplakin antibody to be a patchwork of mononuclear cells and syncytial units. Although the DNA content of the culture decreased by 35 per cent during the 2 week culture, the metabolic capacity and protein content remained relatively constant. Thus, CD9 immuno-elimination gives a high yield of enriched and viable trophoblasts that can be cultured for at least 2 weeks with almost no contamination by stromal cells.

Analysis of the synergistic stimulation of mouse macrophage proliferation by macrophage colony-stimulating factor (CSF-1) and tumor necrosis factor alpha (TNF-alpha).

Tumor necrosis factor alpha (TNF-alpha) more than doubles tritiated thymidine ([3H]TdR) uptake in mouse macrophages stimulated by macrophage colony-stimulating factor (CSF-1). However, nothing is known of how TNF-alpha affects this increase or even whether it is manifested by increased cellular proliferation. Here we characterize the effects of TNF-alpha on CSF-1-stimulated proliferation of both primary cells (bone marrow-derived macrophages, BMMs) and a cloned growth factor-dependent macrophage cell line (S1). We show that the TNF-alpha-induced increase in [3H]TdR uptake of CSF-1-stimulated macrophages is directly proportional to an increase in the DNA content of the culture and that the effects of TNF-alpha are direct and independent of cell number. TNF-alpha decreases the population doubling time of log-phase growing macrophages having quite different growth rates to the same (approximately 30%) extent: the doubling time of BMMs decreases from 24 to 17 h and that of S1 cells from 17 to 13 h. TNF-alpha exerts its effects on log-phase growth by increasing to the same proportion CSF-1-stimulated proliferation at all concentrations of CSF-1; that is, TNF-alpha does not shift, but rather amplifies, the CSF-1 dose-response curve. Although TNF-alpha alone does not stimulate macrophage proliferation, its presence in S1 cell cultures coming to quiescence after withdrawal of CSF-1 greatly increases subsequent CSF-1-stimulated [3H]TdR uptake as the cells reenter the cycle. Finally, we show that both human and mouse TNF-alpha increase CSF-1-stimulated log-phase growth and reentry of quiescent cells into the cycle equally on a molar basis (half-maximal stimulation of approximately 0.3 nM). The latter observation argues that the growth-stimulatory effects of TNF-alpha are mediated via the 55-60-kd TNF receptor. We conclude that TNF-alpha acts directly on growth-competent macrophages to decrease significantly the population doubling time in a manner that enhances the mitogenic effects of CSF-1.

Macrophage colony-stimulating factor enhances the respiratory burst of human monocytes in response to Entamoeba histolytica.

Mononuclear phagocytes (MP) are probably the most capable effector cells of the body in the defense against virulent strains of E. histolytica. Killing of E. histolytica by MP appears to involve both oxidative and non-oxidative mechanisms. Thus, in this study we have investigated whether trophozoites of an axenic virulent strain E. histolytica HM1:IMSS (EH) were capable of eliciting an oxidative response in pure populations of freshly isolated human monocytes. Using a luminol-enhanced chemiluminescence assay we demonstrate that these cells produce a strong respiratory burst when challenged with live amebas over a wide range of MP:EH ratios. Furthermore, pre-incubation of monocytes with recombinant Macrophage Colony Stimulating Factor (M-CSF) could further increase the oxidative metabolism of MP in response to E. histolytica. Our results indicate that, in contrast to what occurs with polymorphonuclear leukocytes, the interaction of E. histolytica with MP leads to the production of reactive oxygen intermediates by this cells. The enhancement of this potent microbicidal mechanism by inflammatory cytokines may further increase the amebicidal capacity of human mononuclear phagocytes.

[Cytokines and their role as health and disease mediators. New approaches to old problems]. / Las citocinas y su papel como mediadores de salud y enfermedad. Nuevos enfoques para viejos problemas.

The cytokines are multifunctional polypeptide hormones, produced by a variety of cells, that participate in the regulation of many biological processes. Essentially acting as intercellular messengers, they play a central role in the maintenance of homeostasis in normal tissues. Cytokines are key mediators of both local and systemic immune-inflammatory responses; therefore, disturbances in their secretion, response and/or regulation have been implicated in the pathophysiology of several conditions in which an exaggerated auto-destructive component appears to play a role. Conversely, deficits in cytokine production probably impair the host's ability to mount an effective immune response and may underlie the increased susceptibility to microbial infection observed, for example, in the neonate or in the malnourished patient. A review of the principal aspects of the biology of cytokines is presented together with the evidence supporting their involvement in several pathological states and the potential therapeutic applications of these mediators as novel immunomodulating agents.