Soluble NS1 Antagonizes IgG- and IgA- Mediated Monocytic Phagocytosis of DENV Infected Cells.

Dengue virus (DENV) is endemic in >100 countries, infecting an estimated 400 million individuals every year. Infection with DENV raises an antibody response primarily targeting viral structural proteins. However, DENV encodes several immunogenic nonstructural (NS) proteins, one of which, NS1, is expressed on the membrane of DENV-infected cells. IgG and IgA isotype antibodies that bind NS1 are abundant in serum following DENV infection. Our study aimed to determine if NS1-binding IgG and IgA isotype antibodies contribute to the clearance of DENV-infected cells by antibody-mediated cellular phagocytosis. We observed that both IgG and IgA isotype antibodies can facilitate monocytic uptake of DENV NS1-expressing cells in an FcγRI- and FcαRI-dependent fashion. Interestingly, this process was antagonized by the presence of soluble NS1, suggesting that the production of soluble NS1 by infected cells may serve as immunological chaff, antagonizing opsonization and clearance of DENV-infected cells.

Early T-bet promotes LFA1 upregulation required for CD8+ effector and memory T cell development.

The T-box transcription factor T-bet is regarded as a "master regulator" of CD4+ Th1 differentiation and IFN-γ production. However, in multiple models of infection, T-bet appears less critical for CD8+ T cell expansion and effector function. Here, we show that following vaccination with a replication-deficient strain of Toxoplasma gondii, CD8+ T cell expression of T-bet is required for optimal expansion of parasite-specific effector CD8+ T cells. Analysis of the early events associated with T cell activation reveals that the α chain of LFA1, CD11a, is a target of T-bet, and T-bet is necessary for CD8+ T cell upregulation of this integrin, which influences the initial priming of CD8+ effector T cells. We propose that the early expression of T-bet represents a T cell-intrinsic factor that optimizes T-DC interactions necessary to generate effector responses.

Mechanically active integrins target lytic secretion at the immune synapse to facilitate cellular cytotoxicity.

Cytotoxic lymphocytes fight pathogens and cancer by forming immune synapses with infected or transformed target cells and then secreting cytotoxic perforin and granzyme into the synaptic space, with potent and specific killing achieved by this focused delivery. The mechanisms that establish the precise location of secretory events, however, remain poorly understood. Here we use single cell biophysical measurements, micropatterning, and functional assays to demonstrate that localized mechanotransduction helps define the position of secretory events within the synapse. Ligand-bound integrins, predominantly the αLß2 isoform LFA-1, function as spatial cues to attract lytic granules containing perforin and granzyme and induce their fusion with the plasma membrane for content release. LFA-1 is subjected to pulling forces within secretory domains, and disruption of these forces via depletion of the adaptor molecule talin abrogates cytotoxicity. We thus conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance their cytotoxic power and fidelity.

TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines.

TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.

Cryptosporidium rhoptry effector protein ROP1 injected during invasion targets the host cytoskeletal modulator LMO7.

The parasite Cryptosporidium invades and replicates in intestinal epithelial cells and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms that underlie infection and pathogenesis are largely unknown. Here, we delineate the events of host cell invasion and uncover a mechanism unique to Cryptosporidium. We developed a screen to identify parasite effectors, finding the injection of multiple parasite proteins into the host from the rhoptry organelle. These factors are targeted to diverse locations within the host cell and its interface with the parasite. One identified effector, rhoptry protein 1 (ROP1), accumulates in the terminal web of enterocytes through direct interaction with the host protein LIM domain only 7 (LMO7) an organizer of epithelial cell polarity and cell-cell adhesion. Genetic ablation of LMO7 or ROP1 in mice or parasites, respectively, impacts parasite burden in vivo in opposite ways. Taken together, these data provide molecular insight into how Cryptosporidium manipulates its intestinal host niche.

Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Lymphocyte egress signal sphingosine-1-phosphate promotes ERM-guided, bleb-based migration.

Ezrin, radixin, and moesin (ERM) family proteins regulate cytoskeletal responses by tethering the plasma membrane to the underlying actin cortex. Mutations in ERM proteins lead to severe combined immunodeficiency, but the function of these proteins in T cells remains poorly defined. Using mice in which T cells lack all ERM proteins, we demonstrate a selective role for these proteins in facilitating S1P-dependent egress from lymphoid organs. ERM-deficient T cells display defective S1P-induced migration in vitro, despite normal responses to standard protein chemokines. Analysis of these defects revealed that S1P promotes a fundamentally different mode of migration than chemokines, characterized by intracellular pressurization and bleb-based motility. ERM proteins facilitate this process, controlling directional migration by limiting blebbing to the leading edge. We propose that the distinct modes of motility induced by S1P and chemokines are specialized to allow T cell migration across lymphatic barriers and through tissue stroma, respectively.

A Murine Model of X-Linked Moesin-Associated Immunodeficiency (X-MAID) Reveals Defects in T Cell Homeostasis and Migration.

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesinR171W). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesinR171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesinR171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesinR171W. Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesinR171W, and will provide new insights into how the actin cortex regulates lymphocyte function.

Automated cell tracking using StarDist and TrackMate.

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images.

LFA-1 signals to promote actin polymerization and upstream migration in T cells.

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the phosphoinositide 3-kinase (PI3K) and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, whereas VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl (also known as CBL), failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior.This article has an associated First Person interview with the first author of the paper.

Ectromelia-encoded virulence factor C15 specifically inhibits antigen presentation to CD4+ T cells post peptide loading.

Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4+ but not CD8+ T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4+ T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4+ T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4+ T cell activation, reflecting the importance of this cell type in control of the virus.

CrkL is required for donor T cell migration to GvHD target organs.

The success of cancer therapies based on allogeneic hematopoietic stem cell transplant relies on the ability to separate graft-versus-host disease (GvHD) from graft-versus-tumor (GVT) responses. Controlling donor T cell migration into peripheral tissues is a viable option to limit unwanted tissue damage, but a lack of specific targets limits progress on this front. Here, we show that the adaptor protein CrkL, but not the closely related family members CrkI or CrkII, is a crucial regulator of T cell migration. In vitro, CrkL-deficient T cells fail to polymerize actin in response to the integrin ligand ICAM-1, resulting in defective migration. Using a mouse model of GvHD/GVT, we found that while CrkL-deficient T cells can efficiently eliminate hematopoietic tumors they are unable to migrate into inflamed organs, such as the liver and small intestine, and thus do not cause GvHD. These results suggest a specific role for CrkL in trafficking to peripheral organs but not the lymphatic system. In line with this, we found that although CrkL-deficient T cells could clear hematopoietic tumors, they failed to clear the same tumor growing subcutaneously, highlighting the role of CrkL in controlling T cell migration into peripheral tissues. Our results define a unique role for CrkL in controlling T cell migration, and suggest that CrkL function could be therapeutically targeted to enhance the efficacy of immunotherapies involving allogeneic donor cells.

Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1.

T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on "outside-in" signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function-associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling-induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.

The Actin Cytoskeleton: A Mechanical Intermediate for Signal Integration at the Immunological Synapse.

The immunological synapse (IS) is a specialized structure that serves as a platform for cell-cell communication between a T cell and an antigen-presenting cell (APC). Engagement of the T cell receptor (TCR) with cognate peptide-MHC complexes on the APC activates the T cell and instructs its differentiation. Proper T cell activation also requires engagement of additional receptor-ligand pairs, which promote sustained adhesion and deliver costimulatory signals. These events are orchestrated by T cell actin dynamics, which organize IS components and facilitate their signaling. The actin network flows from the edge of the cell inward, driving the centralization of TCR microclusters and providing the force to activate the integrin LFA-1. We recently showed that engagement of LFA-1 slows actin flow, and that this affects TCR signaling. This study highlights the physical nature of the IS, and contributes to a growing appreciation in the field that mechanosensing and mechanotransduction are essential for IS function. Additionally, it is becoming clear that there are multiple types of actin structures at the IS that promote signaling in distinct ways. How the different actin structures contribute to force production and mechanotransduction is just beginning to be explored. In this Perspective, we will feature recent work from our lab and others, that collectively points toward a model in which actin dynamics drive mechanical signaling and receptor crosstalk during T cell activation.

Integrins Modulate T Cell Receptor Signaling by Constraining Actin Flow at the Immunological Synapse.

Full T cell activation requires coordination of signals from multiple receptor-ligand pairs that interact in parallel at a specialized cell-cell contact site termed the immunological synapse (IS). Signaling at the IS is intimately associated with actin dynamics; T cell receptor (TCR) engagement induces centripetal flow of the T cell actin network, which in turn enhances the function of ligand-bound integrins by promoting conformational change. Here, we have investigated the effects of integrin engagement on actin flow, and on associated signaling events downstream of the TCR. We show that integrin engagement significantly decelerates centripetal flow of the actin network. In primary CD4+ T cells, engagement of either LFA-1 or VLA-4 by their respective ligands ICAM-1 and VCAM-1 slows actin flow. Slowing is greatest when T cells interact with low mobility integrin ligands, supporting a predominately drag-based mechanism. Using integrin ligands presented on patterned surfaces, we demonstrate that the effects of localized integrin engagement are distributed across the actin network, and that focal adhesion proteins, such as talin, vinculin, and paxillin, are recruited to sites of integrin engagement. Further analysis shows that talin and vinculin are interdependent upon one another for recruitment, and that ongoing actin flow is required. Suppression of vinculin or talin partially relieves integrin-dependent slowing of actin flow, indicating that these proteins serve as molecular clutches that couple engaged integrins to the dynamic actin network. Finally, we found that integrin-dependent slowing of actin flow is associated with reduction in tyrosine phosphorylation downstream of the TCR, and that this modulation of TCR signaling depends on expression of talin and vinculin. More generally, we found that integrin-dependent effects on actin retrograde flow were strongly correlated with effects on TCR signaling. Taken together, these studies support a model in which ligand-bound integrins engage the actin cytoskeletal network via talin and vinculin, and tune TCR signaling events by modulating actin dynamics at the IS.

Murine chronic graft-versus-host disease proteome profiling discovers CCL15 as a novel biomarker in patients.

Improved diagnostic and treatment methods are needed for chronic graft-versus-host disease (cGVHD), the leading cause of late nonrelapse mortality (NRM) in long-term survivors of allogenic hematopoietic cell transplantation. Validated biomarkers that facilitate disease diagnosis and classification generally are lacking in cGVHD. Here, we conducted whole serum proteomics analysis of a well-established murine multiorgan system cGVHD model. We discovered 4 upregulated proteins during cGVHD that are targetable by genetic ablation or blocking antibodies, including the RAS and JUN kinase activator, CRKL, and CXCL7, CCL8, and CCL9 chemokines. Donor T cells lacking CRK/CRKL prevented the generation of cGVHD, germinal center reactions, and macrophage infiltration seen with wild-type T cells. Whereas antibody blockade of CCL8 or CXCL7 was ineffective in treating cGVHD, CCL9 blockade reversed cGVHD clinical manifestations, histopathological changes, and immunopathological hallmarks. Mechanistically, elevated CCL9 expression was present predominantly in vascular smooth muscle cells and uniquely seen in cGVHD mice. Plasma concentrations of CCL15, the human homolog of mouse CCL9, were elevated in a previously published cohort of 211 cGVHD patients compared with controls and associated with NRM. In a cohort of 792 patients, CCL15 measured at day +100 could not predict cGVHD occurring within the next 3 months with clinically relevant sensitivity/specificity. Our findings demonstrate for the first time the utility of preclinical proteomics screening to identify potential new targets for cGVHD and specifically CCL15 as a diagnosis marker for cGVHD. These data warrant prospective biomarker validation studies.

Embracing the Enemy: Cell-to-Cell Force Transmission Enhances Cytotoxicity.

Cells respond to the mechanical properties of their environment, but how biomechanics contributes to intercellular signaling remains unclear. Reporting in Cell, Basu et al. (2016) showed that forces exerted by cytotoxic T lymphocytes enhance the function of the pore-forming protein perforin, thereby leading to more effective target cell killing.

HIV-1-Induced Small T Cell Syncytia Can Transfer Virus Particles to Target Cells through Transient Contacts.

HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing "fusion sinks" are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis.

Vpu is the main determinant for tetraspanin downregulation in HIV-1-infected cells.

UNLABELLED: Tetraspanins constitute a family of cellular proteins that organize various membrane-based processes. Several members of this family, including CD81, are actively recruited by HIV-1 Gag to viral assembly and release sites. Despite their enrichment at viral exit sites, the overall levels of tetraspanins are decreased in HIV-1-infected cells. Here, we identify Vpu as the main viral determinant for tetraspanin downregulation. We also show that reduction of CD81 levels by Vpu is not a by-product of CD4 or BST-2/tetherin elimination from the surfaces of infected cells and likely occurs through an interaction between Vpu and CD81. Finally, we document that Vpu-mediated downregulation of CD81 from the surfaces of infected T cells can contribute to preserving the infectiousness of viral particles, thus revealing a novel Vpu function that promotes virus propagation by modulating the host cell environment. IMPORTANCE: The HIV-1 accessory protein Vpu has previously been shown to downregulate various host cell factors, thus helping the virus to overcome restriction barriers, evade immune attack, and maintain the infectivity of viral particles. Our study identifies tetraspanins as an additional group of host factors whose expression at the surfaces of infected cells is lowered by Vpu. While the downregulation of these integral membrane proteins, including CD81 and CD82, likely affects more than one function of HIV-1-infected cells, we document that Vpu-mediated lowering of CD81 levels in viral particles can be critical to maintaining their infectiousness.