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Journal of Experimental Medicine, The

In this issue, Canault et al. report for the first time a point mutation in the RAS guanyl-releasing protein 2 (RASGRP2) gene that results in a severe...

From multipotent stem cells, the hematopoietic system undergoes continuous differentiation to generate a remarkable array of mature cells throughout life. These hematopoietic stem cells (HSCs) hold promise for cell...

In this issue, Rodgers et al. report a new substrate of the linear ubiquitin assembly complex (LUBAC) and a new function of...

The preexisting HIV-1–specific T cell repertoire must influence both the immunodominance of T cells after infection and immunogenicity of vaccines. We directly compared two methods for measuring the preexisting CD4+ T cell repertoire in healthy HIV-1–negative volunteers, the HLA-peptide tetramer enrichment and T cell library technique, and show high concordance (r = 0.989). Using the library technique, we examined whether naive, central memory, and/or effector memory CD4+ T cells specific for overlapping peptides spanning the entire HIV-1 proteome were detectable in 10 HLA diverse, HIV-1–unexposed, seronegative donors. HIV-1–specific cells were detected in all donors at a mean of 55 cells/million naive cells and 38.9 and 34.1 cells/million in central and effector memory subsets. Remarkably, peptide mapping showed most epitopes recognized by naive (88%) and memory (56%) CD4+ T cells had been previously reported in natural HIV-1 infection. Furthermore, 83% of epitopes identified in preexisting memory subsets shared epitope length matches (8–12 amino acids) with human microbiome proteins, suggestive of a possible cross-reactive mechanism. These results underline the power of a proteome-wide analysis of peptide recognition by human T cells for the identification of dominant antigens and provide a baseline for optimizing HIV-1–specific helper cell responses by vaccination.


Eosinophil accumulation is a defining feature of the immune response to parasitic worm infection. Tissue-resident cells, such as epithelial cells, are thought to initiate eosinophil recruitment. However, direct recognition of worms by eosinophils has not been explored as a mechanism for amplifying eosinophil accumulation. Here, we report that eosinophils rapidly migrate toward diverse nematode species in three-dimensional culture. These include the mammalian parasite Nippostrongylus brasiliensis and the free-living nematode Caenorhabditis elegans. Surprisingly, collective migration toward worms requires paracrine leukotriene B4 signaling between eosinophils. In contrast, neutrophils show a minimal response to nematodes, yet are able to undergo robust leukotriene-dependent migration toward IgG-coated beads. We further demonstrate that eosinophils accumulate around C. elegans in the lungs of mice. This response is not dependent on bacterial products, CCR3, or complement activation. However, mice deficient in leukotriene signaling show markedly attenuated eosinophil accumulation after injection of C. elegans or N. brasiliensis. Our findings establish that nematode-derived signals can directly induce leukotriene production by eosinophils and that leukotriene signaling is a major contributor to nematode-induced eosinophil accumulation in the lung. The similarity of the eosinophil responses to diverse nematode species suggests that conserved features of nematodes are recognized during parasite infection.


Apoptosis is critical for the elimination of activated lymphocytes after viral infection. Proapoptotic factor Bim (Bcl2l11) controls T lymphocyte contraction and the formation of memory T cells after infection. Natural killer (NK) cells also undergo antigen-driven expansion to become long-lived memory cells after mouse cytomegalovirus (MCMV) infection; therefore, we examined the role of Bim in regulating the MCMV-driven memory NK cell pool. Despite responding similarly early after infection, Bcl2l11–/– Ly49H+ NK cells show impaired contraction and significantly outnumber wild-type (WT) cells after the expansion phase. The inability to reduce the effector pool leads to a larger Bcl2l11–/– NK memory subset, which displays a less mature phenotype (CD11blo, CD27+) and lower levels of NK cell memory-associated markers KLRG1 and Ly6C. Bcl2l11–/– memory NK cells demonstrate a reduced response to m157-mediated stimulation and do not protect as effectively as WT memory NK cells in an MCMV challenge model. Thus, Bim-mediated apoptosis drives selective contraction of effector NK cells to generate a pool of mature, MCMV-specific memory cells.


Follicular helper T (Tfh) cells access the B cell follicle to promote antibody responses and are particularly important for germinal center (GC) reactions. However, the molecular mechanisms of how Tfh cells are physically associated with GCs are incompletely understood. We report that the sphingosine-1-phosphate receptor 2 (S1PR2) gene is highly expressed in a subpopulation of Tfh cells that localizes in GCs. S1PR2-deficient Tfh cells exhibited reduced accumulation in GCs due to their impaired retention. T cells deficient in both S1PR2 and CXCR5 were ineffective in supporting GC responses compared with T cells deficient only in CXCR5. These results suggest that S1PR2 and CXCR5 cooperatively regulate localization of Tfh cells in GCs to support GC responses.


Microvascular plasma protein leakage is an essential component of the inflammatory response and serves an important function in local host defense and tissue repair. Mediators such as histamine and bradykinin act directly on venules to increase the permeability of endothelial cell (EC) junctions. Neutrophil chemoattractants also induce leakage, a response that is dependent on neutrophil adhesion to ECs, but the underlying mechanism has proved elusive. Through application of confocal intravital microscopy to the mouse cremaster muscle, we show that neutrophils responding to chemoattractants release TNF when in close proximity of EC junctions. In vitro, neutrophils adherent to ICAM-1 or ICAM-2 rapidly released TNF in response to LTB4, C5a, and KC. Further, in TNFR–/– mice, neutrophils accumulated normally in response to chemoattractants administered to the cremaster muscle or dorsal skin, but neutrophil-dependent plasma protein leakage was abolished. Similar results were obtained in chimeric mice deficient in leukocyte TNF. A locally injected TNF blocking antibody was also able to inhibit neutrophil-dependent plasma leakage, but had no effect on the response induced by bradykinin. The results suggest that TNF mediates neutrophil-dependent microvascular leakage. This mechanism may contribute to the effects of TNF inhibitors in inflammatory diseases and indicates possible applications in life-threatening acute edema.


Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence.


Linear ubiquitination is a newly discovered posttranslational modification that is currently restricted to a small number of known protein substrates. The linear ubiquitination assembly complex (LUBAC), consisting of HOIL-1L, HOIP, and Sharpin, has been reported to activate NF-B–mediated transcription in response to receptor signaling by ligating linear ubiquitin chains to Nemo and Rip1. Despite recent advances, the detailed roles of LUBAC in immune cells remain elusive. We demonstrate a novel HOIL-1L function as an essential regulator of the activation of the NLRP3/ASC inflammasome in primary bone marrow–derived macrophages (BMDMs) independently of NF-B activation. Mechanistically, HOIL-1L is required for assembly of the NLRP3/ASC inflammasome and the linear ubiquitination of ASC, which we identify as a novel LUBAC substrate. Consequently, we find that HOIL-1L–/– mice have reduced IL-1β secretion in response to in vivo NLRP3 stimulation and survive lethal challenge with LPS. Together, these data demonstrate that linear ubiquitination is required for NLRP3 inflammasome activation, defining the molecular events of NLRP3 inflammasome activation and expanding the role of LUBAC as an innate immune regulator. Furthermore, our observation is clinically relevant because patients lacking HOIL-1L expression suffer from pyogenic bacterial immunodeficiency, providing a potential new therapeutic target for enhancing inflammation in immunodeficient patients.


The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet’s ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.


T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c+ acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c+ human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy.


Loss of cell cycle controls is a hallmark of cancer and has a well-established role in aggressive B cell malignancies. However, the role of such lesions in indolent follicular lymphoma (FL) is unclear and individual lesions have been observed with low frequency. By analyzing genomic data from two large cohorts of indolent FLs, we identify a pattern of mutually exclusive (P = 0.003) genomic lesions that impair the retinoblastoma (RB) pathway in nearly 50% of FLs. These alterations include homozygous and heterozygous deletions of the p16/CDKN2a/b (7%) and RB1 (12%) loci, and more frequent gains of chromosome 12 that include CDK4 (29%). These aberrations are associated with high-risk disease by the FL prognostic index (FLIPI), and studies in a murine FL model confirm their pathogenic role in indolent FL. Increased CDK4 kinase activity toward RB1 is readily measured in tumor samples and indicates an opportunity for CDK4 inhibition. We find that dual CDK4 and BCL2 inhibitor treatment is safe and effective against available models of FL. In summary, frequent RB pathway lesions in indolent, high-risk FLs indicate an untapped therapeutic opportunity.


Paneth cells (PCs) are terminally differentiated, highly specialized secretory cells located at the base of the crypts of Lieberkühn in the small intestine. Besides their antimicrobial function, PCs serve as a component of the intestinal stem cell niche. By secreting granules containing bactericidal proteins like defensins/cryptdins and lysozyme, PCs regulate the microbiome of the gut. Here we study the control of PC degranulation in primary epithelial organoids in culture. We show that PC degranulation does not directly occur upon stimulation with microbial antigens or bacteria. In contrast, the pro-inflammatory cytokine Interferon gamma (IFN-) induces rapid and complete loss of granules. Using live cell imaging, we show that degranulation is coupled to luminal extrusion and death of PCs. Transfer of supernatants from in vitro stimulated iNKT cells recapitulates degranulation in an IFN--dependent manner. Furthermore, endogenous IFN- secretion induced by anti-CD3 antibody injection causes Paneth loss and release of goblet cell mucus. The identification of IFN- as a trigger for degranulation and extrusion of PCs establishes a novel effector mechanism by which immune responses may regulate epithelial status and the gut microbiome.


Mast cells play a key role in the induction of anaphylaxis, a life-threatening IgE-dependent allergic reaction, by secreting chemical mediators that are stored in secretory granules. Degranulation of mast cells is triggered by aggregation of the high-affinity IgE receptor, FcRI, and involves dynamic rearrangement of microtubules. Although much is known about proximal signals downstream of FcRI, the distal signaling events controlling microtubule dynamics remain elusive. Here we report that DOCK5, an atypical guanine nucleotide exchange factor (GEF) for Rac, is essential for mast cell degranulation. As such, we found that DOCK5-deficient mice exhibit resistance to systemic and cutaneous anaphylaxis. The Rac GEF activity of DOCK5 is surprisingly not required for mast cell degranulation. Instead, DOCK5 associated with Nck2 and Akt to regulate microtubule dynamics through phosphorylation and inactivation of GSK3β. When DOCK5–Nck2–Akt interactions were disrupted, microtubule formation and degranulation response were severely impaired. Our results thus identify DOCK5 as a key signaling adaptor that orchestrates remodeling of the microtubule network essential for mast cell degranulation.


Natural killer (NK) cell development relies on signals provided from the bone marrow (BM) microenvironment. It is thought that lymphotoxin (LT) α1β2 expressed by the NK cell lineage interacts with BM stromal cells to promote NK cell development. However, we now report that a small number of RORt+ innate lymphoid cells (ILCs), and not CD3NK1.1+ cells, express LT to drive NK development. Similar to LT–/– or RORt–/– mice, the mice conditionally lacking LTα1β2 on RORt+ ILCs experience a developmental arrest at the immature NK stages, between stages of NK development to the mature NK cell stage. This developmental block results in a functional deficiency in the clearance of NK-sensitive tumor cells. Reconstitution of Thy1+ ILCs from BM or purified RORt+ ILCs from lamina propria lymphocytes into LT-deficient RORt+ BM cultures rescues NK cell development. These data highlight a previously undiscovered role of RORt+ ILCs for NK cell development and define LT from ILCs as an essential molecule for the stromal microenvironment supporting NK cell development.


Biologics to TNF family receptors are prime candidates for therapy of immune disease. Whereas recent studies have highlighted a requirement for Fc receptors in enabling the activity of CD40, TRAILR, and GITR when engaged by antibodies, other TNFR molecules may be controlled by additional mechanisms. Antibodies to 4-1BB (CD137) are currently in clinical trials and can both augment immunity in cancer and promote regulatory T cells that inhibit autoimmune disease. We found that the action of agonist anti–4-1BB in suppressing autoimmune and allergic inflammation was completely dependent on Galectin-9 (Gal-9). Gal-9 directly bound to 4-1BB, in a site distinct from the binding site of antibodies and the natural ligand of 4-1BB, and Gal-9 facilitated 4-1BB aggregation, signaling, and functional activity in T cells, dendritic cells, and natural killer cells. Conservation of the Gal-9 interaction in humans has important implications for effective clinical targeting of 4-1BB and possibly other TNFR superfamily molecules.


Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in individuals below the age of 40. We recently reported that IL-17A is required for the development of DCMi. We show a novel pathway connecting IL-17A, cardiac fibroblasts (CFs), GM-CSF, and heart-infiltrating myeloid cells with the pathogenesis of DCMi. Il17ra–/– mice were protected from DCMi, and this was associated with significantly diminished neutrophil and Ly6Chi monocyte/macrophage (MO/M) cardiac infiltrates. Depletion of Ly6Chi MO/M also protected mice from DCMi. Mechanistically, IL-17A stimulated CFs to produce key chemokines and cytokines that are critical downstream effectors in the recruitment and differentiation of myeloid cells. Moreover, IL-17A directs Ly6Chi MO/M in trans toward a more proinflammatory phenotype via CF-derived GM-CSF. Collectively, this IL-17A–fibroblast–GM-CSF–MO/M axis could provide a novel target for the treatment of DCMi and related inflammatory cardiac diseases.


Myelin oligodendrocyte glycoprotein (MOG), a constituent of central nervous system myelin, is an important autoantigen in the neuroinflammatory disease multiple sclerosis (MS). However, its function remains unknown. Here, we show that, in healthy human myelin, MOG is decorated with fucosylated N-glycans that support recognition by the C-type lectin receptor (CLR) DC-specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN) on microglia and DCs. The interaction of MOG with DC-SIGN in the context of simultaneous TLR4 activation resulted in enhanced IL-10 secretion and decreased T cell proliferation in a DC-SIGN-, glycosylation-, and Raf1-dependent manner. Exposure of oligodendrocytes to proinflammatory factors resulted in the down-regulation of fucosyltransferase expression, reflected by altered glycosylation at the MS lesion site. Indeed, removal of fucose on myelin reduced DC-SIGN–dependent homeostatic control, and resulted in inflammasome activation, increased T cell proliferation, and differentiation toward a Th17-prone phenotype. These data demonstrate a new role for myelin glycosylation in the control of immune homeostasis in the healthy human brain through the MOG–DC-SIGN homeostatic regulatory axis, which is comprised by inflammatory insults that affect glycosylation. This phenomenon should be considered as a basis to restore immune tolerance in MS.


Rodent-borne hantaviruses are emerging human pathogens that cause severe human disease. The underlying mechanisms are not well understood, as hantaviruses replicate in endothelial and epithelial cells without causing any cytopathic effect. We demonstrate that hantaviruses strongly stimulated neutrophils to release neutrophil extracellular traps (NETs). Hantavirus infection induced high systemic levels of circulating NETs in patients and this systemic NET overflow was accompanied by production of autoantibodies to nuclear antigens. Analysis of the responsible mechanism using neutrophils from β2 null mice identified β2 integrin receptors as a master switch for NET induction. Further experiments suggested that β2 integrin receptors such as complement receptor 3 (CR3) and 4 (CR4) may act as novel hantavirus entry receptors. Using adenoviruses, we confirmed that viral interaction with β2 integrin induced strong NET formation. Collectively, β2 integrin–mediated systemic NET overflow is a novel viral mechanism of immunopathology that may be responsible for characteristic aspects of hantavirus-associated disease such as kidney and lung damage.


Insight from William Muller

Insight from Margaret Goodell

Insight from Bertrand Boisson (left) and Jean-Laurent Casanova (right)