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Regulatory T cells (Tregs) are plastic cells playing a pivotal role in the maintenance of immune homeostasis. Tregs actively adapt to the microenvironment where they reside; as a consequence, their molecular and functional profiles differ among tissues and pathologies. In tumors, the features acquired by Tregs remains poorly characterized. Here, we observed that human tumor-infiltrating Tregs selectively overexpress CD74, the MHC class II invariant chain. CD74 has been previously described as a regulator of antigen-presenting cell biology, however its function in Tregs remains unknown. CD74 genetic deletion in human primary Tregs revealed that CD74KO Tregs exhibit major defects in the organization of their actin cytoskeleton and intracellular organelles. Additionally, intra-tumoral CD74KO Tregs show a decreased activation, a drop in Foxp3 expression, a low accumulation in the tumor, and consistently, they accelerate the tumor rejection. These observations are unique to tumor conditions as, at steady state, CD74KO-Treg phenotype, survival, and suppressive capacity are unaffected in vitro and in vivo. CD74 therefore emerges as a specific regulator of tumor-infiltrating Tregs and as a novel target to interfere with Treg anti-tumor activity.

Tumor associated macrophages (TAMs), which differentiate from circulating monocytes, are pervasive across human cancers and comprise heterogeneous populations. The contribution of tumor-derived signals to TAM heterogeneity is not well understood. In particular, tumors release both soluble factors and extracellular vesicles (EVs), whose respective impact on TAM precursors may be different. Here, we show that triple negative breast cancer cells (TNBCs) release EVs and soluble molecules promoting monocyte differentiation toward distinct macrophage fates. EVs specifically promoted proinflammatory macrophages bearing an interferon response signature. The combination in TNBC EVs of surface CSF-1 promoting survival and cargoes promoting cGAS/STING or other activation pathways led to differentiation of this particular macrophage subset. Notably, macrophages expressing the EV-induced signature were found among patients’ TAMs. Furthermore, higher expression of this signature was associated with T cell infiltration and extended patient survival. Together, this data indicates that TNBC-released CSF-1-bearing EVs promote a tumor immune microenvironment associated with a better prognosis in TNBC patients.

Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63- or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs’ release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we discovered that: 1) the two drugs act on EVs generated in distinct subcellular compartments and 2) EVs released upon treatment with Homosalate, but not with Bafilomycin A1, conferred anti-anoikis properties to another recipient tumor cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harboring distinct functional properties.

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type., Extracellular vesicles (EVs) play a role in intercellular communication, however the precise biogenesis of different populations of EVs are not clear. Here, the authors follow the intracellular trafficking of two proteins before their secretion in EVs and report the biogenesis and protein markers of EV subtypes: ectosomes budding from the plasma membrane as well as exosomes from late endosomes.

Summary The human immunodeficiency virus (HIV) enters the nucleus to establish infection, but the role of nuclear envelope proteins in this process is incompletely understood. Inner nuclear transmembrane proteins SUN1 and SUN2 connect nuclear lamins to the cytoskeleton and participate in the DNA damage response (DDR). Increased levels of SUN1 or SUN2 potently restrict HIV infection through an unresolved mechanism. Here, we find that the antiviral activities of SUN1 and SUN2 are distinct. HIV-1 and HIV-2 are preferentially inhibited by SUN1 and SUN2, respectively. We identify DNA damage inducers that stimulate HIV-1 infection and show that SUN1, but not SUN2, neutralizes this effect. Finally, we show that chromatin movements and nuclear rotations are associated with the effects of SUN proteins and Lamin A/C on infection. These results reveal an emerging role of chromatin dynamics and the DDR in the control of HIV infection by structural components of the nuclear envelope.

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release., A new proteomic approach allows comprehensive characterisation of exosome heterogeneity in human T cells.

The Human Immunodeficiency Virus (HIV) enters the nucleus to establish infection. HIV interacts with nuclear pore components to cross the nuclear envelope. In contrast, the role of other proteins of the nuclear envelope in HIV infection is not yet understood. The inner nuclear transmembrane proteins SUN1 and SUN2 connect lamins in the interior of the nucleus to the cytoskeleton in the cytoplasm. Increased levels of SUN1 or SUN2 potently restrict HIV infection through an unresolved mechanism. Here, we find that SUN1 and SUN2 exhibit a differential and viral strain-specific antiviral activity HIV-1 and HIV-2. In macrophages and HeLa cells, HIV-1 and HIV-2 are respectively preferentially inhibited by SUN1 and SUN2. This specificity maps to the nucleoplasmic domain of SUN proteins, which associates with Lamin A/C and participates to the DNA damage response. We find that etoposide, a DNA-damaging drug, stimulates infection. Inhibition of the DNA damage signaling kinase ATR, which induces a DNA damage response, also enhances HIV-1 infection. The proviral effect of ATR inhibition on infection requires the HIV-1 Vpr gene. Depletion of endogenous Lamin A/C, which sensitizes cells to DNA damage, also enhances HIV-1 infection in HeLa cells. SUN1 overexpression neutralizes these proviral effects, while the antiviral effect of SUN2 is rescued by etoposide treatment. Finally, we show that inhibition of HIV-1 infection by overexpressed SUN proteins and endogenous Lamin A/C is associated with reduced internal movements of chromatin and reduced rotations of the nucleus. Altogether, these results highlight distinct antiviral activities of SUN1 and SUN2 and reveal an emerging role of nuclear movements and the DNA damage response in the control of HIV infection by structural components of the nuclear envelope.

Despite their important and multiple roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized yet. In particular, how and to what extent EVs form either as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (ectosomes) remains unclear. We followed in HeLa cells the intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment and identified transient co-localization both at the plasma membrane (PM) and in endosomes, before they finally segregate. CD9 was more abundantly released in EVs than CD63. However, when forcing expression of CD63 at the PM, by mutating its lysosome-addressing motive, its secretion in EVs was increased. Thus, in HeLa cells, small ectosomes are more prominently released than exosomes. By comparative proteomic analysis, we identified a few surface proteins likely specific of either exosomes (e.g. LAMP1) or ectosomes (e.g. BSG, SLC3A2), based on their known intracellular location in lysosomes or the PM, and on the different effects on their release of Bafilomycin A1, a drug that neutralizes endosomal pH. Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

A significant proportion of HIV-2-infected patients exhibit natural virological control that is generally absent from HIV-1-infected patients. Along with CD4+ T cells, HIV-1 targets macrophages which may contribute to viral spreading and the latent reservoir. We have studied the relationship between macrophages and HIV-2, focusing on post-entry steps. HIV-2-infected monocyte-derived macrophages (MDMs) produced substantial amounts of viral particles that were largely harbored intracellularly. New viruses assembled at the limiting membrane of internal compartments similar to virus-containing compartments (VCCs) described for HIV-1. VCCs from MDMs infected with either virus shared protein composition and morphology. Strikingly, HIV-2 Gag was mostly absent from the cytosol and almost exclusively localized to the VCCs, whereas HIV-1 Gag was distributed in both locations. Ultrastructural analyses of HIV-2-infected MDMs revealed the presence of numerous VCCs containing both immature and mature particles in the lumen. HIV-2 particles produced de novo by MDMs were poorly infectious in reporter cells and in transmission to activated T cells through a process that appeared independent of BST2 restriction. Rather than being involved in viral spreading, HIV-2-infected macrophages may represent a cell-associated source of viral antigens that can participate in the immune control of HIV-2 infection.

Cells release diverse types of extracellular vesicles (EVs), which can transfer complex signals to surrounding cells. Specific markers to distinguish different classes of EVs (e.g. exosomes, microvesicles, enveloped viruses) are still lacking. We have developed an unbiased quantitative proteomic profiling approach for characterizing EV subtypes, and applied it to human T lymphoma cells under two experimental conditions: uninfected or infected with HIV-1. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Comparing EVs from control and HIV-1-infected cells, we identified altered profiles of numerous cellular proteins including SPN and MOV10, specifically incorporated into virions, and SERINC3, in non-viral EVs. Our unbiased EV profile analysis tool is thus a powerful approach to identify common cargoes of given EV subtypes. Furthermore, it can be used in any in vitro experimental system to investigate physiological or pathological modifications of EV release.

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells at cross-presenting antigens to CD8+ T cells. Although it is generally accepted that dendritic cells take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen-specific CD8+ T cell clones after 5 days of differentiation into monocytes-derived dendritic cells (MoDC). Tagged-long peptides can be visualized in electron-dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from eighteen metastatic melanoma patients and found that CD14+ monocytes from 2 patients effectively activate a MelanA-specific CD8 T cell clone after in vitro differentiation into MoDC. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor-specific immune response and should be taken into account for cancer immunotherapy. This article is protected by copyright. All rights reserved.

The human dendritic cell (DC) lineage has recently been unraveled by high-dimensional mapping, revealing the existence of a discrete new population of blood circulating DC precursors (pre-DCs). Whether this new DC population possesses specific functional features as compared to the other blood DC subset upon pathogen encounter remained to be evaluated. A unique feature of pre-DCs among blood DCs is their constitutive expression of the viral adhesion receptor Siglec-1. Here, we show that pre-DCs, but not other blood DC subsets, are susceptible to infection by HIV-1 in a Siglec-1–dependent manner. Siglec-1 mediates pre-DC infection of CCR5- and CXCR4-tropic strains. Infection of pre-DCs is further enhanced in the presence of HIV-2/SIVmac Vpx, indicating that Siglec-1 does not counteract restriction factors such as SAMHD1. Instead, Siglec-1 promotes attachment and fusion of viral particles. HIV-1–infected pre-DCs produce new infectious viral particles that accumulate in intracellular compartments reminiscent of the virus-containing compartment of macrophages. Pre-DC activation by toll-like receptor (TLR) ligands induces an antiviral state that inhibits HIV-1 fusion and infection, but Siglec-1 remains functional and mediates replication-independent transfer of HIV-1 to activated primary T lymphocytes. Altogether, Siglec-1–mediated susceptibility to HIV-1 infection of pre-DCs constitutes a unique functional feature that might represent a preferential relationship of this emerging cell type with viruses.

Human dendritic cell (DC) lineage has been recently unraveled by high dimensional mapping revealing the existence of a discrete new population of blood circulating DC precursor (pre-DC also referred to as AS DC). Among all blood DC subsets, only pre-DC highly express Siglec-1, a lectin-like receptor able to bind HIV-1. We show that pre-DC are uniquely equipped among blood DC populations to promote HIV-1 replication and dissemination. Pre-DC stands out as the most susceptible DC population to infection by both HIV-1 CXCR4- and CCR5-tropic viral particles in a Siglec-1-dependent manner. HIV-1-infected pre-DC produce new viral progeny and transmit the virus to CD4+T cells. Upon TLR activation, pre-DC become resistant to HIV-1 fusion and thus to infection and switch to a replication-independent mechanism of virus transfer to activated primary T lymphocytes mediated by Siglec-1. Thus, beside their role in DC ontogeny, blood pre-DC possess stage-specific properties that HIV-1 may exploit for viral spreading and modulation of the immune response.

Summary Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells., Graphical Abstract, Highlights • Intracellular trafficking of IL-12 in dendritic cells is mediated by the SNARE VAMP7 • VAMP7 is required for optimal secretion of IL-12 in the extracellular space • IL-12/VAMP7+ vesicles gather at the immune synapse • VAMP7 controls synaptic release of IL-12 and IFN-γ production in T cells, Efficient priming of T cells requires antigenic and soluble cytokine signals. Chiaruttini et al. analyze the intracellular trafficking pathway of IL-12 in dendritic cells and identify the SNARE VAMP7 as a key regulator of cytokine release and T cell activation.

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.

Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. Fil: Liao, Zhaohao. University Johns Hopkins; Estados Unidos Fil: Martin Jaular, Lorena. Inserm; Francia. PSL Research University; Francia Fil: Soueidi, Estelle. Inserm; Francia. PSL Research University; Francia Fil: Jouve, Mabel. PSL Research University; Francia Fil: Muth, Dillon C.. University Johns Hopkins; Estados Unidos Fil: Schøyen, Tine H.. University Johns Hopkins; Estados Unidos Fil: Seale, Tessa. University Johns Hopkins; Estados Unidos Fil: Haughey, Norman J.. University Johns Hopkins; Estados Unidos Fil: Ostrowski, Matias. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Théry, Clotilde. PSL Research University; Francia Fil: Witwer, Kenneth W.. University Johns Hopkins; Estados Unidos

Here we describe a new mouse model that exploits the pattern of expression of the high-affinity IgG receptor (CD64) and allows diphtheria toxin (DT)–mediated ablation of tissue-resident macrophages and monocyte-derived cells. We found that the myeloid cells of the ear skin dermis are dominated by DT-sensitive, melanin-laden cells that have been missed in previous studies and correspond to macrophages that have ingested melanosomes from neighboring melanocytes. Those cells have been referred to as melanophages in humans. We also identified melanophages in melanocytic melanoma. Benefiting of our knowledge on melanophage dynamics, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture–release–recapture without any tattoo vanishing. Therefore, congruent with dermal macrophage dynamics, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity.

INTRODUCTION Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises multiple subsets, including plasmacytoid DC (pDC) and two functionally specialized lineages of conventional DC (cDC1 and cDC2), whose origins and differentiation pathways remain incompletely defined. RATIONALE As DC are essential regulators of the immune response in health and disease, potential intervention strategies aiming at manipulation of these cells will require in-depth insights of their origins, the mechanisms that govern their homeostasis, and their functional properties. Here, we employed two unbiased high-dimensional technologies to characterize the human DC lineage from bone marrow to blood. RESULTS We isolated the DC-containing population (Lineage − HLA − DR + CD135 + cells) from human blood and defined the transcriptomes of 710 individual cells using massively parallel single-cell mRNA sequencing. By combining complementary bioinformatic approaches, we identified a small cluster of cells within this population as putative DC precursors (pre-DC). We then confirmed this finding using cytometry by time-of-flight (CyTOF) to simultaneously measure the expression of a panel of 38 different proteins at the single-cell level on Lineage − HLA − DR + cells and found that pre-DC possessed a CD123 + CD33 + CD45RA + phenotype. We confirmed the precursor potential of pre-DC by establishing their potential to differentiate in vitro into cDC1 and cDC2, but not pDC, in the known proportions found in vivo . Interestingly, pre-DC also express classical pDC markers, including CD123, CD303, and CD304. Thus, any previous studies using these markers to identify or isolate pDC will have inadvertently included CD123 + CD33 + pre-DC. We provide here new markers that can be used to identify unambiguously pre-DC from pDC, including CD33, CX3CR1, CD2, CD5, and CD327. When CD123 + CD33 + pre-DC and CD123 + CD33 − pDC were isolated separately, we observed that pre-DC have unique functional properties that were previously attributed to pDC. Although pDC remain bona fide interferon-α–producing cells, their reported interleukin-12 (IL-12) production and CD4 T cell allostimulatory capacity can likely be attributed to “contaminating” pre-DC. We then asked whether the pre-DC population contained both uncommitted and committed pre-cDC1 and pre-cDC2 precursors, as recently shown in mice. Using microfluidic single-cell mRNA sequencing (scmRNAseq), we showed that the human pre-DC population contains cells exhibiting transcriptomic priming toward cDC1 and cDC2 lineages. Flow cytometry and in vitro DC differentiation experiments further identified CD123 + CADM1 − CD1c − putative uncommitted pre-DC, alongside CADM1 + CD1c − pre-cDC1 and CADM1 − CD1c + pre-cDC2. Finally, we found that pre-DC subsets expressed T cell costimulatory molecules and induced comparable proliferation and polarization of naive CD4 T cells as adult DC. However, exposure to the Toll-like receptor 9 (TLR9) ligand CpG triggered IL-12p40 and tumor necrosis factor–α production by early pre-DC, pre-cDC1, and pre-cDC2, in contrast to differentiated cDC1 and cDC2, which do not express TLR9. CONCLUSION Using unsupervised scmRNAseq and CyTOF analyses, we have unraveled the complexity of the human DC lineage at the single-cell level, revealing a continuous process of differentiation that starts in the bone marrow (BM) with common DC progenitors (CDP), diverges at the point of emergence of pre-DC and pDC potential, and culminates in maturation of both lineages in the blood and spleen. The pre-DC compartment contains functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123 + pre-DC subset and two CD45RA + CD123 lo lineage-committed subsets. The discovery of multiple committed pre-DC populations with unique capabilities opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T-cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question, and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combines a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross-presenting antigens to CD8

In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T-cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question, and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combines a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin® resin, allowing the purification of membranes containing the Strep-tagged molecule of interest. By keeping the membranes intact, this protocol leads to the purification of molecules physically associated with the Strep-tagged protein as well as of molecules present in the same membrane compartment: transmembrane proteins, proteins strongly associated with the membranes, and luminal proteins. The example shown herein is the purification of membrane compartment prepared from T lymphocytes expressing LAT fused to a Strep-tag.

International audience; Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles.

The ontogeny of human Langerhans cells (LCs) remains poorly characterized, in particular the nature of LC precursors and the factors that may drive LC differentiation. Here we report that thymic stromal lymphopoietin (TSLP), a keratinocyte-derived cytokine involved in epithelial inflammation, cooperates with transforming growth factor (TGF)-β for the generation of LCs. We show that primary human blood BDCA-1(+), but not BDCA-3(+), dendritic cells (DCs) stimulated with TSLP and TGF-β harbor a typical CD1a(+)Langerin(+) LC phenotype. Electron microscopy established the presence of Birbeck granules, an intracellular organelle specific to LCs. LC differentiation was not observed from tonsil BDCA-1(+) and BDCA-3(+) subsets. TSLP + TGF-β LCs had a mature phenotype with high surface levels of CD80, CD86, and CD40. They induced a potent CD4(+) T-helper (Th) cell expansion and differentiation into Th2 cells with increased production of tumor necrosis factor-α and interleukin-6 compared with CD34-derived LCs. Our findings establish a novel LC differentiation pathway from BDCA-1(+) blood DCs with potential implications in epithelial inflammation. Therapeutic targeting of TSLP may interfere with tissue LC repopulation from circulating precursors.

Proteins of the Bin/amphiphysin/Rvs (BAR) domain superfamily are essential in controlling the shape and dynamics of intracellular membranes. Here, we present evidence for the unconventional function of a member of the endophilin family of BAR and Src homology 3 domain-containing proteins, namely endophilin B2, in the perinuclear organization of intermediate filaments. Using mass spectrometry analysis based on capturing endophilin B2 partners in in situ pre-established complexes in cells, we unravel the interaction of endophilin B2 with plectin 1, a variant of the cytoskeleton linker protein plectin as well as with vimentin. Endophilin B2 directly binds the N-terminal region of plectin 1 via Src homology 3-mediated interaction and vimentin indirectly via plectin-mediated interaction. The relevance of these interactions is strengthened by the selective and drastic reorganization of vimentin around nuclei upon overexpression of endophilin B2 and by the extensive colocalization of both proteins in a meshwork of perinuclear filamentous structures. By generating mutants of the endophilin B2 BAR domain, we show that this phenotype requires the BAR-mediated membrane binding activity of endophilin B2. Plectin 1 or endophilin B2 knockdown using RNA interference disturbed the perinuclear organization of vimentin. Altogether, these data suggest that the endophilin B2-plectin 1 complex functions as a membrane-anchoring device organizing and stabilizing the perinuclear network of vimentin filaments. Finally, we present evidence for the involvement of endophilin B2 and plectin 1 in nuclear positioning in individual cells. This points to the potential importance of the endophilin B2-plectin complex in the biological functions depending on nuclear migration and positioning.

International audience; Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e., exosomes) and EVs of various sizes budding from the plasma membrane. Specific markers for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation speed, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Extensive quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We identified proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative abundance in distinct EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies.

International audience; Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility and ability to assemble into highly ordered oligomers, contribute to inducing curvature of target membranes. Endophilins, diverging into A and B sub-groups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics and permeabilization during apoptosis. Here, we report on the involvement of the third α-helix of endophilins A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of amino-terminal truncation retaining the high dimerization capacity of the third α-helices of endophilins A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR-sequence mediated dimerization on SH3 domain partnership. In comparison to wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution, exhibit very significant changes in membrane-binding and shaping activities as well as dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact on both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution, impairs the endocytic recycling of transferrin receptors.

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells. However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2.

International audience; Macrophages represent viral reservoirs in HIV-1-infected patients and accumulate viral particles within an endosomal compartment where they remain infectious for long periods of time. To determine how HIV-1 survives in endocytic compartments that become highly acidic and proteolytic and to study the nature of these virus-containing compartments, we carried out an ultrastructural study on HIV-1-infected primary macrophages. The endosomal compartments contain newly formed virions rather than internalized ones. In contrast to endocytic compartments free of viral proteins within the same infected cells, the virus containing compartments do not acidify. The lack of acidification is associated with an inability to recruit the proton pump vacuolar ATPase into the viral assembly compartment. This may prevent its fusion with lysosomes, since acidification is required for the maturation of endosomes. Thus, HIV-1 has developed a strategy for survival within infected macrophages involving prevention of acidification within a devoted endocytic virus assembly compartment.

The initiation of cytotoxic immune responses by dendritic cells (DCs) requires the presentation of antigenic peptides derived from phagocytosed microbes and infected or dead cells to CD8(+) T cells, a process called cross-presentation. Antigen cross-presentation by non-activated DCs, however, is not sufficient for the effective induction of immune responses. Additionally, DCs need to be activated through innate receptors, like Toll-like receptors (TLRs). During DC maturation, cross-presentation efficiency is first upregulated and then turned off. Here we show that during this transient phase of enhanced cross-presentation, phago-lysosome fusion was blocked by the topological re-organization of lysosomes into perinuclear clusters. LPS-induced lysosomal clustering, inhibition of phago-lysosome fusion and enhanced cross-presentation, all required expression of the GTPase Rab34. We conclude that TLR4 engagement induces a Rab34-dependent re-organization of lysosomal distribution that delays antigen degradation to transiently enhance cross-presentation, thereby optimizing the priming of CD8(+) T cell responses against pathogens. Fil: Alloatti, Andrés. Inserm; Francia. Institute Curie; Francia Fil: Kotsias, Fiorella. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Institute Curie; Francia. Inserm; Francia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento Virologia; Argentina Fil: Pauwels, Anne Marie. University of Ghent; Bélgica Fil: Carpier, Jean Marie. Institute Curie; Francia. Inserm; Francia Fil: Jouve, Mabel. Institute Curie; Francia Fil: Timmerman, Evy. University of Ghent; Bélgica Fil: Pace, Luigia. Institute Curie; Francia. Inserm; Francia Fil: Vargas, Pablo. Institute Curie; Francia. Inserm; Francia. Centre National de la Recherche Scientifique; Francia Fil: Maurin, Mathieu. Institute Curie; Francia. Inserm; Francia Fil: Gehrmann, Ulf. Institute Curie; Francia. Inserm; Francia Fil: Joannas, Leonel. Institute Curie; Francia. Inserm; Francia Fil: Vivar, Omar I.. Institute Curie; Francia. Inserm; Francia Fil: Lennon Duménil, Ana Maria. Institute Curie; Francia. Inserm; Francia Fil: Savina, Ariel. Institute Curie; Francia. Inserm; Francia. Roche SAS; Francia Fil: Gevaert, Kris. University of Ghent; Bélgica Fil: Beyaert, Rudi. University of Ghent; Bélgica Fil: Hoffmann, Eik. Inserm; Francia. Institute Curie; Francia. University of Ghent; Bélgica Fil: Amigorena, Sebastian. Inserm; Francia. Institute Curie; Francia

The afa operons from Escherichia coli associated with extra-intestinal and intestinal infections have been characterized and the AfaD protein has been shown to be involved in the low internalization of laboratory strains expressing the afa-3 operon. The aim of this study was to determine the role of the AfaD invasin during the interaction of pathogenic E. coli with epithelial cells. We show that AfaD is implicated in the entry of a clinical isolate into both HeLa and undifferentiated Caco-2 cells. Once in the cytoplasm of these cells, the bacteria formed inclusions in which they were able to survive for at least 72 h. Internalization assays using polystyrene beads coated with His6-tagged purified AfaD (rAfaD) demonstrated that this invasin mediates entry into cells derived from various tissues (intestine and urothelium) that are targets for afa-positive strains. Consistent with the previous observation that an antibody blockade involving anti-alpha5beta1 integrin abolishes bacterial internalization, we show here that the entry of rAfaD-coated beads was dependent on the production and accessibility of beta1 integrins on the cells. The AfaD proteins belong to a family of invasins that are at least 45% identical. Despite their differences, the recombinant rAfaD-III and rAfaD-VIII proteins both bound to beta1 integrins. Our results suggest that beta1 integrin is a common receptor for AfaD invasins and that additional AfaD-type-specific receptors exist.

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048–5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr + adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr − adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr − adhesin) was found to predominate in afa -positive isolates from sepsis patients (75%); it was frequent in afa -positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr + strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa -positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa -positive strains.

The afimbrial adhesive sheath, encoded by the afa-3 gene cluster, is composed of two proteins with different roles in bacterium–HeLa cell interactions. AfaE is required for adhesion and AfaD for internalization. In this study, we found that the AfaD invasin was structurally and functionally conserved among human afa-expressing strains, independently of AfaE subtype and clinical origin of the Escherichia coli isolate. The AggB protein from enteroaggregative E. coli was also found to be an AfaD-related invasin. These data suggest that AfaD is the prototype of a family of invasins encoded by adhesion-associated operons in pathogenic E. coli.

In the versatile single species of Escherichia coli, the diarrheagenic group displays a remarkable array of virulence traits. These comprise microbial attachment, production of secretory endotoxins or cell-destroying cytotoxins, direct epithelial cell invasion, and localized effacement of the epithelium. The knowledge of how enteric E. coli induce disease has become increasingly important in the world, because of new pathogen emergence, increasing threats of drug resistance, and growing awareness of their importance in malnutrition and diarrhea. Numerous research programs have demonstrated various mechanisms of pathogenesis. We point out how some pathogens are able to develop intercourse with their host through subversion of its cytoskeleton and signaling processes without toxin secretion or heavy invasiveness. In that domain, the cellular biology of infected cells owes fundamental data to the electron microscopic approach. Combined with advances in microbiology and molecular biology, this approach may provide answers to many unanswered questions.

The afa gene clusters, which encode proteins involved in adhesion to epithelial cells, from Escherichia coli strains associated with urinary and intestinal infections in humans have been characterized. Pathogenic isolates of bovine and porcine origin that possess afa -related sequences have recently been described. We report in this work the cloning and characterization of the afa-7 and afa-8 gene clusters from bovine isolates. Hybridization and sequencing experiments revealed that despite similarity in genetic organization, the afa-7 and afa-8 genes, and the well-characterized afa-3 operon expressed by human-pathogenic isolates, correspond to three different members of the afa family of gene clusters. However, like the afa-3 gene cluster, both the afa-7 and afa-8 gene clusters were found to encode an afimbrial adhesin (AfaE) and an invasin (AfaD). The AfaD peptides encoded by the three gene clusters were only 45% identical, but functional complementation experiments indicated that they belong to the same family of invasins. Hemagglutination and adhesion assays demonstrated that the AfaE-VII and AfaE-VIII adhesins bind to different receptors and that these receptors are not the human decay-accelerating factor recognized to be the receptor of all previously described AfaE adhesins. The AfaE-VIII adhesin is very similar to the M agglutinin of human-uropathogenic strains. We used PCR assays to screen 25 bovine strains for afaD and afaE genes of either the afa-7 or afa-8 gene cluster. The afa-8 gene cluster was highly prevalent in bovine isolates previously reported to carry afa -related sequences (23 of 24 strains), particularly in strains producing cytotoxic necrotizing factors (16 of 16 strains). The location of the afa-8 gene cluster on the plasmids or chromosome of these isolates suggests that it could be carried by a mobile element, facilitating its dissemination among bovine-pathogenic E. coli strains.

Antibody targeting CD36 on HIV-1–infected macrophages results in rapid retention of virions within virus-containing compartments to inhibit release and viral transmission to T cells., HIV-1–infected macrophages likely represent viral reservoirs, as they accumulate newly formed virions in internal virus-containing compartments (VCCs). However, the nature and biogenesis of VCCs remain poorly defined. We show that upon HIV-1 infection of primary human macrophages, Gag is recruited to preexisting compartments containing the scavenger receptor CD36, which then become VCCs. Silencing of CD36 in HIV-1–infected macrophages decreases the amount of virions released. Strikingly, soluble anti-CD36 antibodies, but not the natural ligands of CD36, inhibit release of virions from HIV-1–infected macrophages and the transmission of virus to CD4+ T cells. The effect of the antibodies is potent, rapid, and induces the retention of virions within VCCs. Ectopic expression of CD36 in HeLa cells renders them susceptible to the inhibitory effect of the anti-CD36 mAb upon HIV-1 infection. We show that the anti-CD36 mAb inhibits HIV-1 release by clustering newly formed virions at their site of budding, and that signaling via CD36 is not required. Thus, HIV-1 reservoirs in macrophages may be tackled therapeutically using anti-CD36 antibodies to prevent viral dissemination.

International audience; During HIV pathogenesis, infected macrophages behave as ‘‘viral reservoirs’’ that accumulate and retain virions withindedicated internal Virus-Containing Compartments (VCCs). The nature of VCCs remains ill characterized and controversial.Using wild-type HIV-1 and a replication-competent HIV-1 carrying GFP internal to the Gag precursor, we analyzed thebiogenesis and evolution of VCCs in primary human macrophages. VCCs appear roughly 14 hours after viral proteinsynthesis is detected, initially contain few motile viral particles, and then mature to fill up with virions that become packedand immobile. The amount of intracellular Gag, the proportion of dense VCCs, and the density of viral particles in theirlumen increased with time post-infection. In contrast, the secretion of virions, their infectivity and their transmission to Tcells decreased overtime, suggesting that HIV-infected macrophages tend to pack and retain newly formed virions intodense compartments. A minor proportion of VCCs remains connected to the plasma membrane overtime. Surprisingly, livecell imaging combined with correlative light and electron microscopy revealed that such connections can be transient,highlighting their dynamic nature. Together, our results shed light on the late phases of the HIV-1 cycle and reveal some ofits macrophage specific features

KIF3A is important for the intracellular transport of HIV-containing compartments and HIV release from infected macrophages., Macrophages are long-lived target cells for HIV infection and are considered viral reservoirs. HIV assembly in macrophages occurs in virus-containing compartments (VCCs) in which virions accumulate and are stored. The regulation of the trafficking and release of these VCCs remains unknown. Using high resolution light and electron microscopy of HIV-1–infected primary human macrophages, we show that the spatial distribution of VCCs depended on the microtubule network and that VCC-limiting membrane was closely associated with KIF3A+ microtubules. Silencing KIF3A strongly decreased virus release from HIV-1–infected macrophages, leading to VCC accumulation intracellularly. Time-lapse microscopy further suggested that VCCs and associated KIF3A move together along microtubules. Importantly, KIF3A does not play a role in HIV release from T cells that do not possess VCCs. These results reveal that HIV-1 requires the molecular motor KIF3 to complete its cycle in primary macrophages. Targeting this step may lead to novel strategies to eliminate this viral reservoir.

International audience; Antigen (Ag) crosspresentation by dendritic cells (DCs) involves the presentation of internalized Ags on MHC class I molecules to initiate CD8+ T cell-mediated immunity in response to certain pathogens and tumor cells. Here, we identify the SNARE Sec22b as a specific regulator of Ag crosspresentation. Sec22b localizes to the ER-Golgi intermediate compartment (ERGIC) and pairs to the plasma membrane SNARE syntaxin 4, which is present in phagosomes (Phgs). Depletion of Sec22b inhibits the recruitment of ER-resident proteins to Phgs and to the vacuole containing the Toxoplasma gondii parasite. In Sec22b-deficient DCs, crosspresentation is compromised after Ag phagocytosis or endocytosis and after invasion by T. gondii. Sec22b silencing inhibited Ag export to the cytosol and increased phagosomal degradation by accelerating lysosomal recruitment. Our findings provide insight into an intracellular traffic pathway required for crosspresentation and show that Sec22b-dependent recruitment of ER proteins to Phgs critically influences phagosomal functions in DCs.

The molecular mechanisms involved in the assembly of newly synthesized Human Immunodeficiency Virus (HIV) particles are poorly understood. Most of the work on HIV-1 assembly has been performed in T cells in which viral particle budding and assembly take place at the plasma membrane. In contrast, few studies have been performed on macrophages, the other major target of HIV-1. Infected macrophages represent a viral reservoir and probably play a key role in HIV-1 physiopathology. Indeed macrophages retain infectious particles for long periods of time, keeping them protected from anti-viral immune response or drug treatments. Here, we present an overview of what is known about HIV-1 assembly in macrophages as compared to T lymphocytes or cell lines. Early electron microscopy studies suggested that viral assembly takes place at the limiting membrane of an intracellular compartment in macrophages and not at the plasma membrane as in T cells. This was first considered as a late endosomal compartment in which viral budding seems to be similar to the process of vesicle release into multi-vesicular bodies. This view was notably supported by a large body of evidence involving the ESCRT (Endosomal Sorting Complex Required for Transport) machinery in HIV-1 budding, the observation of viral budding profiles in such compartments by immuno-electron microscopy, and the presence of late endosomal markers associated with macrophage-derived virions. However, this model needs to be revisited as recent data indicate that the viral compartment has a neutral pH and can be connected to the plasma membrane via very thin micro-channels. To date, the exact nature and biogenesis of the HIV assembly compartment in macrophages remains elusive. Many cellular proteins potentially involved in the late phases of HIV-1 cycle have been identified; and, recently, the list has grown rapidly with the publication of four independent genome-wide screens. However, their respective roles in infected cells and especially in macrophages remain to be characterized. In summary, the complete process of HIV-1 assembly is still poorly understood and will undoubtedly benefit from the ongoing explosion of new imaging techniques allowing better time-lapse and quantitative studies.

We used a recent scanning electron microscope equipped with field emission gun and highly sensitive detectors to develop a fast and simple protocol for double immunogold staining using 10- and 15-nm gold particles. We used this approach to analyse the afimbrial adhesive sheath produced by pathogenic Escherichia coli interacting with the surface of epithelial cells. We demonstrated that AfaE adhesin and AfaD invasin were exposed at the bacterial surface during the interaction. This method could be easily and widely extended to the study of the early invasion process of many bacterial and viral pathogens, by immunocytochemical probing.

In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children ( P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea ( P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors ( P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.